Cytological, genetic and agronomic characterization of a barley reciprocal translocation
Alba Farré Martinez
Cytological, genetic and agronomic characterization of a barley reciprocal
translocation
Alba Farré Martinez
ii
Thesis committee Thesis supervisor Prof dr. F.A. van Eeuwijk Professor of Applied Statistics Wageningen University Thesis co-supervisors Prof dr. I. Romagosa Professor of Crop Science University of Lleida, Spain Dr.ir. J. Jansen Senior Research Scientist, Biometris Wageningen UR Other members
Prof. dr. J. Voltas, University of Lleida, Spain Dr. W.T.B. Thomas, The James Hutton Institute, Dundee, UK Prof. dr. R.G.F. Visser, Wageningen University Prof. dr. J.H.S.G.M. de Jong, Wageningen University This research was conducted under the auspices of the C.T. de Wit Graduate School of Production Ecology and Resource Conservation.
iii
Cytological, genetic and agronomic characterization of a barley reciprocal
translocation
Alba Farré Martinez
Thesis submitted in fulfillment of the requirements for the degree of doctor
at Wageningen University by the authority of the Rector Magnificus
Prof. dr. M.J. Kropff, in the presence of the
Thesis Committee appointed by the Academic Board to be defended in public
on Wednesday 17 October 2012 at 4 p.m. in the Aula.
iv
Alba Farré Martinez Cytological, genetic and agronomic characterization of a barley reciprocal translocation, 130 pages.
PhD thesis Wageningen University, Wageningen, The Netherlands (2012) With references, with summaries in Dutch and English ISBN 978-94-6173-411-2
vii
Contents
Chapter 1 General introduction …………………………………………………….5
Chapter 2 Linkage map construction involving a reciprocal translocation ……….15
Chapter 3 General characterization of a reciprocal translocation present in a widely
grown barley variety …………………………………………………...31
Chapter 4 Agronomic effects of a large reciprocal translocation present in a widely
grown Spanish barley variety …………………………………….…....49
Chapter 5 Construction of an integrated genetic linkage map of barley (Hordeum
vulgare L.) involving a parent carrying a reciprocal translocation .…...55
Chapter 6 QTL analysis in multiple interconnected barley doubled haploid
populations in the presence of a reciprocal translocation …………...…83
Chapter 7 General discussion ………………………………………….…….…..106
References ……………………………………………………………….………..….115
Summary
Samenvatting
Acknowledgements
Curriculum vitae
1
Chapter 1
General introduction
Today, cereals are still the basis of global agriculture by providing more than half the
human food consumption. Among the cereals, barley ranks fourth after maize (Zea
mays), wheat (Triticum ssp.) and rice (Oryza sativa) with the widest range of production
areas in the world (130-140 million tonnes in the recent years; Horsley et al. 2009). In
Spain, barley is the cereal with the largest cultivated area (2.8 million ha), 46% of the
total area used for growing cereals, with an annual production of 9 million tonnes which
corresponds with approximately 51% of the total cereal production (MARM; 2011).
Cultivated barley (Hordeum vulgare) is a diploid species (2n = 2x =14), with some
tetraploid and hexaploid wild relatives (Martin et al. 1999). It has been derived from the
wild progenitor Hordeum vulgare ssp spontaneum, which continues to grow in the
Middle East. Barley is one of the most widely adapted plant species grown in a wide
range of environments from near-arctic to semi-desert. Barley varieties have been
classified according to their morphology and agronomic adaptation. Morphological,
barley varieties can be divided according the number of kernel rows in the head: two-
row (only one spikelet at each node is fertile) and six-row (all three spikelets are fertile).
Considering agronomic adaptation, varieties can be classified into three types: winter,
spring and facultative. Winter barley varieties are tolerant to low temperature, sensitive
to daylenght and require vernalization. Spring barley varieties do not require
vernalization, and are typically insensitive to short day photoperiod. Facultative barley
varieties are tolerant to low temperatures, but do not require vernalization (Zitzewitz et
al. 2005).
Despites its large genome (approximately 5,100 Mb; Arumuganathan and Earle 1991;
Doležel et al. 1998) barley provides an excellent model for other Triticeae species
because it is a diploid self-pollinating species with a huge genetic variability. It has been
found that the large size of its genome is not due to the presence of a greater number of
genes in comparison with other species from the Triticeae tribe (family Poaceae), but to
the presence of highly repetitive DNA (80% of the genome), mostly retrotransposons (
Chapter 1
2
Bennetzen et al. 1998; Ramakrishna et al. 2002; Schulman et al. 2004). In fact, it has
been estimated that barley has around 32,000 genes, which is not very different from
rice (41,000 genes, Jung et al. 2008), Sorghum (34,500 genes, Paterson et al. 2009) or
Brachypodium (30,000 genes, The International Brachypodium Initiative 2010). The
genetic progress in barley has been focused on improving: yield and stability, resistance
against diseases and pests, malting quality and quality as animal feed. Barley is grown
for many purposes, but most barley is used as animal feed, human food and the
production of malt.
The barley variety ‘Albacete’
Conventional plant breeding have been successful in improving yield and yield stability
of some important crops under favourable conditions or to conditions which could be
made favourable by the use of fertilizers, irrigation, pesticides and fungicides. However,
current research is focused on raising yields under low-input conditions, which are more
self-sustaining and less harmful to the environment.
In the semiarid areas of Spain crop productivity is not only limited by drought, but also
by high temperatures at the end of the growth cycle. The increased use of ‘local’
germplasm could lead to the selection of varieties adapted to specific regional
conditions. So, the use of a barley variety like ‘Albacete’, with its proven adaptation to
semiarid conditions, can be an excellent option.
In Spain ‘Albacete’ was the most widely grown barley variety during the last decades
(up to 1 million ha/year), particularly in semiarid rain-fed areas. This variety was
obtained more than 50 years ago by Enrique Sánchez-Monge at the Estación
Experimental Aula Dei (Spanish Research Council) in Zaragoza, as an inbred line in a
local heterogeneous landrace population from the Albacete province. It is a six-row
facultative variety with a relatively long growth cycle but showing lodging at the end of
the cycle. Its yield, in optimal environmental conditions, is average compared to other
cultivars. However, its tolerance to cold and drought makes ‘Albacete’ the best choice
in years with low rainfall. For all these properties, it has been grown for decades,
particularly in drought-prone areas.
General Introduction
3
Lasa et al. (1987) investigated different barley populations under drought conditions in
Spain using ‘Albacete’ cultivar as a check. They found significant differences between
the populations and between the populations and ‘Albacete’ for all the traits (related to
crop development, growth and yield components). For most of the traits, the local
cultivar ‘Albacete’ gave the best results, with the exception of test weight and lodging.
‘Albacete’ was the latest in flowering, and had the shortest grain filling period. Later,
Arnau et al. (1997) studied the effect of water stress on plant water status and net
photosynthetic gas exchange in six barley genotypes differing in productivity and
drought tolerance. ‘Albacete’ showed the highest osmotic adjustment capacity, relative
water content and photosynthetic gas exchange. The high osmotic adjustment capacity
of ‘Albacete’ could explain its high yield under very dry conditions.
Intense breeding efforts to cross ‘Albacete’ with other varieties during the 1970s and
80s failed due to the sterility of the offspring, which gave ‘Albacete’ a poor reputation
as a parent in breeding programmes. Vazquez and Sanchez-Monge (1987) found the
cytological cause of this sterility by carrying out meiotic analyse of semi-sterile F1
hybrids involving ‘Albacete’. The presence at metaphase-anaphase I of five bivalents
and one quadrivalent, representing 75% of PMC (pollen mother cells) (Figure 1),
provided clear evidence of the existence of a reciprocal translocation. This high
percentage indicates that the interchanged segments must be of considerable length.
They also detected that the coorientation of the quadrivalent in the hybrids of ‘Albacete’
was mainly of the adjacent type (74%) and the average fertility of the translocation
heterozygotes was 54%. However, they did not identify the chromosomes involved. A
preliminary study has revealed that chromosomes 1H and 3H are involved in the
reciprocal translocation (Lacasa-Benito et al. 2005). ‘Albacete’ is the only known,
widely cultivated barley variety that carries a reciprocal translocation. Until now, it is
unknown whether the translocation is responsible for the drought tolerance and whether
it has some interesting phenotypic attributes that could be transferred to other barley
genotypes to develop specific germplasm adapted to stress.
Chapter 1
4
Figure 1. Meiotic metaphase of F1 hybrid involving ‘Albacete’ showing 5 II + 1 IV (adjacent
coorientation). (provided by L. Cistué)
Chromosome rearrangements
Chromosome rearrangements include deletions, duplications, inversions and
translocations have played an important role in plant evolution. Each of these events can
be caused by chromosome breakages which are followed by the reunion of broken ends
(Figure 2a) or crossing-over between repetitive DNA segments (Figure 2b). Sometimes
these breaks will occur within genes generally disrupting gene functions.
There are two general types of rearrangements, balanced and imbalanced. Imbalanced
rearrangements change the total amount of chromosomal material of the affected
chromosomes. The two simple classes of imbalanced rearrangements are deletions and
duplications. Some examples in human are the cri du chat (del (5p)) or Wolf-
Hirschhorn (del (18p); del (18q); del (4p)) syndromes. In contrast, balanced
rearrangements change the chromosomal gene order but do not change the total amount
of chromosomal material. The two simple classes of balanced rearrangements are
inversions and reciprocal translocations.
General Introduction
5
Figure 2. Origins of chromosomal rearrangements: (a) by breakage and rejoining; (b) by crossing-
over between repetitive DNA
Reciprocal translocations are produced by an interchange of chromosome segments
between two non-homologous chromosomes. Rieger et al. (1968) classified the
reciprocal translocations into asymmetrical or symmetrical translocations depending on
the way the chromosome segments rejoin after a chromosomal breakage (Figure 3). In
an asymmetrical translocation, the segments carrying the centromeres are joined
together resulting in an acentric (without centromere) and a dicentric (with two
centromeres) chromosomes. Standard monocentric chromosomes are formed in
symmetrical translocations, which are often viable.
(a)
(b)
= break
= repetitive DNA segments
= rejoining
= crossover
Chapter 1
6
Figure 3. Possible products of a reciprocal translocation interchange
When a symmetrical reciprocal translocation occurs, two new chromosomal
arrangements appear, so the individuals can be: (i) homozygotes for the standard
arrangement (Figure 4a); (ii) homozygotes for the interchange (Figure 4b) and (iii)
heterozygotes for the interchange (Figure 4c). For both homozygotes the meiotic
behaviour is regular as they form bivalents. The complex meiosis for the structural
heterozygote is explained below.
Figure 4. Possible individuals resulting from the reciprocal translocation
Meiosis in heterozygotes having two translocated chromosomes and their normal
counterparts causes some important genetic and cytological effects. During meiotic
prophase I homologous chromosomes interact with each other through precise synapsis
and crossover recombination. In the case of a reciprocal translocation heterozygote,
saturated pairing gives rise to a quadrivalent (association of 4 chromosomes) involving
the two translocation chromosomes and their non-translocation counterparts instead of
two independent bivalents in the normal situation (Burnham 1962; Sybenga 1975).
Various configurations can be observed at diakinesis–metaphase I, depending on the
number and positions of the crossing overs: zig-zag shapes, rings and open forms of 2–4
chromosomes, as well as univalents. As shown in Figure 5, crossing overs may occur in
three types of chromosomal segments: the interchanged segment, the interstitial
segment (between translocation points and centromeres) and the non-interchanged
segments (chromosome arms distal to the centromeres).
a) b) c)
Symmetrical
Asymmetrical
General Introduction
7
Figure 5. Pairing diagram of translocation heterozygote (1 and 2 are the normal chromosome
arrengement, 12 and 21 are the translocated chromosomes)
If no crossing over takes place in the interstitial segments, the viability of the resulting
gametes will depend upon the coorientation of the centromeres at metaphase I. These
could be: (i) alternate segregation (corresponding with a zig-zag configuration of the
quadrivalent) or (ii) adjacent segregation (corresponding with a ring configuration of the
quadrivalent). The latter type is further divided into two classes: Adjacent-2 and
Adjacent-1 segregations, according to the separation of homologous centromeres, and
whether they end up into the same Telophase I cell or not, respectively. Adjacent
segregation leads to gametes with a translocation and a non-translocation chromosome,
containing a duplicated and a deleted segment; this leads to non-viable gametes.
Alternate segregation occurs when the chromosomes on opposite sides of the
translocation cross segregate into the same pole. In these cases, four balanced gametes
are formed: 50% carry the normal chromosome arrangement, and 50% have the
translocated chromosomes. Hence, depending on the relative frequency of the
alternative orientation, individuals that are heterozygous for a translocation will produce
a certain proportion of non-viable gametes. This results in a reduction of fertility; it is
called semi-sterility.
If we assume that a crossover occurs in one interstitial segment followed by alternate or
adjacent-1 segregations, 50% of the gametes belong to the non-recombinant parental
type and the other 50%, recombinant, carry a segment deficiency and duplication and
are usually non-viable. The same crossover followed by adjancent-2 will produced non-
viable gametes. It is important to note that, in barley, the alternate segregation in the
interchange heterozygote is more frequent than the others types of separations, therefore
crossing over will further reduce genetic recombination of loci within the interstitial
segment because the chromatids carrying the crossover pass to the unbalanced gametes
(Burnham, 1964). Therefore the presence of a reciprocal translocation reduces
1 21
2 12
× × × ×
×
×
Chapter 1
8
recombination frequencies between the translocation breakpoints (TB) and the
centromere. If the TB is close to or in the centromere region, the reduction in
recombination that may take place will be due to the interference effects in the flanking
TB region.
Lacadena (1989) showed that more complex combinations of crossovers may lead to
balanced gametes carrying recombination within the interstitial segment. For instance, if
a single crossover occurs in both interstitial segments and in non-interchanged
chromosome arms distal to the centromeres, it may happen that with adjacent
segregation some gametes results in balanced recombinant chromatids. Following the
same reasoning, the alternate segregation could generate unbalanced gametes. Thus, the
possibility of crossing-over in the interstitial region can alter the viability of gametes
produced after an adjacent or alternate segregation.
Reciprocal translocations may occur spontaneously or in response to irradiation or
treatments with certain chemicals. The first report on a naturally occurring reciprocal
translocation was by Belling (1914) in the Florida velvet bean (Stizolobium
deeringianum), and Muller (1930) was first to report on X-ray-induced translocations in
Drosophila. In barley, few cases of spontaneous reciprocal translocations have been
described in cultivated barley, although they are induced frequently by mutagen
treatments. Konishi and Linde-Laursen (1988) conducted an exhaustive study which
showed that chromosomal rearrangements such as inversions and translocations rarely
occur spontaneously in barley. They investigated 1,240 cultivated barley lines and 120
wild barley lines to detect spontaneous reciprocal translocations. Of the 1,240 cultivated
barley lines, four Ethiopian landraces carrying a reciprocal translocation had the same
breakpoints at the centromere involving chromosomes 2H and 4H (2HS·4HS and
2HL·4HL), suggesting that the chromosomes involved in the rearrangement had a
common origin. Of the 120 wild barley genotypes, three carried translocations between
chromosomes 2H and 4H, 3H and 5H and 3H and 6H, respectively.
Translocations have been widely utilized in both applied and fundamental scientific
research for chromosome mapping, in particular for assigning linkage groups to
chromosomes, development of physical maps in plants (Kim et al. 1993; Sorokin et al.
1994; Marthe and Künzel 1994; Künzel et al. 2000), and to improve our understanding
General Introduction
9
of meiotic chromosome pairing behaviour (Rickards 1983). They have been reported in
a number of crop species, such as rye (Benito et al. 1994), soybean (Mahama and
Palmer 2003), Prunus (Jáuregui et al. 2001), Lens (Tadmor et al. 1987), pea (Kosterin et
al. 1999), wheat (Naranjo et al. 1987; Badaeva et al. 2007), Brassica napus (Osborn et
al. 2003) and other crops.
Methodological aspects
Some methodological problems arise when dealing with reciprocal translocations:
i. Map construction:
Specific methodology for linkage map construction in the presence of a
reciprocal translocation has to be developed in order to cope with the ‘pseudo-
linkage’ present between the chromosomes involved in the interchanges.
Durrant et al. (2006) introduced QuadMap, a software application, to analyze
linkage maps affected by translocations. Large intermarker distance variance
was proposed as an indication of the presence of a breakpoint. However, high
variances may also be caused by gross deficiencies in the data.
ii. Integrating linkage maps:
Integrating linkage maps of multiple populations requires powerful methods to
handle the huge number of missing data due to markers not segregating in all
populations. A widely used platform for the construction of genetic linkage
maps is provided by JoinMap®. However, the regression algorithm of
JoinMap® fails when it is required to integrate linkage maps involving large
numbers of markers. Besides that, the effect of the reciprocal translocation on
the construction of a linkage map must be taken into account.
iii. QTL analysis in multiple populations:
QTL methods have been developed for multi-trait and multi-environment but
few studies have been published using interconnected mapping populations in
cereals (i.e. Blanc et al. 2006, 2008; Cuesta-Marcos et al. 2008a; Liu et al.
2010). The model used for QTL analysis must involve a correction for the
Chapter 1
10
effect of the translocation as the reciprocal translocation is present in a part of
the populations. In populations involving the translocations we have to deal
with split linkage maps.
Scope of the thesis
The aim of the thesis is to develop an in depth understanding of the reciprocal
translocation present in the widely grown Spanish barley variety ‘Albacete’. To achieve
this objective different complementary approaches will be followed. First, a statistical-
genetic approach will be developed for the construction of a linkage maps in
populations obtained from reciprocal translocation heterozygotes using a double haploid
population (Chapter 2). Second, the reciprocal translocation present in ‘Albacete’ will
be characterized by a combination of cytological and molecular genetic approaches
(Chapter 3). Third, a large number of doubled haploid lines derived from four crosses
involving ‘Albacete’ as one of the parents will be used to phenotypically characterize
the effects of the reciprocal translocation on some interesting agronomic traits at two
locations in two growing seasons (Chapter 4). Fourth, once we know how to construct a
linkage map if one of the parents carries a reciprocal translocation, use will be made of
the statistical-genetic approach to construct an integrated genetic linkage map
combining five interconnected biparental doubled haploid populations involving four
barley varieties (‘Albacete’, ‘Barberousse’, ‘Plaisant’ and ‘Orria’) (Chapter 5). Fifth,
development of specific methodology for the simultaneous analysis of these
interconnected populations for QTL analysis in the presence of a reciprocal
translocation (Chapter 6).
11
Chapter 2
Linkage map construction involving a reciprocal
translocation
A. Farré1,4 I. Lacasa Benito1 L. Cistué2 J.H. de Jong3 I. Romagosa1 J. Jansen4
Published in Theor Appl Genet (2011) 122:1029-1037
Abstract
This paper is concerned with a novel statistical-genetic approach for the construction of
linkage maps in populations obtained from reciprocal translocation heterozygotes of
barley (Hordeum vulgare L.). Using standard linkage analysis, translocations usually
lead to 'pseudo-linkage': the mixing up of markers from the chromosomes involved in
the translocation into a single linkage group. Close to the translocation breakpoints
recombination is severely suppressed and, as a consequence, ordering markers in those
regions is not feasible. The novel strategy presented in this paper is based on (1)
disentangling the "pseudo-linkage" using principal coordinate analysis, (2) separating
individuals into translocated types and normal types and (3) separating markers into
those close to and those more distant from the translocation breakpoints. The methods
make use of a consensus map of the species involved. The final product consists of
integrated linkage maps of the distal parts of the chromosomes involved in the
translocation.
Keywords Linkage maps Reciprocal translocation Translocation breakpoint
Barley Principal coordinate analysis Graphical genotypes
1Department of Plant Production and Forest Science, University of Lleida, Lleida, Spain; 2Estación Experimental de Aula Dei, CSIC, Zaragoza, Spain; 3Laboratory of Genetics, Wageningen University and Research Centre, Wageningen, the Netherlands; 4Biometris, Wageningen University and Research Centre, Wageningen, the Netherlands
Chapter 2
12
Introduction
At present barley (Hordeum vulgare L.) is the fourth most important cereal crop, after
maize, wheat and rice. Barley is a diploid (2n = 14) self-pollinating species which can
be used as a model plant for other Triticeae species providing an excellent system for
genome mapping and map-based analyses (Costa et al. 2001). Well-established linkage
maps containing morphological, chromosomal, protein and molecular markers are
indispensable tools for genetic analysis and for analyzing DNA sequence variation
between the crops and its relative accessions and species. In barley, common molecular
marker technologies include restriction digest based RFLPs (Restriction Fragment
Length Polymorphisms; Graner et al. 1991; Heun et al. 1991; Kleinhofs et al. 1993),
and PCR-based markers like AFLPs (Amplified Fragment Length Polymorphism; Vos
et al. 1995), STS (Sequence Tagged Site; Olson et al. 1989), SSRs (Simple Sequence
Repeat; Liu et al. 1996; Ramsay et al. 2000; Pillen et al. 2000; Li et al. 2003), and
recently, DArT (Diversity Arrays Technology), a technique based on DNA
hybridization (Jaccoud et al. 2001; Wenzl et al. 2004). The more recent SNPs (Single
Nucleotide Polymorphisms) have become popular thanks to a high density within the
genome, easy development from sequence data and high reproducibility. Wenzl et al.
(2006) constructed a high-density linkage map which will be used as a reference for
future mapping work. The map was based on datasets from multiple populations,
comprising 2,935 loci (2,085 DArT, 850 SSR, RFLP and/or STS markers) and spans
1,161 cM. Recently, Varshney et al. (2007) produced a high-density linkage map, using
SSR marker data from six mapping populations. A total of 775 microsatellite loci from
688 primer pairs were used to cover 1,068 cM of the barley genome.
Major complications in the construction of genetic maps arose in populations containing
reciprocal translocation heterozygotes (Lehmensiek et al. 2005). Reciprocal
translocations involve the exchange of two terminal segments between two non-
homologous chromosomes. Due to reciprocal translocations recombination between loci
around the translocation breakpoints will be suppressed. Consequently, markers in these
regions become 'pseudolinked', i.e. linkage is detected between markers lying on
different chromosomes. Reciprocal translocations are induced spontaneously or
artificially by ionizing radiation, mutagens or by transposons and can be confirmed by
aberrant chromosome and banding morphology, by establishing quadrivalent formation
Linkage map construction involving a reciprocal translocation
13
in metaphase I complements, and semisterility of pollen in translocation heterozygotes,
and by linkage analysis (Jáuregui et al. 2001). Translocations are well-documented in
various crops, including rye (Alonso-Blanco et al. 1993; Benito et al. 1994; Catarino et
al. 2006), soybean (Mahama and Palmer. 2003), Prunus (Jáuregui et al. 2001), Lens
(Tadmor et al. 1987) and pea (Kosterin et al. 1999). Many bread wheat cultivars contain
the 1RS-1BL wheat-rye chromosome translocation, introduced to provide resistance to
pests and diseases (Mago et al. 2002; Sharma et al. 2009).
In barley, spontaneous reciprocal translocations were described by Konishi and Linde-
Laursen (1988) who investigated 1,240 barley landraces and varieties and 120 wild
barley lines, using test-cross and Giemsa chromosome banding analyses. Of the 1,240
landraces and varieties, four carried translocations involving chromosomes 2H and 4H
with breakpoints near or at the centromere. Of the 120 wild barley lines, three carried
translocations between chromosomes 2H and 4H, 3H and 5H and 3H and 6H,
respectively. 'Albacete' is a barley variety that it is known to have a reciprocal
chromosomal translocation. Recent results suggest that chromosomes 1H and 3H are
involved in this translocation (Lacasa-Benito et al. 2005).
The consensus map of barley (Wenzl et al. 2006) was used as a reference. Preliminary
linkage analysis of a doubled haploid population obtained from a cross between
'Albacete' and the non-translocation barley variety 'Barberousse' shows that linkage
groups 1H and 3H are indeed involved in the translocation. In order to detect
unexpected relationships between markers at those chromosomes we apply a principal
coordinate analysis which visualizes marker positions in a tridimensional graph. The
next step of our analysis is based on the cytogenetical theory that (1) during meioses
two types of viable gametes are produced: a "normal" type (with a balanced set of non-
translocation chromosomes) and a "translocated" type (with a balanced set of
translocated chromosomes), and (2) that around the translocation breakpoints
recombination is suppressed (Burnham, 1962; Sybenga, 1975). The crucial step consists
of dividing the doubled haploid population into two subpopulations according to the
origin of the alleles at the translocation breakpoints: 'Barberousse' alleles refer to the
"normal" type and 'Albacete' alleles refer to the "translocated" type. The position of the
translocation breakpoint is found by a graphical display of recombination frequencies
versus positions on the consensus map. At this stage we discuss the problem of extreme
Chapter 2
14
segregation distortion around the translocation breakpoints in the two subpopulations,
which is solved by removing markers around the translocation breakpoints with severe
segregation distortion. Linkage maps for the various chromosome arms are then
obtained for each of the subpopulations separately. The final step is integrating the
linkage maps of the corresponding arms of linkage groups 1H and 3H of the two
subpopulations.
Materials and methods
Plant material
Two six-row parental barley varieties, 'Albacete' and 'Barberousse', were crossed and
anther culture lines were produced from the F1 from which 231 spontaneous doubled
haploids were obtained at Estación Experimental de Aula Dei, Zaragoza, Spain. Seed
from each doubled haploid line was retained as a reference stock and used to grow the
plants for DNA extraction. More than 50 years ago, 'Albacete' was found by Enrique
Sánchez-Monge at the Estación Experimental Aula Dei, Spanish Research Council, as
an inbred line in a local heterogeneous landrace population from Albacete (province of
Spain). It is a six-row barley variety, drought tolerant with a stable grain yield. For these
properties, it has been grown for decades on up to 1 million ha/year, especially in
drought-prone areas. 'Barberousse' is a six-row winter barley variety released in France
in 1977. It was obtained from the cross [259 711/(Hatif de Grignon/Ares)]/Ager.
Although it is sensitive to drought, this variety is known for its good productivity and
easy adaptation.
DNA isolation
Genomic DNA of the DHs was extracted using two different protocols. For genomic
and EST-derived SSR markers, genomic DNA was extracted from 0.1 g of fresh leaf
tissue sprayed with liquid nitrogen according to the method of Doyle and Doyle (1990)
with some modifications. DNA was diluted in TE buffer pH8 and preserved for a long
period at a temperature of -20ºC. For DArT markers the genomic DNA was extracted
from fresh leaf tissue using the Qiagen DNeasy 96 Plant Kit.
Linkage map construction involving a reciprocal translocation
15
Genotyping
Genotypic data were first produced for genomic and EST-derived SSR markers. We
refer to the Genomic SSRs markers using the prefix ("Bmac" or "Bmag") followed by
four digits and to the ESTs-derived SSR markers using the prefix ("scssr") followed by
five digits. Genomic and EST-derived SSR analyses were carried out using the
fluorescent fragment detection system on an ABI PRISM® 3100-Avant Genetic
Analyzer using Genescan software from Applied Biosystems. For this method, the
reverse primer was labeled with a fluorochrome at the 5’ end. For all amplifications the
PCR programs were used are published in the UK CropNet databases
(http://ukcrop.net/db.html).
Subsequently, a genome wide scan was carried out using the Diversity Arrays
Technology (DArT, http:\\www.diversityarrays.com). In a single assay, DArT uses
microarrays to detect DNA polymorphisms at several hundred genomic loci spread over
the entire genome. It is based on hybridising labelled genomic representations of
individual DNA samples on a micro-array, which contains a large number of DNA
fragments derived from the total genomic DNA of the species under consideration
(Wenzl et al. 2004). Polymorphisms were scored as presence (= 1) or absence (= 0) of
hybridization to individual array elements. The locus designations of Triticarte Pty. Ltd.
were used. DArT markers consisted of prefix (“bPb” or “CloneID”) followed by a
number corresponding to the particular clone in the genomic representation.
Linkage analysis
For the construction of linkage maps we used the maximum likelihood (ML) algorithm
in Jansen et al. (2001) which enables map construction in batch mode and the
implementation in JoinMap 4.0 (van Ooijen 2006) which enables the use of many
graphical tools. Linkage maps were drawn using MapChart 2.2 (Voorrips 2002).
Statistical techniques
Principal coordinate analysis (Chatfield and Collins 1980), a special form of
multidimensional scaling, was used to obtain a 3D representation of the markers on the
Chapter 2
16
linkage groups involved in the reciprocal translocation. The similarity between markers
was measured by the simple matching coefficient, which for a doubled haploid
population is equal to one minus the recombination frequency. Calculations were
carried with Genstat version 12 (Payne et al. 2009), using the directives FSIMILARITY
and PCO.
Results
Polymorphisms between parents
We applied the following liberal rules for excluding markers: (1) markers with an
unknown location on the consensus map (these were later included again when
constructing our final maps); (2) markers with an ambiguous chromosome assigment on
the consensus map; (3) markers with more than 5% missing data and (4) markers with
low quality readings (Q value less than 77). For linkage analysis, 30 polymorphic
genomic and EST-derived SSR markers and 309 polymorphic DArT were used. This
amounted to an average of 48 markers per chromosome, with an average interlocus
distance of 4.4 cM based the consensus linkage map. A few regions had gaps of over 10
cM. Chromosome 4H showed strikingly low levels of polymorphisms, which is in
agreement with the findings of Qi et al. (1996), Karakousis et al. (2003), Kleinhofs et al.
(1993) and Wenzl et al. (2006).
Preliminary linkage map construction
We obtained six main linkage groups involving 305 markers. Using a recombination
frequency threshold of 0.2, 34 markers could not be assigned to one of these linkage
groups. Five linkage groups could be related to linkage groups of the consensus map
(Wenzl et al. 2006): 2H (48 markers), 4H (20 markers), 5H (48 markers), 6H (49
markers) and 7H (58 markers). The remaining 82 markers had known positions on 1H
and 3H of the consensus map, of which 78 formed one linkage group. Four markers, on
the consensus map located at the very end of the long arm of linkage group 1H, were
found to be unlinked with the 78 markers of 1H and 3H. One marker showing a
consistently high nearest-neighbour stress (Van Ooijen 2006) was also excluded. Figure
1 displays one linkage map with the positions of the remaining 77 markers obtained by
Linkage map construction involving a reciprocal translocation
17
the maximum likelihood algorithm. Many different linkage maps were obtained by
rerunning the maximum likelihood algorithm. This was a clear indication that it was not
possible to obtain a linear order of the markers in an unambiguous way.
Figure 1. Graphical representation of a linkage map obtained for markers of linkage groups 1H
and 3H according to the consensus map of Wenzl et al. (2006); markers of linkage
group 1H and 3H are displayed in blue and ochre, respectively.
A spatial representation of the positions of the markers was obtained using principal
coordinate analysis. Figure 2 shows a three-dimensional plot in which the points
represent the positions of the markers with regard to the first three principal axes; the
percentages variance explained by the first three axes were 27.2, 15.59 and 13.9,
respectively. The points have been joined by straight lines according to their position on
the consensus map of Wenzl et al. (2004). Markers of linkage group 1H and 3H have
been displayed in blue and ochre, respectively. Figure 2 shows that linkage groups 1H
Chapter 2
18
and 3H merge at one point (indicated by the circle) and that the short and long
chromosome arms divert from this point, and from each other, in three-dimensional
space. As a consequence, the translocation breakpoints lie within the circle.
Figure 2. Graphical representation of a principal coordinate analysis using the simple matching
coefficient for the markers of linkage groups 1H and 3H according to the consensus
map of Wenzl et al. (2006). The percentage variance explained by the first three
principal axes are 27.2, 15.5 and 13.9, respectively. The markers have been joined
according to their position on the consensus map of Wenzl et al. (2006). The black
circle encloses markers of linkage groups 1H and 3H that are close to the translocation
breakpoints; they are separated by a small number of recombinations.
Recombination around the translocation breakpoints
To study the recombination behaviour of markers around the translocation breakpoints
we focused on estimates of the recombination frequencies between the markers of 1H
and 3H, in which the allocation of markers to linkage groups as well as their positions
Linkage map construction involving a reciprocal translocation
19
were based on the consensus map. In Figure 3, these recombination frequencies were
plotted against the corresponding positions on the consensus map. In Figure 3a, we
observed that markers close to the translocation breakpoints have recombination
frequencies close to zero. On linkage group 1H the translocation breakpoint was
situated at about 60 cM and on linkage group 3H at about 65 cM based on the consensus
map. The marker pair with the smallest recombination frequency consisted of HvM20
(1H) and Bmag0136 (3H) with a recombination frequency of 0.017 (4 recombinants out
of 231 DHs). Cross-sections of Figure 3a through the positions of markers Bmag0136
and HvM20 are shown in Figure 3b and 3c, respectively.
Figure 3. (a) Graphical representation of the recombination frequencies between markers of
linkage group 1H and linkage group 3H versus their position on the consensus map of
Wenzl et al. (2006). (b) Graphical representation of the recombination frequencies
between markers of linkage group 1H with marker Bmag0136 of linkage group 3H
versus their position on linkage group 1H of the consensus map of Wenzl et al. (2006).
(c) Graphical representation of the recombination frequencies between markers of
Position on 3H(cM)
Position on 1H(cM)
2040
6080
100
020
4060
80100
120140
160180120
0.5
0.4
0.3
0.2
0.1
0.0
Rec
ombi
nati
on f
requ
ency
Position on 3H(cM)
Position on 1H(cM)
2040
6080
100
020
4060
80100
120140
160180120
0.5
0.4
0.3
0.2
0.1
0.0
Rec
ombi
nati
on f
requ
ency
Chapter 2
20
linkage group 3H with marker HvM20 of linkage group 1H versus their position on
linkage group 3H on the consensus map of Wenzl et al. (2006).
Cytogenetical theory suggests that the DH1 population should be split into two
subpopulations: (1) a subpopulation containing individuals in which the alleles close to
the translocation breakpoint are identical to 'Albacete', and (2) a subpopulation
containing individuals in which the alleles close to the translocation breakpoint are
identical to 'Barberousse'. In the first subpopulation the chromosomal arrangement will
be that of 'Albacete' (1HS-3HL, 3HS-1HL), in the second subpopulation the
chromosomal arrangement will be that of 'Barberousse' (1HS-1HL, 3HS-3HL). The
separation of the 231 DH individuals into two subpopulations was based on the data of
markers HvM20 and Bmag0136 (Figure 4). First, the markers were divided into linkage
groups 1H and 3H and subsequently sorted vertically according to their position within
each of the linkage groups based on the consensus map. Secondly, individuals were
sorted according to the origin of their allele on marker HvM20; more or less the same
effect was obtained if the markers were sorted according to Bmag0136. The split of the
individuals into two groups was not perfect; 53 individuals had to be excluded because
they had deviating chromosomal combinations within 5 cM from the presumed
translocation breakpoints. Some of these deviations concerned so-called 'singletons';
they are likely due to observation errors or caused by gene conversions.
Figure 4. Graphical genotypes of individuals markers on linkage 1H and 3H according to the
consensus map of Wenzl et al. (2006). Individuals have been divided into two types
with regard to their alleles at the translocation breakpoints: the 'Albacete' type (shown in
red), carrying the translocation, and the 'Barberousse' type (shown in green), with
'normal' chromosome arrangement.
Linkage map construction involving a reciprocal translocation
21
In a doubled haploid population markers are expected to segregate according to a 1:1
ratio. However, due to the division into subpopulations, markers near the translocation
breakpoints show severe segregation distortion in each of the subpopulations. The
distortion decreases as markers are further away from the translocation breakpoints.
Severe segregation distortion significantly affects estimation of genetic distances, but
will not seriously affect the marker order (Hackett and Broadfoot 2003). Therefore, in
order to construct robust linkage maps, markers with severe segregation distortion (in
this case markers with segregation ratios below 30% or above 70%) were eliminated. In
both subpopulations 37 markers out of 75 were removed, leaving 14 markers for 1H and
24 markers for 3H; these markers were common in both subpopulations.
The two subpopulations were considered separately to construct independent linkage
maps for the chromosomes involved in the reciprocal translocation. Subpopulation 1
(with the 'Albacete' allele near or at the translocation breakpoint) contained 92 DHs;
Subpopulation 2 ('Barberousse' allele near the translocation breakpoint) contained 86
DHs. The two subpopulations were then subjected to independent linkage analyses.
Finally the linkage maps of the two subpopulations were integrated. Results are shown
in Figure 5. The order of the markers for the 'Albacete' and the 'Barberousse' population
are identical, but differences in distance are present.
bPb-3622bPb-1165bPb-1348
bPb-3776
bPb-9414bPb-8973
bPb-7306
1HS-Albacete
bPb-1165bPb-3622bPb-1348
bPb-3776
bPb-9414bPb-8973
bPb-7306
1HS-Integrated
bPb-1165bPb-3622bPb-1348
bPb-3776
bPb-7306bPb-9414bPb-8973
1HS-Barberousse
0
5
10
bPb-5555bPb-4022
bPb-1264bPb-9945bPb-9583
bPb-7199bPb-7770
bPb-7448
bPb-2929
3HS-Albacete
bPb-4022bPb-5555
bPb-9583bPb-9945bPb-1264
bPb-7199bPb-7770
bPb-7448
bPb-2929
3HS-Integrated
bPb-4022bPb-5555
bPb-9583bPb-9945bPb-1264
bPb-7770bPb-7199
bPb-7448
bPb-2929
3HS-Barberousse
0
5
10
15
20
25
30
Chapter 2
22
Figure 5. Linkage maps of the short and long arms of linkage groups 1H and 3H obtained for the
individuals of type 'Albacete' and for type 'Barberousse' at the translocation breakpoints.
The maps of the two types have been connected by their corresponding integrated maps.
The maps were obtained for markers with a segregation ratio between 0.3 and 0.7.
Discussion
The methods developed in this paper provide essential tools for disentangling 'pseudo-
linkage' between markers in a population derived from a translocation heterozygote.
The methods can be implemented using standard software. In this paper a high-quality,
consensus map of 'normal' barley (used as a reference) is essential in several steps of
the analysis: (1) interpretation of the three-dimensional principal coordinate plot (Figure
2), (2) determination of the translocation breakpoints on chromosomes 1H and 3H of
'normal' barley (Figure 3), and (3) assignment of markers to the respective chromosome
arms (1HS, 1HL, 3HS and 3HL) in 'normal' barley (Figure 4). Without a high-quality,
consensus map of 'normal' barley, step (2) will be impossible, and steps (1) and (3) will
be more difficult. The chromosome arms designated 1HS, 1HL, 3HS and 3HL become
arbitrary chromosome arms.
bPb-3389
bPb-4144bPb-1213bPb-5249bPb-3089
bPb-9108bPb-5014
1HL-Albacete
bPb-3389
bPb-3089bPb-4144bPb-1213bPb-5249
bPb-9108
bPb-5014
1HL-Integrated
bPb-3389
bPb-3089bPb-1213bPb-4144bPb-5249
bPb-9108
bPb-5014
1HL-Barberousse
0
5
10
15
20
25
30
35
40
45
50
bPb-8506bPb-2550bPb-8621
bPb-9111
bPb-5666bPb-0789bPb-9599bPb-7827bPb-6383bPb-3899bPb-8419bPb-7815bPb-0200
bPb-0361
bPb-7724
3HL-Albacete
bPb-8506bPb-2550bPb-8621
bPb-9111
bPb-5666bPb-3899bPb-0789bPb-7827bPb-6383bPb-9599bPb-7815bPb-8419bPb-0200
bPb-0361
bPb-7724
3HL-Integrated
bPb-2550bPb-8506bPb-8621
bPb-9111bPb-5666bPb-6383bPb-3899bPb-0789bPb-7827bPb-9599bPb-7815
bPb-8419bPb-0200
bPb-0361
bPb-7724
3HL-Barberousse
0
5
10
15
20
25
30
35
40
45
50
55
Linkage map construction involving a reciprocal translocation
23
In a preliminary linkage analysis, six main linkage groups were detected, one of them
joining markers from chromosomes 1H and 3H. As indicated by an earlier study this
"mixing" of markers of chromosomes 1H and 3H was attributed to the presence of a
reciprocal translocation between these chromosomes. In this study the position of the
translocation breakpoint could be determined at about 60 cM on chromosome 1H and
65 cM on chromosome 3H (positions refer to the consensus map of 'normal' barley),
which correspond with the centromeric regions of these chromosomes. These positions
were confirmed in this study when all available markers from 1H and 3H were used to
determine in more detail which markers were involved in the translocation breakpoints.
In another DH1 population obtained from a cross of ‘Albacete’ and 'Plaisant' (results not
shown), the translocation breakpoints were found at similar positions.
In order to characterize the translocation breakpoint we identified 92 DHs as carrying
the translocation and 86 DHs with the standard chromosome arrangement. Fifty three
genotypes could not be classified in either of the two subpopulations due to deviating
chromosomal combinations which may be caused by scoring errors. In marker studies
like these scoring errors should be avoided by using very accurate marker systems.
Nevertheless, it is interesting to note that, regardless of the true nature of the 53 non-
classified types, the presence of this chromosome rearrangement did not affect the
success rate in the double haploid regeneration process. As a result, two different
genetic linkage maps were obtained, one with the two interchanged chromosomes
together (1HS·3HL + 1HL·3HS, 'Albacete' type) and the other with the two normal
chromosomes together (1HS·1HL + 3HS·3HL, 'Barberousse' type).
It is expected that barley varieties carrying chromosomal segments containing the
reciprocal translocation between chromosomes 1H and 3H, have an advantage under
certain environmental conditions. So, for future breeding purposes, it may be
worthwhile to be able to follow individuals carrying interchanged chromosomes. In our
case, this is the 'Albacete' haplotype. This requires the development of markers close
enough to the translocation breakpoints with allelic combinations that allow
differentiation between translocated and non-translocated origin of the chromosomal
segments around the translocation breakpoints; in our case marker haplotypes that
distinguish between 'Albacete' and all other material used in the breeding program.
Away from the translocation breakpoints, a mixing of haplotypes will occur due to
Chapter 2
24
recombination. In the end, this would allow the construction of a specific linkage map
of the karyotype with a chromosomal rearrangement close to the translocation
breakpoints.
A genetic consequence of reciprocal translocation is that linkage relationships in a
translocation heterozygote are altered as recombination between loci may be
significantly reduced, particularly within the interstitial segment and between genes
close to the translocation breakpoint. If the translocation breakpoint is located at or near
the centromeric region, the interstitial zone becomes really narrow or even null and,
therefore, the recombination that may take place will be due to interference effects near
the translocation breakpoint. In our data we have not found a common set of completely
linked markers from chromosome 1H and 3H, although a few showed very low levels of
recombination. These rare recombinants can be due to scoring errors or, what is more
likely, due to the fact that the interstitial zone is practically nonexistent.
The genomic SSR markers linked closely to the translocation breakpoint on
chromosome 3H were identified flanking the centromere at 55 cM in the Lina ×
Hordeum spontaneum 'Canada Park' genetic map, between Bmac0067, Bmag0136 and
Bmag006 in the short arm and Bmac0209 in long arm covering a genetic distance of 5
cM (Künzel and Waugh 2002). However, this region (with suppressed recombination)
covered 36% of the entire chromosome length on the physical map. For chromosome
1H, no articles were found, which relate physical distance to genetic distance using
genomic SSR markers along the entire chromosome; only one RFLP-based map
observed this phenomenon (Künzel et al. 2000). The gene density around the
translocation breakpoints can be inferred using the synteny observed for barley-rice.
Mayer et al. (2009) mentioned that approximately 20% of all genes on barley
chromosome 1H are located in centromeric and subcentromeric regions with very low
recombination frequencies. A similar gene density can be expected for barley
chromosome 3H; Smilde et al. (2001) reported a 30-fold reduction of recombination
around the barley 3H centromere.
Livingstone et al. (2000) state that karyotypic rearrangements, such as reciprocal
translocations, lead to high variances of inter-marker distances for markers on
chromosomes involved in the reciprocal translocation away from the translocation
Linkage map construction involving a reciprocal translocation
25
breakpoints, in repeated attempts to construct a linkage map (see also Durrant et al.,
2006). Markers close to the translocation breakpoints show small variances of inter-
marker distances. However, high variances may also be caused by deficiencies in the
data. The methods developed in this paper provide a novel combination of methods
available in standard (statistical) software that can be used to identify the true
background of 'pseudo-linkage' in a straightforward manner.
In conclusion, the results of this study confirm that there is a reciprocal translocation as
the result of an interchange of chromosome arms between 1H and 3H. The markers
found to be linked to the translocation breakpoints are HvM20 and EBmac0501 for 1H
and Bmag0136, bPb-9746 and a reduced set of genomic SSR markers for 3H, which are
located around the centromere. Linkage analysis revealed that alternative arrangements
involving the short and long chromosome arms of 1H and 3H may be possible.
However, the most frequent arrangement is 1HS·3HL + 3HS·1HL. In addition,
regardless of the presence of a reciprocal translocation we have produced a linkage map
for the 'Albacete' x 'Barberousse' population that will be used to identify quantitative
trait loci (QTLs) for agronomic traits in future work.
Acknowledgements
This study has been supported by the Spanish Ministry of Science and Innovation
(Project AGL2008-05541-C02 and the Agrogenomics CONSOLIDER). We are grateful
to Dr. Martin Boer who provided us with simulated data which helped us in better
understanding the mapping problems arising from the presence of a reciprocal
translocation. Comments by the Editor and two referees are gratefully acknowledged.
27
Chapter 3
Genetic characterization of a reciprocal translocation present
in a widely grown barley variety
A. Farré1,2 · A. Cuadrado3 · I. Lacasa-Benito1 · L. Cistué4 · I. Schubert5 · J. Comadran6 ·
J. Jansen2 · I. Romagosa1,7
Published in Mol Breeding (2012) DOI: 10.1007/s11032-011-9698-z
Abstract
Artificially induced translocation stocks have been used to physically map the barley
genome; however, natural translocations are extremely uncommon in cultivated
genotypes. ‘Albacete’ is a barley variety widely grown in the last decades in Spain and
carrying a reciprocal translocation which obviously does not affect its agronomical
fitness. This translocation has been characterized by a combination of cytological and
molecular genetic approaches. First, recombination frequencies between markers on
chromosomes 1H and 3H were estimated to determine the boundaries of the reciprocal
interchange. Secondly, 1H-3H wheat barley telosome addition lines were used to assign
selected markers to chromosome arms. Thirdly, fluorescence in situ hybridization
(FISH) with rDNA probes (5S and 18S-5.8S-26S) and microsatellite probes [(ACT)5,
(AAG)5 and (CAG)5] was used to determine the locations of the translocation
breakpoints more precisely. Fourthly, fine-mapping of the regions around the
translocation breakpoints was used to increase the marker density for comparative
genomics. The results obtained in this study indicate that the translocation is quite large
with breakpoints located on the long arms of chromosomes 1H and 3H, between the
pericentromeric (AAG)5 bands and above the (ACT)5 interstitial distal bands, resulting
1Department of Plant Production and Forest Science, University of Lleida, Lleida, Spain; 2Biometris, Wageningen University and Research Centre, Wageningen, the Netherlands; 3Department of Cell Biology and Genetics, University of Alcalá de Henares, Alcalá de Henares, Spain; 4Estación Experimental de Aula Dei, CSIC, Zaragoza, Spain; 5Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany; 6Genetics Programme, The James Hutton Institute , Dundee, Scotland; 7Centre UdL-IRTA, University of Lleida, Lleida, Spain
Chapter 3
28
in the reciprocal translocation 1HS.1HL-3HL and 3HS.3HL-1HL. The gene content
around the translocation breakpoints could be inferred from syntenic relationships
observed among different species from the grass family Poaceae (rice, Sorghum and
Brachypodium) and was estimated at approximately 1,100 and 710 gene models for 1H
and 3H, respectively. Duplicated segments between chromosomes Os01 and Os05 in
rice derived from ancestral duplications within the grass family overlap with the
translocation breakpoints on chromosomes 1H and 3H in the barley variety ‘Albacete’.
Keywords Reciprocal translocation Barley Translocation breakpoint
Fluorescence In Situ Hybridization Comparative genomics
Introduction
Reciprocal translocations, interchanges of chromosome segments between two non-
homologous chromosomes, are one of the most common structural chromosomal
rearrangements occurring in plant species. Translocations in plants have been widely
described by Burnham (1956). Permanent translocation heterozygotes were first
observed by Gates (1908) in Oenothera species which received generous cytological
and genetic studies by Cleland (1922) and Belling and Blakeslee (1926). McClintock
(1930) was the first to supply cytological evidence of interchanges in economically
important crops such as maize. Translocations have been widely utilized in both applied
and fundamental scientific research for chromosome mapping, in particular for
assigning linkage groups to chromosomes, development of physical maps in plants
(Kim et al. 1993; Künzel et al. 2000; Sorokin et al. 1994; Marthe and Künzel 1994), and
to improve our understanding of meiotic chromosome pairing behaviour (Rickards
1983). They have been reported in a number of crop species, such as rye (Benito et al.
1994), soybean (Mahama and Palmer 2003), Prunus (Jáuregui et al. 2001), Lens
(Tadmor et al. 1987), pea (Kosterin et al. 1999), wheat (Naranjo et al. 1987) and
Brassica napus (Osborn et al. 2003).
Chromosomal interchanges, and translocations in general, can be artificially induced in
somatic or meiotic cells by ionizing radiation or mutagens. They may also occur
Genetic characterization of a reciprocal translocation present in a widely grown barley variety
29
spontaneously, although there are few cases of spontaneous reciprocal translocations
described in cultivated barley. Konishi and Linde-Laursen (1988) investigated 1,240
cultivated barley lines and 120 wild barley lines to detect spontaneous reciprocal
translocations, which were identified by both semi-sterility associated to test crosses
and subsequent Giemsa banding technique. Of the 1,240 cultivated barley lines, four
Ethiopian landraces carrying a reciprocal translocation had the same breakpoints at the
centromere involving chromosomes 2H and 4H (2HS·4HS and 2HL·4HL), suggesting
that the chromosomes involved in the rearrangement had a common origin. Of the 120
wild barley genotypes, three carried translocations between chromosomes 2H and 4H,
3H and 5H and 3H and 6H, respectively. Xu and Kasha 1991 identified a chromosomal
interchange between chromosomes 3H and 4H using N-banding and in situ
hybridization techniques in a wild barley cross. The plant heterozygous for the
interchange was derived from the backcross of ‘Su Pie’ with pollen from a triploid
interspecific F1 hibrid of ‘Su Pie’ × tetraploid Hordeum bulbosum accession GBC141.
One of the consequences of a reciprocal translocation is the suppression of genetic
recombination in a translocation heterozygote in the interstitial zone (the chromosome
segment between the centromere and the breakpoint). The suppression depends on the
centromere coorientation frequency at metaphase I (alternate or adjacent segregations)
and upon the chiasmata (crossover) frequency expected in both interstitial segments
(Hanson and Kramer 1949; Burnham and Hagberg 1956; Kasha and Burnham 1965;
Sybenga 1975). Recombination suppression in the interstitial zone affects the linkage
relationships in a translocation heterozygote which results in “pseudo-linkage” between
the genes of the two chromosomes involved in the reciprocal translocation and
subsequent disturbed linkage maps. Another consequence of reciprocal translocations is
the occurrence of gametic sterility of which depends on the frequency of the alternate or
adjacent orientation in the quadrivalent. In barley, an excess of alternate over adjacent
segregation of the chromosomes at meiotic metaphase I would explain the average 25%
sterility caused by the interchange (Kakeda and Miyahara 1995).
Plant accessions carrying chromosome rearrangements such as translocations have been
identified in the past by its effects on partial pollen and seed sterility (i.e. Jáuregui et al.
2001). However, depending on the chromosome breakpoints, a translocation can result
in the disruption or misregulation of normal gene functions. Thus, special interest
Chapter 3
30
resides on the characterization of the physical locations of the translocation breakpoints
on the genome allowing physical mapping of genes involved in the translocation on the
chromosomes. These can be delimited by a combination of cytogenetics and molecular
genetics. In barley, chromosome identification can be achieved by using in situ
hybridization with ribosomal RNA probes (Brown et al. 1999). In addition, with
labelled SSRs it is now possible to cover the physical map with many landmarks
distributed along all chromosome arms (Cuadrado and Jouve 2007). Undoubtedly, this
rich set of chromosome markers should help to identify the barley breakpoints more
precisely than conventional staining techniques.
In the present paper, we aim at determining the positions of the translocation
breakpoints in the Spanish six-row barley variety ‘Albacete’. Translocations may have
dramatic consequences such as modified phenotypes. In fact, ‘Albacete’ is the only
extensively cultivated barley variety known to carry a reciprocal translocation between
chromosomes 1H and 3H without any major reduction in fitness. It is adapted to low-
yielding West Mediterranean areas and it has been the most widely grown cultivar in the
driest Spanish areas for the last decades (over a million ha per year). The translocation
was first identified by Cistué (unpublished) upon meiotic analysis of semi-sterile F1
hybrids involving this variety.
In tracing the possible origin of the reciprocal translocation in ‘Albacete’ we use
syntenic relationships between different families of the grass family Poaceae. For the
location and characterization of the translocation breakpoints we used a combination of
molecular genetical and cytological techniques. Dense genetic linkage maps of the
chromosomes involved in the reciprocal translocation will be used to identify markers
in the vicinity of the translocation. Pseudo-linkage arising from suppressed
recombination in the interstitial space may result in increased similarities between
markers located on the two chromosomes involved in the reciprocal translocation and
can be used to fine-map the recombination breakpoints. Further validation and physical
characterisation of the translocation breakpoints will be achieved by the use of wheat-
barley telosome addition lines and fluorescence in situ hybridization with rDNA probes
(5S and 18S-5.8S-26S) and microsatellite probes [(ACT)5, (AAG)5 and (CAG)5] of
‘Albacete’ and doubled haploid lines derived from crosses between ‘Albacete’ and
cultivars with a standard chromosome arrangement.
Genetic characterization of a reciprocal translocation present in a widely grown barley variety
31
Materials and methods
Linkage analysis
Two bi-parental doubled haploid (DH) mapping populations were used. The first
mapping population, ‘Albacete’ × ‘Barberousse’ (AB), consists of 231 DH lines derived
from anther-culture from the cross between the translocation-carrying six-row winter
variety ‘Albacete’ and the six-row winter variety ‘Barberousse’. DNA isolation and
genotyping data, SSR and DArT®, were performed according to Farré et al. (2011). The
second mapping population, (‘Albacete’ × ‘Plaisant’) × ‘Plaisant’ (APP), consists of 94
DH lines derived from the cross of a DH line produced from the ‘Albacete’ × ‘Plaisant’
F1, which carried the ‘Albacete’ reciprocal translocation, backcrossed to ‘Plaisant’.
DNA was extracted from leaf tissue using kit DNeasy Plant Mini Kit (Quiagen,
Valencia, CA, USA). A set of 3072 EST-based high confidence SNP markers were
genotyped using GoldenGate BeadArray technology (Illumina) as previously described
(Close et al. 2009).
The allocation of markers to linkage groups as well as their genetic map position were
based on the published barley consensus map (Wenzl. et al. 2006) for the AB
population and Close et al. (2009) for the APP population. The position of the
translocation breakpoints was first inferred according to the methods described by Farré
et al (2011).
Wheat-barley telosome addition lines
The chromosome arm locations of seven SSR markers on the chromosomes of the
barley cultivar ‘Betzes’ were checked on the ‘Chinese Spring’-‘Betzes’ (CS-B) wheat-
barley telosome addition lines for chromosomes 1H and 3H. CS-B telosome addition
lines for the chromosome 1H and 3H and chromosome arms 1HS, 3HS and 3HL were
used to assign selected markers to chromosome arms. No telosome addition line for
chromosome arm 1HL was available. Genomic DNA from these addition lines were
kindly provided by Rafiqul Islam (School of Agriculture, Food and Wine. The
University of Adelaide, Australia) and Marion Röder (Leibniz Institute of Plant
Genetics and Crop Plant Research. IPK, Germany).
Chapter 3
32
Fluorescence in situ Hybridization
Plant material and root tip and chromosome preparation
Root tips were obtained from seedlings of Hordeum vulgare cv. ‘Albacete’. Seeds were
germinated on moist filter paper for 24 h at 25ºC and then kept at 4ºC for 72h followed
by 25ºC for 24h to synchronize cell division. The seedlings were then transferred to ice-
cold water for 24h to accumulate metaphases before tissue fixation in ethanol-glacial
acetic acid (3:1) (Cuadrado and Jouve 2007). Chromosome preparations were prepared
as described by Schwarzacher et al. (1989). Briefly, root tips were macerated with an
enzymatic mixture and then squashed in a drop of 45% acetic acid. After removing the
cover slips by quick freezing, the slides were air dried.
DNA probes and in situ hybridization
The three oligodeoxyribonucleotide probes [(ACT)5, (CAG)5 and (AAG)5] were
provided by Roche labelled at their 5’ and 3’ with digoxigenin or biotin. The novel and
rapid non-denaturating FISH (ND-FISH) technique developed by Cuadrado and Jouve
(2010) was carried out to detect SSR-enriched chromosome regions.
The other probes, pTa71 (a plasmid containing the 18S-5.8S-25S rDNA and the
intergenic spacer of Triticum aestivum) and pTa794 (contains a 410-bp BamHI
fragment of 5S rDNA isolated from wheat Triticum aestivum) were labelled by nick
translation and PCR, respectively (Leitch and Heslop-Harrison 1992; Leitch and
Heslop-Harrison 1993; Pedersen and Linde-Laursen 1994; Brown et al. 1999).
Chromosome and probe denaturation and the in situ hybridization steps were carried out
as described in Cuadrado et al. (2000).
Fluorescence microscopy and imaging
Slides were examined with a Zeiss Axiophot epifluorescence microscope. The separate
images from each filter set were captured using a cooled CCD camera (Nikon DS) and
processed using Adobe Photoshop, employing only those functions that are applied
equally to all pixels in the image.
Genetic characterization of a reciprocal translocation present in a widely grown barley variety
33
Results
Similarities between markers on chromosomes 1H and 3H in the AB population
In order to identify DArT and SSR markers in the vicinities of the reciprocal
translocation breakpoints, we explored recombination frequencies, between the markers
located on chromosomes 1H and 3H. The allocation of markers to linkage groups as
well as their genetic map position were based on the published barley consensus map
(Wenzl et al. 2006). A total of 78 out of 305 polymorphic markers were mapped on
chromosomes 1H and 3H for the AB population. A graphical representation of the
recombination frequencies was presented by Farré et al. (2011) which shows that
markers on chromosome 1H and 3H were strongly related. This suggests the presence
of a reciprocal translocation between these chromosomes. So, for the AB population,
the markers with recombination frequencies close to zero were located in the consensus
map around 59-64 cM and 66-70 cM for 1H (HvM20 and EBmac0501) and 3H
(Bmac209, Bmac067, Bmag0006 and Bmag0136), respectively, which corresponds
with the pericentromeric regions of both chromosomes 1H and 3H.
1H and 3H wheat-barley telosome addition lines
Wheat-barley chromosome addition lines were then used to assign the chromosome arm
location of the SSR markers found to be closely linked to the translocation breakpoints
using their presence/absence as detected in hexaploid wheat T. aestivum cv. ‘Chinese
Spring’ (CS) and barley H. vulgare cv. ‘Betzes’ using PCR . For chromosome 3H, 3 out
of 4 SSR markers were found to be located on the short arm and the other one on the
long arm (positions reported by Künzel and Waugh 2002). Using 1H wheat-barley
addition lines the two SSR markers used mapped on different chromosome arms.
HvM20 amplified in both CS and Betzes and, therefore, did not have any diagnostic
value.
Fluorescence in situ hybridization analysis
Further progress in localizing the translocation breakpoints can be expected from direct
cytological observations of barley chromosomes by FISH using probes that were used
Chapter 3
34
successfully as anchored chromosomal markers. The following locations of pTa794
(5SrDNA) were reported: interstitial on chromosome arms 2HL and 3HL, distal on 4HL
and proximal on 7HS (Leitch and Heslop-Harrison 1993). In addition to the two NOR-
bearing barley chromosomes, 5HS and 6HS, the locations of four minor rDNA loci with
pTa71 (18S-5.8S-26S rDNA) were reported on chromosome arms 1HS, 2HS, 4HS and
7HS differentiated by their position and intensity (Pedersen and Linde-Laursen 1994).
Thus the use of these two ribosomal probes should allow easy identification of all
barley chromosomes including chromosomes 1H and 3H.
First, two-colour FISH was carried out with pTa71 and pTa794 in pollen mother cells of
a hybrid between ‘Albacete’בPlaisant’. The expected chromosome pairing occurred
during meiotic I prophase for a heterozygous genotype for the chromosomic
arrangement, in which five bivalents and one quadrivalent were observed (Fig. 1a).
Bivalents for the two satellited chromosomes, 5H and 6H, with the strongest pTa71
signals, and the bivalents formed by chromosomes labelled with both probes, 2H, 4H
and 7H, were identified despite the weak signals observed in the Figure 1. Thus,
chromosome 1H, with the stronger pTa71 signal between the non-satellited
chromosomes, and 3H are the chromosomes involved in the quadrivalent (Fig. 1b,c).
We subsequently analysed mitotic metaphases of ‘Albacete’ (Fig. 1d-h). Chromosomes
2H, 4H, 5H, 6H and 7H showed the expected and distinctive FISH pattern of ribosomal
probes. In addition, one pair of submetacentric chromosomes with a rather strong
pTa71 signal on its short chromosome arm and pTa794 signal on its long chromosome
arm and another pair of metacentric chromosomes without any ribosomal signals,
confirmed the presence of a reciprocal translocation resulting in two chromosomal
combinations 1HS-3HL and 3HS-1HL, respectively (Fig. 1f). These two chromosomes
are shown in Fig. 1i. Therefore, the translocation breakpoints are located below the
pTa71 signal on 1HS and above the pTa794 signal on 3HL.
More information about the exact position of the translocation breakpoints can be
obtained by identifying relocated FISH landmarks on chromosomes 1H and 3H. To this
aim three microsatellites probes, (ACT)5, (CAG)5 and (AAG)5 were chosen for their
characteristic patterns on these chromosomes (Cuadrado and Jouve 2007). As expected,
(ACT)5 resulted in a distinct pattern of FISH signals on chromosomes 2H, 3H, 4H, 5H,
Genetic characterization of a reciprocal translocation present in a widely grown barley variety
35
and 6H. The characteristic signals on the long arm of chromosome 3H were found on
the translocated chromosomes 1HS-3HL (Fig 1g and i). These results suggest that the
translocation breakpoints are located below the pTa71 signal on 1HS and above the
(ACT)5 signal on 3HL (Fig. 1e). The (AAG)5 microsatellite probe facilitates the
identification of all barley chromosomes thanks to a rich pattern of signals of different
intensities located in the pericentromeric and interstitial chromosomal regions (Fig. 1h).
The characteristic patterns obtained in the translocated chromosomes are similar to the
patterns found in other barley varieties with normal 1H and 3H chromosomes and
suggest that the translocation breakpoints are located below the pericentromeric AAG
signals on the long arms of chromosomes 1HL and 3HL (Fig. 1i-j). Results obtained
with the (CAG)5 probe suport the detection and identification of the translocations
1HS.1HL-3HL and 3HS.3HL-1HL. The signals obtained in the centromere of
translocated chromosomes 3HS-1HL were stronger than on 1HS-3HL (Fig 1j).
Chapter 3
36
Figure 1. FISH results with rDNA probes (5S and 18S-5.8S-26S) and microsatellite probes
((ACT)5, (AAG)5 and (CAG)5). (a-c) In situ hybridization of rDNA probes pTa71 (red)
and pTa794 (green) in pollen mother cells of hybrid between ‘Albacete’ × ‘Plaisant’
(Arrows indicate the quadrivalent): (a) DAPI staining for DNA; (b) pTa71 and pTa794
probes; (c) combination of DAPI and pTa71 and pTa794 probes. (d-h) Root-tip
metaphase chromosomes from barley (Hordeum vulgare cv. ‘Albacete’) after DAPI
staining and in situ hybridization with biotin-labelled probes (detected by red Cy3) or
digoxigenin-labelled probes (detected by green FITC): (d) DAPI staining for DNA; (e)
rDNA probes and (ACT)5 (arrows indicate the characteristic ACT signals on 3HL and
arrowheads the 45S rDNA loci on 1HS); (f) pTa71 and pTa794 (arrows indicate the 5S
rDNA loci on 3HL and arrowheads the 45S rDNA loci on 1HS); (g) (ACT)5 (arrows
indicate the characteristic ACT signals on 3HL); (h) (AAG)5 and (ACT)5 (arrowsheads
indicate the characteristic ACT signals on 3HL. Lines indicated the pericentromeric
Genetic characterization of a reciprocal translocation present in a widely grown barley variety
37
AAG signals on chromosomes 1H and 3H); (i-j) translocated chromosomes hybridized
with pTa71, pTa794, (AAG)5, (ACT)5 and (CAG)5. Scale bar represent 10 µm.
Fine-mapping of the translocation breakpoint region
In order to increase the marker density around the translocation breakpoints the APP
population genotyped with SNP was used. As (AAG)5 and (ACT)5 microsatellite probes
have proved to be useful to determine the translocation breakpoints on 1H and 3H
chromosomes, they were also chosen for detailed cytogenetic characterization of 8 out
of 94 DH lines from the APP population (Figure 2). These DH lines were selected
according to the ‘Albacete’ SNP allele distribution along 1H and 3H aiming to narrow
down the position of the translocation breakpoints. The allocation of markers to linkage
groups as well as their genetic map position were based on the published barley
consensus map (Close et al. 2009). 755 out of 3072 SNP markers were polymorphic for
the APP population; of these 212 markers mapped on chromosomes 1Hand 3H. From
Figure 2 it can be observed that 8 markers around 50-52.5 cM on 1H (11_20427,
11_20660, 12_11209, 12_31208, 11_31381, 11_20912, 11_21312 and 12_30350) and
30 markers around 43.2-55.6 cM on 3H (12_21533, 11_11002, 11_21101, 11_11086,
11_11501, 11_10137, 11_10328, 11_20970, 12_30039, 12_30130, 12_30318,
12_31008, 12_31372, 11_10365, 12_10155, 12_31502, 11_10008, 11_20102,
11_10224, 11_20333, 11_20428, 11_20439, 11_10456, 11_20796, 11_20856,
11_20890, 11_21062, 11_21147, 11_11124 and 11_11337) were close to translocation
breakpoints. This set of SNP markers was used to determine the gene content around
the translocation breakpoints with rice, Brachypodium and Sorghum using Strudel
(http://bioinf.scri.ac.uk/strudel/; Bayer et al. 2011). Inferred gene contents for rice,
Brachypodium and Sorghum yielded around 1,100, 600 and 550 for the region on 1H
and 710, 410 and 430 gene models for 3H, respectively.
Chapter 3
38
Figure 2. Fine-mapping of the translocation breakpoint region using eight selected lines from the
APP mapping population. Graphical genotypes of the eight selected DH lines (top).
Blue and yellow colours indicate ‘Albacete’ and ‘Plaisant’ parental alleles respectively.
Genomic region in the vicinity of the reciprocal translocation breakpoints are indicated
with grey colour. Horizontal dashed lines are drawn every 10 cM with the distance
proportional to the number of SNPs markers. Line number are shown using green and
red colours according to the presence or not of the reciprocal translocation, respectively
(line nº30200 was used as a positive control whereas 3.1 24 as a negative). On the
bottom, In situ hybridization of (ACT)5 (red or green) and (AAG)5 (red) in root-tip
metaphase chromosomes 1H and 3H after DAPI staining and in situ hybridization with
biotin-labelled probes (detected by red Cy3) or digoxigenin-labelled probes (detected by
green FITC).
Discussion
The combination of cytogenetics and molecular genetics allowed us to determine the
translocation breakpoints on chromosomes 1H and 3H of the barley variety 'Albacete'
more accurately than with conventional staining techniques. The success of the
combination of methods depends on the locations of the translocation breakpoints and
on the presence of differential signals on the chromosomes involved (Xu and Kasha,
1991).
In the present study, the positions of the translocation breakpoints have been determined
using a number of approaches. First, an analysis involving recombination frequencies
Genetic characterization of a reciprocal translocation present in a widely grown barley variety
39
between markers was used to determine markers located near the translocation
breakpoints with a high degree of precision (Farré et al. 2011). SNP and microsatellite
markers putatively flanking the translocation breakpoints were identified in the AB and
APP mapping populations in the pericentromeric regions of chromosomes 1H and 3H.
Secondly, 1H and 3H wheat-barley telosome addition lines were used to assign a
number of markers closely linked to the translocation breakpoints to their proper
chromosome arms and to validate the genetic mapping results. For the genomic SSR
markers on 3H chromosome, the positions are in good agreement with the positions
reported by Künzel and Waugh (2002). They placed 24 microsatellite loci onto the
physical RFLP map of barley chromosome 3H using the map position of the
translocation breakpoints as reference (Künzel et al. 2000). Our genomic SSR markers
on 3H chromosome were identified to be flanking the centromere (positioned at 55 cM)
in the ‘Lina’×Hordeum spontaneum Canada Park genetic map, where Bmac0067,
Bmag0136 and Bmag0006 on the short arm and Bmac0209 on the long arm cover a
genetic distance of 5 cM. It is well known that genetic map distances between markers
along chromosomes correlate poorly with physical distances, particularly in the large
cereal genomes where closely linked markers which genetically map near the
centromere represent considerable physical distances (Schwarzacher 2003). As a
consequence, most of the chromosomal gene content is trapped in chromosome
segments with severely suppressed recombination. According to Künzel and Waugh
(2002) the region flanked by our four SSR covers 36% of the entire 3H chromosome
length on the physical map. For chromosome 1H, no studies were found relating genetic
to physical distance using the genomic SSR we identified close to the translocation
breakpoint. Although being pericentromeric we may expect results similar to those
reported for 3H. In general, Triticeae centromeres are characterised by suppressed
recombination (Schwarzacher 2003; Rostoks et al. 2002). In barley, suppressed
recombination rates in proximal segments and high recombination rates in distal regions
were demonstrated by Pedersen et al. (1995). Later, Künzel et al. (2000) reported that
most recombination was confined in a relatively small chromosomal region, mostly at
the arm ends, alternating abruptly with regions of severely suppressed recombination.
Our approach allowed us to identify and genetically delimit the physical regions in
which the reciprocal translocation took place. However, as the regions involved in the
translocation are located in the vicinity of the centromeres, given the recombination
Chapter 3
40
suppression, they represent fairly large physical regions on both chromosomes 1H and
3H. FISH was used to validate the positions of the translocation breakpoints on the
chromosomes involved in the chromosomal interchanges, which was found to be a
useful approach to characterize non-recombinant regions.
The proposed positions of the breakpoints on the long arms of chromosomes 1H and 3H
can be attributed to the combined use of rDNA and microsatellite probes. Once the
translocation between chromosome 1H and 3H was confirmed using the ribosomal
probes (pTa71 and pTa794) and (ACT)5, the reasons that led us to concluding that the
breakpoints are on the long arms of chromosomes 1H and 3H (resulting in the
reciprocal translocation 1HS.1HL-3HL and 3HS.3HL-1HL) are multiple. Firstly, the
signals obtained using the (AAG)5 probe (which were similar to banding patterns
obtained by C-banding) on the short arms of both chromosomes are the same as those
observed in other barley varieties which indicates that the short arms are complete until
the centromere and not involved in the reciprocal interchange (Pedersen and Linde-
Laursen 1994; Cuadrado and Jouve 2007). However, a discrepancy in the number of
bands on 3HL among different barley varieties was found; some barley lines such as
‘Plaisant’, ‘Gaelic’ and ‘Hispanic’ carried two bands compared with just one for
varieties as ‘Albacete’, ‘Dobla’ and ‘Golden Promise’ (Á. Cuadrado, personal
communication). The lacking of a band near the centromere could be a polymorphism in
these varieties (unpublished data). Besides that, it is important to note that the signal
intensity of the pericentromeric band present on 3HL is stronger than the one present on
1HL, which is characteristic for all barley varieties. Therefore, these results suggest that
the translocation breakpoints are located below this band. Secondly, the differences in
intensity found in the centromeres of the translocated chromosomes with the
(CAG)5 probe are consistent with those obtained for Plaisant (a variety with an standard
chromosome arrangement). Signals on chromosome 3H were stronger than on 1H
(Cuadrado and Jouve 2007) suggesting that the translocation breakpoints are located
below the centromere on both chromosomes. Thirdly, the interchanged chromosomes
1HS.1HL-3HL and 3HS.3HL-1HL are more similar in total length and more
submetacentric and metacentric, respectively, compared with published ‘normal’ barley
karyotypes in which centromeres of chromosome 1H, the smallest barley chromosome,
and 3H are located at 41 and 44% FL (fraction length), respectively (arm ratios for 1H
and 3H were estimated at 1.4 and 1.3 milliGeNomes (mGN), respectively, and
Genetic characterization of a reciprocal translocation present in a widely grown barley variety
41
chromosome arms are the following: 51 mGN (1HS), 72 mGN (1HL), 64 mGN (3HS)
and 83 mGN (3HL); Taketa et al. 2003). To summarize, the different patterns of these
SSR probes have proven to be of a great value for localizing and validating the
translocation breakpoints on 1H and 3H chromosomes in the Spanish barley variety
‘Albacete’. The results obtained in this study suggest that the translocation breakpoints
are located on the long arms of both chromosomes between the (AAG)5 and
(ACT)5 pericentromeric bands for 3HS.3HL-1HL and below the
(AAG)5 pericentromeric band for 1HS.1HL-3HL leading to a interstitial zone with
suppressed recombination.
For barley and other not yet sequenced species synteny conservation with related
Poaceae species sequenced genomes such as rice, Brachypodium, maize or Sorghum
(Mayer et al. 2011) can be explored and exploited for studying genome evolution and
identification of candidate genes for traits of interest. So, an interesting point is to use
the shared syntenic relationships observed among different species from the grass
family Poaceae to determine the gene content around the translocation breakpoints. To
explore genome co-linearity of barley with rice, Brachypodium and Sorghum we
employed Strudel (http://bioinf.scri.ac.uk/strudel/), a standalone Java desktop
application that allows the simultaneous multi-way comparison of several genomes
(Bayer et al. 2011), on a reduced set of SNP markers selected using a cytogenetical and
molecular characterization of the APP population.
The gene contents, as inferred from rice Os01/Os10 and Os05 chromosomes, around the
translocation breakpoints were estimated as approximately 1,100 and 710 for
chromosomes 1H and 3H, respectively. As a proportion of the genes represent
retroelement-like components and pseudo-genes, the final number of functional genes in
barley must be smaller. Inferred gene contents for Sorghum and Brachypodium yielded
similar figures, around 600 (Sb09/Sb01) and 550 (Bradi02/Bradi03) genes for the
region on 1H and 410 (Sb02) and 430 (Bradi03) gene models for 3H, respectively. This
gene content is in good agreement with the gene content reported by Mayer et al. (2011)
who estimated that the barley genome contains in the order of 32,000 genes. With such
a large gene content the use of a candidate gene approach aiming at identifying which
gene(s) may have been altered by the translocation conferring a higher drought
tolerance to ‘Albacete’ is not practical.
Chapter 3
42
Whole-genome duplication analyses have been documented in grass species. Thiel et al.
(2009) identified shared duplicated segments that originate from the last common
ancestor and that remain in the barley genome. A large degree of synteny existed
between barley chromosome Hv1H and rice chromosome Os05 as well as between
Hv3H and Os01. The barley chromosome Hv3H corresponded to the ancestral
chromosome type of rice chromosome Os01. The structure of chromosome 1H could be
explained by combining the ancestral type of two rice chromosomes, where the first
(A10) appears to be nested in the second (A05). An interesting observation arising from
the comparative mapping of several Poaceae species is the presence of duplicated
segments between chromosomes Os01 and Os05 in rice derived from ancestral
duplications within the grass family (Guyot and Keller 2004) which are overlapping
with the translocation breakpoints on chromosomes 1H and 3H in ‘Albacete’.
Moreover, there are also large grass ancestral genome duplications affecting rice
chromosomes Os03 and Os07 homologous to barley chromosomes 2H and 4H. Smaller
ancestral duplications are present in regions of the rice genome homologous to barley
chromosomes 3H-5H and 3H-6H, which are also involved in spontaneous reciprocal
translocations in barley (Konishi and Linde-Laursen 1988).
In conclusion, the widely grown Spanish barley variety ‘Albacete’ carries a large
pericentric chromosome rearrangement between chromosomes 1H and 3H without any
major change in fitness. SNP and microsatellite markers located in the proximity of the
reciprocal translocation breakpoints were identified in both AB and APP mapping
populations within the pericentromeric regions of both chromosomes. The physical
characterization suggested that the translocation breakpoints are located on the long
arms of both chromosomes between the (AAG)5 and (ACT)5 pericentromeric bands
for 3HS.3HL-1HL and below the (AAG)5 pericentromeric band for 1HS.1HL-3HL. The
gene content in this region was estimated at approximately 1,100 and 710 gene models
for 1H and 3H respectively. Presence of duplicated segments between chromosomes
Os01 and Os05 in rice derived from ancestral duplications within the grass family
(Guyot and Keller 2004) overlaps with the translocation breakpoints on 1H and 3H
chromosomes in the barley variety ‘Albacete’. However, no current evidence is
available of whether similar gene contents on different chromosomes arising from
Genetic characterization of a reciprocal translocation present in a widely grown barley variety
43
ancestral duplications play a role in spontaneous chromosomal exchanges through non-
homologous chromosome pairing.
Acknowledgments
This study has been supported by the Spanish Ministry of Science and Innovation
(Project AGL2008-05541-C02 and the Agrogenomics CONSOLIDER Center).
45
Chapter 4
Agronomic effects of a large reciprocal translocation present in a widely grown Spanish barley variety
A. Farré1,2 · A. Visioni1 · I. Lacasa-Benito1 · L. Cistué3 · J. Jansen2 · I. Romagosa1,4
Published in Euphytica (2012) 185:119-122
Abstract
A large spontaneous reciprocal translocation is present in a widely grown Spanish
barley cv. 'Albacete'. It has been hypothesized that high popularity of ‘Albacete’ with
farmers, particularly in semi-arid areas where barley is grown under rainfed conditions,
may be due to the presence of this translocation. Agronomic effects of this translocation
were studied at two locations and two growing seasons in a set of 245 doubled haploid
lines derived from the F1s of four crosses involving 'Albacete'. The results have shown a
significant positive main effect of the translocation on the thousand kernel weight and a
significant environment by translocation interaction for the thousand kernel weight,
lodging and tiller number. However, the results do not support the hypothesis that this
chromosomal structural change alone provides an increased adaptation to low-yielding
sites.
Keywords Reciprocal translocation Barley Breeding Adaptation
1Department of Plant Production and Forest Science, University of Lleida, Lleida, Spain; 2Biometris, Wageningen University and Research Centre, Wageningen, the Netherlands; 3Estación Experimental de Aula Dei, CSIC, Zaragoza, Spain; 4Centre UdL-IRTA, University of Lleida, Lleida, Spain
Chapter 4
46
Introduction
Spontaneous reciprocal translocations seldomly occur in cultivated barley; only a few
cases have been described (Konishi and Linde-Laursen 1988). Translocations usually
reduce the agronomic value; ‘Albacete’ is the only extensively cultivated barley variety
carrying a reciprocal translocation without apparent loss of agronomic value. In Spain it
has been grown for decades on up to 1 million ha/year. The reciprocal translocation was
identified in a meiotic analysis of semi-sterile F1 hybrids involving ‘Albacete’ (Vazquez
and Sanchez-Monge 1989; see also Farré et al. (2011)). Farré et al. (2012) performed a
molecular and cytogenetic characterization of the reciprocal translocation and
determined the position of the translocation breakpoints. Drought is the main factor
limiting the yield of cereals in environments with high temperatures and limited rain
during the grain-filling period (López-Castañeda and Richards 1994). It is unknown
whether the reciprocal translocation has a positive effect on drought tolerance and other
traits that make it worth to be introduced in the barley germplasm. In the present study,
248 doubled haploid (DH) lines from four crosses involving ‘Albacete’ as one of the
parents will be used to phenotypically characterize the effects of the presence of the
reciprocal translocation.
Materials and methods
Plant material
Different agronomic traits were evaluated in 245 DH lines of barley derived from the
F1s between ‘Albacete’ and ‘Barberousse’, ‘Plaisant’ and ‘Orria’ and a DH line derived
from ‘Plaisant’בOrria’. ‘Albacete’ is a variety with a long cycle and an alternative
growth habit. It is drought tolerant with a stable grain yield production. ‘Barberousse’ is
known for its good productivity and easy adaptation; it is sensitive to drought. ‘Plaisant’
shows good adaptation and high-yield under Spanish conditions. ‘Orria’ is a Spanish
variety of CIMMYT origin, well adapted to fertile, rainfed environments. The DH lines
were scored for the presence of the reciprocal translocation using molecular data (Farré
et al. 2011). The number of lines carrying/not carrying the translocation were 41/54,
18/20, 40/27, 36/9 for A×B, A×O, A×P and A×(P×O), respectively.
Phenotyping
Agronomic effects of a large reciprocal translocation present in a widely grown Spanish barley variety
47
Four field trials were carried out at two rainfed locations in North-Eastern Spain in
2008/2009 and 2009/2010: Gimenells (41º 37’N, 0º 22’E, 248m) and Foradada (41º
51’N, 1º 0’E, 407m). Experiments contained one or two replicates per DH line
augmented by four replicated checks in a rectangular set-up.
Data analysis
For each population the average broad sense heritability was estimated. For each trait,
Best Linear Unbiased Estimates (BLUEs) of DH individuals were estimated by
removing spatial effects. The BLUEs were further analysed using the mixed model
facilities of Genstat version 13 (Payne et al. 2009), heterogeneous variances within
population were corrected.
Results and Discussion
(Table 1) Broad sense heritabilities (H2) ranged from 0.27 to 0.84. A highly significant
main effect of the reciprocal translocation was obtained for TKW; DH lines carrying the
reciprocal translocation had a greater TKW than those with a standard chromosome
arrangement (34.8 vs 32.9 gr, respectively). No significant main effects were found for
the other traits. For TKW, lodging and till number a significant environment by
translocation interaction was found. More lodging was recorded at Gimenells and for
the RT genotypes. Differences in the response of the RT to till number may be
associated to specific meteorological conditions. In conclusion, the results do not
support the hypothesis that the reciprocal translocation alone provides an increased
adaptation to low-yielding sites; TKW is the only trait which is clearly enhanced by the
reciprocal translocation. Future work combining the results from this study with QTL
analysis will be carried out to characterize the effects of the reciprocal translocation and
QTL simultaneously.
Chapter 4
48
Tab
le 1
. (a
) S
umm
ary
of t
he m
ixed
mod
el a
naly
sis
perf
orm
ed f
or a
ll t
he a
gron
omic
tra
its
stud
ied
com
pris
ing
982
geno
type
s ca
rryi
ng a
rec
ipro
cal
tran
sloc
atio
n (R
T)
or th
e st
anda
rd c
hrom
osom
e ar
rang
emen
t (no
RT
) in
fou
r tr
ials
(th
e si
gnif
ican
t lev
els
are
base
d on
the
Wal
d te
st).
(b)
Ave
rage
val
ues
for
the
two
grou
ps in
4
tria
ls (
carr
ying
or
not t
he r
ecip
roca
l tra
nslo
cati
on).
1 T
he d
egre
es o
f fr
eedo
m f
or D
ays
to J
oint
ing,
num
ber
of s
pike
s in
50
cm, E
arly
Gro
wth
and
Til
l Num
ber
shou
ld b
e eq
ual t
o tw
o, a
s da
ta w
as n
ot r
ecor
ded
in o
ne tr
ial
(a)
Sou
rce
of v
aria
tion
d.f.
1
Tra
its
asso
ciat
ed t
o d
evel
opm
ent
Yie
ld a
nd
yie
ld c
omp
onen
ts
D
ays
to J
oin
tin
g D
ays
to H
ead
ing
Day
s to
Mat
uri
ty
Nº
spik
es 5
0 cm
Y
ield
(T
/ha)
T
KW
(g)
Wal
d-te
st
p-va
lue
Wal
d-te
stp-
valu
e W
ald-
test
p-
valu
e W
ald-
test
p-
valu
e W
ald-
test
p-
valu
e W
ald-
test
p-
valu
e
Env
iron
men
t (E
) 3
1468
9.0
<0.
001
6287
1.4
<0.
001
7336
0.5
<0.
001
195.
2 <
0.00
1 55
9.6
<0.
001
2982
.0
<0.
001
P
opul
atio
n (P
op)
3 3.
8 0.
292
27.0
<
0.00
1 4.
1 0.
263
19.7
<
0.00
1 11
.0
0.01
6 37
.4
<0.
001
R
T
1 0.
2 0.
625
0.2
0.66
7 1.
5 0.
218
2.7
0.10
3 0.
7 0.
402
21.0
<
0.00
1
Pop
.RT
3
2.3
0.51
2 2.
3 0.
518
1.7
0.64
1 4.
8 0.
200
1.2
0.76
8 2.
9 0.
411
E
.Pop
9
13.3
0.
040
24.1
0.
005
13.3
0.
152
14.7
0.
024
6.4
0.69
4 62
.8
<0.
001
E
.RT
3
3.6
0.16
5 1.
7 0.
625
1.4
0.71
3 5.
0 0.
082
10.6
0.
014
17.6
<
0.00
1
E.P
op.R
T
9 17
.7
0.00
8 13
.5
0.14
2 1.
6 0.
996
5.1
0.53
4 10
.6
0.30
9 23
.9
0.00
5
H2
0.
73
0.84
0.
57
0.27
0.
60
0.81
(b
)
Env
iron
men
t
no R
T
RT
no
RT
R
T
no R
T
RT
no
RT
R
T
no R
T
RT
no
RT
R
T
F
-200
9/20
10
14
9.5
148.
1 18
3.0
183.
1 22
3.0
223.
4 44
.5
45.6
4.
9 5.
1 35
.6
37.9
F-2
008/
2009
- -
166.
6 16
6.8
196.
0 19
6.3
57.7
55
.8
5.4
5.6
29.8
32
.3
G
-200
9/20
10
12
6.9
127.
1 16
1.8
161.
9 20
2.0
202.
4 56
.4
59.5
6.
0 6.
3 38
.3
40.0
G-2
008/
2009
109.
4 10
9.2
151.
3 15
1.2
184.
6 18
4.8
- -
4.7
4.4
27.8
28
.4
A
vera
ge s
.e.d
0.63
0.
35
0.27
1.
80
0.13
0.
51
Agronomic effects of a large reciprocal translocation present in a widely grown Spanish barley variety
49
(Tab
le 1
. Con
tinu
ed)
1 T
he d
egre
es o
f fr
eedo
m f
or D
ays
to J
oint
ing,
num
ber
of s
pike
s in
50
cm, E
arly
Gro
wth
and
Til
l Num
ber
shou
ld b
e eq
ual t
o tw
o, a
s da
ta w
as n
ot r
ecor
ded
in o
ne tr
ial
(a)
Sour
ce o
f va
riat
ion
d.f.
1
Arc
hite
ctu
re t
rait
s
E
arly
Gro
wth
T
ill N
um
ber
T
otal
Hei
ght
(cm
) L
odgi
ng
W
ald-
test
p-
valu
e W
ald-
test
p-
valu
e W
ald-
test
p-
valu
e W
ald-
test
p-
valu
e
Env
iron
men
t (E
) 3
393.
4 <
0.00
113
57.0
<0.
001
176.
5<
0.00
123
1.0
<0.
001
P
opul
atio
n (P
op)
3 4.
1 0.
263
13.9
0.
005
19.3
<
0.00
1 36
.0
<0.
001
R
T
1 1.
3 0.
262
1.3
0.26
3 0.
0 0.
923
0.1
0.74
5
Pop
.RT
3
1.6
0.66
2 6.
0 0.
123
2.0
0.57
9 1.
6 0.
654
E
.Pop
9
13.0
0.
044
20.3
0.
003
17.4
0.
045
18.3
0.
034
E
.RT
3
0.5
0.76
5 12
.6
0.00
2 1.
1 0.
781
13.7
0.
004
E
.Pop
.RT
9
6.0
0.42
54.
70.
584
7.1
0.62
47.
80.
559
H
2
0.46
0.
49
0.41
0.
60
(b)
E
nvir
onm
ent
no
RT
R
T
no R
T
RT
no
RT
R
T
no R
T
RT
F-20
09/2
010
3.
1 3.
1 2.
7 2.
8 90
.1
88.7
4.
3 4.
3
F-20
08/2
009
-
- -
- 10
2.8
104.
8 4.
78
3.9
G
-200
9/20
10
2.
7 2.
7 5.
2 5.
8 93
.8
94.1
4.
6 5.
2
G-2
008/
2009
3.8
3.7
7.4
7.0
99.0
97
.0
6.8
7.2
A
vera
ge s
.e.d
0.09
0.23
1.82
0.41
Chapter 4
50
Acknowledgements
This study has been supported by the Spanish Ministry of Science and Innovation
(Project AGL2008-05541-C02 and the Agrogenomics CONSOLIDER Center).
51
Chapter 5
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal
translocation
A. Farré1,2, I. Romagosa1,3, L. Cistué4, J. Jansen2
Abstract
Using DArT® markers, an integrated genetic linkage map has been constructed by
combining five interconnected biparental doubled haploid (DH) populations of barley
involving four barley varieties. Three populations share 'Albacete' as a parent. 'Albacete'
carries a reciprocal translocation between chromosomes 1H and 3H which complicates
the construction of genetic linkage maps due to ‘pseudo-linkage’ between markers on
the chromosomes involved in the translocation. Levels of polymorphism varied
considerably among populations; the highest level was observed for ‘Albacete’ ×
‘Barberousse’ (65.68%) and ‘Albacete’ × ‘Plaisant’ (60.86%) whereas ‘Barberousse’ ×
‘Plaisant’ (22.08%) showed the lowest level. Only markers polymorphic in at least two
populations were used for constructing the integrated map. As a consequence,
construction of an integrated linkage map involves huge numbers of missing data. The
algorithm used for constructing the integrated map is based on minimization of the
number of recombination events between adjacent markers using hidden inheritance
vectors. This algorithm enabled us to develop a robust integrated linkage map involving
complex chromosomal rearrangements. The integrated map consists of 310 markers
covering 938 cM for all chromosomes except 1H and 3H. The number of markers per
chromosome varied between 17 (4H) and 92 (7H).
Keywords Integrated map DArT Reciprocal translocation Barley
1Department of Plant Production and Forest Science, University of Lleida, Lleida, Spain; 2Biometris, Wageningen University and Research Centre, Wageningen, the Netherlands; 3Centre UdL-IRTA, University of Lleida, Lleida, Spain; 4Estación Experimental de Aula Dei, CSIC, Zaragoza, Spain
Chapter 5
52
Introduction
High-density genetic linkage maps have been constructed for many crop species,
including barley (Hordeum vulgare L.). They are increasingly used in plant research to
understand genomic organization and evolution, the determination of synteny
relationships, gene tagging and map-based cloning. One of the most important
objectives for developing high-density genetic maps is to identify precise locations of
genes and quantitative trait loci (QTL) associated with traits of interest. This objective
can be achieved by constructing consensus maps using information from multiple
populations. In principle, the use of multiple populations has many advantages over the
use of a single population. In particular, a larger number of markers can be placed on a
single map providing greater genome coverage. Marker order and intermarker distances
can be estimated more accurately, and possible chromosomal rearrangements and gene
duplication can be determined.
Although many consensus maps have been produced in barley (Karakousis et al. 2003;
Langridge et al. 1995; Marcel et al. 2007; Qi et al. 1996; Stein et al. 2007; Varshney et
al. 2007; Rostoks et al. 2005), the consensus map which provided us the opportunity to
compare the order of the DArT markers on our integrate linkage maps with the order of
the same markers on a high-density genetic map was constructed by Wenzl et al. (2006).
This map was based on segregation data from seven DH populations and three
populations of RIL, comprising 2,935 loci (2,085 DArT, 850 SSR, RFLP and/or STS
markers) and spans 1,161 cM. Later, this map was improved by incorporating 607
newly developed DArT markers to create a new integrated map of the barley genome
containing 3542 markers (Alsop et al. 2010). Other saturated genetic maps were
developed using DArT markers in barley for instance the one which was constructed by
Hearnden et al. (2007). This was developed from an F1-derived doubled haploid
population generated from a cross between cultivated barley (Hordeum vulgare) and the
subspecies H. vulgare ssp. spontaneum. The map comprises 1,000 loci, amplified using
536 SSR (558 loci) and 442 DArT markers.
The construction of high-density genetic linkage map requires powerful methods, which
can perform the necessary calculations in a limited amount of time. There are several
programs that can be used to build consensus maps, including JoinMap, Carthagene and
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
53
PhenoMap (Stam 1993; De Givry et al. 2005; (GeneFlow Inc., Centreville, VA.). The
most important point to keep in mind when building a consensus map is that markers
are sorted correctly. For that, two aspects have to be considered: the mapping criterion
and the mapping algorithm (Van Os et al. 2005). The mapping criteria that have been
proposed include the maximum likelihood, the minimum sum of adjacent recombination
fractions (SARF), the maximum sum of adjacent LOD scores (SALOD), the minimum
number of crossovers and the ‘least square locus order’. For the mapping algorithms
several approaches have been proposed like branch-and-bound, seriation and simulated
annealing. A widely used platform for the construction of genetic linkage maps is
provided by JoinMap®. The map integration facilities were developed in a period that
segregating markers were scarce and many populations had to be combined to obtain
adequate genome coverage. However, the regression algorithm of JoinMap® fails when
it is required to integrate linkage maps involving large numbers of markers.
Jansen (2005) introduced an algorithm for constructing genetic linkage maps for full-sib
families of outbreeding species, which typically involves the integration of maternal and
paternal maps. In outbreeding species, markers may have different segregation types,
i.e. some markers only segregate only in the mother, and other markers only segregate
in the father. This leads to missing information about the inheritance of genomic
fragments. The algorithm is based on minimization of the number of recombination
events between adjacent markers using hidden inheritance vectors. Inheritance vectors
are used to indicate the grandparental origins of alleles.
The aim of the present study was to adapt the algorithm developed by Jansen (2005) to
cope with multiple populations derived from crosses between pure lines. In this
situation missing data occur due to the fact that markers may segregate in some
populations but not in others. This paper presents the results of integrating marker data
obtained using DArT technology from five interconnected DH populations of barley
into a single consensus map. Three of these DH populations have one parent in
common: ‘Albacete’ which is known to carry a reciprocal chromosomal translocation
between chromosomes 1H and 3H. To construct linkage maps in the populations
obtained from reciprocal translocation heterozygotes we followed the novel statistical-
genetic approach developed by Farré et al. (2011) which disentangled the 'pseudo-
Chapter 5
54
linkage' between the markers from the chromosomes involved in the reciprocal
translocation making use of the consensus map obtained by Wenzl et al. (2006).
Materials and methods
Mapping populations
Five connected DH populations were derived from crosses between four parents:
‘Albacete’, ‘Barberousse’, ‘Plaisant’ and ‘Orria’. ‘Albacete’ is a six-row facultative
variety selection from a local Spanish landraces with a relatively long cycle and an
alternative growth habit. It is drought tolerant showing a relatively stable grain yield
production. ‘Barberousse’ ('Ager' × ['W.259-711' × ('Hatif' × 'Ares')]), a six-row feed
winter variety released in France in 1977, is known for its good productivity and easy
adaptation although it is more sensitive to drought. ‘Plaisant’ ('Ager' × 'Nymphe') is a
six-row French winter malting cultivar released in 1979. ‘Orria’ ({[('Api' × 'Kristina') ×
'M66.85'] × 'Sigfrido's} × '79W40762') is a Spanish six-row facultative variety obtained
from a cross made at CIMMYT; it is well adapted to fertile, rainfed environments.
DH lines were obtained from the F1s using the anther-culture technique (Cistué et al.
2003). The five DH populations that will be used to construct the integrated linkage
map are shown in Figure 1, together with the numbers of DH lines per population and
polymorphic DArT markers.
Figure 1. Set-up of the five DH populations with the number of DH lines (top) and the number of
DArT markers (bottom).
DNA isolation and genotyping
Albacete Plaisant
Barbarrousse Orria
181 DHL709 DArT®
39 DHL729 DArT®
54 DHL276 DArT®231 DHL
709 DArT®186 DHL
472 DArT®
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
55
Genomic DNA of the DH lines was extracted from fresh leaf tissue using the Qiagen
DNeasy 96 plant Kit. A genome wide scan was carried out using Diversity Arrays
Technology (DArT®) (www.diversityarrays.com). This technique is based on DNA
hybridization, does not require a priori sequence information and is carried out on a
microarray platform which allows high-throughput screening of hundreds of molecular
markers simultaneously (Jaccoud et al. 2001, Wenzl et al. 2004). The polymorphisms
scored are the presence (1) or absence (0) of hybridizations to individual array elements.
The locus designations used by Triticarte Pty. Ltd. were adopted in this paper. DArT
marker names consist of the prefix “bPb”, followed by numbers corresponding to a
particular clone in the genomic representation, where b stands for barley, P for PstI
(primary restriction enzyme used) and b for BstNI (secondary restriction enzyme).
Genetic linkage map construction
The first step in the construction of the consensus map was to assess each DH
population separately. For each DH population quality-filtered datasets were prepared
by removing markers and DH lines in two different steps: pre-mapping and post-
mapping diagnostics. For pre-mapping diagnostics markers were removed according to
the following criteria: (1) Q value (estimate of marker quality) less than 77, (2) markers
with more than 10% missing data and (3) markers with segregation ratio less than 0.2 or
greater than 0.8. DH lines were discarded according to high segregation distortion ratio
(above 80%). For post-mapping diagnostics, JoinMap® 4.0 (Van Ooijen, 2006) was
used to: (1) determine the number of linkage groups present in each population using a
recombination frequency of 0.20, (2) remove markers that do not fit well by the
“nearest-neighbour stress” criterion and (3) discard DH lines with large number of
recombinations per individual (more than 20).
The novel statistical-genetic approach developed by Farré et al (2011) was followed to
construct linkage maps in the populations obtained from reciprocal-translocation
heterozygotes making use of the consensus map obtained by Wenzl et al. (2006).
Briefly, the five chromosomes showing a standard arrangement were analyzed
separately. Then, for chromosomes 1H and 3H four stages were followed: (i) the
recombination frequencies between the markers from 1H and 3H were calculated, which
Chapter 5
56
allowed us to select the markers close to the TB; (ii) based on the genotype for those
markers the DH lines were separated into two distinct subpopulations: S1 which around
the translocation breakpoint the DH lines are identical to ‘Albacete’, so they are
expected to carry the translocation between 1H and 3H chromosomes (1HS·3HL +
3HS·1HL) and S2 which close to the TB the alleles are identical to standard type
(‘Plaisant’, ‘Barberousse’ or ‘Orria’), this indicates that they have a standard
chromosomal arrangement (1HS·1HL + 3HS·3HL); (iii) the segregation ratios for each
marker were calculated for each of the two subpopulations, markers showing
segregation ration less than 0.3 or greater than 0.7 were removed; (iv) the two
subpopulations were considered separately to obtain independent linkage maps for the
short and long arms of 1H and 3H. The final product consists of integrated linkage maps
of the chromosome parts away from the translocation breakpoint.
The markers used for constructing the consensus map were those which segregated in
more than 300 DH lines for all chromosomes except for 1H and 3H; in that case, the
markers selected were those that mapped in more than 150 DH lines. Therefore, all
markers used were segregating in at least two mapping populations.
Integrated linkage maps were obtained by a simplified version of the algorithm
described by Jansen (2005) and developed for full-sib families of outbreeding species.
The algorithm has also been applied succesfully in F2 populations with dominant
markers (Jansen 2009). The algorithm aims at obtaining the marker order with the
smallest number of recombinations between adjacent markers on hidden inheritance
vectors. Inheritance vectors represent the grandparental origin of alleles in binary form:
0 = allele of DH line is identical to allele of grandmother (= female parent of cross), 1 =
allele of DH line is identical to allele of grandfather (= male parent of cross). Since DH
lines contain information from a single meiosis, inheritance vectors for DH lines consist
of a single digit. If the parents differ with regard to hybridization with DArT probes (i.e
polymorphic parents) and hybridization data are present for the DH lines, hybridization
data can be converted uniquely into inheritance vectors according to the scheme in
Table 1. In other cases information about the transmission of alleles is missing and the
inheritance vectors may assume the value zero or one. Two steps were performed to
obtain the integrated maps for each linkage group separately. In the first step, a
framework map is constructed for a set of markers which are selected because they
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
57
cover the whole linkage map using the spatial sampling procedure. In the next step, all
non-selected markers will be attached on the previous map close to the nearest selected
marker. Linkage maps were drawn using MapChart 2.2 (Voorrips, 2002).
Table 1. Conversion of hybridization data into inheritance vectors
Results
Single populations
Pre-mapping diagnostics
Linkage maps are strongly affected by scoring errors. Therefore, high quality genotypic
data is critical. Thus, in order to get robust estimates very stringent criteria were used
for data filtering. For each DH population separately, markers and DH lines were
excluded according to different criteria. In the case of the markers, although a total of
2,895 DArT® polymorphic markers were found by the Australian company Triticarte
Pty for the five DH populations, 545 markers were removed according to: Q value (195
markers), missing data (230 markers) and high segregation distortion ratios (120
markers). The results are shown in Table 2. Population A×O has a remarkably large
number of markers with an extreme segregation ratio. This may be due to the small
population size (only 39 DH lines). As a result, 2,350 DArT markers can be used for the
map construction. In the case of DH lines, a total of 691 DH lines were available for the
construction of linkage maps but 7 individuals were discarded due to the high
segregation distortion ratios (above 80%) caused by very limited recombination as they
consisted almost entirely of one parental haplotype (5 like ‘Albacete’ and 2 like
‘Plaisant’ from A×B and P×O crosses, respectively).
DH line: Hybridization
DH line: No-hybridization
DH line: Missing data
Grandmother: hybridization Grandfather: no hybridization
0 1 0, 1
Grandmother: no hybridization Grandfather: hybridization
1 0 0, 1
Chapter 5
58
Table 2. Summary of individual mapping data used to construct the consensus map (* = in the
case of A×O population markers with more than 5% of missing data were removed; Pop
= population).
Post-mapping diagnostics
Markers that according to the information supplied by the Australian company Triticarte
Pty belonged to more than one chromosome or mapped on different loci within the
same chromosome were not removed and location was attempted in the correct
chromosome (16, 5, 13, 13 and 8 markers from A×B, A×O, A×P, P×O and B×P crosses,
respectively). 38 out of 684 DH lines were discarded because of an extremely large
number of recombinations per individual (6, 2, 1, 28 and 1 for A×B, A×P, A×O, P×O
and B×P crosses, respectively). As a result, a total of 646 DH lines were used for the
linkage maps construction.
Polymorphisms between parents
The first step required in the construction of a linkage map is to check the parental lines
for polymorphism. A high level of polymorphism between parents facilitates the
construction of high-density consensus map. The level of polymorphism among
populations for each chromosome is represented in Figure 2 and SEM I_Table 3. The
location of markers to chromosomes and their positions was obtained from the
consensus map of barley (Wenzl et al. 2006). This clearly shows the highest level of
polymorphism revealed by DArT markers between parents of the A×B (65.68%) and
A×P (60.86%) mapping populations. However, B×P exhibits the lowest level (22.08%),
in almost all chromosomes followed by A×O (38.66%). For the parental lines ‘Plaisant’
Pop. No. polymorphic Markers
Number of markers excluded per criterion
Available polymorphic markers
Q value Missing data Segregation ratio
A×B 709 16 85 8 600 A×P 709 64 51 2 592 A×O 729 51 34* 108 536 P×O 472 50 40 0 382 B×P 276 14 20 2 240 Total 2,895 195 230 120 2,350
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
59
and ‘Orria’ (52.89%), 1H was the highest level (92.23%) while the lowest is observed
on 4H (9.62%). In the present study, the percentage of polymorphism of the individual
chromosomes for all mapping populations ranged from 13% (4H) to 95.8% (6H).
Figure 2. Polymorphism among populations for each chromosome. The allocation of markers as
well as their position were estimated based on the consensus map of barley (Wenzl et al.
2006) (horizontal lines are drawn every 10 cM; red colour indicated the regions of the
consensus map without coverage).
Chapter 5
60
Individual maps
In all populations and for the recombination value used (r = 0.2), more than seven
linkage groups corresponding to each of the barley chromosomes were obtained because
in a lot of cases each chromosome was split in more than one linkage group. It is
important to note that markers near the centromeric region for chromosomes 1H and 3H
based on the consensus map appeared together in the same linkage group for the DH
lines derived from the cross between ‘Albacete’ and another parental (A×B, A×P, and
A×O). This confirms the presence of the reciprocal translocation previously studied in
the DH population derived from the cross between ‘Albacete’ and ‘Barberousse’ (Farré
et al. 2012). Table 4 shows the selected markers close to the translocation breakpoint
used to divide the DH line for each population into two distinct subpopulations; not all
selected markers coincide among populations, this is because some markers were
removed according to the criteria described in Farré et al. 2011 or they were not within
the 5 cM from the presumed translocation breakpoints.
The mapping populations with the highest number of mapped loci were A×B (521
markers) and A×P (514 markers) while the lowest was observed for B×P (225 markers).
The total length of the individual maps ranged from 1,623 cM (A×P) to 683 cM (B×P).
For the three populations that share ‘Albacete’ as a parent, 5H was the individual
linkage group with the highest map length (252, 296 and 243 cM from A×B, A×P and
A×O crosses, respectively); for P×O and B×P population was 7H (243 cM) and 3H (221
cM), respectively; whereas the smallest linkage group varied among mapping
populations. In the case of A×B and A×P, 1H was the linkage group with the smallest
map length (149 and 160 cM, respectively). For A×O and P×O it corresponded with 7H
(85 cM) and 4H (92 cM), respectively. And for B×P, the map length for the linkage
groups 5H and 6H was 0 cM because they had 4 markers which mapped in the same
position. The greatest gap in all mapping populations was on chromosome 3H (104, 76,
130, 81 and 60 cM from A×B, A×P, A×O, B×P and P×O crosses, respectively).
It is important to note that some markers that according to the information supplied by
the Australian company Triticarte Pty could be mapped in more than one chromosome
or multiple loci on the same chromosome were located correctly (9, 5, 6, 2 and 2
markers from A×B, A×P, A×O, B×P and P×O population, respectively). However,
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
61
some markers mapped on different chromosomes according to the consensus map (bPb-
0413, bPb-6063, bPb-5389 and bPb-4963 mapped on 5H instead of 2H; bPb-6109 and
bPb-7181 on 7H instead of 3H and 6H, respectively).
Comparison of the individual maps with the consensus map of Wenzl et al (2006)
The individual map distances generated by the algorithm developed by Jansen et al.
(2001, 2005 and 2009) are larger than those on the consensus map of barley (Wenzl et
al. 2006). For population, the overestimation of distances relative to the consensus map
distance was calculated. The A×P and A×B populations had the highest percentage, 40
and 22%, respectively, while the smallest percentage was for A×O (4%) and B×P (-
41%).
Chapter 5
62
Table 4. Summary of the selected markers used to divide the DH lines for each mapping
population and subpopulation data (S1: 1HS·3HL + 3HS·1HL and S2: 1HS·1HL +
3HS·3HL).
Population
S1 S2 Linkage group
1H 3H
No. lines
Markers with segregation distortion (<30%, >70%)
No. lines
Markers with segregation distortion (<30%, >70%)
Marker loci
Consensus map position (cM)
Marker loci
Consensus map position (cM)
A×B 88 65 85 69 bPb-9717 58.7 bPb-6329 66.5
bPb-6621 59.1 bPb-0094 69.3
bPb-4949 59.2 bPb-5771 69.3
bPb-0910 59.4 bPb-5012 69.8
bPb-9957 63.3 bPb-8321 70.4
bPb-9360 64.4
A×P 66 50 80 60 bPb-5749 63.3 bPb-0094 69.3
bPb-9767 63.3 bPb-5771 69.3
bPb-9957 63.3 bPb-8283 69.6
bPb-1193 62.2 bPb-5012 69.8
bPb-3382 62.2 bPb-8321 70.4
bPb-1791 67.1
A×O 18 61 15 55 bPb-5749 63.3 bPb-0040 72.2
bPb-9767 63.3 bPb-3805 72.2
bPb-9957 63.3 bPb-3317 78.6
bPb-1193 62.2 bPb-8410 78.6
bPb-3382 62.2
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
63
Multiple populations
Integrated maps
The construction of the consensus map was carried out for each chromosome separately
using the algorithm developed by Jansen et al. (2001, 2005, and 2009). For
chromosomes 1H and 3H two independent integrated maps were obtained using the
markers from A×B, A×P, A×O and B×P, P×O mapping populations separately.
The integrated maps of seven barley chromosomes are presented by Figure 4 and Table
5. This map has a total length of 938 cM with 310 markers for all chromosomes except
1H and 3H providing an average density of one marker every 3.02 cM. The individual
linkage groups ranged from 228 cM (5H) to 148 cM (4H). The numbers of common
markers varied greatly between linkage groups: 17 on 4H compared to 92 on 7H. On the
integrated map, linkage group 7H had the greatest number of markers (92) with an
average marker density of 2.19 cM followed by linkage group 6H (75) with an average
marker density of 2.14 cM. Linkage group 4H had the smallest number of markers (17)
and the highest marker density (8.70 cM). The highest gap is in linkage group 4H (41
cM). The resulting maps for each population compared with the integrated are shown on
SEM II_Figure 3 and Table 5.
Comparison of integrated map with the consensus map of Wenzl et al (2006)
At completion of construction of the map a comparison was made between the order of
loci on the consensus map and the integrated map which showed a high level of
conservation of DArT marker order with the exception on 4H and for bPb-7335 marker
on 3HS for A×O/A×P/A×B integrated map which is located on 3HL on the consensus
map. The linkage group distances are larger than those on the consensus map. Linkage
group 1H obtained using markers data from P×O and B×P mapping populations had the
highest percentage (27%) followed by 7H (22%). The smallest percentage was in 1H
obtained using markers data from A×B, A×P and A×O mapping populations (-27%).
Chapter 5
64
Table 5. Summary of individual and integrated mapping data (I = integrated and CN =
consensus) (length of disjoint intergroup segments within a linkage group determined
based on the consensus map; a= map obtained using marker data from A×O, A×B and
A×P populations; b= map obtained using markers data from P×O and B×P mapping
populations).
Pop 1H 2H 3H 4H 5H 6H 7H Total
A×B
No. of segments 3 2 2 3 2 1 3 Length segments (cM)
87 238 82 104 240 207 170 1,128
No. of markers 41 98 55 43 79 101 104 521 Consensus intergap
62 7 105 58 12 - 45
Estimate total 149 245 187 162 252 207 215 1,417
A×O
No. of segments 3 3 2 3 4 2 4 Length segments (cM)
25 192 20 92 180 133 59 701
No. of markers 14 91 16 38 61 59 65 344 Consensus intergap
115 33 130 104 63 32 26
Estimate total 140 225 150 196 243 165 85 1,204
A×P
No. of segments 3 3 2 3 2 2 3 Length segments (cM)
62 243 146 101 278 206 235 1,271
No. of markers 41 95 54 37 82 94 111 514 Consensus intergap
98 39 76 74 18 14 33
Estimate total 160 282 222 175 296 220 268 1,623
P×O
No. of segments 1 3 3 2 3 1 2 Length segments (cM)
226 120 135 53 143 126 236 1,039
No. of markers 73 41 44 20 41 51 108 378 Consensus intergap
- 60 88 39 100 - 14
Estimate total 226 180 223 92 243 126 250 1,340
B×P
No. of segments 3 3 2 1 1 1 2 Length segments (cM)
131 33 140 22 0 0 86 412
No. of markers 61 40 28 16 4 4 72 225 Consensus intergap
59 104 81 - - - 27
Estimate total 190 137 221 22 0 0 113 683
Integrated
No. of segments 3a 1b 2 2a 2b 3 2 1 1 Length segments (cM)
45a 193b 180 86a 114b 80 203 161 202
No. of markers 40a 63b 64 51a 30b 17 62 75 92 Consensus intergap
66a - 19 104a 41b 68 25 - -
Estimate total 111a 193b 199 190a 155b 148 228 161 202 I/CN (%) -27a 27b 19 3a -5b 0 17 9 22
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
65
Fig
ure
4.
Com
pari
son
of t
he i
nteg
rate
d m
aps
wit
h th
e co
nsen
sus
map
s co
nstr
ucte
d by
Wen
zl e
t al
. 20
06 (
only
com
mon
mar
kers
are
ill
ustr
ated
). C
hrom
osom
es a
re
orie
nted
wit
h th
e sh
ort a
rm a
t the
top.
The
gen
etic
dis
tanc
es a
re e
xpre
ssed
in m
ap d
ista
nces
(cM
). (
TB
= tr
ansl
ocat
ion
brea
kpoi
nt).
bPb-
8559
bPb-1
165
bPb-3
622
bPb-
1348
bPb-
3776
bPb-
8973
bPb-
0442
bPb-
7306
bPb-
9288
bPb-
1226
Clon
eID-
2221
91bP
b-94
15Cl
oneI
D-22
1017
bPb-0
851
bPb-
0760
Clon
eID-
2217
26bP
b-030
1bP
b-93
37
bPb-
3389
bPb-
9656
bPb-
8983
bPb-
3089
bPb-
9401
bPb-
8605
bPb-
4144
bPb-1
213
bPb-5
249
Clon
eID-
2216
87bP
b-91
08bP
b-003
9Cl
oneID
-221
039
bPb-
0340
bPb-
5014
Clon
eID-
2219
02
bPb-
8619
bPb-
0699
bPb-
3660
bPb-
1381
bPb-
1487
bPb-
2260
bPb-
0405
bPb-
1165
bPb-1
348
bPb-3
622
bPb-
3776
bPb-
2723
bPb-
9608
bPb-7
043
bPb-
7137
bPb-
8973
bPb-
1318
bPb-
9604
bPb-
9952
bPb-
4657
bPb-
2183
bPb-
7306
bPb-
9337
bPb-
9423
bPb-
9333
bPb-
2175
bPb-
9717
bPb-
9675
bPb-
0910
bPb-4
980
bPb-
0468
bPb-
2813
bPb-7
325
bPb-7
859
bPb-
8960
bPb-
4531
bPb-
6853
bPb-
3605
bPb-
5683
bPb-
9611
bPb-1
922
bPb-5
292
bPb-
6133
bPb-
4590
bPb-5
334
bPb-8
897
bPb-
5486
bPb-
8107
bPb-
3389
bPb-
7609
bPb-
9116
bPb-
3089
bPb-
4144
bPb-
5249
bPb-9
121
bPb-4
898
bPb-
6901
bPb-
6911
bPb-
1213
bPb-
1366
bPb-
1419
bPb-
4515
bPb-9
180
bPb-
9108
bPb-
5014
bPb-
5198
bPb-
2240
bPb-
8112
bPb-
0699
bPb-
3660
bPb-
1487
bPb-
2260
bPb-
0405
Clon
eID-2
2156
3bP
b-27
23bP
b-13
18bP
b-71
37bP
b-96
08bP
b-70
43bP
b-99
52bP
b-96
04Cl
oneID
-222
789
Clon
eID-2
2104
0bP
b-46
57bP
b-21
83
bPb-
9337
bPb-
9423
bPb-
9333
bPb-
2175
Clon
eID-2
2110
8bP
b-51
64bP
b-97
17bP
b-09
10bP
b-56
83bP
b-04
68bP
b-89
60bP
b-785
9Cl
oneID
-223
899
bPb-
9675
bPb-
3605
Clon
eID-
2208
14bP
b-73
25bP
b-28
13bP
b-68
53bP
b-498
0bP
b-45
31bP
b-52
92bP
b-96
11bP
b-19
22bP
b-53
34bP
b-45
90bP
b-88
97bP
b-72
89bP
b-54
86bP
b-61
33bP
b-81
07bP
b-76
09bP
b-30
89bP
b-91
16bP
b-91
21bP
b-48
98bP
b-69
01bP
b-13
66bP
b-12
13bP
b-69
11bP
b-52
49
bPb-
4515
bPb-
1419
bPb-
9180
Clon
eID-2
2103
9
bPb-
5198
bPb-
5014
bPb-
2240
Clon
eID-2
2421
3
bPb-
8112
bPb-7
803
bPb-3
598
bPb-
6466
bPb-7
626
bPb-8
399
bPb-5
444
bPb-
9220
bPb-8
292
bPb-3
050
bPb-
6963
bPb-
5884
bPb-1
098
bPb-4
523
bPb-
8750
bPb-8
038
bPb-4
261
bPb-
9682
bPb-4
877
bPb-3
519
bPb-8
449
bPb-2
230
bPb-9
992
bPb-
3677
bPb-
2219
bPb-3
575
bPb-6
088
bPb-4
040
bPb-9
754
bPb-7
991
bPb-1
926
bPb-3
563
bPb-
6194
bPb-4
184
bPb-9
458
bPb-7
124
bPb-5
755
bPb-3
858
bPb-7
816
bPb-6
087
bPb-2
244
bPb-
9199
bPb-1
986
bPb-
7208
bPb-8
530
bPb-1
566
bPb-
5460
bPb-
5619
bPb-8
240
bPb-
0689
bPb-6
048
bPb-
9909
bPb-
3598
bPb-
6466
bPb-
7803
bPb-
7626
bPb-
3459
bPb-
8399
bPb-
6235
Clon
eID-
2212
38Cl
oneI
D-22
0974
bPb-
5444
bPb-
9220
bPb-
8292
bPb-
3050
bPb-
6963
bPb-
5884
bPb-
4523
bPb-
8750
bPb-
1098
bPb-
8038
bPb-
4261
bPb-
9682
bPb-
4877
bPb-
9802
bPb-
9131
bPb-
2230
Clon
eID-
2227
60bP
b-35
72bP
b-39
85bP
b-20
13bP
b-36
77bP
b-99
92bP
b-22
19bP
b-35
75bP
b-60
88bP
b-404
0bP
b-975
4Cl
oneI
D-22
1652
bPb-
6194
bPb-
3563
bPb-1
926
bPb-
7991
Clon
eID-
2212
27bP
b-41
84bP
b-94
58bP
b-71
24bP
b-38
58bP
b-57
55bP
b-78
16bP
b-91
99bP
b-22
44bP
b-60
87bP
b-56
19bP
b-72
08bP
b-19
86bP
b-85
30bP
b-15
66bP
b-54
60bP
b-06
89bP
b-82
40Cl
oneI
D-22
0851
bPb-
9909
bPb-
6048
2233
37
Clon
eID-
1051
69bP
b-40
22Cl
oneI
D-10
5031
bPb-5
555
Clon
eID-
2220
55bP
b-84
57bP
b-12
64bP
b-95
83bP
b-99
45Cl
oneI
D-22
0951
bPb-
0476
Clon
eID-
2235
77bP
b-77
70bP
b-71
99bP
b-36
49Cl
oneI
D-22
3396
Clon
eID-
2208
82Cl
oneI
D-22
1142
bPb-
9745
Clon
eID-
2218
98bP
b-74
48bP
b-13
02bP
b-72
85Cl
oneI
D-22
4036
bPb-
0331
Clon
eID-
2212
74bP
b-63
61bP
b-73
35bP
b-292
9
bPb-
8506
bPb-
8621
bPb-
2550
bPb-
1609
bPb-
9111
bPb-
6249
bPb-
3867
bPb-
5666
bPb-
3899
bPb-
6383
bPb-
7827
bPb-0
789
bPb-
9599
bPb-
0945
bPb-
7815
bPb-
8419
bPb-
0200
bPb-
0735
bPb-
0361
bPb-
9965
bPb-
5795
bPb-
7724
bPb-
4022
bPb-
5555
bPb-
1264
bPb-
9583
bPb-
9945
bPb-
7770
bPb-
7199
bPb-
7448
bPb-
2929
bPb-
0312
bPb-
6347
bPb-
2440
bPb-
7872
bPb-
1681
bPb-
8506
bPb-
8621
bPb-
2550
bPb-1
609
bPb-
9111
bPb-
6249
bPb-
7335
bPb-
3899
bPb-
8557
bPb-
5666
bPb-
6383
bPb-
9118
bPb-7
827
bPb-
0789
bPb-
9599
bPb-
7815
bPb-
8419
bPb-
6631
bPb-
0200
bPb-
1928
bPb-
7164
bPb-
8322
bPb-8
907
bPb-
1411
bPb-
0361
bPb-
0164
bPb-
7724
bPb-7
481
bPb-
7705
bPb-
7918
Clon
eID-
2209
51bP
b-940
2bP
b-71
99bP
b-61
27Cl
oneID
-221
142
Clon
eID-2
2088
2
bPb-0
312
bPb-6
782
bPb-6
347
bPb-7
937
bPb-2
440
bPb-7
872
Clon
eID-2
2235
8bP
b-168
1Cl
oneID
-222
616
bPb-7
827
bPb-
6383
bPb-
9118
bPb-
8557
Clon
eID-2
2087
8bP
b-663
1
bPb-7
164
bPb-
1928
bPb-
8907
bPb-
8322
bPb-1
411
bPb-0
164
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
2051H
-AxB/AxP/AxO
1H-consensus
1H-PxO/BxP
2H-consensus
2H3H-AxB/AxP/AxO
3H-consensus
3H-PxO/BxP
TB
TB
Chapter 5
66
Fig
ure
4. (
cont
inue
d)
bPb-
9304
bPb-
4699
bPb-
1408
bPb-
8437
bPb-
6640
bPb-
9039
bPb-
4183
bPb-
3512
bPb-
9504
bPb-
6949
bPb-
9998
bPb-
5265
bPb-
9304
Clon
eID-
2226
50bP
b-46
99
bPb-
8437
bPb-
1408
bPb-
6640
Clon
eID-
2223
52
bPb-
6949
Clon
eID-
2237
51bP
b-95
04
Clon
eID-
2225
09
bPb-
4183
bPb-
9039
bPb-
3512
bPb-
9998
Clon
eID-
2216
36
bPb-
5265
bPb-
6485
bPb-
9562
bPb-
6051
bPb-
1909
bPb-
4814
bPb-
8072
bPb-
2460
bPb-
9632
bPb-
0050
bPb-
6067
bPb-
6495
bPb-
6363
bPb-
7348
bPb-
3412
bPb-
3792
bPb-
6710
bPb-
3852
bPb-
7561
bPb-
0709
bPb-
2497
bPb-
6288
bPb-
4721
bPb-
7395
bPb-
5596
bPb-
4494
bPb-
2580
bPb-
5845
bPb-
4318
bPb-
1420
bPb-
4970
bPb-
2006
bPb-
0171
bPb-
6179
bPb-
8070
bPb-
4595
bPb-
0835
bPb-
1965
bPb-
4971
bPb-
1217
bPb-
9660
bPb-
4809
bPb-
6485
bPb-
9562
bPb-
6051
bPb-
1909
Clon
eID-
2232
99bP
b-48
14bP
b-80
72bP
b-24
60
bPb-
0349
bPb-
9632
bPb-
0050
bPb-
6495
bPb-
6067
bPb-
6363
Clon
eID-
2242
19bP
b-73
48bP
b-34
12bP
b-37
92
bPb-
3852
bPb-
6710
bPb-
0709
bPb-
7561
bPb-
4721
bPb-
6288
bPb-
2497
bPb-
9032
bPb-
5516
Clon
eID-
2232
33
bPb-
7395
bPb-
5596
bPb-
0780
bPb-
5389
bPb-
6063
bPb-
8449
bPb-
3519
bPb-
0413
Clon
eID-
2221
96bP
b-70
22bP
b-04
86bP
b-25
80bP
b-88
54bP
b-44
94bP
b-58
45bP
b-43
18bP
b-14
20bP
b-49
70bP
b-20
06Cl
oneI
D-22
2499
bPb-
8809
bPb-
0621
bPb-
0171
Clon
eID-
2213
74bP
b-80
70bP
b-45
95bP
b-08
35bP
b-61
79bP
b-19
65bP
b-49
71Cl
oneI
D-22
2075
bPb-
1217
bPb-
9660
bPb-
4809
bPb-
3780
bPb-
0426
bPb-
1009
bPb-
7193
bPb-
7362
bPb-
3586
bPb-
8528
bPb-
2930
bPb-
9852
bPb-
2672
bPb-
6419
bPb-
6069
bPb-
9651
bPb-
6661
bPb-
5252
bPb-
9807
bPb-
6457
bPb-
3427
bPb-
2881
bPb-
0245
bPb-
5910
bPb-
7179
bPb-
3746
bPb-
2464
bPb-
5822
bPb-
3487
bPb-
9114
bPb-
1666
bPb-
6567
bPb-
9082
bPb-
2593
bPb-
4750
bPb-
9051
bPb-
9835
bPb-
3068
bPb-
0451
bPb-
4178
bPb-
0606
bPb-
9063
bPb-
6477
bPb-
3470
bPb-
9349
bPb-
3144
bPb-
1029
bPb-
2304
bPb-
2863
bPb-
6677
bPb-
2940
bPb-
1621
bPb-
0403
bPb-
7146
bPb-
4626
bPb-
2137
bPb-
0426
bPb-
3780
Clon
eID-
2227
74bP
b-10
09bP
b-71
93bP
b-35
86bP
b-98
52bP
b-29
30bP
b-85
28bP
b-73
62bP
b-64
19bP
b-60
69bP
b-26
72bP
b-07
42bP
b-96
51Cl
oneI
D-22
1373
bPb-
5252
bPb-
6661
bPb-
3427
bPb-
6457
bPb-
2881
bPb-
9807
bPb-
0245
Clon
eID-
2239
14bP
b-59
10Cl
oneI
D-22
1002
Clon
eID-
2233
86bP
b-37
46bP
b-71
79bP
b-03
96Cl
oneI
D-22
0959
bPb-
2464
bPb-
5822
bPb-
6567
bPb-
1666
bPb-
9114
bPb-
3487
bPb-
4750
bPb-
2593
bPb-
9835
bPb-
9051
bPb-
9082
Clon
eID-
2215
14Cl
oneI
D-22
2255
Clon
eID-
2223
06bP
b-30
68Cl
oneI
D-22
1627
Clon
eID-
2211
05bP
b-33
09bP
b-27
55bP
b-87
70bP
b-21
85Cl
oneI
D-22
3391
Clon
eID-
2219
46bP
b-41
78bP
b-04
51bP
b-06
06bP
b-64
77bP
b-34
70bP
b-90
63bP
b-29
40bP
b-66
77bP
b-28
63Cl
oneI
D-22
2519
bPb-
3144
bPb-
9349
Clon
eID-
2237
21bP
b-02
98bP
b-21
37bP
b-46
26bP
b-23
04bP
b-71
46bP
b-04
03bP
b-16
21bP
b-10
29
bPb-
8921
bPb-
8161
bPb-
2718
bPb-
6170
bPb-
6976
bPb-
5259
bPb-
0043
bPb-
7038
bPb-
4064
bPb-
2595
bPb-
0179
bPb-
3718
bPb-
4097
bPb-
3727
bPb-
7417
bPb-
7183
bPb-
5494
bPb-
1806
bPb-
2478
bPb-
9601
bPb-
9898
bPb-
6821
bPb-
5091
bPb-
2866
bPb-
7835
bPb-
1209
bPb-
0366
bPb-
8524
bPb-
0037
bPb-
6706
bPb-
4541
bPb-
4597
bPb-
7952
bPb-
4219
bPb-
1952
bPb-
5599
bPb-
8956
bPb-
2097
bPb-
1770
bPb-
7603
bPb-
7437
bPb-
2188
bPb-
7915
bPb-
9912
bPb-
5296
bPb-
7399
bPb-
8074
bPb-
5747
bPb-
1596
bPb-
0202
bPb-
4191
bPb-
7628
bPb-
6167
bPb-
0758
bPb-
3980
bPb-
1631
bPb-
0917
bPb-
0889
bPb-
5923
bPb-
0773
bPb-
1336
bPb-
7983
bPb-
8833
bPb-
3226
bPb-
2897
bPb-
2693
bPb-
2854
bPb-
0375
bPb-
2718
bPb-
6170
Clon
eID-
2209
12bP
b-00
43bP
b-52
59bP
b-69
76bP
b-81
61bP
b-89
21bP
b-70
38bP
b-40
64bP
b-48
86bP
b-25
95Cl
oneI
D-22
1452
Clon
eID-
2224
70Cl
oneI
D-22
1538
bPb-
0179
bPb-
3718
bPb-
4097
bPb-
7417
bPb-
3727
bPb-
2478
Clon
eID-
2210
61bP
b-18
06bP
b-54
94bP
b-71
83bP
b-96
01bP
b-98
98Cl
oneI
D-22
2477
bPb-
6821
bPb-
5091
bPb-
0366
bPb-
2866
bPb-
7835
bPb-
1209
bPb-
0037
bPb-
7728
bPb-
6706
bPb-
9803
bPb-
8524
bPb-
4541
bPb-
4597
bPb-
1952
bPb-
4219
bPb-
7952
bPb-
2188
bPb-
7915
bPb-
1770
bPb-
7437
bPb-
8956
bPb-
5599
bPb-
7603
bPb-
2097
bPb-
7399
bPb-
5296
bPb-
9912
bPb-
7150
bPb-
1596
bPb-
5747
bPb-
8074
Clon
eID-
2234
44bP
b-41
91bP
b-02
02Cl
oneI
D-22
1054
Clon
eID-
2224
65Cl
oneI
D-22
2751
bPb-
0773
bPb-
7628
bPb-
1336
bPb-
5923
bPb-
0889
Clon
eID-
2221
59bP
b-09
17bP
b-61
67bP
b-07
58Cl
oneI
D-22
0949
bPb-
7983
bPb-
1631
bPb-
3980
bPb-
8833
Clon
eID-
2225
28bP
b-21
21bP
b-99
48bP
b-55
48bP
b-28
54bP
b-26
93bP
b-32
26bP
b-28
97bP
b-03
75
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
210
4H-consensus
4H5H-consensus
5H6H-consensus
6H7H-consensus
7H
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
67
Discussion
High density genetic linkage maps provide a greater probability that the entire genome
is covered with molecular markers avoiding large gaps in the map without markers. For
the construction of high-density genetic linkage maps is of particular importance to
have several loci in common between populations. This requires a high level of
polymorphism between parents. In the present study, the highest level of polymorphism
among populations was observed for A×B and A×P whereas B×P showed the lowest
level. This appears to be in good agreement with the results obtained by Casas et al.
(1998). They estimated the genetic resemblance among a representative set of 37 barley
cultivars, including our four parents, grown in Spain using RFLP markers. A
dendrogram constructed from UPGMA cluster analysis split the 37 barley cultivars into
five distinct germplasm groups that were consistent with the history of the cultivars:
winter European (including ‘Barberousse’ and ‘Plaisant’), spring European, CIMMYT-
ICARDA materials (including ‘Orria’), the single cultivar 'Dobla' and Spanish local
materials (including ‘Albacete’). Parental lines ‘Plaisant’ and ‘Barberousse’
demonstrated the highest level of similarity in the dendrogram compared with the other
parents. This could be explained because they share ‘Ager’ as parent. The level of
polymorphism between populations is determined by the level of genetic divergence
among parents.
The integrated map developed in this paper was obtained by assembling marker data
from five interconnected DH populations which allowed us to fill a number of gaps
present in maps of individual populations. The order of the markers on the integrated
map as well as on maps of individual populations are consistent with the order on the
consensus map (Wenzl et al. 2006) except for some markers on chromosome 4H and
one marker on chromosome 3H. In our study, map distances are slightly larger than
those on the consensus map. The discrepancy in map lengths may be due to difference
in “data curation” possibly in combination to the use of different mapping criteria and
mapping algorithms.
For map integration it is very important to create high-quality datasets free of errors
because genotyping errors (which produce unexpected crossovers) and missing data
lead to map expansion, especially in regions with a high marker density. The filtered
Chapter 5
68
high quality datasets from the consensus map used in this paper to verify our integrated
map were obtained following different steps: (i) JoinMap 4.0 was used to distribute loci
into linkage groups and remove redundant lines; (ii) loci order within individual
population was determined using RECORD programme which employs a marker-
ordering criteria based on minimization of the total number of recombination events and
the algorithm applied combine elements of branch-and-bound with local reshuffling
(Van Os et al. 2005a) and (iii) lines and loci with an excess of singletons were identified
and removed using a purpose-built Perl script. Map expansion could also be reduced by
removing markers that do not fit well by “nearest-neighbour stress” and/or had high
segregation ratios. However, a more accurate method will be necessary to remove the
appropriate DH lines.
Differences in map lengths could also arise due to the different methods used by the
software packages to calculate map lengths. Whereas the consensus map was derived
using the regression mapping algorithm we focused on a multipoint maximum
likelihood (ML) mapping algorithm developed by Jansen et al. (2001, 2005 and 2009).
JoinMap® algorithm fails when it is required for integrating linkage maps with large
numbers of markers, missing data, typing errors and pairwise recombination frequencies
are not present for all marker combinations. Therefore, the algorithm applied in this
study is an efficient method for constructing good-quality dense linkage maps. Spatial
sampling used to obtain a small set of markers homogenously distributed in the linkage
group provided an efficient way to reduce map expansion.
The percentage of genome coverage of our integrated map compared with the consensus
map of Wenzl et al. 2006 from the three populations with the highest number of
individuals (A×B, A×P and P×O) are 82.0, 10.6, 86.6, 94.5 and 95.5% for 2H, 4H, 5H,
6H and 7H chromosomes respectively, this percentage improved for chromosome 2H
up to 7% more when B×P and A×O populations with the smallest number of individuals
were included in the analysis.
In conclusion, the consensus map constructed with the useful method develop by Jansen
(2005) is consistent compared with the consensus map of barley (Wenzl et al. 2006).
This will be used for QTL mapping for quantitative agronomic characters allowing us to
compare QTL between different genetic backgrounds. A high density genetic linkage
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
69
map allows a greater precision of QTL location than in a sparse map (fewer markers)
because the greater the distance between markers, the greater the chance of
recombination between the marker and the QTL. These cause that the magnitude of the
effect of a QTL to be underestimated (Collard et al. 2005).
Acknowledgments
This study has been supported by the Spanish Ministry of Science and Innovation
(Project AGL2008-05541-C02 and the Agrogenomics CONSOLIDER).
Chapter 5
70
SUPPLEMENTARY MATERIAL I - POLYMORPHISMS AMONG PARENTS
Table 3. Level of polymorphism among populations and chromosomes (values in brackets are
expressed as percentages).
Chromosome Consensus
(cM) A×B (cM)
A×P (cM)
A×O (cM)
P×O (cM)
B×P (cM)
1H 151.90 40.80 (26.86)
39.30 (25.87)
14.60 (9.61)
140.10 (92.23)
82.10 (54.05)
2H 166.70 144.60 (86.74)
132.60 (79.54)
118.40 (71.03)
63.50 (38.09)
42.87 (25.72)
3H 184.59 74.10 (40.14)
88.30 (47.84)
17.40 (9.43)
76.50 (41.44)
63.20 (34.24)
4H 148.58 63.90 (43.01)
46.40 (31.23)
14.90 (10.03)
14.30 (9.62)
8.10 (5.45)
5H 194.15 185.30 (95.44)
168.20 (86.63)
103.0 (53.05)
89.50 (46.1)
0.30 (0.15)
6H 147.08 139.0 (94.51)
117.60 (79.96)
108.80 (73.97)
86.80 (59.02)
2.40 (1.63)
7H 165.42 113.20 (68.43)
112.60 (68.07)
70.80 (42.8)
142.0 (85.84)
56.80 (34.34)
Total (%) 65.68 60.86 38.66 52.89 22.08
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
71
SUPPLEMENTARY MATERIAL II- COMPARISON OF THE INTEGRATED MAP WITH THE INDIVIDUAL MAPS
Figure 3. (A-G). Individual maps and their integrated map of the seven barley chromosomes.
Chromosomes are oriented with the short arm at the top. Lines are indicated the common markers, which are presented in black and underline.
(A) Chromosome 1H
bPb-9608 bPb-0473 CloneID-104922bPb-5638CloneID-224191
bPb-9108 CloneID-221039 CloneID-105006
bPb-8935 bPb-8453 bPb-0617
CloneID-105054 bPb-1487 bPb-2260
bPb-1348 bPb-3622 bPb-1165bPb-8559bPb-3776bPb-9414bPb-8973bPb-7306 bPb-0442bPb-0851 CloneID-221726 bPb-0301bPb-0760 CloneID-221017 bPb-9415bPb-1226 bPb-9288 CloneID-222191bPb-9337
bPb-3389bPb-9656bPb-8983
bPb-3089 bPb-9401bPb-1213 bPb-5249 bPb-4144bPb-8605
CloneID-221687bPb-9108 CloneID-221039 bPb-0039bPb-0340 bPb-5014CloneID-221902
bPb-8619bPb-0699bPb-3660 bPb-1487 bPb-1381bPb-2260
bPb-8559 bPb-1165 bPb-3622bPb-1348bPb-3776bPb-8973bPb-0442bPb-7306bPb-9288bPb-1226 CloneID-222191bPb-9415 CloneID-221017 bPb-0851bPb-0760CloneID-221726 bPb-0301bPb-9337bPb-3389bPb-9656bPb-8983bPb-3089bPb-9401bPb-8605bPb-4144 bPb-1213 bPb-5249CloneID-221687bPb-9108 bPb-0039 CloneID-221039bPb-0340bPb-5014CloneID-221902bPb-8619bPb-0699bPb-3660bPb-1381bPb-1487bPb-2260
bPb-5064bPb-0405CloneID-221563 bPb-6451CloneID-222381 bPb-0487 bPb-3249bPb-1562CloneID-222534CloneID-222789CloneID-221040bPb-7306 bPb-4657 bPb-2183bPb-0851CloneID-221726 CloneID-221017 bPb-0760
bPb-9116bPb-3089bPb-1213 bPb-4898 bPb-6901bPb-9121 bPb-1366 bPb-5249bPb-6911bPb-4515 bPb-9180 bPb-1419bPb-7429
bPb-3660bPb-1487 bPb-2260bPb-5550bPb-8307 bPb-9762bPb-0589 bPb-5201 bPb-8112bPb-3116
05101520253035404550556065707580859095100105110115120125130135140145150155160165170175180185190195200205210215220225
bPb-6451 bPb-0405 bPb-2883bPb-2289 bPb-2208 bPb-5064bPb-8593bPb-8559 bPb-8212 bPb-1348bPb-1165 bPb-5242bPb-8345 bPb-3249 bPb-3579bPb-0374 bPb-0487bPb-5297 bPb-8973 bPb-9414bPb-0915 bPb-2055bPb-5638bPb-6273 bPb-4793bPb-9243 bPb-45000bPb-5866 bPb-9418 bPb-3217bPb-8359bPb-2175bPb-5164bPb-1604 bPb-4949 bPb-0910bPb-4980 bPb-9717 bPb-5962bPb-8509bPb-4170bPb-4271bPb-5425 bPb-1150bPb-4041bPb-1133
bPb-7949 bPb-8983
bPb-4515 bPb-1419 bPb-9180bPb-8570bPb-0039
bPb-8615bPb-0589 bPb-5201 bPb-9410bPb-8112bPb-0699 bPb-6873 bPb-8308
bPb-0405CloneID-221563bPb-2723bPb-1318 bPb-7137bPb-9608bPb-7043bPb-9952 bPb-9604CloneID-222789CloneID-221040bPb-4657bPb-2183bPb-9337bPb-9423bPb-9333bPb-2175CloneID-221108bPb-5164bPb-9717bPb-0910bPb-5683 bPb-0468 bPb-8960bPb-7859 CloneID-223899 bPb-9675bPb-3605 CloneID-220814 bPb-7325bPb-2813 bPb-6853 bPb-4980bPb-4531bPb-5292 bPb-9611bPb-1922bPb-5334 bPb-4590bPb-8897bPb-7289bPb-5486bPb-6133bPb-8107bPb-7609bPb-3089bPb-9116bPb-9121bPb-4898 bPb-6901bPb-1366 bPb-1213 bPb-6911bPb-5249bPb-4515 bPb-1419 bPb-9180CloneID-221039bPb-5198bPb-5014bPb-2240CloneID-224213bPb-8112
bPb-0405CloneID-221563bPb-2723bPb-1318 bPb-9608 bPb-7137bPb-7043bPb-9604bPb-9952CloneID-222789CloneID-221040bPb-2183bPb-4657bPb-9337bPb-0851bPb-9423bPb-8294 bPb-9333bPb-2175CloneID-221108bPb-5164bPb-0910bPb-9717bPb-4980 bPb-9675 bPb-9057bPb-4531 CloneID-223899 bPb-3605bPb-4662 bPb-7325 bPb-8960bPb-5683 bPb-6853 bPb-7859bPb-0468 bPb-6358 CloneID-220814bPb-2813bPb-9611bPb-1922 bPb-5292bPb-9360bPb-5486bPb-6133 bPb-4590 bPb-4614bPb-8897 bPb-5334 bPb-7289bPb-8107bPb-5339bPb-7609bPb-3089 bPb-9116bPb-1213 bPb-1366 bPb-4898bPb-6901 bPb-9121 bPb-6911bPb-5249bPb-1419 bPb-9180 bPb-4515bPb-9108 CloneID-221039bPb-5198 bPb-5014bPb-2240CloneID-224213bPb-8112bPb-3756
05101520253035404550556065707580859095100105110115120125130135140145150155160165170175180185190195200205210215220225230235
AxO AxB 1H-AxB/AxP/AxO AxP
BxP 1H-PxO/BxP PxO
Chapter 5
72
Figure 3. (continued)
Fig
ure
3. (
cont
inue
d)
(
B)
Chr
omos
ome
2H
bPb
-359
8C
lone
ID-2
223
67b
Pb
-646
6bP
b-7
803
Clo
neI
D-2
24
196
Clo
neID
-22
3332
Clo
neI
D-2
2357
6
Clo
neI
D-2
24
040
bPb-
7626
Clo
neI
D-2
2315
9bP
b-9
788
bPb
-109
8b
Pb-
5884
bP
b-4
523
bPb
-875
0C
lone
ID-2
212
38b
Pb
-835
4C
lon
eID
-10
510
5C
lone
ID-1
049
25bP
b-8
038
bPb
-121
2bP
b-7
975
bP
b-56
29bP
b-4
261
bP
b-60
63b
Pb
-968
2bP
b-8
449
bP
b-48
77C
lon
eID
-220
996
bPb
-351
9b
Pb-
5389
Clo
neI
D-2
24
250
1049
56b
Pb
-980
2bP
b-9
131
Clo
neID
-22
4126
bPb
-286
7
bPb
-683
5
bPb
-223
0C
lone
ID-2
229
64C
lon
eID
-222
760
bPb
-992
5
2Clo
neID
-22
968
bPb-
6055
bP
b-7
991
bPb
-192
6b
Pb-
3563
bP
b-6
194
Clo
neI
D-2
24
177
Clo
neID
-22
4007
bP
b-0
771
Clo
neI
D-2
22
905
bPb
-827
4
bPb
-418
4C
lone
ID-2
212
27C
lon
eID
-105
191
bPb
-803
9b
Pb-
3719
bPb
-054
1C
lon
eID
-22
143
5C
lone
ID-1
049
87
bPb
-575
5C
lon
eID
-10
485
5C
lone
ID-1
048
78b
Pb
-781
6C
lon
eID
-10
485
4C
lone
ID-2
219
83C
lon
eID
-105
190
Clo
neI
D-1
05
064
bPb
-224
4b
Pb-
9199
bPb
-608
7bP
b-1
566
bP
b-19
86b
Pb
-561
9bP
b-8
530
bP
b-54
60b
Pb
-720
8
bPb
-990
9bP
b-8
698
bP
b-62
96b
Pb
-310
2bP
b-1
085
bPb
-677
7b
Pb-
8734
bP
b-6
222
bPb
-610
5
Clo
neI
D-1
05
025
bPb-
9467
bP
b-7
890
Clo
neI
D-2
22
957
Clo
neI
D-2
2303
5b
Pb-
051
6b
Pb-
646
6b
Pb-
359
8C
lone
ID-2
223
67
bP
b-7
803
Clo
neI
D-2
2419
6C
lon
eID
-223
972
bP
b-6
297
Clo
neI
D-2
2333
2C
lon
eID
-223
147
Clo
neI
D-2
2363
1C
lon
eID
-223
665
bP
b-9
788
Clo
neI
D-2
2404
0b
Pb-
762
6b
Pb-
345
9C
lon
eID
-221
238
bP
b-5
884
Clo
neI
D-1
0492
5bP
b-8
35
4b
Pb-
310
8b
Pb-
109
8bP
b-8
75
0b
Pb-
45
23b
Pb-
803
8b
Pb-
968
2bP
b-4
26
1b
Pb-
487
7b
Pb-
224
3bP
b-9
13
1C
lon
eID
-224
126
bP
b-9
802
bP
b-2
867
Clo
neI
D-2
2276
0b
Pb-
223
0b
Pb-
357
2bP
b-3
98
5
bP
b-3
056
bPb
-64
38
bP
b-6
881
bP
b-3
575
Clo
neI
D-2
2330
3bP
b-3
67
7b
Pb-
99
92b
Pb-
221
9
bP
b-4
040
bP
b-9
754
bPb
-60
88
bP
b-3
318
Clo
neI
D-2
2165
2b
Pb-
805
2
bP
b-0
858
bP
b-3
563
bPb
-19
26
bP
b-7
991
bP
b-6
194
bP
b-8
260
bP
b-8
274
bP
b-4
184
bPb
-88
51
Clo
ne
ID-2
212
27
bP
b-9
458
bP
b-7
160
bP
b-1
967
bP
b-3
858
bP
b-5
755
Clo
neI
D-2
2198
3b
Pb-
150
5b
Pb-
601
2bP
b-3
97
8b
Pb-
77
23b
Pb-
232
2bP
b-7
81
6b
Pb-
81
73b
Pb-
521
6b
Pb-
919
9bP
b-2
24
4b
Pb-
608
7b
Pb-
561
9bP
b-8
53
0b
Pb-
72
08b
Pb-
616
9bP
b-1
98
6b
Pb-
118
4bP
b-1
56
6b
Pb-
54
60b
Pb-
159
3bP
b-8
25
5C
lon
eID
-223
260
bP
b-0
946
bP
b-1
051
bPb
-14
15
Clo
neI
D-2
2085
1C
lon
eID
-223
821
bPb
-99
09
bP
b-2
408
bPb
-46
91
bP
b-78
92C
lone
ID-2
230
35b
Pb-
6466
bP
b-35
98b
Pb-
8445
bP
b-78
03C
lone
ID-2
239
72C
lone
ID-2
231
47
bP
b-76
26
bP
b-34
59C
lone
ID-2
212
38b
Pb-
6235
bP
b-8
399
Clo
neID
-22
0974
bP
b-9
220
bPb-
5444
bP
b-61
28b
Pb-
8292
bP
b-30
50b
Pb
-696
3b
Pb-
5884
bP
b-80
38b
Pb-
9682
bP
b-4
261
bP
b-48
77C
lon
eID
-222
314
bP
b-91
31b
Pb
-166
4b
Pb-
0671
bP
b-98
02C
lon
eID
-222
357
Clo
neID
-22
4126
Clo
neID
-22
2760
bP
b-22
30b
Pb-
3985
bP
b-3
572
bPb-
2013
bP
b-02
20
Clo
neID
-22
3503
bP
b-35
75b
Pb-
9992
bP
b-36
77b
Pb-
2219
bP
b-97
54b
Pb
-404
0bP
b-60
88C
lone
ID-2
227
71b
Pb-
5440
Clo
neID
-22
1652
bP
b-81
00b
Pb-
6055
bP
b-7
991
bPb-
1926
bP
b-35
63b
Pb
-619
4
Clo
neID
-22
2810
Clo
neID
-22
0896
Clo
neI
D-2
2122
7bP
b-4
184
bP
b-26
80
bP
b-94
58b
Pb-
7124
bP
b-38
58b
Pb-
5755
Clo
neID
-22
1983
bP
b-31
65b
Pb-
7816
bP
b-0
659
bP
b-78
90b
Pb-
3334
bP
b-91
99b
Pb
-224
4b
Pb-
6087
bP
b-85
30b
Pb
-720
8bP
b-19
86b
Pb-
0303
bP
b-5
619
bP
b-15
66b
Pb-
5460
bP
b-06
89b
Pb-
8240
Clo
neID
-22
0851
bP
b-86
98b
Pb
-108
5bP
b-62
96b
Pb-
6048
Clo
neI
D-2
2333
7b
Pb-
9794
bP
b-40
93b
Pb
-745
5b
Pb-
9467
bP
b-95
20C
lon
eID
-223
912
Clo
neID
-22
3655
Clo
neID
-22
303
5bP
b-3
59
8bP
b-6
46
6bP
b-7
80
3bP
b-7
62
6bP
b-3
45
9bP
b-8
39
9bP
b-6
23
5C
lone
ID-2
212
38
Clo
neID
-22
097
4bP
b-5
44
4bP
b-9
22
0bP
b-8
29
2bP
b-3
05
0bP
b-6
96
3bP
b-5
88
4bP
b-4
52
3bP
b-8
75
0bP
b-1
09
8bP
b-8
03
8bP
b-4
26
1bP
b-9
68
2bP
b-4
87
7bP
b-9
80
2bP
b-9
13
1bP
b-2
23
0C
lone
ID-2
227
60
bPb
-35
72
bPb
-39
85
bPb
-20
13
bPb
-36
77
bPb
-99
92
bPb
-22
19
bPb
-35
75
bPb
-60
88
bP
b-4
040
bPb
-975
4C
lone
ID-2
216
52
bPb
-61
94
bPb
-35
63
bP
b-1
926
bPb
-79
91
Clo
neID
-22
122
7bP
b-4
18
4
bPb
-94
58
bPb
-71
24
bPb
-38
58
bPb
-57
55
bPb
-78
16
bPb
-91
99
bPb
-22
44
bPb
-60
87
bPb
-56
19
bPb
-72
08
bPb
-19
86
bPb
-85
30
bPb
-15
66
bPb
-54
60
bPb
-06
89
bPb
-82
40
Clo
neID
-22
085
1bP
b-9
90
9bP
b-6
04
8C
lone
ID-2
233
37
bP
b-8
399
bP
b-6
235
bP
b-5
444
bPb-
9220
bP
b-8
292
Clo
neI
D-2
2097
4bP
b-00
03b
Pb
-305
0bP
b-69
63b
Pb
-452
3b
Pb
-875
0
Clo
neI
D-2
2409
5
bP
b-3
574
bPb-
6992
bP
b-5
629
bP
b-3
572
bP
b-2
013
bPb-
3985
bP
b-9
925
bP
b-1
836
bP
b-3
575
bP
b-2
219
bP
b-8
700
Clo
neI
D-2
2290
5bP
b-76
71b
Pb
-803
9b
Pb
-371
9
bP
b-6
122
bPb-
1266
bP
b-8
949
bP
b-9
266
bP
b-9
458
bP
b-7
124
bP
b-3
858
bP
b-8
240
bPb-
0689
bP
b-8
734
bPb-
6777
bP
b-9
909
bP
b-6
048
Clo
neI
D-2
2333
7
bP
b-6
235
bP
b-0
003
bP
b-9
220
bPb
-61
28
bP
b-1
098
bPb
-82
92
bP
b-8
750
bP
b-4
523
bPb
-96
20
bP
b-9
594
bPb
-30
50
bP
b-2
107
7b
Pb-
998
4bP
b-3
23
9b
Pb-
11
89
bP
b-7
418
bPb
-68
81
bP
b-6
438
bP
b-3
056
bP
b-0
946
bPb
-15
93
bP
b-8
255
bP
b-9
395
bP
b-1
051
bPb
-14
15
bP
b-5
015
bP
b-8
698
bPb
-79
30
bP
b-6
199
bP
b-1
085
bPb
-78
90
bP
b-9
467
bP
b-6
048
bPb
-62
96
bP
b-7
434
bP
b-4
093
bPb
-68
94
bP
b-5
416
bP
b-6
280
bPb
-95
20
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
210
215
220
225
230
235
240
245
250
255
260
AxO
AxB
AxP
Integrated
PxO
BxP
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
73
(C) Chromosome 3H
AxO AxB 3H-AxB/AxP/AxO AxP
CloneID-221259 bPb-1264 bPb-9583bPb-9945 bPb-0476
bPb-1609
bPb-6249 bPb-9111bPb-5796 bPb-8621 bPb-8506bPb-7245 bPb-2550
bPb-0789 bPb-7815
bPb-6631
bPb-5555 CloneID-105031 bPb-4022bPb-6884 bPb-4472 CloneID-105169CloneID-222055bPb-8457bPb-1264bPb-9583 CloneID-220951 CloneID-223577bPb-9945bPb-0476bPb-7770 bPb-7199 bPb-3649CloneID-223396 CloneID-220882 CloneID-221142bPb-9745CloneID-221898 bPb-7448
bPb-1302bPb-7285 CloneID-224036CloneID-221274 bPb-0331bPb-6361 bPb-2929 bPb-5289bPb-7350 bPb-7335bPb-9961
bPb-8506bPb-8621 bPb-2550
bPb-9111bPb-6249
bPb-3867 bPb-5666bPb-3899 bPb-7827 bPb-0945bPb-9599 bPb-0789 bPb-6383bPb-7815bPb-0200 bPb-8419bPb-0735
bPb-0361
bPb-9965
bPb-7724 bPb-5795
CloneID-105169 bPb-4022 CloneID-105031bPb-5555CloneID-222055bPb-8457bPb-1264bPb-9583bPb-9945CloneID-220951bPb-0476CloneID-223577bPb-7770bPb-7199bPb-3649CloneID-223396CloneID-220882CloneID-221142bPb-9745CloneID-221898bPb-7448bPb-1302bPb-7285 CloneID-224036bPb-0331 CloneID-221274bPb-6361 bPb-7335 bPb-2929
bPb-8506bPb-8621bPb-2550
bPb-1609
bPb-9111bPb-6249bPb-3867bPb-5666bPb-3899bPb-6383 bPb-7827 bPb-0789bPb-9599bPb-0945bPb-7815bPb-8419bPb-0200bPb-0735
bPb-0361
bPb-9965
bPb-5795bPb-7724
bPb-6884
bPb-7481 bPb-7705 CloneID-220951bPb-7918bPb-1264
bPb-9945
CloneID-221622bPb-9745CloneID-221551CloneID-221898bPb-7448 CloneID-221552bPb-0654bPb-1302bPb-7285bPb-1077bPb-3565bPb-5289 bPb-7350 bPb-9903bPb-2929
bPb-3630 bPb-4209
CloneID-220818
bPb-2550 bPb-8621bPb-8894
bPb-1609
bPb-2640bPb-9111
bPb-6249
bPb-0049
bPb-8021
bPb-9599bPb-2888bPb-0789CloneID-223538bPb-6383bPb-8557bPb-7827 bPb-9118bPb-7815
bPb-1822
bPb-0361
bPb-6221
bPb-7724 bPb-5256bPb-5355bPb-7247CloneID-224240bPb-9923 CloneID-221232bPb-5569
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
210
215
Chapter 5
74
Figure 3. (continued)
PxO 3H-PxO/BxP BxP
bPb-7918 CloneID-220951 bPb-7705bPb-7481
bPb-9797bPb-9402 bPb-7199 bPb-6127CloneID-220882CloneID-221142
bPb-7002
bPb-0312
bPb-6782
bPb-6347
bPb-7937bPb-2440
bPb-7872
CloneID-222616CloneID-222358 bPb-1681
bPb-6383 bPb-7827 bPb-8557bPb-9118
CloneID-220878 bPb-6631CloneID-222327
bPb-1928 bPb-7164 bPb-8907bPb-8322
bPb-1411
bPb-5864bPb-0164
bPb-7481 bPb-7705 bPb-7918CloneID-220951
bPb-9402 bPb-7199 bPb-6127CloneID-221142CloneID-220882
bPb-0312
bPb-6782
bPb-6347
bPb-7937bPb-2440
bPb-7872
CloneID-222358 bPb-1681 CloneID-222616
bPb-7827 bPb-6383 bPb-9118bPb-8557
CloneID-220878 bPb-6631
bPb-7164 bPb-1928 bPb-8907bPb-8322
bPb-1411
bPb-0164
bPb-48285
bPb-7918 bPb-7705 bPb-2018bPb-7481
bPb-7770 bPb-8441 bPb-3649
bPb-3603
bPb-8697 bPb-6722
bPb-4616 bPb-9336
bPb-3630 bPb-4209
bPb-5246bPb-2640 bPb-1609bPb-6695
bPb-3907bPb-0049
bPb-8419bPb-0200 bPb-1114 bPb-42312
bPb-5568 bPb-7911
bPb-6536
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
210
215
220
225
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
75
Fig
ure
3. (
cont
inue
d)
(
D)
Chr
omos
ome
4H
AxO
AxB
AxP
Integrated
PxO
BxP
bP
b-2
83
7bP
b-8
56
9bP
b-1
469
bP
b-7
27
5
bP
b-4
69
922
26
50
22
29
03
22
40
48
22
34
13
10
48
83
bP
b-8
43
7bP
b-1
40
8b
Pb-6
64
02
210
13
bP
b-3
51
2bP
b-4
33
3bP
b-0
513
bP
b-2
42
7bP
b-3
04
5bP
b-0
365
bP
b-0
13
0bP
b-3
68
4b
Pb-6
77
510
51
70
22
22
94
bP
b-4
50
122
35
44
bP
b-6
07
4b
Pb-1
49
6bP
b-0
03
6
22
14
40
bP
b-6
87
2bP
b-6
94
910
512
4
10
48
57
22
41
31
22
16
36
bP
b-9
82
0
22
379
5bP
b-9
304
222
65
0b
Pb
-46
99
22
402
0
bP
b-4
990
bP
b-2
427
bP
b-3
045
bP
b-0
365
bP
b-3
684
bP
b-4
333
bP
b-0
130
bP
b-0
513
bP
b-3
512
bP
b-0
996
22
250
9bP
b-4
501
bP
b-9
134
bP
b-6
074
bP
b-1
496
bP
b-0
036
bP
b-6
775
22
229
4bP
b-3
34
722
410
1
bP
b-7
987
bP
b-0
059
bP
b-3
829
bP
b-4
214
22
160
8
bP
b-8
701
bP
b-1
329
bP
b-9
998
bP
b-9
048
bP
b-9
440
bP
b-5
711
22
413
1bP
b-1
166
22
163
6bP
b-6
101
bP
b-9
668
bP
b-5
265
bP
b-4
765
222
650
bP
b-9
304
bP
b-4
69
9
bP
b-0
99
6b
Pb
-24
27
bP
b-3
68
4b
Pb-0
13
0b
Pb-0
36
5b
Pb
-05
13
bP
b-3
04
5b
Pb-4
33
3
222
509
bP
b-6
77
5b
Pb-6
07
4b
Pb-4
50
1b
Pb
-14
96
bP
b-9
57
9b
Pb
-00
36
bP
b-4
18
32
225
47
bP
b-9
03
9b
Pb
-32
68
bP
b-1
27
8
bP
b-9
50
42
23
751
bP
b-2
30
5b
Pb-6
94
9b
Pb-6
87
2
222
352
bP
b-4
21
4
bP
b-1
32
9b
Pb
-87
01
bP
b-9
99
8
221
636
bP
b-3
46
8
bP
b-9
82
0b
Pb
-52
65
bP
b-9
30
422
26
50
bP
b-4
69
9
bP
b-8
43
7b
Pb-1
40
8b
Pb-6
64
0
22
23
52
bP
b-6
94
9
22
37
51
bP
b-9
50
4
22
25
09
bP
b-4
18
3
bP
b-9
03
9
bP
b-3
51
2
bP
b-9
99
8
22
16
36
bP
b-5
26
5
bP
b-1
46
9bP
b-8
56
9bP
b-2
837
bP
b-7
27
5
22
40
48
22
34
13
bP
b-1
14
8
bP
b-1
40
8b
Pb-8
43
7bP
b-6
64
0
bP
b-1
76
2bP
b-8
89
6
22
25
09
22
08
46
22
22
94
22
37
51
bP
b-9
50
4
22
40
01
22
23
52
bP
b-9
99
8
bP
b-4
990
bP
b-3
512
bP
b-3
725
3bP
b-4
18
3bP
b-9
039
bP
b-6
155
bP
b-9
504
bP
b-3
34
7
bP
b-6
949
bP
b-9
340
bP
b-6
87
2bP
b-7
987
bP
b-0
059
bP
b-3
82
9
bP
b-4
240
bP
b-3
047
0 5 10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
130
135
140
145
150
155
160
Chapter 5
76
Fig
ure
3. (
cont
inue
d)
(E
) C
hrom
osom
e 5H
AxO
AxB
AxP
Integrated
PxO
BxP
bP
b-5
31
7
bP
b-1
90
9C
loneID
-2232
99
bP
b-8
07
2b
Pb-2
460
Clo
neID
-2217
73
bP
b-0
091
bP
b-3
830
bP
b-6
56
8b
Pb-7
407
bP
b-1
82
0
bP
b-6
36
3
Clo
neID
-1049
20
Clo
neID
-2213
41
Clo
neID
-22421
9b
Pb-7
348
bP
b-3
79
2b
Pb-3
412
bP
b-6
805
bP
b-0
90
9
bP
b-6
26
0b
Pb-6
710
Clo
neID
-2229
00
Clo
neID
-22247
3C
loneID
-223686
bP
b-1
81
3C
loneID
-222163
Clo
ne
ID-2
24143
bP
b-0
48
6
bP
b-3
56
5b
Pb-2
325
Clo
neID
-2214
75
bP
b-3
11
4b
Pb-5
845
bP
b-7
32
0b
Pb-7
214
bP
b-4
058
bP
b-3
88
7bP
b-7
27
7C
loneID
-2217
14
bP
b-2
006
bP
b-1
420
Clo
neID
-2224
99
bP
b-4
970
bP
b-8
80
9
Clo
neID
-2229
06
bP
b-1
965
bP
b-6
17
9b
Pb-4
595
bP
b-0
835
bP
b-8
07
0C
loneID
-2213
74
bP
b-0
171
Clo
ne
ID-1
05144
Clo
neID
-2233
26
bP
b-5
76
6b
Pb-4
725
bP
b-6
124
bP
b-7
29
2b
Pb-7
360
bP
b-9
244
bP
b-9
562
bP
b-6
485
bP
b-6
051
bP
b-1
909
Clo
neID
-223299
bP
b-4
814
bP
b-8
072
bP
b-2
460
bP
b-6
067
bP
b-0
050
bP
b-9
632
bP
b-6
495
bP
b-6
363
bP
b-9
680
Clo
neID
-224219
Clo
neID
-221341
bP
b-7
348
bP
b-3
412
bP
b-3
792
bP
b-6
805
bP
b-5
727
bP
b-7
881
bP
b-2
762
bP
b-3
852
bP
b-6
710
bP
b-7
561
bP
b-0
709
bP
b-4
721
bP
b-2
497
bP
b-6
288
bP
b-9
032
bP
b-5
516
Clo
neID
-223
233
bP
b-5
229
bP
b-0
780
bP
b-2
171
bP
b-3
519
bP
b-0
413
bP
b-6
063
bP
b-5
389
bP
b-8
449
bP
b-7
022
Clo
neID
-222
196
bP
b-0
486
bP
b-4
494
bP
b-2
757
Clo
neID
-221475
bP
b-5
845
bP
b-4
970
bP
b-4
318
bP
b-1
420
bP
b-2
006
Clo
neID
-222
499
bP
b-8
809
bP
b-0
621
bP
b-9
085
bP
b-0
171
Clo
neID
-221374
bP
b-8
070
bP
b-4
595
bP
b-0
835
bP
b-6
179
bP
b-1
965
bP
b-1
224
bP
b-9
868
bP
b-4
971
bP
b-8
754
Clo
neID
-222075
bP
b-6
239
bP
b-1
217
bP
b-9
660
bP
b-4
809
Clo
neID
-223220
Clo
neID
-221474
Clo
neID
-224112
Clo
neID
-224153
bP
b-9
732
bP
b-9
088
bP
b-3
170
bP
b-6
051
bP
b-6
485
bP
b-9
56
2bP
b-0
085
bP
b-1
909
Clo
neID
-223299
bP
b-4
814
bP
b-8
072
bP
b-2
460
bP
b-9
632
bP
b-0
34
9bP
b-6
067
bP
b-0
050
bP
b-6
49
5
bP
b-6
363
Clo
neID
-224219
bP
b-7
348
bP
b-6
805
bP
b-0
909
bP
b-3
79
2bP
b-3
412
bP
b-0
899
bP
b-3
852
bP
b-7
627
bP
b-6
26
0bP
b-6
710
bP
b-0
709
bP
b-7
561
bP
b-6
288
bP
b-2
49
7bP
b-4
721
bP
b-9
032
Clo
neID
-1050
44
Clo
neID
-223233
bP
b-5
516
bP
b-5
596
bP
b-7
395
bP
b-8
449
bP
b-0
413
bP
b-6
06
3bP
b-0
653
bP
b-3
519
bP
b-5
38
9bP
b-8
553
bP
b-0
78
0C
loneID
-222196
bP
b-7
022
bP
b-0
486
bP
b-3
603
bP
b-2
580
bP
b-8
854
bP
b-4
494
bP
b-8
462
bP
b-3
70
0bP
b-3
910
bP
b-5
845
Clo
neID
-222215
bP
b-6
578
bP
b-1
420
bP
b-4
318
Clo
neID
-2224
99
bP
b-4
970
bP
b-2
006
bP
b-8
319
bP
b-8
809
Clo
neID
-223857
bP
b-0
621
bP
b-4
753
bP
b-3
590
bP
b-0
171
Clo
neID
-221374
bP
b-8
070
bP
b-0
835
bP
b-6
17
9bP
b-4
595
bP
b-1
965
bP
b-4
971
Clo
neID
-222075
bP
b-1
217
bP
b-9
660
bP
b-4
809
Clo
neID
-224160
bP
b-6
485
bP
b-9
562
bP
b-6
051
bP
b-1
909
Clo
neID
-223299
bP
b-4
814
bP
b-8
072
bP
b-2
460
bP
b-0
349
bP
b-9
632
bP
b-0
050
bP
b-6
495
bP
b-6
067
bP
b-6
363
Clo
neID
-224219
bP
b-7
348
bP
b-3
412
bP
b-3
792
bP
b-3
852
bP
b-6
710
bP
b-0
709
bP
b-7
561
bP
b-4
721
bP
b-6
288
bP
b-2
497
bP
b-9
032
bP
b-5
516
Clo
neID
-223233
bP
b-7
395
bP
b-5
596
bP
b-0
780
bP
b-5
389
bP
b-6
063
bP
b-8
449
bP
b-3
519
bP
b-0
413
Clo
neID
-222196
bP
b-7
022
bP
b-0
486
bP
b-2
580
bP
b-8
854
bP
b-4
494
bP
b-5
845
bP
b-4
318
bP
b-1
420
bP
b-4
970
bP
b-2
006
Clo
neID
-222499
bP
b-8
809
bP
b-0
621
bP
b-0
171
Clo
neID
-221374
bP
b-8
070
bP
b-4
595
bP
b-0
835
bP
b-6
179
bP
b-1
965
bP
b-4
971
Clo
neID
-222075
bP
b-1
217
bP
b-9
660
bP
b-4
809
bP
b-9
562
bP
b-7
599
bP
b-6
485
bP
b-5
317
bP
b-8
580
bP
b-4
814
Clo
neID
-221773
bP
b-7
407
bP
b-1
807
bP
b-6
568
bP
b-0
091
bP
b-1
820
bP
b-0
349
bP
b-6
067
bP
b-0
050
bP
b-9
632
bP
b-6
495
bP
b-5
532
bP
b-5
516
Clo
neID
-223
233
bP
b-7
395
bP
b-5
596
Clo
neID
-222196
bP
b-7
022
bP
b-7
854
bP
b-8
854
bP
b-4
494
bP
b-2
580
Clo
neID
-221714
bP
b-4
058
bP
b-7
214
bP
b-3
887
bP
b-7
277
bP
b-4
971
Clo
neID
-222075
bP
b-4
809
bP
b-5
766
bP
b-4
725
bP
b-6
124
bP
b-9
244
bP
b-7
360
bP
b-7
763
bP
b-4
863
bP
b-4
067
bP
b-0
686
0 5 10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
210
215
220
225
230
235
240
245
250
255
260
265
270
275
280
285
290
295
300
305
Construction of an integrated genetic linkage map of barley (Hordeum vulgare L.) involving a parent carrying a reciprocal translocation
77
Fig
ure
3. (
cont
inue
d)
(E)
Chr
omos
ome
6H
bPb-
042
6b
Pb
-37
80
bPb-
051
9C
lon
eID
-104
934
Clo
neID
-22
196
1C
lone
ID-2
2352
9bP
b-35
86
bPb-
666
1b
Pb
-52
52bP
b-2
058
bPb-
775
5bP
b-39
21
bPb-
374
6
bPb-
970
2b
Pb
-37
22bP
b-5
698
Clo
neI
D-2
224
84bP
b-0
055
bPb-
475
0b
Pb
-00
18
Clo
neI
D-2
232
73
Clo
neI
D-2
215
14C
lon
eID
-222
306
Clo
neI
D-2
222
55bP
b-38
83
bPb-
125
6b
Pb
-83
47bP
b-3
230
Clo
neI
D-2
232
11bP
b-97
96
bPb-
590
3b
Pb
-57
78C
lone
ID-2
225
67
bPb-
412
5C
lone
ID-2
2260
0
bPb-
347
0C
lon
eID
-104
954
bPb-
314
4b
Pb
-24
10bP
b-9
285
bPb-
230
4b
Pb
-21
37bP
b-5
167
bPb-
286
3C
lone
ID-2
2368
7bP
b-93
49
Clo
neI
D-2
209
34C
lone
ID-2
225
19
bP
b-6
677
bPb-
040
3b
Pb
-46
26bP
b-1
621
Clo
neI
D-1
051
72bP
b-9
817
bPb-
714
6bP
b-10
29
Clo
neID
-223
721
bPb-
989
0bP
b-53
46
AxO
bPb-
227
6bP
b-04
26
bP
b-3
780
Clo
neID
-222
774
bPb-
220
9bP
b-05
19
Clo
neI
D-2
235
87C
lone
ID-2
235
29
bP
b-1
009
bPb-
719
3bP
b-35
86
bPb-
736
2b
Pb
-29
30bP
b-85
28bP
b-98
52
bP
b-5
275
bPb-
267
2b
Pb
-35
54bP
b-64
19bP
b-60
69
bPb-
074
2C
lone
ID-2
2137
3bP
b-74
67
bP
b-9
651
bPb-
027
1bP
b-81
50
bPb-
716
5bP
b-52
52
bPb-
775
5bP
b-66
61
Clo
neI
D-2
232
03bP
b-64
57
bP
b-3
427
bPb-
980
7b
Pb
-28
81C
lone
ID-2
2099
8C
lon
eID
-223
914
bPb-
024
5bP
b-79
98
bPb-
455
5C
lone
ID-2
2100
2bP
b-74
92
bPb-
591
0bP
b-37
46
bPb-
717
9bP
b-03
96
Clo
neID
-220
959
bPb-
602
3bP
b-24
64
bPb-
582
2bP
b-78
21
bP
b-0
934
bPb-
656
7b
Pb
-34
87bP
b-16
66bP
b-91
14
bPb-
475
0b
Pb
-25
93C
lon
eID
-222
255
Clo
neID
-22
151
4C
lone
ID-2
2230
6C
lon
eID
-221
627
Clo
neID
-22
110
5b
Pb
-67
35bP
b-37
73
bP
b-3
908
bPb-
306
8C
lon
eID
-222
508
bPb-
877
0b
Pb
-21
85C
lon
eID
-223
391
Clo
neID
-22
194
6b
Pb
-04
32bP
b-17
24
bPb-
417
8b
Pb
-04
51bP
b-19
82
bP
b-6
477
bPb-
347
0bP
b-92
14
bPb-
491
7bP
b-28
63
bPb-
667
7b
Pb
-45
65bP
b-29
40bP
b-31
44
Clo
neID
-222
519
bPb-
934
9bP
b-51
67
bPb-
534
6C
lone
ID-2
2372
1bP
b-02
98
bPb-
102
9bP
b-21
37
Clo
neID
-223
519
bPb-
230
4bP
b-16
21
bP
b-0
403
bPb-
7146
bPb-
338
1bP
b-46
26
AxB
bPb-
0426
bP
b-3
965
Clo
neI
D-2
2360
8bP
b-37
80C
lon
eID
-222
774
Clo
neI
D-2
2352
9bP
b-10
09C
lone
ID-2
2358
7bP
b-71
93bP
b-73
62bP
b-85
28bP
b-29
30b
Pb-
985
2bP
b-35
86bP
b-84
73bP
b-64
19bP
b-26
72b
Pb-
606
9bP
b-96
51bP
b-07
42b
Pb-
883
6C
lon
eID
-221
373
bPb-
5252
bP
b-6
661
bPb-
0245
Clo
neID
-22
391
4bP
b-20
58bP
b-98
07bP
b-34
27bP
b-64
57C
lon
eID
-221
002
bPb-
5910
Clo
neI
D-2
2338
6bP
b-37
46bP
b-71
79C
lon
eID
-220
959
bPb-
0396
bPb-
2464
bPb-
3120
bP
b-5
822
bPb-
9922
bPb-
6567
bPb-
3487
bPb-
9114
bP
b-1
666
bPb-
4750
bPb-
2593
bPb-
9082
bP
b-9
835
bPb-
9051
Clo
neI
D-2
2230
6C
lone
ID-2
2151
4C
lone
ID-2
222
55bP
b-33
09bP
b-67
35C
lone
ID-2
216
27
bPb
-27
55C
lon
eID
-221
105
bPb-
3068
bPb-
2185
bP
b-8
770
Clo
neI
D-2
2194
6C
lone
ID-2
2339
1C
lon
eID
-221
935
bPb-
0451
bPb-
0606
bPb-
4178
bPb-
9063
bP
b-6
477
bPb-
3470
bPb-
8371
bPb-
9413
bPb-
7446
bPb-
2940
bPb-
6677
bP
b-3
760
bPb-
286
3C
lon
eID
-222
519
bPb-
3144
bPb-
9349
bPb-
9285
Clo
neI
D-2
2372
1bP
b-02
98bP
b-21
37bP
b-46
26bP
b-04
03b
Pb-
230
4C
lon
eID
-105
090
bPb-
7146
bPb-
1621
bPb-
1029
bPb-
9817
bP
b-9
890
bPb-
241
0
AxP
bPb
-04
26bP
b-3
780
Clo
neID
-222
774
bPb
-10
09bP
b-7
193
bPb
-35
86bP
b-9
852
bPb
-29
30bP
b-8
528
bPb
-73
62bP
b-6
419
bPb
-60
69bP
b-2
672
bPb
-07
42bP
b-9
651
Clo
neID
-221
373
bPb
-52
52bP
b-6
661
bPb
-34
27bP
b-6
457
bPb
-28
81bP
b-9
807
bPb
-02
45C
lone
ID-2
2391
4bP
b-5
910
Clo
neID
-221
002
Clo
neID
-223
386
bPb
-37
46bP
b-7
179
bPb
-03
96C
lone
ID-2
2095
9bP
b-2
464
bPb
-58
22bP
b-6
567
bPb
-16
66bP
b-9
114
bPb
-34
87bP
b-4
750
bPb
-25
93bP
b-9
835
bPb
-90
51bP
b-9
082
Clo
neID
-221
514
Clo
neID
-222
255
Clo
neID
-222
306
bPb
-30
68C
lone
ID-2
2162
7C
lone
ID-2
2110
5bP
b-3
309
bPb
-27
55bP
b-8
770
bPb
-21
85C
lone
ID-2
2339
1C
lone
ID-2
2194
6bP
b-4
178
bPb-
045
1bP
b-0
606
bPb
-64
77bP
b-3
470
bPb
-90
63bP
b-2
940
bPb
-66
77bP
b-2
863
Clo
neID
-222
519
bPb
-31
44bP
b-9
349
Clo
neID
-223
721
bPb
-02
98bP
b-2
137
bPb
-46
26bP
b-2
304
bPb
-71
46bP
b-0
403
bPb
-16
21bP
b-1
029
Integrated
Clo
neI
D-2
220
53bP
b-0
572
bPb-
074
2bP
b-64
57
Clo
neID
-223
203
bPb-
342
7bP
b-28
81
bPb-
980
7bP
b-64
21
Clo
neID
-223
914
Clo
neI
D-2
233
86C
lon
eID
-221
002
Clo
neI
D-2
218
23bP
b-71
79
bPb-
537
9C
lon
eID
-223
115
bPb-
005
5bP
b-37
22
Clo
neID
-222
484
bPb-
970
2bP
b-56
98
bP
b-5
196
bPb-
908
2b
Pb
-98
35bP
b-90
51bP
b-27
55
bPb-
330
9C
lon
eID
-221
627
Clo
neI
D-2
211
05bP
b-43
69
bP
b-6
721
bPb-
4409
bPb-
306
8b
Pb
-83
47bP
b-12
56
bPb-
979
6bP
b-57
78
bP
b-5
903
Clo
neID
-222
567
bPb-
412
5b
Pb
-66
07C
lon
eID
-222
600
bPb-
877
0b
Pb
-21
85bP
b-06
06
bP
b-0
451
bPb-
4178
bPb-
085
7bP
b-90
63
bP
b-7
015
bPb-
6477
PxO
bP
b-9
051
bPb-
919
8b
Pb
-69
59b
Pb-
908
2
BxP
0 5 10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
210
215
220
225
230
235
240
245
250
255
260
265
270
Chapter 5
78
Fig
ure
3. (
cont
inue
d)
(E)
Chr
omos
ome
7H
AxO
AxB
AxP
Integrated
PxO
BxP
bPb-
0043
2209
1210
5125
1051
67bP
b-75
5922
1337
2241
58bP
b-80
8422
3746
1048
47bP
b-07
14bP
b-25
95bP
b-85
5822
2470
2214
52bP
b-99
8622
1538
bPb-
3718
bPb-
0179
bPb-
2076
bPb-
4097
1051
21bP
b-49
29bP
b-78
63bP
b-19
94bP
b-80
43bP
b-46
3422
1830
2242
0222
2301
bPb-
6091
bPb-
3727
bPb-
7417
bPb-
4532
bPb-
7521
bPb-
9601
bPb-
9898
bPb-
0037
bPb-
8524
bPb-
7728
bPb-
6706
bPb-
9803
bPb-
1952
bPb-
4219
1049
10bP
b-79
52bP
b-52
94
1049
7910
5109
2211
9722
2266
bPb-
5599
bPb-
8956
bPb-
2097
bPb-
1770
bPb-
2379
bPb-
7437
bPb-
7603
bPb-
2188
bPb-
7915
bPb-
6110
bPb-
1596
bPb-
5747
bPb-
8074
bPb-
4321
bPb-
6170
bPb-
2718
bPb-
5259
2212
93bP
b-69
7622
2060
2209
12bP
b-89
21bP
b-22
1622
3528
bPb-
8272
bPb-
7038
bPb-
3732
bPb-
8161
bPb-
0043
bPb-
4064
bPb-
4886
bPb-
0956
2241
5822
3746
bPb-
5935
bPb-
2595
2213
37bP
b-11
40bP
b-92
0122
1452
2215
38bP
b-37
18bP
b-01
7922
2470
bPb-
4097
bPb-
6091
bPb-
7417
bPb-
6453
bPb-
3727
2210
49bP
b-21
91bP
b-90
27bP
b-54
94bP
b-24
78bP
b-18
06bP
b-71
8322
1061
2218
32bP
b-96
01bP
b-98
9822
2477
bPb-
9181
bPb-
6821
bPb-
5091
2209
64bP
b-03
66bP
b-28
66bP
b-12
09bP
b-78
35bP
b-98
03bP
b-77
28bP
b-85
24bP
b-00
37bP
b-67
06bP
b-45
41bP
b-45
97bP
b-85
68bP
b-35
89bP
b-11
05bP
b-84
60bP
b-42
19bP
b-79
52bP
b-19
52bP
b-89
00bP
b-52
94bP
b-55
97bP
b-21
8822
1197
bPb-
7437
bPb-
7603
bPb-
3323
bPb-
1770
bPb-
7915
bPb-
6819
2222
66bP
b-55
99bP
b-89
56bP
b-17
48bP
b-20
97
bPb-
8074
bPb-
1596
bPb-
6110
bPb-
5747
bPb-
0757
bPb-
6214
bPb-
0027
bPb-
9908
bPb-
2855
bPb-
0773
bPb-
4394
bPb-
1556
bPb-
0758
bPb-
9948
bPb-
2121
2210
34bP
b-03
7510
5003
2234
76bP
b-61
70bP
b-70
04bP
b-27
18bP
b-00
4322
0912
bPb-
3127
2220
6022
1293
bPb-
6976
bPb-
5259
bPb-
7038
bPb-
4064
bPb-
0108
bPb-
9729
bPb-
8161
bPb-
8921
bPb-
4886
2215
76bP
b-25
95bP
b-44
45bP
b-58
16bP
b-58
97bP
b-37
1822
2470
2215
38bP
b-01
79bP
b-97
8322
1452
bPb-
2076
bPb-
6029
bPb-
4097
bPb-
7417
bPb-
3727
bPb-
4584
bPb-
5769
2210
61bP
b-24
78bP
b-18
06bP
b-54
94bP
b-71
83bP
b-96
01bP
b-98
9822
2477
bPb-
5091
bPb-
6821
2209
64bP
b-03
66bP
b-12
09bP
b-28
66bP
b-78
35bP
b-85
24bP
b-67
06bP
b-77
28bP
b-00
37bP
b-98
03bP
b-45
41bP
b-79
52bP
b-42
19bP
b-19
52bP
b-35
61
bPb-
7150
bPb-
7399
bPb-
5296
bPb-
9912
bPb-
1847
bPb-
6386
2234
4422
2913
2229
8022
2465
2227
51bP
b-65
95bP
b-02
02bP
b-41
9122
1054
2241
88bP
b-49
2422
4212
bPb-
8860
bPb-
2855
bPb-
4071
bPb-
0773
bPb-
7628
bPb-
1336
1050
43bP
b-09
17bP
b-59
23bP
b-08
89bP
b-61
6722
2159
bPb-
0758
bPb-
3980
2209
49bP
b-16
31bP
b-79
83bP
b-09
81bP
b-88
3322
2528
bPb-
9948
bPb-
5548
bPb-
2121
bPb-
2897
bPb-
2693
bPb-
2854
bPb-
3226
2219
34bP
b-07
8322
1600
2219
0722
3518
bPb-
2718
bPb-
6170
2209
12bP
b-00
43bP
b-52
59bP
b-69
76bP
b-81
61bP
b-89
21bP
b-70
38bP
b-40
64bP
b-48
86bP
b-25
9522
1452
2224
7022
1538
bPb-
0179
bPb-
3718
bPb-
4097
bPb-
7417
bPb-
3727
bPb-
2478
2210
61bP
b-18
06bP
b-54
94bP
b-71
83bP
b-96
01bP
b-98
9822
2477
bPb-
6821
bPb-
5091
bPb-
0366
bPb-
2866
bPb-
7835
bPb-
1209
bPb-
0037
bPb-
7728
bPb-
6706
bPb-
9803
bPb-
8524
bPb-
4541
bPb-
4597
bPb-
1952
bPb-
4219
bPb-
7952
bPb-
2188
bPb-
7915
bPb-
1770
bPb-
7437
bPb-
8956
bPb-
5599
bPb-
7603
bPb-
2097
bPb-
7399
bPb-
5296
bPb-
9912
bPb-
7150
bPb-
1596
bPb-
5747
bPb-
8074
2234
44bP
b-41
91bP
b-02
0222
1054
2224
6522
2751
bPb-
0773
bPb-
7628
bPb-
1336
bPb-
5923
bPb-
0889
2221
59bP
b-09
17bP
b-61
67bP
b-07
5822
0949
bPb-
7983
bPb-
1631
bPb-
3980
bPb-
8833
2225
28bP
b-21
21bP
b-99
48bP
b-55
48bP
b-28
54bP
b-26
93bP
b-32
26bP
b-28
97bP
b-03
75
2220
60bP
b-68
68bP
b-69
76bP
b-89
21bP
b-97
29bP
b-37
32bP
b-40
64bP
b-80
84bP
b-81
61bP
b-08
44bP
b-07
14
bPb-
1994
bPb-
7863
2218
3022
1832
bPb-
2478
2210
61bP
b-18
06bP
b-28
28bP
b-54
94bP
b-51
72bP
b-61
56bP
b-25
33bP
b-50
74bP
b-03
24bP
b-89
39bP
b-13
60bP
b-67
47bP
b-58
5222
2477
bPb-
6821
bPb-
7835
bPb-
2866
bPb-
4541
bPb-
4597
bPb-
5126
bPb-
3227
bPb-
8396
bPb-
7915
bPb-
1770
bPb-
2188
bPb-
7437
bPb-
5599
bPb-
6975
bPb-
8956
2224
56bP
b-73
99bP
b-52
96bP
b-25
61bP
b-71
50bP
b-99
12bP
b-15
96bP
b-80
74bP
b-57
47bP
b-61
1022
3444
2241
2122
3782
2210
2722
1941
2230
15bP
b-41
91bP
b-02
0222
1054
2227
5122
2465
2234
00bP
b-06
39bP
b-88
60bP
b-28
55bP
b-43
89bP
b-51
88bP
b-88
67bP
b-16
69bP
b-61
86bP
b-17
67bP
b-77
3922
2295
bPb-
3145
1026
07bP
b-03
6422
0913
2221
60bP
b-08
89bP
b-09
1722
2159
bPb-
6167
bPb-
5923
bPb-
1336
1050
43bP
b-76
28bP
b-07
58bP
b-79
8322
0949
bPb-
3980
bPb-
1631
2225
28bP
b-88
33bP
b-55
48bP
b-99
48bP
b-32
32bP
b-58
98bP
b-28
54bP
b-28
97bP
b-26
93bP
b-32
2622
1034
bPb-
0375
bPb-
8157
bPb-
9971
bPb-
2188
bPb-
2379
bPb-
5597
bPb-
7915
bPb-
1770
bPb-
8956
bPb-
5599
bPb-
6819
bPb-
2097
bPb-
8213
bPb-
7603
bPb-
7437
bPb-
1748
bPb-
7686
bPb-
3227
bPb-
1079
bPb-
5768
bPb-
8005
bPb-
5747
bPb-
1596
bPb-
6110
bPb-
8074
bPb-
0757
bPb-
7150
bPb-
2561
bPb-
5296
bPb-
9912
bPb-
6386
bPb-
1847
bPb-
0202
bPb-
8658
bPb-
7181
bPb-
9662
bPb-
4191
bPb-
9329
bPb-
6595
bPb-
8901
bPb-
4488
bPb-
6214
bPb-
4743
bPb-
0027
bPb-
3055
bPb-
9908
bPb-
4394
bPb-
6166
bPb-
2328
bPb-
7983
bPb-
9944
bPb-
4090
bPb-
0917
bPb-
8778
bPb-
5923
bPb-
7628
bPb-
7345
bPb-
0981
bPb-
1336
bPb-
6626
bPb-
8833
bPb-
9866
bPb-
4552
bPb-
8103
bPb-
2693
bPb-
6109
bPb-
2854
bPb-
5099
bPb-
7882
bPb-
1232
bPb-
6476
bPb-
0375
bPb-
9884
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
210
215
220
225
230
235
240
245
250
255
260
265
270
275
280
285
290
295
300
305
310
315
79
Chapter 6
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal
translocation
A. Farré1,2, A.M. Casas3, L. Cistué3, I. Lacasa-Benito1, P Hedley5, M Macaulay5, WTB
Thomas5, I. Romagosa1,6, F.A. van Eeuwijk2 and J. Jansen2
Abstract
Multiple connected populations address broader diversity than biparental populations
which have been widely used for QTL analysis. The use of multiple connected
populations allows a more global comparison of the effects of all QTL alleles
segregating in the populations simultaneously and allows investigation of interactions
between QTL and genetic background. In this study, a new approach was developed for
QTL detection in a multi-population and a multi-environment analysis. Five
interconnected biparental doubled haploid (DH) populations of barley derived from
crosses between pure lines were used. Three of the populations have a common parent
which is known to carry a reciprocal chromosomal translocation between chromosomes
1H and 3H. Days do heading (DtH) and thousand kernel weight (TKW) will be used to
develop the methods. The QTL found for DtH and TKW are consistent with the results
of previous QTL studies. The integral approach used in this study provided a direct
comparison of the effects of the four parental alleles. Only for TKW was a direct effect
of the reciprocal translocation was found; DH lines carrying the reciprocal translocation
had a greater TKW than those with a standard chromosome arrangement.
Keywords Reciprocal translocation Barley Translocation breakpoint Connected
populations Quantitative trait locus
1Department of Plant Production and Forest Science, University of Lleida, Lleida, Spain; 2Biometris, Wageningen University and Research Centre, Wageningen, the Netherlands; 3Estación Experimental de Aula Dei, CSIC, Zaragoza, Spain; 5James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK; 6Centre UdL-IRTA, University of Lleida, Lleida, Spain
Chapter 6
80
Introduction
In breeding programs most traits of agronomic interest display continuous variation due
to the contributions of many genes with small effects. Such genes are usually called
quantitative trait loci (QTL). Quantitative traits, also called complex traits in contrast to
simple Mendelian traits, are traits where the relation between genotype and phenotype
cannot be observed directly. Examples are yield, quality or some forms of disease
resistance. QTL mapping has been widely used during the last two decades thanks to the
abundance of molecular markers in combination with the development of powerful
methods for statistical analysis (Kearsey and Farquhar, 1998; Bernardo 2008, Zhu et al.
2008) implemented in user-friendly software (i.e. MapMaker/QTL (Lincoln and Lander
1990), QTLCartographer (Basten et al. 1999), MapQTL®5 (Van Ooijien 2004), Genstat
(Payne et al. 2011). QTL mapping tools are useful for dissecting complex traits and for
identifying favourable alleles in diverse germplasm, to estimate several genetic
parameters that underlie phenotypic variation, including the number of loci, the type and
magnitude of their effects, interactions between genes (epistasis), and gene-by-
environment interactions (Symonds et al. 2005). Understanding the response of QTLs in
different environments or genetic backgrounds can lead to the development of improved
crop varieties through marker-assisted selection (MAS) which is the most evident
application of QTL analysis together with QTL cloning. But while thousands of marker-
trait association studies have been conducted in many plant species for many traits
(Holland 2007), only a few QTLs have been cloned (Liu et al. 2002, 2003; Price 2006;
Salvi et al. 2005; Ramsay et al. 2011). Until recently mainly biparental populations
(derived from single crosses of two inbred lines) were used to detect and map QTLs in
plants. Genetic analysis of biparental populations only allows differentiation between
alleles for which the parents differ. In general unrelated parents were carefully selected
to maximise marker polymorphism and trait divergence, i.e., Oregon Wolfe barley
mapping population (Costa et al. 2001), barley crosses between wild and cultivated
varieties (Backes et al. 2003), winter and spring varieties (Laurie et. 1995; Hayes et al.
1993; Francia et al. 2004) or resistant and susceptible (Arru et al. 2003). As a result,
some QTLs explaining quantitative variability in the population can be missed because
only the effect of major genes can be studied in such biparental populations (Edwards et
al. 1992). Moreover, results based on biparental populations are based on a small part of
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal translocation
81
the germplasm, results may not apply to other genetic backgrounds (Parisseaux and
Bernardo 2004). So, following this strategy only a small part of the total diversity
available to breeders is explored.
As an alternative, multiple connected biparental populations may be developed. These
are particularly interesting because: (i) the genetic variability addressed is increased, (ii)
they allow a global comparison of the effects of all QTL alleles segregating in the
populations simultaneously thereby increasing the power of QTL detection, but also
allowing the identification of the parental origins of favourable alleles, and (iii)
interactions between QTL and genetic background, which may be an indication of
epistatis, may be investigated (Muranty et al. 1997). For all these advantages, studies
involving connected populations have been reported in cereals. For example, in barley
Cuesta-Marcos et al. (2008a) used 281 doubled haploid lines derived from 17 F1
involving 14 different parental cultivars, to validate the effect of the main QTL
determining heading date. In maize, studies based on multiparental connected
populations were carried out to compare multiparental connected designs with
biparental populations with regard to MAS and phenotypic selection (Blanc et al. 2006,
2008). Here a diallel cross design consisting of six connected F2 populations, derived
from four parental inbreds, was used. Recently, Liu et al. (2010) used three connected
RIL populations derived from the crosses among three parents to find QTLs for the
number of spikelets per panicle and 1,000-grain weight in rice. Other more complex
populations have been proposed, for example, MAGIC (multiparent advanced
generation intercross, Cavanagh et al. 2008), NAM (nested association mapping; Yu et
al. 2008; Buckler et al. 2009) and AMPRIL (a multiparent recombinant inbred line
population, Huang et al. 2011). These studies require genome-wide a high density of
markers and use re-sequencing technology.
The aim of the present paper is to develop an approach for a multi-population and multi-
environment QTL analysis in the presence of a reciprocal translocation. To illustrate our
approach, we analysed days to heading (DtH) and thousand kernel weight (TKW) using
a large number of doubled haploid lines from the F1 of five crosses produced from
Chapter 6
82
different combinations of four parents, one of which was ‘Albacete’. DtH was selected
because is independent of the reciprocal translocation and TKW because it was the only
trait showing a significant positive effect of the reciprocal translocation in ‘Albacete’
(Farré et al 2012b). ‘Albacete’ is known to carry a reciprocal chromosomal
translocation between chromosomes 1H and 3H (Vazquez and Sanchez-Monge, 1987;
Lacasa-Benito et al. 2005; Farré et al. 2011; Farré et al. 2012). This complicates the
QTL analysis due to ‘pseudo-linkage’ between markers on chromosomes involved in
the interchange. Close to the translocation breakpoints recombination is suppressed
(Burnham, 1962; Sybenga, 1975) and, as a consequence, markers in those regions were
removed; see also Farré et al. (2011). The final linkage maps consist of the distal parts
of the chromosomes involved in the translocation (1HS-1HL and 3HS-3HL). The
genotyping was performed using Diversity Arrays Technology (DArT®). The DArT
markers were positioned on the barley chromosomes according to the barley consensus
map (Wenzl et al. 2006).
Materials and methods
Plant material
Agronomic traits were evaluated on anther-culture derived DH lines of barley. The lines
were derived from five F1s obtained by crossing four parents: ‘Albacete’, ‘Barberousse’,
‘Orria’ and ‘Plaisant’. ‘Albacete’ is a landrace variety with a relatively long cycle and
an alternative growth habit. It is drought tolerant showing relatively stable grain yield
production under Spanish conditions. ‘Albacete’ carries a reciprocal translocation
between chromosomes 1H and 3H (Farré et al 2011; Farré et al. 2012a). ‘Barberousse’
is known for its good productivity and easy adaptation, although it is sensitive to
drought in Spain. ‘Orria’ is a Spanish variety of CIMMYT origin that is well adapted to
fertile, rainfed environments. ‘Plaisant’ shows good adaptation and has a high yield
under Spanish conditions. The vernalisation and photoperiod characteristics of the
parents are given in SEM_Table1. The allele representation within each population for
‘Albacete’, ’Barberousse’, ‘Plaisant’ and ‘Orria’ were 336, 166, 138 and 218
descendants, respectively.
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal translocation
83
Genotyping
Genomic DNA of the DH lines was extracted from fresh leaf tissue using the Qiagen
DNeasy 96 plant Kit. A genome wide scan was carried out using Diversity Arrays
Technology (DArT®) (www.diversityarrays.com). In a single assay, DArT uses
microarrays to detect DNA polymorphisms at several hundred genomic loci spread over
the entire genome. It is based on hybridising labelled genomic representations of
individual DNA samples on a micro-array, which contains a large number of DNA
fragments derived from the total genomic DNA of the species under consideration
(Wenzl et al. 2004). Polymorphisms are scored as the presence (= 1) or absence (= 0) of
hybridization to individual array elements. The locus designations used by Triticarte
Pty. Ltd. were adopted in this paper. DArT marker names consist of the prefix “bPb”,
followed by numbers corresponding to a particular clone in the genomic representation,
where b stands for barley, P for PstI (primary restriction enzyme used) and b for BstNI
(secondary restriction enzyme). For each DH population quality-filtered datasets were
prepared by removing markers and DH lines according to Farré et al. 2011. Individual
linkage maps were constructed for the three largest populations (A×B, A×P and P×O).
For A×B and A×P, linkage groups 1H and 3H were split in two as they carry the
reciprocal translocation (Farré et al. 2011). For P×O, 1H and 3H formed a single linkage
group, respectively. DArT markers were located on the barley chromosomes according
to the barley consensus map (Wenzl et al. 2006). The markers used for constructing the
consensus map were those which segregated in at least two mapping populations. DArT
markers located around the translocation breakpoints were removed according to Farré
et al. (2011) resulting in two sub-linkage groups for chromosome 1H and 3H (1HS-1HL
and 3HS-3HL).
Experimental design
Four field trials were carried out at two locations (Gimenells (41º 37’N, 0º 22’E, 248m,
dry, rainfed); Foradada (F; 41º 51’N, 1º 0’E, 407m, dry, rainfed) in North-Eastern Spain
(Catalonia) in two growing seasons (2008/2009 and 2009/2010). Details of the
environmental conditions are given in SEM_Table2. Experimental designs followed a
Chapter 6
84
latinized row and column alpha design with a single replication for most DH lines
augmented by four replicated checks (parents) that were randomly included in a
diagonal fashion along rows and columns. The experiments contained a total of 600
plots arranged in 30 rows and 20 columns, except Foradada-2008/2009 which contained
630 plots arranged in 15 rows and 42 columns. Plots consisted of eight rows, 4 m long
and 15 cm apart and were sown at a density of 300 seeds m2 and an accoding with
standard crop practise in terms of plant density, herbicide treatment and fertilizanty
rates.
Phenotyping
To illustrate our approach we analysed two traits: days to heading and thousand kernel
weight. Days to heading (measured as the number of days between 1st of January until
50% of spikes on a plot had extruding anthers) was selected because it is a major
selection criterion in all plant breeding programs of barley as it determines crop
adaptation. Besides that, it is a highly heritable trait with significant QTLs and its
biological basis has been well characterised in model plants (Arabidopsis; Greenup et
al. 2009) with many barley homologues identified (Higgins et al. 2010). None of the
major genes known to affect flowering time in barley are located in the region of the
reciprocal translocation (Laurie et al. 1995) so days to heading should be independent of
the reciprocal translocation. Thousand kernel weight (measured in grams) was selected
because it was the only trait showing a significant positive effect of the reciprocal
translocation in ‘Albacete’ (Farré et al 2012b).
Data analysis
The observations of each experiment (different site and year combinations) were
analysed by means of a mixed model using Genstat 14 procedures (Payne et al. 2011).
Spatial effects were eliminated by incorporating rows and columns in the random part
of the mixed model. We allowed the mixed model to have unequal variances for
populations, which made it possible to estimate the heritabilities of the traits for all
combinations of sites, years and populations separately. Finally, for each trait Best
Linear Unbiased Estimates (BLUEs) of the contributions of the genotypes were
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal translocation
85
obtained for further analysis. The BLUEs were subjected to QTL analysis. The BLUEs
were also used to calculate for each genotype the difference between the two locations,
the two years and the interaction between locations and years. These constrasts were
also subjected to QTL analysis.
QTL analysis
QTL analysis has been carried out using Genstat 14 (Payne et al. 2011). For all
individuals the probabilities associated with the descent states 0 (maternal allele) and 1
(paternal allele) at marker loci were obtained using the procedure
QIBDPROBABILITIES, based on the DArT genetic linkage map (Wenzl et al. 2006).
Here, maternal and paternal refer to the parents of the cross (F1) from which each DH
population was derived. The descent probabilities for individual j (= 1, 2 ... Ni) of
population i (= 1, 2 ... 5) at locus k were arranged in vectors p0k and p1k, respectively; k
= 1 ... M. Subsequently, the descent probabilities were used to calculate regressors xik,
accounting for allelic differences within populations (irrespective of the parents). Here,
xik= p0k – p1k if the corresponding individual belongs to population i and xik= 0,
elsewhere. For each marker, the regressors x1k, x2k ... x5k represent 4 degrees of freedom.
The regressors for the general (additive) effect of the four parents, denoted by apk (p = 1,
2, 3, 4), were obtained as follows:
a1k = x1k+x2k+x3k,
a2k = -x1k +x4k,
a3k = -x2k -x5k,
a4k = -x3k-x4k +x5k.
Parent 1 (‘Albacete’) is the mother of populations 1, 2 and 3, parent 2 (‘Barberousse’) is
the father of population 1 and the mother of population 4, parent 3 (‘Orria’) is the father
of populations 2 and 5, and finally parent 4 (‘Plaisant’) is the father of populations 3 and
4 and the mother of population 5. It should be noticed that , i.e. the
regressors for the general additive effects of the parents represent 3 degrees of freedom.
Differences in ‘fit’ between the sets of regressors {x1k, x2k ... x5k} on the one hand, and
the sets of regressors {a1k, a2k, a3k, a4k} are due to additive x genetic background
Chapter 6
86
interaction or specific additive effects, and may be an indication of epistasis (Jannink
and Jansen, 2001; Jansen et al. 2009).
Interval mapping was carried out using a mixed model. The mixed model accounted for:
(i) the presence of the reciprocal translocation in the populations that were derived from
‘Albacete’, (ii) differences between population means and (iii) allowed for
heterogeneous variances between populations. Tests for putative QTL were based on the
Wald statistic with the appropriate numbers of degrees of freedom. Initially a forward
selection of marker loci was carried out using a liberal threshold of 3.0 for minus the
logarithm of the p-value associated with the Wald statistic. Only the most significant
marker loci selected in this way were added to the set of cofactors used in the mixed
model. This was followed by backward elimination of the marker loci selected in the
previous step using a threshold of 3.5 (which conforms to the usual threshold obtained
by Li and Ji (2005) for this situation). It should be noticed that in a standard QTL
analysis the Wald statistics involves one degree of freedom for the numerator; here the
number equals three). In the output, contrasts between parents were presented as
differences from parent 1 (‘Albacete’) and appropriated standard errors were calculated.
The effect of the reciprocal translocation was tested by incorporating the effects of the
final set of cofactors obtained by backward elimination.
Results
Genetic map
A total of 364 previously mapped markers (Wenzl et al. 2006) were polymorphic in at
least two of the DH populations with between 75 (B×P) and 274 markers (P×O)
polymorphic in individual populations. No markers were segregating in all five
populations; 14 markers were polymorphic in four populations although the highest
number of polymorphic markers was found across three populations (171 markers). The
number of markers per chromosome varied between 30 (1H) and 86 (4H). The map
used in this study covered 1,111 cM for all seven chromosomes. Chromosomes 1H and
3H were split in two linkage groups due to the presence of the reciprocal translocation
(1HS-1HL and 3HS-3HL; Farré et al. 2011). The DArT markers located around the
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal translocation
87
translocation breakpoints were removed according to Farré et al. (2011). The greatest
gap was on chromosome 4H (64 cM).
Figure 1. Genetic linkage map. Map distances based on the barley consensus map (Wenzl et al. 2006). Linkage groups 1H and 3H were split in two sub-linkage groups: 1HS-1HL and 3HS-3HL.
Agronomic results
For DtH, Barberousse was on average the earliest and Albacete the latest parent, with a
difference of three days. For TKW, Albacete had on average the highest parental value
and Barberousse the lowest, with a difference of 3.1 g.
Means of the DH lines for DtH values ranged from 109.5 to 124.7 days at G9 and F10,
respectively. For TKW, the values ranged from 27.2 gr at G9 to 39.5 g at G10. For both
traits, the magnitude of the correlations varied across environments but not the sign
which is always positive for all the trials. The highest correlation coefficient (0.76) was
observed between G10 and F9 while the lowest (0.29) was between G9 and F10 which
could be explained by the extremely different mean values for DtH. However, for TKW
the lowest values were observed between G9 and the other environments (0.40 to 0.46).
The correlation between traits (DtH and TKW) for the various environments was -0.22
indicating that late flowering genotypes were associated with higher TKW.
1HS
0
2H
25
3HL
50
5H
75
7H
100
125
150
175
200
3HS 6H 1HL 4H
Pos
ition
(cM
)
Chromosome
Chapter 6
88
QTL analysis in single populations across environments
First a multi-environment QTL mapping was performed for the three largest populations
(A×B, A×P and P×O) for DtH and TKW to detect QTL and QTL×E interaction. The
results are presented in Table 1 and Figure 2. For DtH, the numbers of QTL detected
were 4, 2 and 4 for A×B, A×P and P×O, respectively. None of the QTL were present in
all three populations. In A×B, four QTLs were detected on chromosomes 2H, 3H, 5H
and 6H with Albacete alleles delaying flowering date. Only one QTL found on
chromosome 2H at 47 cM (consensus map of Wenzl et al. 2006) was detected in all four
environments (with effects ranging from -0.37 to -1.00 days). For A×P, two QTLs were
detected on chromosome 2H (68cM) and 3H (10 cM) with delayed flowering time for
Albacete alleles. Both are stable QTLs as they did not show any difference for additive
effects between trials. For P×O, four QTLs were determined on chromosomes 2H(2x),
3H and 5H. When more than one peak was found on a chromosome, the peaks were
well spaced; the two peaks on chromosome 2H were located at 49 and 122 cM. The first
one presented an effect in opposite directions for F10 and G9 showing QTL×E
interaction. The delay of flowering time in F10 is due to the Orria allele and in G9 due
to the Plaisant allele. Apart from this QTL all the others did not show any difference
between trial for additive effects (stable QTLs). The QTLs on 2H (49 cM) and 5H (127
cM) coincide with the ones detected in A×B population.
For TKW, QTLs were only detected in A×B and P×O. In A×B, only one QTL was
detected on 3H chromosome with the Barberousse allele giving an increasing of TKW.
In P×O, four QTLs were found: on chromosomes 3H, 5H(2x) and 6H. The QTLs on 3H
(97 cM) and 5H (120 cM) showed effects that were opposite to the effects for the other
QTLs in which Plaisant alleles gave an increase of TKW.
Table 1. QTLs, their locations and effects for DtH and TKW from the three DH populations (A×B, A×P and P×O) for 4 trials in Gimenells (G) and Foradada (F), 2009 and 2010 (Pop = population; CR = chromosome; se = standard error; additive effect: a positive value means that the second parent allele (B, P or O) increases the trait; the chromosome locations were referred to Wenzl et al. 2006).
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal translocation
89
Tra
it
Pop
C
R
cM
Mar
ker
-Log
10(P
) A
ddit
ive
effe
ct (
se)
F10
F
9 G
10
G9
DtH
A
×B
2H
47
bP
b-48
77
9.48
-0
.37
(0.1
41)
-0.8
8 (0
.137
) -0
.79
(0.1
30)
-1.0
0 (0
.175
)
3H
19
bPb-
9745
5.
46
-0.0
6 (0
.160
) -0
.55
(0.1
55)
-0.7
7 (0
.147
) -0
.70
(0.1
98)
5H
12
7 bP
b-44
94
6.02
-0
.31
(0.1
47)
-0.4
2 (0
.142
) -0
.52
(0.1
35)
-0.0
8 (0
.182
)
6H
64
bPb-
5822
4.
41
-0.1
0 (0
.142
) -0
.62
(0.1
38)
-0.4
3 (0
.131
) -0
.78
(0.1
77)
A×
P
2H
68
bPb-
0220
6.
63
-0.6
4 (0
.124
) -0
.64
(0.1
24)
-0.6
4 (0
.124
) -0
.64
(0.1
24)
P
×O
3H
10
bP
b-99
45
5.52
-0
.58
(0.1
24)
-0.5
8 (0
.124
) -0
.58
(0.1
24)
-0.5
8 (0
.124
) 2H
49
bP
b-69
92
10.7
1 0.
75 (
0.20
2)
0.29
(0.
145)
0.
16 (
0.15
9)
-0.8
1 (0
.200
)
2H
122
bPb-
1266
4.
25
0.62
(0.
154)
0.
62 (
0.15
4)
0.62
(0.
154)
0.
62 (
0.15
4)
3H
66
bP
b-24
40
4.11
0.
58 (
0.14
6)
0.58
(0.
146)
0.
58 (
0.14
6)
0.58
(0.
146)
5H
127
bPb-
4494
6.
92
-0.7
3(0.
137)
-0
.73(
0.13
7)
-0.7
3(0.
137)
-0
.73(
0.13
7)
TK
W
A×
B
3H
1 bP
b-55
55
4.31
0.
73 (
0.34
0)
1.27
(0.
366)
2.
01 (
0.40
6)
0.77
(0.
375)
A
×P
-
- -
- -
- -
- P
×O
3H
97
bP
b-67
22
5.08
1.
08 (
0.24
2)
1.08
(0.
242)
1.
08 (
0.24
2)
1.08
(0.
242)
5H
1 bP
b-64
85
10.1
0 -1
.67
(0.3
62)
-1.8
2 (0
.371
) -2
.48
(0.3
55)
-0.9
6 (0
.285
)
5H
120
bPb-
7854
8.
33
1.44
(0.
245)
1.
44 (
0.24
5)
1.44
(0.
245)
1.
44 (
0.24
5)
6H
68
bP
b-56
98
7.82
-1
.27
(0.2
24)
-1.2
7 (0
.224
) -1
.27
(0.2
24)
-1.2
7 (0
.224
)
Chapter 6
90
Figure 2. Example of multi-environment QTL analysis using the data from P×O population for 4 trials in Gimenells (G) and Foradada (F), 2009 and 2010 which was analyzed using Genstat 14 ((a) DtH and (b) TKW). Top panel shows the genome-wide profile, the blue line indicates the profile of –log10 (P-value) for composite interval mapping scan where test is for QTL expression in at least one environment. The red horizontal line shows the threshold value for significance. Low panel shows the representation of QTL allele effects in individual environments. Blue and yellow indicate the size and direction of the QTL effect in each environment for the first and the second parent, respectively. The green line at the top shows the significance for the overall test on QTL presence in the upper panel
10
1H 3H 5H 7H
QTL effects
G9
G10
F9
F10
0
4
8
2H 6H
7H 6H
2
5H 4H 3H 2H
4H
6
1H
Env
ironm
ents
Chromosomes
-log
10(P
)
(a)
p
1H 3H 5H 7H
QTL effects
G9
G10
F9
F10
2
4
6
10
2H 6H
7H
8
6H 5H 4H
4H
3H 2H
0
1H
-log
10(P
)
Chromosomes
Env
ironm
ents
(b)
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal translocation
91
QTL analysis across environments
For the multi-population QTL analysis across environments, the data from the five
populations and the seven chromosomes were used together to detect QTLs using the
overall mean for each genotype from the BLUEs obtained for each trial and the contrast
between locations, years and the interaction (Table 2).
For DtH, a total of 4 QTLs were detected. Three were found using the mean and the
other one was detected using the contrast between locations and years. In the first case,
all three QTLs showed negative additive effects for the three parental alleles (ranged
from -0.09 to -0.80 days) indicating an early flowering time compared to Albacete
allele. The QTL with the biggest effect was located on 3H at 10 cM. In the second case,
one peak was found on chromosome 2H at 148 cM. For the contrast between the two
locations, compared to Albacete the other parental alleles had negative effects (-0.01 to
-1.75 days). For the contrast between the two years, Barberousse and Plaisant had
positive parental allele effects whereas Orria showed earlier flowering time.
For TKW, two QTL was detected for the overall mean one chromosomes 5H at 17 cM.
In this case the effect was highly dependent on the parental allele: two increasing TKW
alleles (0.44 to 1.10 g) were specific to Plaisant and Barberousse and one decreasing
TKW allele (-0.96 g) came from Orria. No QTL×E interaction was found for TKW. No
QTLs were found for both traits in the same position.
Chapter 6
92
Table 2. Comparison of QTLs for DtH and TKW using multi-population multi-environment analysis (CR= chromosome; the chromosome location were referred to Wenzl et al. 2006; additive effect: a positive value means that the parent allele increase the trait compared to Albacete; se = standard error; G vs F = contrast between Gimenells and Foradada; 09 vs 10 = contrast between 2009 and 2010).
Effect of the reciprocal translocation
For the populations in which Albacete was one of the parents (A×B, A×P and A×O), the
presence of the reciprocal translocation was included in the QTL analyses to check if
the reciprocal translocation had an effect on DtH and TKW. Table 3 shows that there
was no evidence of a significant effect of the reciprocal translocation on DtH but for
TKW the lines carrying the reciprocal translocation showed higher TKW than the ones
without it (0.47 to 2.19 g). A positive additive effect of the reciprocal translocation was
found in F9, F10 and G10 for TKW.
Table 3. Estimates of the effects of the reciprocal translocation using the data from A×B, A×O
and A×P populations (se = standard error).
Trait -log10(P) RT vs no RT (se)DtH F9 0.487 0.27 (0.275)
F10 0.323 -0.17 (0.245) G9 0.148 -0.12 (0.337) G10 0.251 0.16 (0.272)
Mean 0.024 0.02 (0.244) PCA1 0.105 -0.00 (0.005)
TKW F9 5.187 2.16 (0.479) F10 5.750 2.19 (0.459) G9 - - G10 0.349 0.47 (0.616)
Mean 3.331 1.39 (0.396) PCA1 - -
Trait mm CR cM -log10(P) Parental additive effects (se)
Barberousse Orria Plaisant DtH Mean bPb9925 2H 64 6.04 -0.77 (0.166) -0.09 (0.202) -0.43 (0.149)
bPb9945 3H 10 9.24 -0.50 (0.134) -0.63 (0.194) -0.79 (0.125) bPb5596 5H 101 4.23 -0.58 (0.209) -0.80 (0.211) -0.37 (0.140) G vs F bPb1986 2H 148 7.14 -0.01 (0.262) -1.75 (0.159) -0.16 (0.206) 09 vs 10 bPb1986 2H 148 4.60 0.03 (0.243) -1.30 (0.347) 0.05 (0.235) interaction - - - - - - -
TKW Mean bPb7407 5H 17 7.31 1.10 (0.298) -0.96 (0.339) 0.44 (0.281) bPb-3512 4H 68 4.52 1.21 (0.272) 0.59 (0.338) 0.11 (0.253) G vs F - - - - - - - 09 vs 10 - - - - - - - interaction - - - - - - -
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal translocation
93
So far the general additive effects of parents were estimated. Specific additive effects
were estimated to analyse background interaction. No interactions between QTL and
genetic background were found.
Discussion
In the Mediterranean climate, characterized by low rainfall, high temperatures and high
light intensity leading to high evaporation, it is important to develop cultivars well
adapted to drought-prone conditions by improving characters related to yield. The
differences observed across environments for DtH and TKW can be attributed to
differences in climatic and other environmental conditions, especially differences in
rainfall. Usually, Foradada is colder and wetter than Gimenells. G9 was the earliest to
flower (12-30 April) whereas F10 the latest (2-14 May). The lowest values for both DtH
and TKW were found in G9, whereas the highest values were obtained in F10 and G10
for DtH and TKW, respectively. The main differences between years and locations were
due to the distribution of rainfall during the growth cycle. During the grain-filling
period (May-June), 2010 had a higher cumulated rainfall compared with 2009 which
could explain the higher TKW values obtained in 2010. Besides, the grain-filling period
was longer in 2010 than in 2009 (40 and 32 days, respectively). This difference in the
days of grain-filling, combined with limited rainfall and higher temperatures could
explain the low TKW values obtained for G9 (27 g) compared to the other
environments (30, 36 and 39 g for F9, F10 and G10, respectively).
The four parents (‘Albacete’, ‘Barberousse’, ‘Orria’ and ‘Plaisant’) used in the present
study show differences in the vernalization and photoperiod genes (Table 1,
supplementary material). All of them are winter varieties with a full or partial
vernalization requirement, performing similar when sown in autumn. Under these
conditions, ‘Albacete’ shows slightly later heading dates than the other parents (Igartua
et al. 1999). Under winter or spring sowing conditions, ‘Barberousse’ and ‘Plaisant’ are
clearly delayed. So, under the conditions of the experiments carried out in this study
major effects of flowering genes cannot be expected.
Chapter 6
94
It is known that genetic variation for genes controlling flowering time has provided
adaptation to different environments maximizing grain yield in cereal varieties
(Cockram et al. 2007). The genetic factors determining flowering time in barley have
been exhaustively studied and can be divided into: photoperiod-responsive genes (day
length), vernalization-responsive genes (response to a low temperature period to induce
transition from vegetative to reproductive stage) and ‘earliness per se’ genes (plant
response to a temperature sum over a certain period) (Boyd et al. 2003). In the QTL
analysis for each population separately, no QTLs were common among all three
populations. A×B and P×O had two QTL in common and A×B and A×P had one. The
most significant QTL for DtH was located on 2HS at 47-49 cM. By comparing genetic
maps (Wenzl et al. 2006; Cuesta-Marcos et al. 2008a; Close et al. 2009; Aghnoum et al.
2010) we assumed that this QTL corresponded to the location of PpdH1, one of the
major genes responsible for photoperiodic response expressed under long days (Laurie
et al. 1995; Turner et al. 2005; Sameri et al. 2011; Borràs-Gelonch et al. 2012). Yet, in
another study in the Mediterranean area, using a large panel of genotypes, Comadran et
al. (2008) concluded that under autumn sowing and latitudes under 43ºN it is unlikely
that PpdH1 might be effective. In P×O, PpdH1 had an opposite effect depending on the
environment. In F10 the delay of flowering time is due to ‘Orria’ allele whereas in G9 is
because of ‘Plaisant’. Similar findings were reported for the ‘SBCC145’×’Beatrix’
population (Ponce-Molina et al. 2012). They found an effect of this gene in an autumn
sowing with the insensitive allele (‘Beatrix’) conferring earliness; the effect changed in
winter sowing where the sensitive allele (‘SBCC145’) led to earlier flowering. This
QTL was also found in other studies using ‘Scarlett’×’ISR42-8’ (Wang et al. 2010) and
‘Steptoe’×’Morex’ (Borràs-Gelonch et al. 2012). An interesting finding was observed
for PpdH1 in A×B population in which ‘Albacete’ allele somewhat delayed flowering
time. However, both parents carry the dominant, long photoperiod responsive allele,
with the same polymorphism in the SNP that is used to differentiate the
dominant/recessive alleles (Turner et al. 2005). This result suggests that, there may be
more alleles for PpdH1. Resequencing the PpdH1 gene, Jones et al. (2008) identified 83
polymorphisms in a set of 266 barley accessions (194 European landraces and 72 wild
barley accesions). Among them 10 Spanish landraces were present. Although they carry
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal translocation
95
the dominant, long photoperiod responsive SNP allele they show other polymorphisms
along the PpdH1 gene which could be related with different phenotypic effects.
The other common QTL was detected on 5HL at 127 cM , where is located Vrn-H1, a
major gene which determines the vernalization requirement (Laurie et al. 1995;
Trevaskis et al. 2003; Cuesta-Marcos et al. 2008a,b). In our study, ‘Barberousse’ and
‘Plaisant’ are two typical winter varieties with full vernalization requirement as they
carry a recessive allele at vrnH1 and a dominant allele at VrnH2. In contrast, ‘Albacete’
and ‘Orria’ carry an intermediate (VRNH1-4) allele, which has been found to be
associated with a reduced vernalization requirement (Casao et al. 2011). In P×O, ‘Orria’
alleles conferred earliness. Different results were obtained in A×B population in which
‘Albacete’ alleles delayed flowering time. The flowering difference in A×B is between
0.16 to 1 day whereas in P×O is 1.5 days. For A×B and A×P, the common QTL was
found on 3HS at 10 cM, which we predict to be in the region of QHea.S42-3H.a (von
Korff et al. 2006). A QTL on chromosome 2H (122 cM detected in P×O) is in the same
region that a QTL found in two association mapping studies (Comadran et al. 2011;
Wang et al. 2012). Other QTLs such as that found on 3H (66 cM detected in P×O) or
6H (64 cM detected in A×B) could be specific to backgrounds of these populations.
Concerning TKW, no QTL were found in common between populations, although the
greatest number of QTL was found in the P×O population. Two other QTL on 5H (120
cM) and 6H (68 cM) are in similar regions to those reported by von Korff et al. (2008)
in the ‘Tadmor’ × ‘ER/Apm’ population.
Until recently the populations used to detect and map QTLs in plants were biparental
populations which have been widely employed by selecting two contrasting parents to
guarantee marker polymorphism and trait divergence (e.g. Backes et al. 2003). This
approach is not particularly applicable to plant breeding because QTLs can be missed as
they do not segregate in the particular cross. Nowadays, the development of multiple
connected populations for QTL analysis is increasing because it: (i) improves the power
to detect QTL and the accuracy of the estimation of their effects and positions; (ii)
increases variability addressed; (iii) permits direct comparison of the epistatic
Chapter 6
96
interactions between parental alleles at specific genome regions across genetic
backgrounds (Rebaï et al. 1997; Verhoeven et al. 2006; Blanc et al. 2006, 2008; Cuesta-
Marcos et al. 2008a; Liu et al. 2010). For that reason, it is necessary to develop new
QTL mapping methodologies for populations derived from more than two parents due
to the fact that most current statistical methods developed for QTL mapping are based
on bi-parental populations (Hackett 2002).
In this study, a new approach was developed for QTL detection in multi-population
analysis in the presence of a reciprocal translocation. This approach was applied using
DtH and TKW providing us with the opportunity to directly compare general additive
effects among four parental alleles. In this paper we mainly looked at the contrast of
Albacete with the other parents involved in creating the populations. For DtH, three
major QTLs, whose positions agree with previously studies, were found at the following
locations: on chromosome arm 2HS at 64-72 cM in the region of Eam6, identified as the
main heading date QTL in Mediterranean conditions (Cuesta-Marcos et al. 2008a,b; von
Korff et al. 2008; Comadran et al. 2011; Wang et al. 2012); on chromosome 3HS at 10
cM, which we predict to be in the region of QHea.S42-3H.a (von Korff et al. 2006) and
finally on chromosome 5HL at 101 cM, in the region of a minor QTL found in ‘Beka’ ×
‘Mogador’ (Cuesta-Marcos et al. 2008b). For TKW, two QTLs were found. One on the
short arm of chromosome 5H could be related to that on detected in
‘TX9425’×’Franklin’ (Wang et al. 2010b) and another one on the chromosome arm
4HS at 68 cM which corresponds with the QTL found by Teulat et al. (2001). QTLs
only found in the present study could be specific to one environment or, when detected
across environments, specific to the genetic backgrounds employed (i.e. for DtH, the
QTL found on chromosome 2HL at 148cM). None QTL were found for both traits at
the same position.
The results obtained when we included the presence of the reciprocal translocation in
the QTL analyses are in agreement with the results obtained by Farré et al. (2012b) in
which the reciprocal translocation affected only TKW. Other studies in
‘Blenheim’×’E224/3’ detected QTL in the region of the translocation (Thomas et al.
QTL analysis in multiple interconnected barley doubled haploid populations in the presence of a reciprocal translocation
97
1995; Powell et al. 1997). DH lines carrying the reciprocal translocation had a greater
TKW than those with a standard chromosome arrangement. Identifying the regions that
improve traits related to yield (i.e. TKW) allows the breeders to use marker-assisted
selection for the introgression of favourable alleles into their germplasm (in our case the
‘Albacete’ haplotype). All genes present in the interstitial zone of a translocation
heterozygote are tightly linked due to the suppression of genetic recombination, and as a
consequence they can be transferred as a block to other backgrounds. In breeding
programs, the use of hybrids derived from one parent carrying a reciprocal translocation
in breeding programs could lead to problems in seed production as a consequence of
semi-sterility. Although semi-sterility disappears once the individuals become
homozygous for the translocation.
Acknowledgments
This study has been supported by the Spanish Ministry of Science and Innovation
(Project AGL2011-30529-C02-02 and the Agrogenomics CONSOLIDER Center). The
James Hutton Institute is supported by the Scottish Government Rural and Environment
Science and Analysis Services (RESAS) Division.
Chapter 6
98
SUPPLEMENTARY MATERIAL
Table 1. Summary of the vernalization and photoperiod gene structure of the parents used in the
production of the doubled haploid populations.
Table 2. Means of temperature (T) and cumulated rainfall (R) during the months of the growth cycle
of the four trials: F9= Foradada 2008/2009, F10= Foradada 2009/2010, G9= Gimenells
2008/2009 and G10= Gimenells 2009/2010 (http://xarxes.meteocat.com).
Growth type
HvFT3 (PpdH2;1H)
SNP22 (PpdH1;2H)
HvZCCT (VrnH2;4H)
HvBM5 (VrnH1;5H)
HvFT1 (VrnH3;7H)
‘Albacete’ Facultative dominant dominant dominant intermediate recessive ‘Barberousse’ Winter recessive dominant dominant recessive recessive ‘Orria’ Facultative recessive recessive dominant intermediate recessive ‘Plaisant’ Winter recessive dominant dominant recessive recessive
Trial Nov Dec Jan Feb Mar Apr May June July Total
F9 R (mm) 33.8 30.6 50.6 34.6 52.2 115.2 9.4 29.5 28.7 384.6 T (ºC) 5.4 3.1 3.5 4.9 8.1 10.2 17.0 21.6 24.1 10.9
F10 R (mm) 10.0 67.8 61.6 32.6 64.1 14.6 54.0 102.1 4.9 411.7 T (ºC) 8.3 3.7 2.8 3.9 6.6 11.3 13.7 18.8 24.4 10.4
G9 R (mm) 23.6 25.6 21.6 13.6 31.0 90.4 8.6 16.4 31.2 262.0 T (ºC) 6.8 4.4 4.4 6.8 9.8 12.1 18.9 22.9 24.6 12.3
G10 R (mm) 5.5 45.5 65.8 26.7 38.4 19.1 27.3 54.3 0.2 282.8 T (ºC) 10.4 5.5 5.1 5.7 8.8 13.2 15.8 20.3 25.5 12.3
99
Chapter 7
General discussion
Alba Farré
Breeding programs in Mediterranean environments are focused on the development of
varieties with high yield and yield stability across environments. However, crop
productivity is limited by drought, one of the most important abiotic stress factors in the
Mediterranean basin (Araus et al. 2003). A Spanish barley variety, ‘Albacete’, which is
drought tolerant and shows adaptation to low-yielding sites was the basis of the present
thesis. ‘Albacete’ was selected because it is an extensively cultivated variety in the
poorest Spanish areas. It is known to carry a reciprocal translocation between
chromosomes 1H and 3H without apparent loss of agronomic value. One of the main
objectives of the present thesis was to investigate whether the reciprocal translocation
has a positive effect on drought tolerance and other traits that make it worth to introduce
genes in the vicinity of the translocation breakpoints into the barley germplasm.
Balanced chromosomal rearrangements such as reciprocal translocations or inversions
seem to be the most common rearrangements in plants (see interchanges in individual
species in the summary by Burnham, 1956, pp.459-522) which not always produce
phenotypic effects either in the heterozygous or homozygous state, other than reducing
heterozygote fertility as a result of duplications and deficiencies. Their maintenance
largely depends on their effects upon two properties: (i) the survival of individuals and
(ii) their fertility (Rees 1961). Wallace (1959) postulates that at the margin of a species
distribution, a translocation heterozygote with one of the better marginal genotypes may
have a relative advantage that compensates for the expected semi-sterility. Furthermore,
the semi-sterility disappears once the individuals become homozygous for the
translocation, i.e. once a pure line is produced. Deleterious effects may arise when a
breakpoint occurs within genes, generally disrupting gene functions; this is called a
‘position effect’. The best established example of a position effect in plants is in
General Discussion
100
Oenothera and involves the factors Ps (broad red and narrow green stripes on the sepals)
and Pr (red or rubricalyx); the translocation of a segment carrying the factor for broad
red calyx stripes (Ps) to a new position resulted in variegated patches of red (Pr). When
the locus was transferred back to the normal chromosome by crossing-over, the normal
phenotype was restored. (Catcheside, 1947). Other examples were found in Drosophila
(Ephrussi and Sutton, 1944; Lewis, 1950; Muller, 1954) and maize (Brink, 1932, 1954;
Roberts, 1942; Jones, 1944; McClintock; 1951, 1953).
The emergence of new species is often accompanied by structural chromosome changes
(Stebbins, 1971; Levin, 2002) which have played an important role in plant evolution
showing adaptive features of many natural populations. For instance, some species of
the genus Clarkia are notorious for a high frequency of translocation heterozygotes (C.
williamsonii (Wedberg et al. 1968); C. amoena (Snow, 1963); C. unguiculata (Mooring,
1958); C. exilis (Vasek, 1960); C. elegans (Lewis, 1951); C. dudleyana (Snow, 1960)).
The frequency of translocation heterozygotes in wild populations of Clarkia
williamsonii was found to be strongly correlated with habitat; heterozygotes are
abundant in populations in foothill woodlands and infrequent in populations at higher
elevations in the yellow pine forest. The constancy of the distributions of heterozygotes
strongly suggests that they have an adaptive role in populations (Wedberg et al. 1968).
This adaptive role has not only been studied in plants but also in other species like the
yeast Saccharomyces cerevisiae. Colson et al. (2004) investigated the effect of a
reciprocal translocation involving chromosomes VI, VII and XVI on the whole
organism’s evolutionary fitness. They found that this naturally occurring rearrangement
had the potential to provide a selective advantage in certain environmental conditions.
These results support the idea of chromosomal evolution involving fixation of major
rearrangements by natural selection. In barley, Konishi and Linde-Laursen (1988)
identified four Ethiopian cultivated landraces carrying a reciprocal translocation with
the translocation breakpoints at the centromere region involving chromosomes 2H and
4H, suggesting a common origin.
General Discussion
101
An interesting example is given by the T5B:7B-1 translocation which is frequent in
hexaploid wheat. It was first identified in French bread wheat (T. aestivum ‘Cappelle-
Desprez’; Riley et al. 1967) and was found later in many other varieties of T. aestivum
(Baier et al. 1974; Bourgeois et al. 1978; Seal, 1982; Vega and Lacadena 1983; Lange et
al. 1987; Schlegel and Schelgel 1989; Friebe and Gill 1994; Hohmann et al. 1996;
Schlegel 1996). Its broad distribution can result from selective advantages of rearranged
genotypes in diverse environmental conditions which may have significant breeding
potential (Miura et al. 1992; Law and Worland 1997). For instance, Law and Worland
(1997) indicate that the 5BS-7BS translocation was responsible for a substantial part of
the adult plant yellow rust resistance of these varieties although it cannot be solely
responsible for the resistance. These authors suggest that the gene(s) involved in
resistance to yellow rust are closely linked to the translocation breakpoints adding an
important attribute to the translocation favouring its selection. They also found some
indication for a yield advantage associated with the 5BL arm. Yasumuro et al. (1998)
suggest that most commercial bread wheat cultivars inherited translocations from their
landrace ancestors; the maintenance of these translocations in landraces and their
transmission to commercial varieties may reflect the significant breeding potential of
translocated chromosomes. A reciprocal chromosomal transposition in Brassica napus
between interstitial homeologous regions on linkage groups N7 and N16 gave
significantly higher seed yields (Osborn et al. 2003).
In the present study, the agronomic effects of the presence of the reciprocal
translocation on some interesting agronomic traits (associated to development, yield and
yield components and architecture traits) were tested using a mixed model procedure
dividing a set of 248 doubled haploid lines in two groups (carrying or not the reciprocal
translocation) (Chapter 4). A highly significant main effect of the translocation was
obtained for TKW; no significant main effects could be detected for other agronomic
traits. It has been hypothesized that the high level of farmer’s acceptance of ‘Albacete’
particularly for semi-arid rainfed areas may be due to the presence of this translocation.
However, the adaptation to low-yielding sites cannot be attributed to this chromosomal
structural change alone.
General Discussion
102
Plant accessions carrying chromosome rearrangements such as translocations have been
identified by the resulting partial pollen and seed sterility and by the apparent linkage of
genes whose normal loci are on separate chromosomes (i.e. Jáuregui et al. 2001).
Special interest resides in the identification of chromosomes involved in the
interchanges and determination of the translocation breakpoints as it will provide new
information on the origin of chromosomal rearrangements. It will also allow physical
mapping of genes involved in the translocation on the chromosomes. These can be
achieved by a combination of cytogenetics and molecular genetics (Chapter 3). The
translocation breakpoints on 1H and 3H chromosomes in the barley variety ‘Albacete’
overlaps with duplicated segments between chromosomes Os01 and Os05 in rice
derived from ancestral duplications within the grass family (Guyot and Keller 2004;
Thiel et al. 2009). Among other causes the original interchange may have originated
from a crossing-over between duplicated segments in non-homologous chromosomes
(Burnham, 1956). Identification of structural changes is crucial in utilizing genetic pools
of wild and cultivated varieties in breeding programs (Badaeva et al. 2007). Due to the
suppression of genetic recombination, the genes in the interstitial zone of a translocation
heterozygote are linked and can be transferred as a block to other backgrounds. So,
chromosome segments carrying the reciprocal translocation are worth to be transferred
in the barley germplasm. The time needed to incorporate favourable alleles into the
germplasm can be reduced due to the development of specific molecular markers.
The incorporation of molecular technologies in plant breeding programs allows linkage
analysis of agronomically important traits. For that purpose, linkage map construction
using data from molecular markers is an indispensable tool. Major complications arose
in populations based on reciprocal translocation heterozygotes as recombination
between loci around the translocation breakpoints is suppressed (Burnham, 1962;
Sybenga, 1975). Consequently, markers in these regions become ‘pseudo-linked’. In
order to disentangling this ‘pseudo-linkage’ a novel statistical-genetic approach was
developed making use of a high-quality consensus map of ‘normal’ barley (Wenzl et al.
2006) (Chapter 2). An alternative study investigated the effects of karyotypic
differences on the construction of linkage maps (Livingstone et al. 2000). They used
Mapmaker/EXP to create several genetic maps to examine intermarker distances using
General Discussion
103
two simulated datasets for F2 progeny which differed by a reciprocal translocation or a
reciprocal translocation and an inversion. They found alternative maps corresponding to
different chromosomal rearrangements around the translocation breakpoints. Using this
study as a reference, Durrant et al. (2006) introduced QuadMap, a software application,
to analyze linkage maps affected by translocations. Quadmap constructs and compares
multiple maps using Mapmaker/EXP. It generates multiple maps, permutates the marker
order, and analyzes the data from the linkage groups involved in the translocation
simultaneously using a large number of iterations. It gives the frequencies, average
intermarker centimorgan distance and centimorgan distance variance of each marker
pair. Large intermarker distance variance was proposed as an indication of the presence
of a breakpoint. However, high variances may also be caused by deficiencies in the
data. The methods developed in the present study can be implemented using standard
statistical software. When a consensus map is not available the minimum spanning tree
method (MST) can be used as an alternative to the principal coordinate analysis. This
allows us to separate the markers around the translocation breakpoint from the short and
long chromosome arms. This alternative strategy is based on (1) disentangling the
"pseudo-linkage" using the minimum spanning ; (2) remove markers around the TBs
according to a define threshold in terms of recombination frequencies and (3) four
independent marker sets will be used to construct the final linkage maps of the distal
parts of the chromosomes involved in the translocation.
The first steps for the introgression of favourable alleles into germplasm are the
construction of a high-quality linkage maps (Chapter 5) followed by QTL analysis
(Chapter 6) which allows the identification of the precise location of chromosome
regions associated with traits of interest. This goal can be achieved by constructing
consensus maps using information from multiple populations which provides greater
genome coverage avoiding large gaps in the map without markers and the marker order
and intermarker distances can be estimated more accurately. This requires having
common markers among populations which depends on the high level of polymorphism
between parents. In the present study, the highest level of polymorphism among
populations was observed for A×B and A×P whereas B×P showed the lowest level. This
appears to be in good agreement with the results obtained by Casas et al. (1998). They
General Discussion
104
demonstrated that parental lines ‘Plaisant’ and ‘Barberousse’ showed the highest level
of genetic similarity compared with the other parents. This could be explained because
they share the cultivar ‘Ager’ as one the parents. So, to enhance the chance of detecting
more polymorphic markers the parents should be divergent. But this can result in
populations that are not representative of germplasm used in breeding programs. It also
has a direct consequence for the QTL analysis because if marker alleles are non-
segregating in the B×P population also QTL will have a higher chance of being non-
segregating.
High-quality genetic linkage maps are of great importance for robust QTL mapping. For
that, high-quality datasets free of errors are essential as genotyping errors and missing
data lead to map expansion, especially in regions with a high marker density. In our
study discrepancies in map lengths, compared with the reference consensus map (Wenzl
et al. 2006), may be due to difference in “data curation” possibly in combination to the
use of different mapping criteria and mapping algorithms (Van Os et al. 2005). The
algorithm applied in this study which was adapted from the algorithm developed by
Jansen (2005), is an efficient method for constructing high-quality dense, integrated
linkage maps. This algorithm enabled us to develop a robust integrated linkage map
involving complex chromosomal rearrangements.
New QTL mapping methodologies have to be developed to cope with multi-population,
multi-environment analysis. Until now, most QTL studies were performed using
biparental populations (F2 or F× families, backcross, recombinant inbred lines, near-
isogenic lines and double haploids) which were obtained by crossing two inbred parents
with contrasting difference in phenotypic traits of interest (Semagn et al. 2010).
Consequently, a small part of the total variability for breeders is explored. So, the
development of multiple connected populations has become useful to: (i) increase the
variability addressed; (ii) allow global comparison of the effects of all QTL alleles
segregating in the different populations increasing the power to detect QTL, the
accuracy of the estimation of their effects and position and identifying the parental
origin(s) of favorable alleles at each QTL; and (iii) addressed interactions between QTL
and the genetic backgrounds (Muranty et al. 1997). As an alternative, other more
General Discussion
105
complex populations have been proposed to exploit the advantages of both linkage
analysis and association mapping such as the MAGIC populations (multiparent
advanced generation intercross) (Cavanagh et al. 2008), the nested association mapping
populations (NAM) design based on a huge set of recombinant inbred lines derived
from a large number of founder genotypes (Yu et al. 2008; Buckler et al. 2009) and
AMPRIL populations (a multiparent recombinant inbred line population) (Huang et al.
2011). For instance, NAM population captured approximately 136,000 crossover
events, corresponding, on average, to three crossover events per gene (McMullen et al.
2009). This NAM was derived from the crosses between 25 diverse inbred maize lines
and a reference line (B73) comprising 5000 RILs, 200 RILS per family. This allows
genetic factors to be mapped to very specific regions of the genome. These studies
genome-wide require a high density of markers and use re-sequencing technology.
The results obtained using the new methodology implemented in this study for a multi-
population, multi-environment QTL analysis was found useful to directly compare
additive effects among four parental alleles (Albacete, Barberousse, Orria and Plaisant)
(Chapter 6). The reciprocal translocation affected thousand kernel weight. DH lines
carrying the reciprocal translocation had a greater TKW than those with a standard
chromosome arrangement. These results are in concordance with the results found by
Farré et al. 2012 (Chapter 3).
The progress made with new marker technologies (such as DArT and SNP markers)
allow high throughput and low cost genotyping which provides the opportunity to
develop high-quality linkage maps. Currently, the new challenge in plant science is the
plant phenotyping which is considered to be the new bottleneck. The ‘phenotyping
bottleneck’ can be addressed by combining novel technologies such as robotics,
noninvasive imaging and analysis using powerful computing. This brings us to the age
of ‘phenomics’.
General Discussion
106
The present study was focused on the characterization of a reciprocal translocation
present in the widely grown Spanish barley variety ‘Albacete’. The combination of
different complementary approaches (cytogenetic, linkage and QTL mapping) led us to
develop an in-depth understanding of the reciprocal translocation and its consequences.
107
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Summary
Reciprocal translocations (RT) are one of the most common structural chromosomal
rearrangements occurring in plant species. Spontaneous RT are extremely uncommon in
cultivated barley. In fact, ‘Albacete’ is the only extensively cultivated barley variety
known to carry a RT without any major reduction in fitness. Previous results have
shown that 1H and 3H chromosomes are involved in this translocation which may have
a potential interest for plant breeding. A depth understanding of this RT could allowed
us to know whether this translocation is involved or not in their drought tolerance and
find some interesting phenotypic attributes that could be transfer to other barley
genotypes to develop specific germoplasm adapted to stress.
In a first step, a set of 230 doubled haploid lines derived from the cross between
‘Albacete’ and the non-translocation barley variety ‘Barberousse’ was used to develop a
statistical-genetic approach for the construction of a linkage maps in populations
obtained from RT heterozygotes. A preliminary linkage study revealed six main linkage
groups, compared to the seven linkage groups on the barley consensus map. One of the
linkage groups contained markers of chromosomes 1H and 3H. This union was due to
the presence of a RT between chromosomes 1H and 3H in the variety ‘Albacete’. The
results of this study revealed that the position of the translocation breakpoints was
placed around the centromeric regions for both chromosomes.
In a second step, a combination of cytological and molecular genetic approaches was
used in order to know how large the segments are that are involved in the interchange
between the two chromosomes. The results obtained in this study indicate that the
translocation is quite large with breakpoints located on the long arms of chromosomes
1H and 3H resulting in the RT 1HS.1HL-3HL and 3HS.3HL-1HL. The gene content
around the translocation breakpoints was estimated at approximately 1,100 and 710
gene models for 1H and 3H, respectively.
Summary
126
A large number of doubled haploid lines derived from four crosses involving ‘Albacete’
as one of the parents were used to phenotypically characterize the effects of the RT on
some interesting agronomic traits. The results suggested that the RT alone is not causing
the drought tolerance of ‘Albacete’; thousand kernel weight is the only trait which is
clearly enhanced in the lines carrying the RT. Further work combining the results from
this study with QTL analysis was carried out to phenotypically characterize the effects
of the reciprocal translocation and QTL simultaneously. For that, an integrated map was
constructed making used of a modified version of an algorithm developed by Jansen
(2005). Finally, specific QTL methodology for the simultaneous analysis of these
interconnected populations was developed; two traits: days to heading (independent of
the RT) and thousand kernel weight (affected by the RT) were used as an example.
127
Samenvatting
Reciproke translocaties behoren tot de meest voorkomende structurele herschikkingen
van de chromosomen bij planten. In gecultiveerde gerst komen spontane reciproke
translocaties zelden voor. In feite is ‘Albacete’ het enige veel geteelde gerstras waarvan
bekend is dat het drager is van een reciproke translocatie zonder dat het leidt tot een
sterke afname van ‘fitness’. Eerdere resultaten hebben aangetoond dat de chromosomen
1H en 3H betrokken zijn bij de translocatie die van potentieel belang kan zijn voor de
plantenveredeling. Meer kennis van de werking van deze reciproke translocatie zou
kunnen aantonen dat deze translocatie betrokken is bij droogtetolerantie en bij andere
interessante eigenschappen. Deze eigenschappen zouden kunnen worden overgedragen
naar andere gerstrassen met als doel het creëren van germplasm dat speciaal is
aangepast aan stress condities.
In een eerste stap zijn 230 verdubbelde haploïde lijnen afkomstig van de kruising tussen
‘Albacete’ en het gerstras ‘Barberousse’, dat geen drager is van de reciproke
translocatie, gebruikt voor ontwikkelen van een statistisch-genetische methode voor het
construeren van een genetische kaarten in populaties verkregen uit heterozygoten
waarvan een van de ouders drager is van een reciproke translocatie. Een eerste
koppelings studie liet zien dat er zes koppelingsgroepen zijn, in vergelijking met de
zeven groepen op de consensus kaart van gerst. Een van de groepen bevatte merkers van
de chromosomen 1H en 3H. Deze ‘clustering’ van merkers werd veroorzaakt door de
aanwezigheid van een reciproke translocatie tussen de chromosomen 1H en 3H in
‘Albacete’. De resultaten van deze lieten zien dat de posities breekpunten van de
translocatie zich op beide chromosomen bevonden in de centromere gebieden.
In een tweede stap is een combinatie van cytologische en moleculair-genetische
methoden gebruikt voor het bepalen van de grootte van de segmenten die betrokken zijn
bij de uitwisseling tussen de twee chromosomen. De resultaten van deze studie laten
zien dat de translocatie aanzienlijk van omvang is en waarbij de breekpunten zich
128
bevinden op de lange armen van de chromosomen 1H en 3H. Dit leidt tot de translocatie
1HS.1HL-3HL en 3HS.3HL-1HL. Het aantal genmodellen rond de translocatie
breekpunten werd bij benadering geschat op 1100 en 710 voor respectievelijk
chromosoom 1H en 3H.
Een groot aantal verdubbelde haploïde lijnen afkomstig van vier kruisingen met
‘Albacete’ als een van de ouders zijn vervolgens gebruikt voor de fenotypische
karakterisering van de effecten van de reciproke translocatie op enkele belangrijke
landbouwkundige eigenschappen. De resultaten laten zien dat de reciproke translocatie
niet alleen verantwoordelijk is voor de droogtetolerantie van ‘Albacete’. Het
duizendkorrelgewicht is de enige eigenschap die hogere waarden heeft in lijnen die de
reciproke translocatie dragen. Deze studie is daarna uitgebreid met een QTL analyse
om de gezamenlijke effecten van de reciproke translocatie en QTLs te karakteriseren.
Voor dat doel is eerst een geïntegreerde koppelingskaart geconstrueerd en vergeleken
met de consensus kaart van gerst. Tenslotte zijn er specifieke QTL methoden voor de
simultane analyse van de kruisingspopulatie ontwikkeld en toegepast. Twee
eigenschappen, het aantal dagen tot doorschieten (niet beïnvloed door de reciproke
translocatie) en duizendkorrelgewicht (wel beïnvloed door de reciproke translocatie),
werden geanalyseerd waarbij een aantal QTL werden gevonden.
129
Acknowledgements
The completion of this thesis is a result of the efforts of several people. I would like to
acknowledge those who contributed and recognize the inestimable value of their work.
First of all I would like to show my gratitude to my supervisors Ignacio Romagosa, Johannes
Jansen and Fred van Eeuwijk for their excellent work, guidance, availability, support and for
giving me this great opportunity. I would have not achieved the successful completion of this
thesis without you.
I would like to thank the people from my lab: Marian, Pilar, Andrea and particularly Eva (for
helping me getting started in the lab with her useful assistance in lab techniques, her excellent
friendship, shared lovely and fun time).
I would like to express my special thanks to the people from Biometris: Joao, Marcos, Martin,
Tahira, Patricia, Sabine, Willem, Marian, Apri, Santosh, Laura, Maggie and Paulo for their
support and kindness. Thanks Thomas and family, I really enjoyed the time I spend with you.
I warmly thank the biotech colleagues who treated me as one of them: Ariadna, Koreen, Sonia,
Svetlana, Gina, Sol, Dawey, Maite, Bruna, Chao, Griselda, Uxue, Edu, Bing, Evangelia, Judit,
Gemma, Changfu, Ludovic and other colleagues from the PVCF department. Also thanks
Teresa and Paul for their support and help when I needed.
I would like to thank co-authors of our publications for the fruitful collaboration and valuable
discussion of manuscripts: Prof. Schubert (IPK, Germany), Dr. Cistué (Estación Experimental
de Aula Dei, Spain), Prof. de Jong (WUR, The Netherlands), Dr. Cuadrado (Department of Cell
Biology and Genetics, Spain), Dr. Comadran (The James Hutton Institute, Scotland), Dr.
Lacasa-Benito, Dr. Casas (Estación Experimental de Aula Dei, Spain) and Dr. Thomas (The
James Hutton Institute, Scotland).
Many thanks go to Marian and Izak Bos. I will never forget your kind help and your warm and
generous hospitality during my stay in Wageningen.
Thanks María, Diana, Merche, Paula and Izabel, your friendship mean a lot for me.
I wish to thank my entire family for their love and encouragement. And last but not least Gerard was remarkably supportive. Thanks so much for always being there for me.
131
PE&RC PhD Education Certificate With the educational activities listed below the PhD candidate has complied with the educational requirements set by the C.T. de Wit Graduate School for Production Ecology and Resource Conservation (PE&RC) which comprises of a minimum total of 32 ECTS (= 22 weeks of activities) Review of literature (4.5 ECTS)
- General introduction
Writing of project proposal (4.5 ECTS) - Development of materials and techniques for the study of adaption to stresses in barley (Hordeum
vulgare L.) Post-graduate courses (3 ECTS)
- Applied bioinformatics in plant science (2010) - Detection and use of QTLs (2011)
Laboratory training and working visits (4.5 ECTS)
- Breakpoint localization of a reciprocal translocation present in the Spanish barley variety “Albacete”
Invited review of (unpublished) journal manuscript (2 ECTS) - Molecular Breeding: eQTLs (2009) - Theoretical and Applied Genetics: resistance in barley cultivars (2010)
Deficiency, refresh, brush-up courses (3 ECTS)
- Modern Statistics for Life Science (2008)
Competence strengthening / skills courses (1.9 ECTS) - Career assessment (2011) - Communication with the media and the general public (2011) - Information literacy including EndNote introduction (2011)
PE&RC Annual meetings, seminars and the PE&RC weekend (1.2 ECTS)
- PE&RC Weekend (2011) - PE&RC Day (2011)
Discussion groups / local seminars / other scientific meetings (7.5 ECTS)
- Biometris Statgen colloquium (2010-2011) - Biometris lunch colloquium (2010-2011) - Plant production and forest science department seminars; intermittent (2007-2011)
International symposia, workshops and conferences (4.4 ECTS) - 19th International Triticeae Mapping Initiative – 3rd COST Tritigen (2009) - Congreso de la sociedad española de genética (SEG) (2011)
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