Using diversity of the chloroplast genome to examine evolutionary history of wheat species

RESEARCH ARTICLE

Using diversity of the chloroplast genome to examineevolutionary history of wheat species

Fiona J. Leigh • Ian Mackay • Hugo R. Oliveira • Nicholas E. Gosman •

Richard A. Horsnell • Huw Jones • Jon White • Wayne Powell •

Terence A. Brown

Received: 10 August 2012 / Accepted: 7 January 2013 / Published online: 11 February 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Chloroplast microsatellites (SSRs) are

conserved within wheat species, yet are sufficiently

polymorphic between and within species to be useful

for evolutionary studies. This study describes the

relationships among a very large set of accessions of

Triticum urartu Thum. ex Gandil., T. dicoccoides

(Korn. ex Asch. et Graebn.) Schweinf., T. dicoccon

Schrank, T. durum Desf., T. spelta L., and T. aestivum

L. s. str. based on their cpSSR genotypes. By

characterising the chloroplast diversity in each wheat

species in the evolutionary series, the impact on

diversity of major evolutionary events such as domes-

tication and polyploidyisation was assessed. We

detected bottlenecks associated with domestication,

polyploidisation and selection, yet these constrictions

were partially offset by mutations in the chloroplast

SSR loci that generated new alleles. The discrete

cpSSR alleles and haplotypes observed in T. urartu

and Aegilops tauschii, combined with other species

specific polymorphisms, provide very strong evidence

that concur with current opinion that neither species

was the maternal and thus cytoplasmic donor for

polyploid wheats. Synthetic hexaploid wheats pos-

sessed the same chloroplast haplotypes as their

tetraploid progenitors demonstrating how the novel

synthetic wheat lines have captured chloroplast diver-

sity from the maternal parents, the chloroplast is

maternally inherited and novel alleles are not created

by genomic rearrangements triggered by the polyplo-

idisation event.

Keywords Chloroplast SSRs � cpSSRs � Genetic

diversity � Polyploid wheat �Wheat �Wheat evolution

Electronic supplementary material The online version ofthis article (doi:10.1007/s10722-013-9957-4) contains supple-mentary material, which is available to authorized users.

F. J. Leigh (&) � I. Mackay � N. E. Gosman �R. A. Horsnell � H. Jones � J. White � W. Powell

John Bingham Laboratory, National Institute

of Agricultural Botany, Huntingdon Road,

Cambridge CB3 0LE, UK

e-mail: [email protected]

H. R. Oliveira

Department of Archaeology, University of Cambridge,

Downing Street, Cambridge CB2 3DZ, UK

Present Address:

H. R. Oliveira

IFM—Biology, Linkopings Universitet,

581 83 Linkoping, Sweden

Present Address:

J. White

UCL Genetics Institute, UCL, Gower Street,

London WC1E 6BT, UK

Present Address:

W. Powell

IBERS, Aberystwyth University, Gogerddan,

Aberystwyth, Ceredigion SY23 3EB, UK

T. A. Brown

Faculty of Life Sciences, Manchester Institute

of Biotechnology, University of Manchester,

Manchester M1 7DN, UK

123

Genet Resour Crop Evol (2013) 60:1831–1842

DOI 10.1007/s10722-013-9957-4

http://dx.doi.org/10.1007/s10722-013-9957-4

Introduction

The wheat genus (Triticum) is one of the most

important families of agricultural crops. It is com-

posed of diploid, tetraploid and hexaploid species with

complex inter-relationships and evolutionary histo-

ries. Cultivated wheats were domesticated from wild

relatives and different species have predominated in

agricultural history. For example, emmer wheat (Trit-

icum dicoccon) was one of the founder crops of

agriculture and became the principal cereal of the

European Neolithic and Bronze age (Zohary et al.

2012). Although hexaploid wheats (Triticum aestivum

and Triticum spelta) replaced emmer as the predom-

inant wheat species around 2000 years ago, emmer

was retained as a minor crop in some areas.

The evolution of wheat species and their relation-

ships to each other have been investigated using tools

including morphological characteristics, molecular

markers and archaeobotany (Hirosawa et al. 2004).

Many factors have influenced the genetic diversity of

wheat species. Events such as domestication, poly-

ploidisation and selection have introduced population

bottlenecks that have constricted the available diver-

sity (Haudry et al. 2007). The wild, or unimproved,

species of wheat therefore harbour alleles that are not

found in the domesticated genepool.

The wheat chloroplast genome is non-recombining

and inherited through the maternal line in wheat. The

mutation rate of the chloroplast genome is lower than

that of the nuclear genome (Provan et al. 1999c); this

low evolutionary rate makes the chloroplast genome

useful in assessing population structure (Allender et al.

2007) whilst chloroplast markers have also been used

for evolutionary, ecological and phylogeographic

plant studies (Matsuoka et al. 2005). Despite the

conserved nature of the chloroplast genome, chloro-

plast SSRs (cpSSRs) have been shown to be poly-

morphic within and between wheat species and have

been applied to the wheat complex in order to

understand the evolutionary relationships between

species (Provan et al. 2004; Hirosawa et al. 2004 and

Ishii et al. 2001). Chloroplast markers have the

potential to be especially useful for studies involving

ancient or historic specimens where DNA preservation

may be poor due to the high copy number of the

chloroplast genome in the plant cell (Schlumbaum

et al. 2008). Preserved plant tissue such as cereal

grains in archaeological or herbarium collections

(Schlumbaum et al. 2008) may provide sufficient

DNA for PCR amplification of cpSSR loci.

The aim of this study was to interrogate cpSSR

diversity to discover the relationships among 1265

accessions members of the wheat tribe including of

T. urartu (171 accessions), T. dicoccoides (106),

T. dicoccon (276), T. durum (113), T. spelta (10),

T. aestivum (542), and Aegilops tauschii (47) and to

study the diversity in the T. dicoccon landraces for

evidence of geographic structure.

Materials and methods

Modern wheat material

Accessions of seven wheat species were obtained from

germplasm collections (the National Small Grains

Collection (NSGC) USA, the John Innes Centre (JIC)

UK, the International Center for Agricultural Research

in the Dry Areas (ICARDA), Syria, Leibniz Institute

of Plant Genetics and Crop Plant Research (IPK)

Germany) according to Jones et al. (2008a), from

collections held by Prof. Salamini and the Interna-

tional Maize and Wheat Improvement Center (CI-

MMYT), and from field collections. All wheat

botanical names are from the classification system

described by Hammer et al. (2011). The Ae. tauschii,

T. urartu and T. dicoccoides accessions are wild lines

originally collected from the Near East whilst the

T. dicoccon accessions are landraces from throughout

Europe. The T. durum material was composed of

Ethiopian landraces, European landraces and culti-

vars, and accessions used in the resynthesis of

synthetic hexaploid wheat by CYMMT. Likewise,

the T. aestivum material included three distinct types;

modern wheat varieties from the UK and the USA, old

varieties or selections from landraces from the UK and

Europe, and synthetic wheats. In total, 1,265 individ-

uals were genotyped.

DNA extraction and genotyping

Genomic DNA was extracted from coleoptile or leaf

material from one randomly selected individual from

each accession using the Qiagen DNeasy Plant96 kit,

except for the Ae. tauschii accessions which were

extracted using a modified Tanksley extraction

method (Fulton et al. 1995).

1832 Genet Resour Crop Evol (2013) 60:1831–1842

123

Five cpSSRs were selected from Ishii et al. (2001)

that had previously identified polymorphisms in

tetraploid wheat. Each forward primer was labelled

at the 50 end with a 6-FAM fluorophore. Target

sequences were amplified in two multiplex PCRs,

WCt12 ? WCt15 were amplified in one reaction,

whilst WCt2 ? WCt13 ? WCt22 were amplified in

the second. Each 10 ll PCR consisted of 20 ng DNA,

0.2 lM each primer, 100 lM each dNTP, 19 PCR

buffer, 1.5 mM MgCl2, 0.2 U Taq (Roche) and was

amplified as detailed in Ishii et al. (2001). Chinese

Spring (CS) was included as a size control.

Allele sequencing

A representative sub-sample of alleles was selected to

create an allele ladder for each marker. Primers were

designed to amplify a 500–600 base pair fragment that

encompassed each SSR marker. These ‘external’

primer sequences are provided in Supplementary

Table 1; reaction conditions were as described above

with an annealing temperature of 60 �C. Amplicons

were sequenced using the forward and reverse external

primers using BigDye v3.1 (Applied Biosystems)

according to the manufacturer’s instructions.

Sequences were aligned using PreGap and Gap, and

polymorphisms identified.

Data analysis

For each individual, the alleles at each of the 5 SSRs

were combined to create a ‘haplotype’. The polymor-

phism information content (PIC) value of each marker

and each haplotype was calculated for each species.

The relationships between each haplotype and

between each sequence variant were illustrated using

DARwin (Perrier et al. 2003; Perrier and Jacquemoud-

Collet 2006).

Results

Allele distribution in wheat species

The simultaneous amplification of multiple markers in

one PCR was an efficient and effective method of

genotyping a large number of samples. The genotypes

were high quality, robust and reproducible; null alleles

were not observed. As the SSRs are mononucleotide

repeats, the differences between alleles were evident

as single base increments. All five SSR markers were

polymorphic, with a wide allele range observed across

species. For each species, the allelic frequency and

range in the five SSRs is displayed in Table 1. All

alleles were sized as being smaller or larger by a

number of bases than Chinese Spring which was

designated to be size 0. In polyploid wheats, the

predominant allele at all five loci (shown in Table 1 in

bold) was the same as that observed in Chinese Spring.

T. urartu and Ae. tauschii exhibited a different allele

range to the polyploid wheats studied though there

were alleles in common. The PIC values of each

marker in each species are shown in Table 2.

Allele composition

A high level of polymorphism was observed at the

SSR loci. From the allele length alone, it was not clear

whether the size variation of the amplicons was due

solely to SSR length polymorphisms or if it was

influenced by other sequence variations in these

alleles. Reliably sequencing the SSR amplicons was

difficult as the published primers (Ishii et al. 2001)

amplify short PCR fragments (all less than 200 bases

in length) and contain a large mononucleotide repeat.

The amplification of larger, external fragments that

encompassed the SSR alleles allowed accurate

sequencing and thus quantification of repeat number

as well as identification of polymorphisms in the

flanking sequences. Examples of sequence variants are

shown in Table 3.

Sequence analysis revealed that the variation in

amplicon size identified by genotyping was due only

to the changes in SSR length. Differences in repeat

number of the SSR correlated exactly with the changes

in allele length; within each species there were no

other size variations in the regions between the SSR

and the published WCt primers. Sequencing revealed

length variation in two SSRs in the WCt12 amplicon

(see Table 3). A T7 SSR at base 43233 was observed in

all species except Ae. tauschii where it was present as a

T6 SSR. All species have several alleles at the second

Tx repeat at base 43444. The WCt12 allele is a product

of both these repeats thus the same fragment size may

be achieved through different combinations of SSR

length, with each combination having a different

evolutionary history. An example of such pseudo

homoplasy, is the 146 bp amplicon from T. aestivum

Genet Resour Crop Evol (2013) 60:1831–1842 1833

123

Table 1 Alleles observed in each species at each SSR locus; the Chinese Spring (CS) allele is shown in bold

Wct2 Allele (bp)

Species 122 123 124 125 126 127 128 129 130

T. urartu 0.023 0.52 0.433 0.018 0.006

T. dicoccoides 0.009 0.009 0.057 0.057 0.651 0.132 0.085

T. dicoccon 0.004 0.134 0.022 0.011 0.819 0.011

T. durum 1

T. aestivum 1

T. spelta 1

Ae. tauschii 0.021 0.064 0.787 0.106 0.021

Wct12 Allele (bp)

Species 145 146 147 148 149 150 151 152

T. urartu 0.012 0.187 0.363 0.094 0.345

T. dicoccoides 0.151 0.745 0.094 0.009

T. dicoccon 0.159 0.833 0.007

T. durum 1

T. aestivum 1

T. spelta 1

Ae. tauschii 0.191 0.426 0.34 0.043

Wct13 Allele (bp)

Species 99 100 101 102 103 104 105 106

T. urartu 0.041 0.012 0.585 0.357 0.006

T. dicoccoides 0.123 0.047 0.811 0.019

T. dicoccon 0.134 0.018 0.841 0.007

T. durum 1

T. aestivum 1

T. spelta 1

Ae. tauschii 0.553 0.404 0.043

Wct15 Allele (bp)

Species 95 96 97 98 99 100 101 102 103 104

T. urartu 0.035 0.854 0.111

T. dicoccoides 0.009 0.132 0.66 0.066 0.123 0.009

T. dicoccon 0.004 0.833 0.004 0.134 0.025

T. durum 1

T. aestivum 0.007 0.991 0.002

T. spelta 1

Ae. tauschii 1

Wct22 Allele (bp)

Species 192 193 194 195 196 197 200

T. urartu 0.006 0.018 0.678 0.123 0.175

T. dicoccoides 0.009 0.236 0.557 0.198

T. dicoccon 0.004 0.058 0.812 0.123 0.004


123

which is composed of T7 at SSR 43233 and T10 at the

Wct12 SSR whilst a 146 bp fragment amplified in Ae.

tasuchii is composed of T6 at SSR 43233 and T11 at the

Wct12 SSR. As we observed variation only in the SSR

at position 43233 in Ae. tauschii, alleles displaying

size based homoplasy would not be observed within a

species though they could be observed between

species.

Sequence variation between the WCt genotyping

primers was observed (see Table 3). Two SNPs were

identified in the WCt2 amplicon. The T14 repeat in

WCt15 is part of a compound SSR; this SSR was

observed in the forms (T)xA(T)6, (T)6A(T)9A(T)6, and

(T)6A(T)x (where x varies to generate different alleles).

These variations were species specific; (T)6A(T)x

forms were observed in T. urartu and Ae. tauschii,

whilst (T)xA(T)6 and (T)6A(T)9A(T)6 were observed in

polyploid wheats. Variation was also observed in

the regions external to the fragments amplified by the

published WCt primers. An insertion of 126 bp in the

region flanking the WCt13 amplicon was observed in

all the T. urartu and Ae. tauschii sequences, but was not

found in any of the polyploid wheats.

The variants revealed by sequencing fell into 4

groups; 2 groups were observed in the polyploid

wheats, whilst the third and fourth were composed

entirely of T. urartu and Ae. tauschii accessions

respectively. The diploid wheats were characterised

by the 126 bp insertion external to the WCt13

amplicon, the (T)6A(T)x forms of the WCt15 SSR

and 3 SNPs around the WCt22 allele. Ae. tauschii was

further distinguished by SNPs at base 7801 in WCt2

and T6 at 43233 in WCt12. Sequence variations

detailed in Table 3 were used to create a radial tree

shown in Fig. 1. This illustrates that the diploid wheat

species are distinct from each other and from the

polyploid wheat species.

Hirosawa et al. (2004) described two groups of

haplotypes in polyploid wheat cytoplasm and pro-

posed that these groups are discrete chloroplast

genome types or ‘plastotypes’ that may represent

differentiated maternal lineages. The profile described

in Chinese Spring represents the most common

plastotype in the wheat species studied by Ishii et al.

(2001) and may be considered to be plastotype 1. The

alleles WCt13: -4 bp and WCt15: ?2 bp are diag-

nostic of a second, rare plastotype (plastotype 2) which

was observed in only one of the 18 T. dicoccon

accessions studied by Ishii et al. (2001) and Hirosawa

et al. (2004). We observed plastotype 2 alleles at a

Table 2 The polymorphism information content (PIC) of markers and haplotypes in each wheat species

Species No. individuals No. ht No. single occurrence ht PIC value

2 12 13 15 22 ht

T. urartu 171 27 19 0.541 0.706 0.529 0.257 0.494 0.781

T. dicoccoides 106 31 20 0.545 0.413 0.324 0.527 0.595 0.866

T. dicoccon 276 15 8 0.311 0.28 0.275 0.287 0.323 0.506

T. durum 113 3 1 0 0 0 0 0.499 0.499

T. aestivum 524 4 1 0 0 0 0.018 0.228 0.244

T. spelta 10 1 0 0 0 0 0 0 0

Ae. tauschii 47 16 9 0.364 0.665 0.529 0 0.678 0.826

Table 1 continued

Wct22 Allele (bp)

Species 192 193 194 195 196 197 200

T. durum 0.009 0.566 0.425

T. aestivum 0.869 0.131

T. spelta 1

Ae. tauschii 0.426 0.043 0.319 0.191 0.021


123

Table 3 Sequence analysis of wheat chloroplast SSR Alleles; the number of mononucleotide repeats in each SSR is shown along

with any polymorphisms found in the regions surrounding the SSR when sequenced with the external primers

Species Ht plastotypea WCt2 WCT12 WCt13

SNP

7795

SNP

7801

SSR

WCt2

SSR

43233

SSR

WCt12

SSR

WCt13

126

bp?

6 bp insertionin the

126 bp insertion

SNP 58 in the

126 bp insertion

SNP

48246

SNP

48364

T. aestivum* 59 1 T C T15 T7 T10 A15 No * * C C

T. dicoccoides-

1

18 2 A C T17 T7 T9 A11 No * * C C

T. dicoccoides-

2

54 1 T C T15 T7 T10 A15 No * * C C

T. dicoccon-1 59 1 T C T15 T7 T10 A15 No * * C C



T. dicoccon-4 9 2 A C T12 T7 T9 A11 No * * C C

T. durum-1 59 1 T C T15 T7 T10 A15 No * * C C

T. durum-2 65 1 T C T15 T7 T10 A15 No * * C C

T. spelta 59 1 T C T15 T7 T10 A15 No * * C C

T. urartu-1 22 A C T9 T7 T13 A14 Yes CTTTTT T C T




T. urartu-5 41 A C T10 T7 T14 A15 Yes CTTTTT A C T

Ae. tauschii-1 83 A A T13 T6 T11 A12 Yes No T C T

Ae. tauschii-2 86 A A T12 T6 T10 A10 Yes No T T T

Species WCt15 WCt22

SSR WCt15a SSR WCt15b SSR WCt15c SSR WCt15d SSR WCt15e SNP 76788 SSR WCt22 SNP 77011 SNP 77080

T. aestivum* * * T14 A T6 C T12 A C

T. dicoccoides-1 T6 A T9 A T6 C T11 A C

T. dicoccoides-2 * * T15 A T6 C T11 A C

T. dicoccon-1 * * T14 A T6 C T12 A C



T. dicoccon-4 T6 A T9 A T6 C T12 A C

T. durum-1 * * T14 A T6 C T12 A C

T. durum-2 * * T14 A T6 C T13 A C

T. spelta * * T14 A T6 C T12 A C

T. urartu-1 * * T6 A T10 A T13 G T





Ae. tauschii-1 * * T6 A T8 A T13 G T

Ae. tauschii-2 * T6 A T8 A T14 G T

The presence of the 126 bp insertion (126?) is shown as yes/no. The sequence of T. aestivum cv. Chinese Spring (T. aestivum*) as published by Ishii et al.

(2001) is included as a reference

Variants observed within genotyping primers are shown in bold

a Denotes plastotype as described by Ishii et al


123

frequency of 0.047 in T. dicoccoides and 0.134 in

T. dicoccon. The additional variation revealed in our

study by sequencing (the SNP at position 7795 in

Wct2 and the (T)6A(T)9A(T)6 form of the WCt15

SSR) support the existence of discrete plastotypes in

wheat cytoplasm.

Fig. 1 The relationships between sequence variants detailed in Table 3


123

Haplotype analysis

As there is no genetic recombination in the chloroplast

genome and sequencing revealed no evidence of size

based homoplasy within species, the alleles at each of

the five loci were combined to generate a haplotype. In

total, 75 haplotypes were observed; 48 of these were

observed only once in our study. The number of

haplotypes observed in each species, the number of

haplotypes with single occurrence in that species and

the PIC values of the haplotypes are shown in Table 2.

In order to distinguish between each haplotype,

haplotypes were numbered 1–75. Figure 2 shows the

number of haplotypes that are found in multiple

species and those that are species specific. T. urartu

and Ae. tauschii had no haplotypes in common with

polyploid wheats or with each other, though there were

shared alleles at several loci. Haplotype 59 is the most

common allelic profile observed in polyploid wheats.

It is also the pattern described by Ishii et al. in bread

wheat (cultivar Chinese Spring). The second most

common haplotype in the tetraploids is haplotype 65,

which differs by one base at one SSR locus from

haplotype 59. Together, there are 923 examples of

these haplotypes in polyploid wheat individuals.

There was a constriction in diversity (in terms of the

number of haplotypes observed and PIC value) associated

with domestication of emmer wheat from wild emmer

and the creation of hexaploid wheat from tetraploids.

Subsets of accessions within species contained different

levels of diversity; T. durum landraces from Ethiopia

were more diverse than those from Europe and synthetic

hexaploid wheats showed the same number of alleles as

their tetraploid parents (data not shown).

Distribution of diversity

The distribution of haplotypes that occur more than

once in the sample of T. dicoccoides and T. dicoccon

are shown in Figs. 3 and 4 respectively. The haplo-

types found only in T. dicoccoides are referred to as

‘private’ haplotypes whilst those plastotypes found in

multiple species are labelled as ‘shared’ haploytpes in

Fig. 3. The T. dicoccoides accessions studied largely

fall into two geographic regions referred to by Ozkan

et al. (2005) as Western and Central Eastern popula-

tions and as Southern and Northern populations by

Luo et al. (2007). Our most frequently observed

haplotype, Ht59 was ubiquitous in both regions. The

high frequency of Ht59 in the wild population is

reflected in its wide occurrence through Europe in

polyploid wheat species. The second most common

haplotype in domesticated wheats is Ht65 which was

found in both regions.

Examples of plastotype 2 haplotypes were found in

Northern and Southern populations but the haplotypes

that are also detected in T. dicoccon were only found in

the Northern accessions. Of the six haplotypes iden-

tified in Northern populations, four (Ht8, 9, 52 and 69)

are specific to this region and all are found in other

domesticated wheat species. High representation of

the available chloroplast diversity in domesticated

emmers supports assertions by many researchers (for

example Ozkan et al. 2005, 2010; Luo et al. 2007) that

germplasm from this region made significant contri-

butions during the domestication of tetraploid wheat.

‘Private’ haplotypes were only recorded in T. dic-

occoides accessions from the Southern populations.

Diversity of T. dicoccon was high near this centre of

origin (Turkey) and also in Italy. Clustering of

haplotypes is evident for Ht21 and Ht9 in Italy and

Spain. As neither haplotype is common, they may be

useful tools for studying geneflow or population

structure in these locations (Leigh et al. 2012). The

observation of high levels of diversity in Italian

landraces compared to those from the rest of Europe

T. dicoccoides T. aestivum

T. dicoccon T. durum

T. urartu Ae. tauschii

5 ht(9)

1 ht(9)

2 ht(923)

7 ht(60)

2 ht(12)

15 ht(25)

27 ht(171)

16 ht(47)

T. aestivum

T. dicoccon T. durum

T. urartu Ae. tauschii

5 ht(9)

1 ht(9)

2 ht(923)

7 ht(60)

2 ht(12)

15 ht(25)

27 ht(171)

16 ht(47)

Fig. 2 Shared and species specific haplotypes. The number of

observations is shown in brackets beneath the number of

haplotypes


123

may be an accurate representation of Italian germ-

plasm, but its apparent allelic richness may also be a

product of our sampling strategy. Emmer landraces

were widespread and commonly cultivated in Italy

until recently and there are large numbers of acces-

sions available in germplasm collections; our sample

therefore contains a disproportionally high number of

Italian landraces.

Fig. 3 Distribution of

chloroplast haplotypes in

T. dicoccoides. Private

haplotypes are marked as

triangles

Fig. 4 Distribution of

T. dicoccon haplotypes

though Europe


123

Discussion

Chloroplast diversity reveals relationships

between wheat species

Polymorphisms in the regions flanking SSRs have been

reported previously in plants (Matsuoka et al. 2002)

and the importance of sequencing chloroplast alleles to

reveal ‘the complex genetic variation association with

hypervariable chloroplast DNA regions’ was identified

by Ebert and Peakall (2009) and Provan et al. (2004).

We observed SNPs flanking 3 of the 5 SSRs studied, a

126 bp insertion and unpublished SSR variants. These

polymorphisms defined 4 groups of cytoplasmic

diversity; plastotypes 1 and 2 observed in polyploid

wheats and the two groups observed in T. urartu and

Ae. tauschii. The persistence of these polymorphisms

in each group infers that they are stable, ancient and

conserved events.

The presence of two distinct chloroplast genotypes in

polyploid wheat has been reported previously by Ishii

et al. (2001) who proposed that they reflect two

cytoplasms that may have arisen independently in

emmer wheat. Two of the five SSRs applied in our study

are diagnostic for these plastotypes; a 100 bp allele at

Wct13 and a 103 bp allele at Wct15 identify the less

frequent ‘group 2’ cytoplasm. We identified additional

variations near the SSRs that reinforce the existence of

these discrete plastotypes. The group 2 alleles are

observed in a small number of European T. dicoccon

accessions and one Turkish T. dicoccoides accession.

The detection of cytoplasmic groups such as these may

be highly informative in long term evolutionary studies

and reinforces the importance of obtaining sequence

data in order to understand the mutation processes that

occur at these loci (Matsuoka et al. 2005).

The discrete cpSSR alleles and haplotypes

observed in T. urartu and Ae. tauschii, combined with

the species specific polymorphisms provide very

strong evidence that concur with current opinion that

neither species was the maternal parent and thus the

cytoplasmic donor for polyploid wheats. That the

synthetic hexaploid wheats possessed the same chlo-

roplast haplotypes as their tetraploid progenitors

demonstrates how the novel synthetic wheat lines

have captured chloroplast diversity from the maternal

parents, the chloroplast is maternally inherited and

novel alleles are not created by genomic rearrange-

ments triggered by the polyploidisation event.

The impact of wheat evolution on chloroplast

diversity

The sequential loss of diversity from wild progenitors

through to cultivated species has been observed in

many crops including rice (Tanksley and McCouch

1997), barley (Provan et al. 1999a), potato (Provan

et al. 1999b) and wheat (Haudry et al. 2007; Provan

et al. 2004). Thus, domestication may be considered as

a population bottleneck in most crop species (Buckler

et al. 2001). We observed a constriction in chloroplast

diversity that accompanies domestication and dis-

persal of tetraploid wheats and creation of hexaploid

wheat by polyploidisation. This loss of diversity was

illustrated by both the number of alleles and haplo-

types in each wheat species, and the PIC values which

reflect both the number of alleles observed and their

frequencies in the populations.

The founder effects that accompany domestication

and dispersal may be offset by a number of mecha-

nisms. The diversity of emmer at the centre of origin

may have been augmented by geneflow between wild

and cultivated emmer. Luo et al. (2007) reported that

‘gene flow between wild and domesticated emmer

took place across the entire area of wild emmers

distribution’ and proposed that this accounted for the

presence of domesticated wheat chloroplast haplo-

types in wild populations. Gene flow or migration

between natural stands of wheat species may therefore

account for some of the rare chloroplast haplotypes

recorded in accessions near the centre of origin of this

species.

We observed 2 examples of haplotypes that were

detected in wild emmer and in bread wheat, but were

absent in the cultivated emmer and durum accessions

genotyped in our study. Firstly, six bread wheat

varieties contained a haplotype (Ht61) only previously

seen in T. dicoccoides, which is distinguished from the

Chinese Spring haplotype Ht59 by a 1 bp increase in

Wct15. Secondly, Ht56 (which differs from Ht59 by

a 1 bp decrease at Wct15) was detected in 1

T. dicoccoides and 4 T. aestivum varieties.

There are several possible explanations for these

observations. It is possible in both cases that the

microsatellite at Wct15 in a Ht59 plant in the

T. aestivum lineage has mutated and increased/

decreased by 1 bp creating a novel allele. This would

make the Ht61 and Ht56 alleles in T. dicoccoides and

T. aestivum examples of size based homoplasy.


123

Alternatively, the T. aestivum accessions with HT61

and Ht56 haplotypes may be descended from hexpap-

loids that are the products of novel polyploidisation

events involving the T. dicoccoides lines with which

they share chloroplast haplotypes. Wild emmers with

the haplotype Ht61 are found in Southern populations

which are not reported to have made significant

contributions to the domesticated genepool. However,

Luo et al. found T. durum to be more related to

Southern wild emmers than Northern wild emmers,

and durum wheats are putatively the female parents of

bread wheat. The identification of sites of domestica-

tion and polyploidisation events are not, however, the

focus of this study.

It is also possible that the HT61 and Ht56

T. aestivum lines could be descended from hexaploids

that have undergone gene flow with wild tetraploid

populations that contain these haplotypes. Alterna-

tively, these haplotypes may have been lost from

contemporary domesticated tetraploid accessions. The

final explanation for the absence of these haplotypes in

domesticated tetraploids is the composition of our

germplasm sample. Although we studied 276 emmer

wheat and 113 durum wheat accessions, our dataset is

not exhaustive and these haplotypes may be present in

accessions that we did not sample.

Finally, we recorded a chloroplast haplotype (Ht9) in

this study that is unique to domesticated wheats in

Asturias, Northern Spain. This haplotype is not

observed in T. dicoccon sampled from the rest of

Europe, but it is found in wild emmer from one location

in Turkey. Again, it is probable that our novel haplotype

arose following a mutation in the WCt22 SSR of a Ht8

plant, which is also found locally, creating a single base

pair increase in the length of the WCt22 SSR and thus a

novel haplotype. This would make the Turkish example

of Ht9 and the Asturian Ht9 additional examples of size

based homoplasy where two alleles of the same

composition have arisen through different routes or

mechanisms and reflect different evolutionary histories.

Further sequence analysis could verify this theory.

Other possible explanations for the presence of this

novel haplotype in Asturias, including the direct

introduction of this germplasm from the centre of

origin and the Asturian populations representing a relic

population subsequently lost in all other areas, are

discussed in Leigh et al. (2012).

The extent to which SSR mutations offset bottlenecks

by creating new alleles and generating novel diversity is

unclear but our findings suggest that it is a detectable

phenomenon. The mutation rate of SSRs in the chloro-

plast genome is reported to be lower than that of the

nuclear genome. Provan et al. (1999c) found chloroplast

SSRs of Pinus torreyana mutated at a rate of between

3.2 9 10-5 and 7.9 9 10-5 whilst the mutation rate of

Zea mays nuclear SSRs was quantified as 7.7 9 10-4 by

Vigouroux et al. (2002). Haudry et al. (2007) reported

that the impact of wheat domestication was less when

measured using SSRs than when measured using

sequence data, reflecting the higher mutation rate of

SSRs. The creation of novel SSR alleles through

mutation occurs at a higher rate than other sequence

changes and may obscure diversity constrictions asso-

ciated with evolutionary events such as polyploidisa-

tion, domestication and selective breeding.

When multiple individuals from a single accession

of emmer wheat were genotyped, some of the acces-

sions were found to be heterogenous. The random

selection of a non-typical individual for characterisa-

tion may result in erroneous conclusions about the

genetic composition of an accession. However, this is

problem that will confound the use of any germplasm

that is collected from natural sources, as it is likely to be

a population or an ecotype rather than a selected (and

thus more uniform) variety (Jones et al. 2008a).

In conclusion, wheat chloroplast SSRs are robust

genotyping tools that reveal diversity within and

between wheat species. Constrictions in diversity asso-

ciated with major evolutionary events such as polyplo-

idisation and domestication may be detected. However,

the markers do not appear to be particularly useful for

tracking the movement of domesticated wheats through

Europe because the low levels of polymorphisms in

domesticated species did not reveal trajectories that

could be traced back to the centre of origin.

Acknowledgments This work was funded by the Natural

Environment Research Council as part of the consortium project

‘The Domestication of Europe’. We thank the project partners at

the University of Cambridge and University of Sheffield for

helpful advice.

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http://dx.doi.org/10.1007/s10722-012-9840-8

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Using diversity of the chloroplast genome to examine evolutionary history of wheat species

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