FEATURED ARTICLE

Next-generation sequencing and syntenic integration offlow-sorted arms of wheat chromosome 4A exposes thechromosome structure and gene content

Pilar Hernandez1, Mihaela Martis2, Gabriel Dorado3, Matthias Pfeifer2, Sergio Galvez4, Sebastian Schaaf2, Nicolas Jouve5,

Hana Simkova6, Miroslav Valarik6, Jaroslav Dolezel6 and Klaus F.X. Mayer2,*

1Instituto de Agricultura Sostenible (IAS-CSIC), Alameda del Obispo s/n, 14080 Cordoba, Spain,2Institute for Bioinformatics and Systems Biology, Helmholtz Center Munich, Ingolstadter Landstraße 1, 85764 Neuherberg,

Germany,3Departamento de Bioquımica y Biologıa Molecular, Campus Rabanales C6-1-E17, Campus de Excelencia Internacional

Agroalimentario, Universidad de Cordoba, 14071 Cordoba, Spain,4Departamento de Lenguajes y Ciencias de la Computacion, ETSI Informatica, Campus de Teatinos,

Universidad de Malaga, Bulevar Louis Pasteur 35, 29071 Malaga, Spain,5Departamento de Biologıa Celular y Genetica, Universidad de Alcala, Campus Universitario, 28871 Alcala de Henares,

Madrid, Spain, and6Centre of the Hana Region for Biotechnological and Agricultural Research, Institute of Experimental Botany,

Sokolovska 6, 77200 Olomouc, Czech Republic

Received 3 August 2011; revised 29 September 2011; accepted 30 September 2011; published online 25 November 2011.

*For correspondence (fax +49 89 3187 2225; e-mail k.mayer@helmholtz-muenchen.de).

SUMMARY

Wheat is the third most important crop for human nutrition in the world. The availability of high-resolution

genetic and physical maps and ultimately a complete genome sequence holds great promise for breeding

improved varieties to cope with increasing food demand under the conditions of changing global climate.

However, the large size of the bread wheat (Triticum aestivum) genome (approximately 17 Gb/1C) and the

triplication of genic sequence resulting from its hexaploid status have impeded genome sequencing of this

important crop species. Here we describe the use of mitotic chromosome flow sorting to separately purify and

then shotgun-sequence a pair of telocentric chromosomes that together form chromosome 4A (856 Mb/1C) of

wheat. The isolation of this much reduced template and the consequent avoidance of the problem of sequence

duplication, in conjunction with synteny-based comparisons with other grass genomes, have facilitated

construction of an ordered gene map of chromosome 4A, embracing ‡85% of its total gene content, and have

enabled precise localization of the various translocation and inversion breakpoints on chromosome 4A that

differentiate it from its progenitor chromosome in the A genome diploid donor. The gene map of

chromosome 4A, together with the emerging sequences of homoeologous wheat chromosome groups 4, 5

and 7, represent unique resources that will allow us to obtain new insights into the evolutionary dynamics

between homoeologous chromosomes and syntenic chromosomal regions.

Keywords: wheat genome, chromosome sorting, genome zipper, grass comparative genomics, wheat

shotgun chromosome, Triticeae genome

INTRODUCTION

Bread wheat (Triticum aestivum) provides approximately

20% of mankind’s dietary energy supply (http://www.fao.

org), but, despite its importance as a crop, acquisition of its

genome sequence remains a major challenge. The biologi-

cal features responsible for the slowness of progress

towards this goal are its large genome size (1C is

ª 2011 The Authors 377The Plant Journal ª 2011 Blackwell Publishing Ltd

The Plant Journal (2012) 69, 377–386 doi: 10.1111/j.1365-313X.2011.04808.x

approximately 17 Gb), its hexaploid status, and its high

content of repetitive DNA (approximately 80%) (Flavell,

1986). Each wheat chromosome is larger than the entire

genome of rice (Oryza sativa), and the whole genome is

more than one hundred times larger than that of Arabidopsis

thaliana. The species arose from two separate hybridization

and allopolyploidization events, the first involving a hybrid

between the A genome donor Triticum urartu (closely

related to the cultivated species Triticum monococcum)

(Dvorak et al., 1993) and the B genome donor, thought to be

an ancestor of Aegilops speltoides (Sarkar and Stebbins,

1956; Dvorak and Zhang, 1990; Wang et al., 1997; Kilian

et al., 2007). This formed the wild tetraploid Triticum dic-

occoides, which was the ancestor of the cultivated tetraploid

parent of bread wheat Triticum turgidum. The second, much

more recent, event involved T. turgidum and the D genome

diploid Aegilops tauschii (McFadden and Sears, 1946).

A successful strategy that is frequently adopted to

circumvent many of the difficulties created by polyploidy

has been to rely on diploid, and in some cases tetraploid,

progenitors as surrogates (Feuillet et al., 2003). The avail-

ability of the genome sequences of rice and Brachypodium

distachyon (Brachypodium) has been of particular value in

providing saturation of the genetic map in specific regions of

the wheat genome (International Rice Genome Sequencing

Project 2005, International Brachypodium Initiative 2010). As

an alternative, Dolezel et al. (2007) proposed that genome

sequencing be based on flow-sorted individual chromo-

somes or chromosome arms, an approach that simplifies

genome analysis by simultaneously reducing the template

to a manageable size, and crucially avoids all of the

complications introduced by the triplication of genic

sequence arising from wheat’s hexaploid status (Kubala-

kova et al., 2002). Next-generation sequencing of chromo-

somal DNA provides a powerful approach to identify most

of the genes and low-copy regions on a chromosome and to

produce annotated syntenic builds whereby the majority of

genes are placed in an approximate order and orientation

(Berkman et al., 2011; Mayer et al., 2011, 2009; Wicker et al.,

2011). The so-called GenomeZipper approach (Mayer et al.,

2011) relies on comparisons of chromosomal shotgun

sequences with reference grass genomes (typically rice,

sorghum (Sorghum bicolor) and Brachypodium) to detect

syntenic regions in these reference genomes. Genes in the

detected regions are selected to generate a genomic build

along a marker scaffold that takes into account the sequen-

tial order of sequence-tagged genes in the reference

genomes as well as the ordering deduced from the marker

scaffold.

Although most of the bread wheat chromosomes have

maintained the structure of ancestral species, that of chro-

mosome 4A underwent a series of re-arrangements. Previ-

ous analyses revealed that the chromosome harbors two

translocations from chromosome arms 5AL and 7BS, and

that it has undergone a pericentric inversion (Figure 1)

(Devos et al., 1995; Miftahudin et al., 2004; Naranjo et al.,

1987) (Figure 1). The 5A translocation occurred at the diploid

level in a common ancestor as it is present in wheats of all

ploidy levels, including diploid wheat progenitors and

related species such as T. monococcum. On the other hand,

the 7BS translocation is detected in tetraploid and hexaploid

wheat only, indicating its occurrence after or at the time of

origin of T. dicoccoides (Devos et al., 1995). Interestingly,

most of the studies on bread wheat also report the presence

of a small region on the most distal part of 4AS that was not

affected by the large pericentric inversion that placed most

of the ancestral short arm on the modern long arm (4AL),

and, as a result, large proportions of the ancestral 4AL now

constitute the modern 4AS.

As many as 40 genes of interest have been mapped to this

chromosome to date, including some encoding resistance/

tolerance to biotic and abiotic stress (Chen et al., 1995, 2005;

Effertz et al., 2001; Nga et al., 2009; Paull et al., 1998; Talbert

et al., 1996), and various agronomic traits (Araki et al., 1999;

Bai et al., 2008; Borner et al., 2002; Keller et al., 1999;

McCartney et al., 2005; Sourdille et al., 2002). Detailed

information on the chromosome gene order would greatly

enhance effective use of the genes in breeding programs

and ultimately in their cloning and functional analysis.

Here, we report a high-resolution gene map of this

chromosome, based on DNA sequence obtained from

flow-sorted chromosome arms. Use has been made of the

genetic marker content present in homoeologous portions

of the barley genome (Hordeum vulgare) and the reference

grass genomes to provide a detailed insight into gene

composition and order along the length of the chromosome.

Figure 1. The structure of wheat chromosome 4A.

The structure of bread wheat chromosome 4A, as inferred by Devos et al. (1995) and Miftahudin et al. (2004). During its evolution, the chromosome first underwent a

pericentric inversion, which resulted in much of the ancient long arm (excluding segment C) becoming the modern short arm. Subsequent translocations from 5AL

(segment D) and 7BS (segment E) completed the rearrangement of the chromosome. The five individual segments A–E are color-coded. The additional small

structural rearrangements proposed by Miftahudin et al. (2004) are not shown as they could not be confirmed in the present study.

378 Pilar Hernandez et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 377–386

This is a powerful approach for production of a high-

resolution draft of gene space for the complex genome of

bread wheat, including its highly rearranged chromosome

4A. The approach has important implications for the

whole-genome analysis of both bread wheat and other

large genomes of agriculturally important grasses such as

rye (Secale cereale), fescue (Festuca ssp.) and ryegrass

(Lolium ssp.).

RESULTS

Preparation of chromosomal DNA and shotgun sequencing

Two separate DNA bulks were prepared from the mitotically

dividing cells of the double di-telosomic 4A stock. The 4AS

preparation contained approximately 78 000 flow-sorted

telosomes, and the 4AL one contained approximately

50 000. The level of purity of these preparations, as esti-

mated by fluorescent in situ hybridization, was 86.9% and

89.0%, respectively. Chromosome 1D comprised approxi-

mately 50% of the contaminants in the 4AL preparation, but

no single chromosome predominated in the 4AS prepara-

tion. The 4AS bulk yielded 29.5 ng DNA, which was

amplified in three independent multiple displacement

amplification reactions to generate 16 lg DNA; similarly, the

4AL bulk produced 44.6 ng DNA, which was amplified in

four reactions to yield 22.7 lg DNA. The individual multiple

displacement amplification reactions for each template were

combined to reduce the probability of bias introduced by

multiple displacement amplification itself. The amplified

DNA was used for 454 shotgun sequencing (Table 1), which

produced 2 181 649 4AS reads of mean length 324 bp,

representing a total of 707 Mb of sequence (NCBI sequence

read archive, http://www.ncbi.nlm.nih.gov/sra, reference

SRA038898.1). Given the estimated length of this arm

(317 Mbp; Safar et al., 2010), this is equivalent to a

sequencing depth of approximately 2.2-fold. For chromo-

some 4AL, the 2 987 571 reads (mean length 302 bp) yielded

901 Mb of sequence (NCBI sequence read archive, reference

SRA034928.1), equivalent to a sequencing depth of approx-

imately 1.7-fold. Sequencing details are summarized in

Table 1.

Identification of syntenic regions in related grass genomes

The full genome sequences of Brachypodium, rice and

sorghum (International Rice Genome Sequencing Project

2005, Paterson et al., 2009; International Brachypodium

Initiative 2010) were used to identify regions of synteny in

order to take advantage of the GenomeZipper approach

(Mayer et al., 2011). The 4AS and 4AL sequences were

compared by BLAST analysis against the genomic

sequences of Brachypodium, rice and sorghum, as well as

against the virtual barley genome (Mayer et al., 2011), to

identify syntenic regions (Figures 2 and 3). The 4AL com-

parison highlighted regions on Brachypodium chromo-

Table 1 Shotgun sequences of wheat chromosome arms 4AS and 4AL

Parameter

4AS 4AL

Raw dataRepeat-maskedand filtereda Raw data

Repeat-maskedand filtereda

Number of sequences 2 181 649 420 739 2 987 571 752 981Number of base pairs 707 234 947 146 653 961 901 236 013 239 649 872Minimal length (bp) 18 100 29 100Maximal length (bp) 826 826 982 982Mean length (bp) 324 349 302 318Repeat content (%) 79.5 72.8GC content (%) 44.7 46.4 41.4 41

aThe filter applied ensured the retention of sequences longer than 100 bp that contained at least 100 bp of non-repetitive sequence.

Figure 2. Comparison of the 4A shotgun sequence with that of barley.

Repeat-masked 4AS and 4AL shotgun sequence reads were compared against the sequence of virtual barley chromosomes (Mayer et al., 2011). Syntenic regions on

chromosomes 4H, 7H and 5H are colored red; non-syntenic regions are colored blue. Centromeres are indicated by black triangles and the arms of the chromosomes

are labeled S and L. Connectors/joins indicate corresponding segments and orientation of the individual segments.

GenomeZipper analysis of wheat chromosome 4A 379

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 377–386

somes 1 and 4, rice chromosomes 3, 6, 11 and 12, and sor-

ghum chromosomes 1 and 10 (Figure 3). In the same way, 4AS

syntenic regions were identified on Brachypodium chromo-

some 1, rice chromosome 3 and sorghum chromosome 1.

Comparison with the barley chromosome reference pro-

duced hits on chromosomes 4H, 5H and 7H (Figure 2). The

4AS sequences identified part of chromosome arm 4HL,

while the 4AL sequences matched the entire 4HS arm, as

well as identifying regions on 5HL and 7HS and a small

region of 4HL (Figure 2). The gene content in these regions

was collated, and the syntenic boundaries were located with

high precision (Tables 4 and 5). The resulting data were then

used to generate a GenomeZipper-based alignment and a

high-resolution genetic map of chromosome 4A.

Gene content of chromosome 4A

In order to estimate the number of genes present on each 4A

chromosome arm, TBLASTX comparisons were made with

the Brachypodium, rice and sorghum genome sequences,

based on a stringency level of at least 75% over at least 30

amino acids (Table 2). This exercise produced between 3278

and 3805 hits for 4AS, and between 3956 and 4523 for 4AL.

The numbers of non-redundant matches were 4383 and

5188, respectively, giving a total of 9571 non-redundant

gene matches on 4A. Given the estimated size of 4A of

856 Mb and a gene density representative of the complete

wheat genome (Qi et al., 2004), this scales up to at least

61 500 genes for the A genome and >180 000 genes for

bread wheat. This result contrasts with recent estimates for

barley (‡32 000 genes; Mayer et al., 2011) and the B genome

of wheat (38 000 genes; Choulet et al., 2010), and with our

own estimate of ‡3000 genes on 4A based on a conservative

synteny-driven integration approach (Table 3).

The structure of chromosome 4A

On the basis of synteny with barley, Brachypodium, rice and

sorghum, it was possible to recognize five distinct regions

Figure 3. Comparison of the 4A shotgun sequence with that of Brachypodium, rice and sorghum.

Repeat-masked 4AS and 4AL shotgun sequence reads were compared with the genome sequences of Brachypodium (Bd), rice (Os) and sorghum (Sb). Syntenic

regions are colored red; non-syntenic regions are colored blue. Centromeres are indicated by black triangles and the arms of the chromosomes are labeled S and L.

Connectors/joins indicate corresponding regions and the orientation of the individual segments.

Table 2 Tagged genes in the reference genomes

Chromosomearm

Non-redundant genesa

Non-redundantgenes (total)Brachypodium Rice Sorghum

4AS 3805 3278 3365 43834AL 4523 3956 4069 5188

aThe number of sequence-tagged genes located on chromosome 4Aas deduced from similarity comparisons (sequence identity ‡75% and‡30 amino acids) with reference genomes.

380 Pilar Hernandez et al.

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(A–E) on chromosome 4A. There are 120 (5!) ways in which

five independent segments can be ordered, but as each

segment can be present in one of two possible orientations,

the true number of possible arrangements is 3840 (120 · 25).

To resolve the actual ordering, advantage was taken of

published genetic mapping data (Devos et al., 1995; Mayer

et al., 2011; Miftahudin et al., 2004). The 4AS sequence-

identified a syntenic region on 4HL, while 4AL sequence-

identified 4HS and a small segment of 4HL (Figures 2 and 3).

Chromosome 4A is known to carry a pericentromeric inver-

sion (Devos et al., 1995; Miftahudin et al., 2004) involving a

portion of the ancient long arm (4ALanc; segment A) and the

complete ancient short arm (4ASanc; segment B); this con-

verted 4ALanc into the modern 4AS, and 4ASanc into the

distal part of the modern 4AL. In addition, a small region of

4AL (segment C) appears to have not been involved in the

pericentromeric inversion (Figure 2). Consequently, the

gene order in segments A and B was reversed with respect

to barley, but that in segment C was conserved. The segment

D sequences on 4AL show homology with a distal portion of

5HL (Figure 2), consistent with genetic mapping data (Devos

et al., 1995; Mayer et al., 2011; Miftahudin et al., 2004). Fi-

nally, genetic data indicated that a further translocation must

have occurred between a distal segment of chromosome

arm 7BS and 4A (Devos et al., 1995; Mayer et al., 2011;

MickelsonYoung et al., 1995; Miftahudin et al., 2004). The

evolutionary scenario proposed by Devos et al. (1995) and

Miftahudin et al. (2004) allowed the orientation of segments

D and E to be determined. On the basis of meiotic pairing

between the distal segments of 4AS, 4BS and 4DS, Naranjo

et al. (1987) have suggested retention of a small segment of

4ASancient in the distal part of modern 4AS; although no

genetic evidence for this was obtained by Devos et al. (1995),

two relevant EST sequences were located within this region

by bin mapping (Miftahudin et al., 2004). A BLASTN com-

parison of these two ESTs against the present set of 4AS and

4AL sequences produced either no hits or hits with restricted

sequence similarity and sequence alignment length (data

not shown), so it was not possible to confirm the presence of

this distal 4AS segment.

A virtual map of chromosome 4A

A map of chromosome 4A was assembled using the

GenomeZipper protocol (Mayer et al., 2011) from the

sequence data and synteny-based deductions (Figure 3,

Table 4 and Table S1). The ordering of segments A–E was

determined based on the marker map of barley (Close et al.,

2009). The number of markers involved in this process ran-

ged from 16 (segment C) to 127 (segment A) (Table 3). The

4AS arm is associated with 127 markers, and the 4AL arm

with 233. Between 79 genes (segment C) and 1182 genes

(segment A) were thus assigned to each of the segments,

resulting in the placement of approximately 3000 genes over

the whole chromosome (Table 3). The five segments varied

considerably in the extent of synteny with the other grass

genomes, and overall just 29% of the genes were conserved

across wheat and all three sequenced genomes (Figure 4).

Almost half of the genes in segment A were present in the

expected location in all three reference genomes, compared

to only approximately one-eighth in segment B. When the

criterion for support was reduced to just one of the three

heterologous genomes, the frequency of conservation

across the whole chromosome with Brachypodium was

22.3%, that with rice was 22.8%, but that with sorghum was

only 7.7%. At the level of the individual segments, the fre-

quency of conservation varied by as much as threefold, with

no evidence that the segments resulting from the two known

translocation events (segments D and E) showed a lower

level of conservation. The clearly unequal level of conser-

vation across the various Pooideae lineages underlines the

value of using more than one reference genome when

attempting synteny-based deduction of gene order.

Table 3 Wheat chromosome 4A GenomeZipper statistics

Parameter

Chromosome 4A segment

4AS 4AL 4AA B C D E

Number of markers 127 107 16 46 64 127 233 360Number of markers with associated genefrom reference genome(s)

92 52 8 27 27 92 114 206

Number of matched barley fl-cDNAs 600 393 29 123 114 600 659 1256Number of non-redundant sequence reads 10 628 18 415 969 5556 5126 10 628 26 584 37 212Number of non-redundant ESTs 935 700 60 217 204 935 1068 1996Number of Brachypodium genes 905 606 49 157 151 905 963 1865Number of rice genes 763 649 31 190 124 763 994 1754Number of sorghum genes 785 248 32 110 103 785 493 1278Number of anchored gene loci 1182 1110 79 300 262 1182 1751 2933

Overview of non-redundant data points anchored along chromosome 4A. The numbers refer to the chromosomal segments A–E, which form thechromosomal zippers for 4AS and 4AL, as well as for the whole chromosome 4A. Numbers given in the individual columns give non-redundantnumbers for each category. Due to small overlaps in segment and arm assignment few cases don’t have an unbiased assignment and can’t beresolved. Thus the non-redundant sums do not always match with the sum of individual values.

GenomeZipper analysis of wheat chromosome 4A 381

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 377–386

Translocation and inversion breakpoints

Alignment of the 4A sequence against that of the barley,

Brachypodium, rice and sorghum genomes allowed precise

localization of the breakpoints associated with the various

rearrangements that have determined the structure of

chromosome 4A (Figures 2 and 3, and Tables 4 and 5).

Sequence comparison against a genome built of the barley

genome as well as against the reference genomes of Brac-

hypodium, rice and sorghum allow precise delineation and

detection of the regions where the rearrangements

occurred, with an almost single-gene resolution (Figures 2

and 3, and Tables 4 and 5). We analyzed the corresponding

regions in the reference genomes for syntenic intervals and

syntenic borders. Based on gene detection by sequence

comparisons of 4AS and 4AL, reads bordering syntenic

regions could be accurately identified. The regions range

between 370 kb and 28 Mb, and contain between 905 genes

and only 31 genes in the respective syntenic regions

(Table 4). The exact positioning of syntenic borders also

allows definition of the bordering genes for the individual

segments and their orthologous counterparts in the

reference genomes (Table 5). The approach based on

flow sorting-facilitated isolation of chromosome arms,

Table 4 Regions in Brachypodium, rice and sorghum sharing synteny with 4AS and 4AL, as deduced from in silico mapping

Reference genome Chromosome Start (Mp) Stop (Mp) Number of genes

Wheat chromosome arm 4ASSegment A Brachypodium 1 60.2 71.8 905

Rice 3 2.6 14.2 763Sorghum 1 58.8 69.7 785

Wheat chromosome arm 4ALSegment B Brachypodium 1 6.5 11.5 240

4 7.9 10.3 754 22.5 31.8 291

Rice 3 23.3 30.8 23311 2.3 11.9 20111 14.1 30.7 215

Sorghum 1 7.5 13.9 248

Segment C Brachypodium 1 71.5 72.6 49Rice 3 1.9 2.7 31Sorghum 1 70.0 71.1 32

Segment D Brachypodium 1 0.3 1.8 157Rice 3 35.3 37.3 123

12 0.1 2.1 67Sorghum 1 0.1 1.9 110

Segment E Brachypodium 1 48.1 50.5 151Rice 6 0.1 2.6 124Sorghum 10 0.6 2.3 103

Based on detected syntenic segments, the table gives the chromosome, start and stop coordinates on the respective reference genome and thenumber of genes located in these regions.

Figure 4. Conservation of synteny between chromosome 4A and rice, Brachypodium and sorghum.

The gene content of segments A–E of chromosome 4A was compared with that of the homologous regions in Brachypodium (Bd), rice (Os) and sorghum (Sb). The

Venn diagrams show the numbers of genes shared between wheat and the reference genomes.

382 Pilar Hernandez et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 377–386

high-throughput sequencing and comparative genome

analysis is thus capable of reconstructing genomes and

identifying evolutionary translocation breakpoints.

DISCUSSION

The structure of chromosome 4A

The large size and polyploidy of the bread wheat genome

pose a considerable challenge for its sequencing. Current

sequencing technology has the ability to acquire large

amounts of sequence economically, but sequence assem-

bly, and, most importantly, establishment of the gene order

within each of the 21 chromosomes are particularly difficult,

mostly because of the presence of homoeologous copies of

most genes and the extent of repetitive DNA present. For-

tunately, much synteny has been retained among Poaceae

species in general, and among the Triticeae species in par-

ticular (Bolot et al., 2009; Devos and Gale, 2000; Moore,

1995; Salse and Feuillet, 2007). A small number of major

chromosomal rearrangements are known among the Triti-

ceae (Devos et al., 1995), but collinearity has largely been

retained across the wheat and barley genomes, despite

their divergence approximately 12 Myr (Gaut, 2002). Within

wheat itself, chromosome 4A suffered the most significant

overall re-arrangement (Figures 1 and 2) (Devos et al., 1995;

Miftahudin et al., 2004), as confirmed by an extensive com-

parative study of the gene content of wheat and barley

(Mayer et al., 2011; Qi et al., 2004). Integration of sequence

and mapping data allowed recognition that chromosome 4A

comprises five separate segments. Segments A, B and C

originated from the pericentromeric inversion, while seg-

ments D and E arose from later interchanges with chromo-

somes 5A and 7B. All five segments were successfully

ordered and oriented to allow subsequent GenomeZipper-

based gene integration and positioning.

Identification of translocations and inversions

While assignment of 4AS-derived sequences to corre-

sponding syntenic segments in the reference genomes and

barley was relatively straightforward, the assignment was

much more complex for 4AL (Figure 3). Integration of the

resulting patterns and comparison with barley chromo-

somes 4H, 5H and 7H led to identification of five syntenic

segments A–E. The orientation of segments A, B and C was

evident from comparisons based on the most parsimonious

single pericentromeric inversion event. On the other hand,

the positioning and orientation of segments D and E, which

resulted from translocations from chromosomes 5A and 7B,

respectively, could not be deduced from synteny patterns

alone. In conjunction with genetic mapping data and a

derived order of segments and their orientation (Devos

et al., 1995; Miftahudin et al., 2004), all five segments were

ordered and oriented for GenomeZipper-based gene inte-

gration and positioning. Thus, by integrating genetic data

with our molecular and comparative data, a conclusive order

and orientation of segments and accordingly a linear order

of genes could be established. This demonstrates the power

of combining and integrating genetic data with chromo-

some next-generation sequencing-derived shotgun

sequence data and comparative and bioinformatic analysis.

Gene content of wheat chromosome 4A

A rather stringent comparison between the 4A sequences

and the various annotated reference genomes produced an

estimated gene content on chromosome 4A of >9500 genes, a

number that is rather higher than has been suggested for

either barley chromosome 4H (4000) or wheat chromosome

3B (6360) (Mayer et al., 2011; Paux et al., 2008). Other

estimates of gene number based on the analysis of individual

chromosomes have also diverged from those based on

Table 5 Overview of the breakpoints between the five chromosome4A segments

Segment Brachypodium Rice Sorghum

A Bradi1g72080.1 Os03g0187500 Sb01g044730.1Bradi1g72086.1 Os03g0187400 Sb01g044740.1Bradi1g72092.1 Os03g0187300 Sb01g044750.1

… … …Bradi1g65190.1 Os03g0296700 Sb01g038210.1Bradi1g65197.1 Os03g0296600 Sb01g038220.1Bradi1g65210.1 Os03g0296400 Sb01g038230.1

B Bradi4g26690.1 Os11g0150450 Sb01g013770.1Bradi4g26670.3 Os11g0151600 Sb01g013780.1Bradi4g26640.1 Os11g0152700 Sb01g013830.1

… … …Bradi1g13777.1 Os03g0652100 Sb01g013490.1Bradi1g13850.1 Os03g0648200 Sb01g013540.1Bradi1g13870.1 Os03g0645100 Sb01g013650.1

C Bradi1g75740.1 Os03g0138200 Sb01g047640.1Bradi1g75720.1 Os03g0140100 Sb01g047630.1Bradi1g75707.1 Os03g0141100 Sb01g047610.1

… … …Bradi1g75960.1 Os03g0147900 Sb01g047070.1Bradi1g75970.1 Os03g0147700 Sb01g047850.1Bradi1g76227.1 Os03g0136900 Sb01g047860.1

D Bradi1g00227.1 Os03g0861800 Sb01g000210.1Bradi1g00237.1 Os03g0860900 Sb01g000220.1Bradi1g00247.1 Os03g0860700 Sb01g000300.1

… … …Bradi1g02940.1 Os03g0823800 Sb01g002280.1Bradi1g02950.1 Os03g0822100 Sb01g002300.1Bradi1g02980.1 Os03g0821633 Sb01g002410.1

E Bradi1g49450.1 Os06g0122200 Sb10g001470.1Bradi1g49460.1 Os06g0125000 Sb10g001520.1Bradi1g49470.1 Os06g0125300 Sb10g001530.1

… … …Bradi1g52060.1 Os06g0103300 Sb10g000300.1Bradi1g52090.1 Os06g0102900 Sb10g000270.1Bradi1g52110.1 Os06g0102700 Sb10g000260.1

Only the first and last three syntenic genes anchored in each segmentare shown.

GenomeZipper analysis of wheat chromosome 4A 383

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 377–386

whole-genome analyses (Mayer et al., 2009; Wicker et al.,

2011). One explanation is that individual chromosomes/

chromosome arms are compared with complete reference

genomes, which may result in a higher rate of false-positive

gene identifications due to the presence of cross-matching

paralogous sequences. In an analysis of shotgun sequences

from wheat homoeologous group 1 chromosomes, Wicker

et al. (2011) identified a significant number of potential

pseudogenes (similar to, or even exceeding the number of

functional genes) that shared homology with various known

genes but were not present in the syntenic regions of either

Brachypodium or rice. This underlines the value of the com-

parative approach when attempting estimation of the num-

ber of genes present on a particular wheat chromosome.

The GenomeZipper method identified 1182 genes on

4AS and 1751 on 4AL, so a total of approximately 3000 genes

supported by synteny was placed on the entire chromosome.

Important in understanding the context of this estimate are

(i) the sequencing depth achieved (2.2-fold for 4AS; 1.7-fold

for 4AL), (ii) the expected gene detection rate (85%, based

on the method described by Lander and Waterman, 1988),

and (iii) that 20–25% of wheat genic sequences fail to detect a

close homolog in the reference genomes (Mayer et al., 2011).

Based on these considerations, we estimate the gene con-

tent of chromosome 4A to be approximately 4300 (2933/

0.85 · 100/80). Assuming that the gene density on chromo-

some 4A is representative of the A genome as a whole, and

given that its physical length is 15.6% of the entire genome,

the A genome contains approximately 28 000 genes, a

number largely in line with estimates for both the B genome

(38 000; Choulet et al., 2010) and for barley (32 000; Mayer

et al., 2011). However, due to the series of translocations of

presumably gene-rich telomeric regions that shaped the

modern chromosome 4A, the gene content of chromosome

4A may deviate from that of other less rearranged wheat

chromosomes. Thus chromosomal shotgun sequences for

other chromosomes will be helpful to refine gene estimates

for the individual wheat sub-genomes.

Limitations in resolution

A high-resolution EST map has been constructed for both

Ae. tauschii, the D genome donor species (Luo et al., 2009),

and chromosome 3B (Paux et al., 2008). At present, only

binned EST markers (Qi et al., 2004) are available for the

other wheat chromosomes. Bin maps lack sufficient reso-

lution to be used for syntenic integration and genome zip-

ping, which is why it was necessary here to rely on the barley

genetic map. The validity of this approach depends on the

retention of a high degree of synteny between barley and

wheat; any small-scale rearrangements will not be detected

until a dense marker map of the wheat genome has been

generated. Nevertheless, it was still possible to identify with

high precision the boundaries between the five segments

that arose as a result of the evolutionary inversion and

translocations. Earlier research based on mapping of cDNA

RFLP loci (Devos et al., 1995) and bin mapping (Miftahudin

et al., 2004) suggested the presence of at least two other

segments on chromosome 4A, but we have not been able to

confirm the presence of either of these. The availability of

higher-resolution genetic maps (which are certainly attain-

able given the volume of relevant sequence data now

available) will enable confirmation of the veracity of these

proposed additional structural rearrangements. A full com-

parative analysis awaits acquisition of genomic sequence

from chromosomes 4B and 4D, and from the translocated

portions of 5A and 7B. These data will enable determination

of the degree of similarity between homoeologs with respect

to gene content and potential loss of genes. Identification of

the translocation breakpoints on chromosomes 7B and 5A

may also allow recognition of molecular signatures and the

molecular environment that marks these translocations.

CONCLUSION

We have demonstrated here that fractionation of the com-

plex wheat genome into single chromosome arms, coupled

with the analysis of shotgun sequences using GenomeZip-

per, provides a successful strategy for constructing a high-

resolution gene-based chromosome map. The acquisition of

a complete ordered gene map, and ultimately of the genome

sequence itself, requires the development of a reliable

physical map, the construction of which is presently being

coordinated by the International Wheat Genome Sequenc-

ing Consortium. A physical map, together with chromosome

survey sequences, offers an ideal means of performing a

detailed analysis of chromosomal rearrangements. Further

developments in sequencing efficiency should also provide

opportunities to improve both chromosome coverage and

gene detection rate. This will eventually enable discovery of

the full genomic gene territories to enable study of the gene

structure and associated non-transcribed elements such as

cis elements.

EXPERIMENTAL PROCEDURES

Plant material

The 4A double di-telosomic stock of bread wheat cv. Chinese Springis a stable line in which chromosome 4A is represented by a pair oftelosomes, one of which is the short arm (4AS) and the other thelong arm (4AL) (Sears and Sears, 1978). Grain of this stock waskindly provided by Dr Bikram Gill (Department of Plant Pathology,Kansas State University, Manhattan, KS).

Chromosome sorting and DNA amplification

Liquid suspensions of mitotic chromosomes were prepared fromseedling root tips as described by Vrana et al. (2000). Telosomeswere isolated and sorted using a FACSVantage SE flow cytometer(Becton Dickinson) into 40 ll sterile deionized water. The level ofpurity of the sorted material was determined using fluorescencein situ hybridization based on a probe containing either the telo-meric repeat Afa or [GAA]n, as described by Kubalakova et al.

384 Pilar Hernandez et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 377–386

(2003). The flow-sorted chromosomes were treated with proteinase,and DNA was then extracted using a Microcon YM-100 column(Millipore, http://www.millipore.com/), as described by Simkovaet al. (2008). Chromosomal DNA was amplified by multiple dis-placement amplification using an Illustra GenomiPhi V2 DNAamplification kit (GE Healthcare, http://www.gehealthcare.com),and a Roche shotgun library (http://www.roche.com) was thencreated for each chromosome arm based on 5 lg multipledisplacement-amplified DNA.

DNA sequencing and analysis

Sequencing of the 4AS and 4AL libraries was performed at theLifesequencing S.L. facilities in Valencia (Spain) (http://www.lifesequencing.com/) on a Genome Sequencer FLX instrument (Roche),using titanium chemistry 454 Life Sciences Technology (Roche).Three full sequencing runs were performed for the 4AL library andtwo for the 4AS library. Repetitive DNA was masked using VMATCH

software (http://www.vmatch.de/), using the MIPS-REDAT POA-CEAE version 8.6.2 repeat library as a reference (http://mips.helmholtz-muenchen.de/plant/genomes.jsp). The following para-meters were applied: 70% identity cut-off, 100 bp minimal length,seed length 14, exdrop 5, e-value 0.001. To estimate the number ofgenes present, the repeat-filtered sequence reads were comparedby TBLASTX against the coding sequences for Brachypodium(ftp://ftpmips.helmholtz-muenchen.de/plants/brachypodium/v1.2),rice (rice RAP-DB genome build 4, http://rapdb.dna.affrc.go.jp)and SORGHUM (version 1.4, http://genome.jgi-psf.org/Sorbi1/Sorbi1.download.ftp.html).

GenomeZipper analysis

The GenomeZipper workflow described by Mayer et al. (2011) wasused, with some adjustments. Comparison and integration of theshotgun sequence into a linear gene order reference were achievedby exploiting synteny with barley, Brachypodium, rice and sor-ghum. The 4A segments were delineated by a BLASTN comparisonof the shotgun sequence data with that of barley artificial chromo-somes (Mayer et al., 2011). Only hits showing at least 85% identityand a minimum alignment of 100 bp were considered. BLASTX wasused to identify homologs in the reference genomes, applying acriterion of >70% similarity and a minimum length of 30 aminoacids. To position and orient genes, a selection of genes present inboth the five 4A segments and the relevant syntenic regions of theother grass genome(s) was aligned using a marker-based map ofbarley chromosomes 4H, 5H and 7H.

ACKNOWLEDGEMENTS

We warmly acknowledge the help provided by Jarmila Cıhalıkova,Romana Sperkova and Zdenka Dubska in chromosome sorting, aswell as the helpful comments of two anonymous reviewers. Thisresearch was financially supported by the Spanish Ministry of Sci-ence and Innovation (grant numbers BIO2009–07443, BIO2011–15237 and AGL2010–17316), the German Ministry of Education andResearch GABI Barlex project, the European Commission FP7-212019 Triticeae Genome grant, the Czech Science Foundation(awards 521/08/1629 and P501/10/1740), and the Czech RepublicMinistry of Education, Youth and Sports/European RegionalDevelopment Fund (Operational Programme Research and Devel-opment for Innovations grant number CZ.1.05/2.1.00/01.0007).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Table S1. GenomeZipper analysis of wheat chromosome 4A.

Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

REFERENCES

Araki, E., Miura, H. and Sawada, S. (1999) Identification of genetic loci

affecting amylose content and agronomic traits on chromosome 4A of

wheat. Theor. Appl. Genet. 98, 977–984.

Bai, G.H., Chen, C.X. and Cai, S.B. (2008) A major QTL controlling seed dor-

mancy and pre-harvest sprouting resistance on chromosome 4A in a Chi-

nese wheat landrace. Mol. Breeding, 21, 351–358.

Berkman, P.J., Skarshewski, A., Lorenc, M.T. et al. (2011) Sequencing and

assembly of low copy and genic regions of isolated Triticum aestivum

chromosome arm 7DS. Plant Biotechnol. J. 9, 768–775.

Bolot, S., Abrouk, M., Masood-Quraishi, U., Stein, N., Messing, J., Feuillet, C.

and Salse, J. (2009) The ‘inner circle’ of the cereal genomes. Curr. Opin.

Plant Biol. 12, 119–125.

Borner, A., Schumann, E., Furste, A., Coster, H., Leithold, B., Roder, M.S. and

Weber, W.E. (2002) Mapping of quantitative trait loci determining agro-

nomic important characters in hexaploid wheat (Triticum aestivum L.).

Theor. Appl. Genet. 105, 921–936.

Chen, X.M., Line, R.F. and Jones, S.S. (1995) Chromosomal location of genes

for resistance to Puccinia striiformis in winter-wheat cultivars Heines-Vii,

Clement, Moro, Tyee, Ikes, and Daws. Phytopathology, 85, 1362–1367.

Chen, X.M., Luo, Y.H., Xia, X.C., Xia, L.Q., Chen, X., Ren, Z.L., He, Z.H. and Jia,

J.Z. (2005) Chromosomal location of powdery mildew resistance gene

Pm16 in wheat using SSR marker analysis. Plant Breeding, 124, 225–228.

Choulet, F., Wicker, T., Rustenholz, C. et al. (2010) Megabase level sequenc-

ing reveals contrasted organization and evolution patterns of the wheat

gene and transposable element spaces. Plant Cell, 22, 1686–1701.

Close, T.J., Bhat, P.R., Lonardi, S. et al. (2009) Development and implemen-

tation of high-throughput SNP genotyping in barley. BMC Genomics, 10,

582.

Devos, K. and Gale, M. (2000) Genome relationships: the grass model in

current research. Plant Cell, 12, 637–646.

Devos, K.M., Dubcovsky, J., Dvorak, J., Chinoy, C.N. and Gale, M.D. (1995)

Structural evolution of wheat chromosomes 4A, 5A, and 7B and its impact

on recombination. Theor. Appl. Genet. 91, 282–288.

Dolezel, J., Kubalakova, M., Paux, E., Bartos, J. and Feuillet, C. (2007) Chro-

mosome-based genomics in the cereals. Chromosome Res. 15, 51–66.

Dvorak, J. and Zhang, H.B. (1990) Variation in repeated nucleotide sequences

sheds light on the phylogeny of the wheat B and G genomes. Proc. Natl

Acad. Sci. USA, 87, 9640–9644.

Dvorak, J., Terlizzi, P., Zhang, H.B. and Resta, P. (1993) The evolution of

polyploid wheats: identification of the A genome donor species. Genome,

36, 21–31.

Effertz, R.J., Anderson, J.A. and Francl, L.J. (2001) Restriction fragment length

polymorphism mapping of resistance to two races of Pyrenophora tritici-

repentis in adult and seedling wheat. Phytopathology, 91, 572–578.

Feuillet, C., Travella, S., Stein, N., Albar, L., Nublat, A. and Keller, B. (2003)

Map-based isolation of the leaf rust disease resistance gene Lr10 from the

hexaploid wheat (Triticum aestivum L.) genome. Proc. Natl Acad. Sci. USA,

100, 15253–15258.

Flavell, R.B. (1986) Repetitive DNA and chromosome evolution in plants.

Philos. Trans. R. Soc. Lond. B Biol. Sci. 312, 227–242.

Gaut, B.S. (2002) Evolutionary dynamics of grass genomes. New Phytol. 154,

15–28.

International Brachypodium Initiative (2010) Genome sequencing and anal-

ysis of the model grass Brachypodium distachyon. Nature, 463, 763–768.

International Rice Genome Sequencing Project (2005) The map-based se-

quence of the rice genome. Nature, 436, 793–800.

Keller, M., Karutz, C., Schmid, J.E., Stamp, P., Winzeler, M., Keller, B. and

Messmer, M.M. (1999) Quantitative trait loci for lodging resistance in a

segregating wheat x spelt population. Theor. Appl. Genet. 98, 1171–1182.

Kilian, B., Ozkan, H., Deusch, O., Effgen, S., Brandolini, A., Kohl, J., Martin, W.

and Salamini, F. (2007) Independent wheat B and G genome origins in

outcrossing Aegilops progenitor haplotypes. Mol. Biol. Evol. 24, 217–227.

GenomeZipper analysis of wheat chromosome 4A 385

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 377–386

Kubalakova, M., Vrana, J., Cıhalıkova, J., Simkova, H. and Dolezel, J. (2002)

Flow karyotyping and chromosome sorting in bread wheat (Triticum aes-

tivum L.). Theor. Appl. Genet. 104, 1362–1372.

Kubalakova, M., Valarik, M., Bartos, J., Vrana, J., Cıhalıkova, J., Molnar-Lang,

M. and Dolezel, J. (2003) Analysis and sorting of rye (Secale cereale L.)

chromosomes using flow cytometry. Genome, 46, 893–905.

Lander, E.S. and Waterman, M.S. (1988) Genomic mapping by fingerprinting

random clones: a mathematical analysis. Genomics, 2, 231–239.

Luo, M.C., Deal, K.R., Akhunov, E.D. et al. (2009) Genome comparisons reveal

a dominant mechanism of chromosome number reduction in grasses and

accelerated genome evolution in Triticeae. Proc. Natl Acad. Sci. USA, 106,

15780–15785.

Mayer, K.F., Taudien, S., Martis, M. et al. (2009) Gene content and virtual gene

order of barley chromosome 1H. Plant Physiol. 151, 496–505.

Mayer, K.F., Martis, M., Hedley, P.E. et al. (2011) Unlocking the barley

genome by chromosomal and comparative genomics. Plant Cell, 23,

1249–1263.

McCartney, C.A., Somers, D.J., Humphreys, D.G., Lukow, O., Ames, N., Noll,

J., Cloutier, S. and McCallum, B.D. (2005) Mapping quantitative trait loci

controlling agronomic traits in the spring wheat cross RL4452 x ‘AC

Domain’. Genome, 48, 870–883.

McFadden, E. and Sears, E. (1946) The origin of Triticum spelta and its free-

threshing hexaploid relatives. J. Hered. 37, 107–116.

MickelsonYoung, L., Endo, T.R. and Gill, B.S. (1995) A cytogenetic ladder-map

of the wheat homoeologous group-4 chromosomes. Theor. Appl. Genet.

90, 1007–1011.

Miftahudin, R.K., Ma, X.F., Mahmood, A.A. et al. (2004) Analysis of

expressed sequence tag loci on wheat chromosome group 4. Genetics,

168, 651–663.

Moore, G. (1995) Cereal genome evolution – pastoral pursuits with lego

genomes. Curr. Opin. Genet. Dev. 5, 717–724.

Naranjo, T., Roca, A., Goicoechea, P.G. and Giraldez, R. (1987) Arm homo-

eology of wheat and rye chromosomes. Genome, 29, 873–882.

Nga, N.T.T., Hau, V.T.B. and Tosa, Y. (2009) Identification of genes for resis-

tance to a Digitaria isolate of Magnaporthe grisea in common wheat cul-

tivars. Genome, 52, 801–809.

Paterson, A.H., Bowers, J.E., Bruggmann, R. et al. (2009) The Sorghum

bicolor genome and the diversification of grasses. Nature, 457, 551–556.

Paull, J.G., Chalmers, K.J., Karakousis, A., Kretschmer, J.M., Manning, S.

and Langridge, P. (1998) Genetic diversity in Australian wheat varieties

and breeding material based on RFLP data. Theor. Appl. Genet. 96,

435–446.

Paux, E., Sourdille, P., Salse, J. et al. (2008) A physical map of the 1-gigabase

bread wheat chromosome 3B. Science, 322, 101–104.

Qi, L.L., Echalier, B., Chao, S. et al. (2004) A chromosome bin map of 16,000

expressed sequence tag loci and distribution of genes among the three

genomes of polyploid wheat. Genetics, 168, 701–712.

Safar, J., Simkova, H., Kubalakova, M., Cıhalıkova, J., Suchankova, P., Bartos,

J. and Dolezel, J. (2010) Development of chromosome-specific BAC

resources for genomics of bread wheat. Cytogenet. Genome Res. 129,

211–223.

Salse, J. and Feuillet, C. (2007) Comparative genomics of cereals. In

Genomics-Assisted Crop Improvement (Rajeev, K. and Varshney, R.T.,

eds). New York: Springer, pp. 177–205.

Sarkar, P. and Stebbins, G.L. (1956) Morphological evidence concerning the

origin of the B genome in wheat. Am. J. Bot. 43, 297–304.

Sears, E.R. and Sears, L.M.S. (1978) The telocentric chromosomes of common

wheat. In Proceedings of the 5th International Wheat Genetics Symposium

(Ramanujam, S. ed.). New Dehli: Indian Soc. Genet Plant Breed, pp. 389–

407.

Simkova, H., Svensson, J.T., Condamine, P., Hribova, E., Suchankova, P.,

Bhat, P.R., Bartos, J., Safar, J., Close, T.J. and Dolezel, J. (2008) Coupling

amplified DNA from flow-sorted chromosomes to high-density SNP map-

ping in barley. BMC Genomics, 9, 294.

Sourdille, P., Cadalen, T., Gay, G., Gill, B. and Bernard, M. (2002) Molecular

and physical mapping of genes affecting awning in wheat. Plant Breeding,

121, 320–324.

Talbert, L.E., Bruckner, P.L., Smith, L.Y., Sears, R. and Martin, T.J. (1996)

Development of PCR markers linked to resistance to wheat streak mosaic

virus in wheat. Theor. Appl. Genet. 93, 463–467.

Vrana, J., Kubalakova, M., Simkova, H., Cıhalıkova, J., Lysak, M.A. and

Dolezel, J. (2000) Flow sorting of mitotic chromosomes in common wheat

(Triticum aestivum L.). Genetics, 156, 2033–2041.

Wang, G.Z., Miyashita, N.T. and Tsunewaki, K. (1997) Plasmon analyses of

Triticum (wheat) and Aegilops: PCR-single-strand conformational poly-

morphism (PCR-SSCP) analyses of organellar DNAs. Proc. Natl Acad. Sci.

USA, 94, 14570–14577.

Wicker, T., Mayer, K.F., Gundlach, H. et al. (2011) Frequent gene movement

and pseudogene evolution is common to the large and complex genomes

of wheat, barley, and their relatives. Plant Cell, 23, 1706–1718.

386 Pilar Hernandez et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 377–386