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Akhunov et al. BMC Genomics 2010, 11:702
http://www.biomedcentral.com/1471-2164/11/702

RESEARCH ARTICLE

Open Access

Nucleotide diversity maps reveal variation in
diversity among wheat genomes and chromosomes
Eduard D Akhunov1,2, Alina R Akhunova1,2, Olin D Anderson3, James A Anderson4, Nancy Blake5, Michael T Clegg6,
Devin Coleman-Derr3, Emily J Conley4, Curt C Crossman3, Karin R Deal1, Jorge Dubcovsky1, Bikram S Gill7, Yong Q Gu3,
Jakub Hadam7, Hwayoung Heo5, Naxin Huo3, Gerard R Lazo3, Ming-Cheng Luo1, Yaqin Q Ma1,8, David E Matthews9,
Patrick E McGuire1, Peter L Morrell4, Calvin O Qualset1, James Renfro3, Dindo Tabanao4,10, Luther E Talbert5, Chao Tian1,
Donna M Toleno6, Marilyn L Warburton11,12, Frank M You1, Wenjun Zhang1, Jan Dvorak1*

Abstract
Background: A genome-wide assessment of nucleotide diversity in a polyploid species must minimize the
inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into their
respective genomes. The same requirements complicate the development and deployment of single nucleotide
polymorphism (SNP) markers in polyploid species. We report here a strategy that satisfies these requirements and
deploy it in the sequencing of genes in cultivated hexaploid wheat (Triticum aestivum, genomes AABBDD) and wild
tetraploid wheat (Triticum turgidum ssp. dicoccoides, genomes AABB) from the putative site of wheat domestication
in Turkey. Data are used to assess the distribution of diversity among and within wheat genomes and to develop a
panel of SNP markers for polyploid wheat.
Results: Nucleotide diversity was estimated in 2114 wheat genes and was similar between the A and B genomes
and reduced in the D genome. Within a genome, diversity was diminished on some chromosomes. Low diversity
was always accompanied by an excess of rare alleles. A total of 5,471 SNPs was discovered in 1791 wheat genes.
Totals of 1,271, 1,218, and 2,203 SNPs were discovered in 488, 463, and 641 genes of wheat putative diploid
ancestors, T. urartu, Aegilops speltoides, and Ae. tauschii, respectively. A public database containing genome-specific
primers, SNPs, and other information was constructed. A total of 987 genes with nucleotide diversity estimated in
one or more of the wheat genomes was placed on an Ae. tauschii genetic map, and the map was superimposed
on wheat deletion-bin maps. The agreement between the maps was assessed.
Conclusions: In a young polyploid, exemplified by T. aestivum, ancestral species are the primary source of genetic
diversity. Low effective recombination due to self-pollination and a genetic mechanism precluding homoeologous
chromosome pairing during polyploid meiosis can lead to the loss of diversity from large chromosomal regions.
The net effect of these factors in T. aestivum is large variation in diversity among genomes and chromosomes,
which impacts the development of SNP markers and their practical utility. Accumulation of new mutations in older
polyploid species, such as wild emmer, results in increased diversity and its more uniform distribution across the
genome.

Background
While nucleotide diversity studies and the development
and deployment of single nucleotide polymorphism
(SNP) markers are straightforward in diploid and paleopolyploid species, such as maize or soybean [1-3], they
* Correspondence: jdvorak@ucdavis.edu
1
Department of Plant Sciences, University of California, Davis, CA 95616, USA
Full list of author information is available at the end of the article

are complicated in recently evolved polyploid species by
high levels of orthologous gene similarity. Sequence
similarity makes sequencing of single genes and allocation of sequences into respective genomes difficult. Special strategies are therefore required for nucleotide
diversity studies and the development of SNP markers
for young polyploid species, which include wheat and
other economically important plants.

© 2010 Akhunov et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

Akhunov et al. BMC Genomics 2010, 11:702
http://www.biomedcentral.com/1471-2164/11/702

Wheat forms an allopolyploid series at three ploidy
levels: diploid (2x = 14), tetraploid (4x = 28), and hexaploid
(6x = 42). Wild tetraploid emmer wheat (Triticum turgidum ssp. dicoccoides, henceforth shortened to T. dicoccoides, genomes AABB) evolved between 0.2 and 0.5
million years ago [4,5] via hybridization of wild T. urartu
(genomes AA) and an extinct or undiscovered species in
the lineage of Aegilops speltoides (genomes SS, where S is
closely related but not identical to the wheat B genome)
[4,6-9]. Hexaploid wheat (T. aestivum, genomes AABBDD)
evolved about 8,500 years ago [10] via hybridization of
T. turgidum with diploid Ae. tauschii (genomes DD) [11,12].
A possible strategy for nucleotide diversity studies and
SNP discovery in young polyploid species, such as
wheat, is to find diverged regions in orthologous genes
and use them for the design of polymerase chain reaction (PCR) primers that anneal to only a single DNA
target. These genome-specific primers (GSPs) amplify
DNA from only a single genome and facilitate gene
sequencing and SNP discovery [13]. An alternative strategy is to shotgun-sequence cDNAs and then allocate
each sequence to a genome. Both approaches have been
used in polyploid wheat [13-15] although those studies
were of limited scope and genome coverage [14-17] and
none mapped the markers.
A domestication bottleneck at the tetraploid level and
a polyploidy bottleneck during the transition from the
tetraploid to hexaploid level are expected to have
reduced the diversity of polyploid wheat compared to
wild emmer. Nucleotide diversity θπ [18] was reported
to be 2.7 × 10 -3 in 24 A- and B-genome wild emmer
genes [16]. For comparison, θπ was estimated to be 9.7
× 10-3 in teosinte genes (Zea mays ssp. parviglumis) [3]
and 7.7 to 8.1 × 10-3 in wild barley genes (Hordeum vulgare ssp. spontaneum) [19,20]. The diversity of emmer
was reduced by the domestication bottleneck but, curiously, no further diversity loss took place in the A and
B genomes during the polyploidy bottleneck accompanying the evolution of T. aestivum from domesticated tetraploid wheat [16]. Levels of diversity in the T. aestivum
D genome are unknown.
Genetic evidence suggests that wild emmer was
domesticated in the Diyarbakir region in southeastern
Turkey [21,22]. The result was hulled domesticated
emmer (T. turgidum ssp. dicoccon), which was then the
primary source of free-threshing tetraploid wheat, such
as durum (T. turgidum ssp. durum, henceforth
T. durum). Transcaucasia and northwestern Caspian
Iran appear to be the primary sites of the evolution of
T. aestivum [23]. Gene flow from wild to domesticated
tetraploid wheat and from tetraploid wheat and
Ae. tauschii to T. aestivum has been experimentally
documented [23-27] but its impact on the evolution of
the T. aestivum A, B, and D genomes is not clear.

Page 2 of 22

We report here the development of GSPs for T. aestivum and their use in sequencing of T. aestivum genes
with the goal of characterizing the nucleotide diversity
of the wheat genomes and discovering SNPs. To make
the GSP development possible, a set of primers
anchored in conserved exons flanking one or several
introns was developed and is also reported. We refer to
these as conserved primers (CPs), as in [13]. Primers of
this type have also been known as conserved orthologous sets (COS) [28]. A map of genes bearing SNPs
constructed in diploid Ae. tauschii is presented and
compared with wheat deletion-bin gene maps [29].
Nucleotide diversity in individual chromosomes in a
wild emmer population from the Diyarbakir region in
Turkey and in T. aestivum was computed and the distribution of diversity among and within wild emmer and
T. aestivum genomes was used to analyze the early
stages of polyploid evolution.

Results
GSP development and SNP discovery

The process of GSP and SNP development is summarized in Figure 1. A total of 6,045 wheat ESTs was downloaded from the wEST database into the pipeline and
CPs anchored in exons and flanking one or two introns
were developed. The Southern hybridization profiles of
the ESTs were examined in the wEST database http://
wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi and
CPs for those that showed a complex profile were eliminated. Amplicons were obtained with CPs for 1,599
T. urartu genes, 1,583 Ae. speltoides genes, and 1,574
Ae. tauschii genes and were sequenced. A total of 1,442
genes was cloned and sequenced from Langdon durum
wheat. A total of 11,764 GSPs was designed and tested
for genome specificity by PCR amplification of T. aestivum nullisomic-tetrasomic (N-T) lines. GSPs derived
from 1,102 EST unigenes (705 in the A genome, 703 in
the B genome, and 706 in the D genome) were validated
by PCR with N-T lines.
Target DNA was PCR amplified in 32 wheat lines
(Tables 1 and 2) using GSPs. A total of 41,065,555 bp of
the amplicons was sequenced (14,734,124 bp in the A
genome, 14,554,737 bp in the B genome, and 11,776,694
bp in the D genome) using GSP pairs as sequencing primers, and 5,471 SNPs at 1,791 loci were discovered.
SNP database

An online SNP database http://probes.pw.usda.gov:8080/
snpworld/Search was constructed. It contains sequences
of GSPs for the amplification and sequencing of 2114
loci and other relevant information about the ESTs and
SNPs (such as deletion-bin mapping of each EST), top
ten blast hits of each EST, alignments of nucleotide
sequences generated with primers derived from each

Akhunov et al. BMC Genomics 2010, 11:702
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Page 3 of 22

Flow of the project
CP design

Download bin-mapped ESTs from the wEST database into the
ConservedPrimers 2.0 pipeline.
(6,045 ESTs downloaded)
Design primers with the pipeline and manually.
(Primer pairs for 2111 unigenes designed)

GSP design

PCR amplify and sequence T. urartu, Ae. tauschii, and Ae. speltoides
amplicons and at lest 12 clones of each 'Langdon' durum amplicon.
Align sequences, allocate them to the A, B, and D genomes
and find divergent nucleotides.
Use a divergent nucleotide to design a genome-specific primer
and pair it with one of the CP primers (11,764 GSPs designed and tested)

GSP validation

Perform PCR using GSPs with relevant wheat nullisomic-tetrasomic lines
and validate primer genome specificity (GSPs for 2114 loci belonging to
1,102 unigenes validated)

SNP discovery

Amplify gene targets using GSPs in the panel of 32 wheat lines.
(2,114 genes amplified)
Align sequences and search for SNPs
(5,471 SNPs discovered at 1,791 gene loci)

Data base
construction

Submit the alignments with SNPs to the project database
(http://probes.pw.usda.gov:8080/snpworld/Search)

Figure 1 Project flow chart.

EST, a reference sequence for a locus and its source,
and graphical and numerical displays of each SNP.
Reference sequences were used to specify the positions
of SNPs. For the majority of the loci, the cv ‘Chinese
Spring’ (code Ta21, Table 1) sequence was used as a
reference sequence because of the central position of
Chinese Spring in the unrooted phylogenetic tree of 468
T. aestivum lines (Additional file 1, Figure S1). If the
sequence from Chinese Spring was unavailable, the next
most complete sequence for the locus was used. SNPs
can be viewed in the context of the entire reference
sequence in the expanded view window for each EST.
The database also contains data for portions of 1,651
genes amplified and sequenced with CPs in T. urartu,

Ae. speltoides, and Ae. tauschii http://probes.pw.usda.
gov:8080/snpworld/Search. The accession used as a
reference sequence for a locus is indicated for each
species. Data in the database include 488 polymorphic
loci containing 1,271 SNPs for T. urartu, 463 polymorphic loci containing 1,218 SNPs for Ae. speltoides,
and 641 polymorphic loci containing 2,203 SNPs for
Ae. tauschii. Additional SNPs for Ae. tauschii can be
found in the database for the D genomes of the synthetic wheats.
Diversity maps

A single Ae. tauschii EST linkage map [30] was used as
the backbone of the diversity maps. The Ae. tauschii

Akhunov et al. BMC Genomics 2010, 11:702
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Page 4 of 22

Table 1 Lines of tetraploid and hexaploid wheat used for
SNP discovery
Species

Database
code

Line

Origin

T. turg. ssp.
dicoccoides

Td01

PI 428020

Diyarbakir,
Turkey

T. turg. ssp.
dicoccoides

Td02

PI 428027

Diyarbakir,
Turkey

T. turg. ssp.
dicoccoides

Td03

PI 428053

Diyarbakir,
Turkey

T. turg. ssp.
dicoccoides

Td04

PI 428073

Diyarbakir,
Turkey

T. turg. ssp.
dicoccoides

Td05

PI 428064

Diyarbakir,
Turkey

T. turg. ssp.
dicoccoides

Td06

PI 428082

Diyarbakir,
Turkey

T. turg. ssp.
dicoccoides

Td07

PI 428083

Diyarbakir,
Turkey

T. turg. ssp.
dicoccoides

Td08

PI 428086

Diyarbakir,
Turkey

T. turg. ssp.
dicoccoides

Td09

G 2844

Diyarbakir,
Turkey

T. turg. ssp.
dicoccoides

Td10

G 2040

Diyarbakir,
Turkey

T. aest. ssp.
aestivum

Ta11

PI 166698

Turkey

T. aest. ssp.
aestivum

Ta13

PI 166792

Turkey

T. aest. ssp.
aestivum

Ta16

PI 622268

Iran,

T. aest. ssp.
aestivum

Ta17

Yangxian
Yangqianmai

Shaanxi (5660)

T. aest. ssp.
aestivum
T. aest. ssp.
aestivum
T. aest. ssp.
aestivum
T. aest. ssp.
aestivum
T. aest. ssp.
aestivum

Ta18

Yecora Rojo

California

Ta19

PI 119325

Turkey

Ta20

PI 622233

Iran

Ta21

Chinese Spring

China

Ta23

Opata 85

CIMMYT

T. aest. ssp.
compactum

Ta12

PI 166305

Turkey

T. aest. ssp.
compactum

Ta14

PI 350731

Austria

T. aest. ssp.
compactum

Ta15

PI 410595

Pakistan

T. aest. ssp. spelta

Ta22

405a (DV1132)

Iran

map backbone contained 870 loci (Table 3). Cosegregating genes were allocated into “recombination blocks”
which were sequentially numbered (Additional file 2,
Table S1). The order of orthologous genes in rice was
used to order genes within a recombination block.
Synteny of the Ae. tauschii genetic map with the rice
genome sequence [30] was exploited in mapping additional
loci for which the parents of the Ae. tauschii mapping
population were not polymorphic (Table 3) and which met

the conditions detailed in Materials and Methods (Map
construction). In a few cases, in which an ambiguity was
encountered in the rice genome sequence, sorghum and
Brachypodium distachyon genome sequences were
employed [30,31]. Consider, for example locus BG313769
located on the short arm of chromosome 1 D (Additional
file 2, Table S1). This locus was mapped to bins 1AS1,
1BS9, and 1DS1 [32]http://wheat.pw.usda.gov/cgi-bin/
westsql/map_locus.cgi. The locus with the highest
sequence similarity in rice is on pseudomolecule Os5 starting at nucleotide 1,903,106. Os5 is homoeologous with
1DS and mapping of the locus in the 1AS1, 1BS9, and
1DS1 bins is consistent with the position of the locus in
Os5 (Additional file 2, Table S1). PCR using genomic DNA
of N1A-T1B and N1A-T1 D as templates with the
BG313769 A-genome GSPs showed that the locus used for
the diversity study was on chromosome 1A http://probes.
pw.usda.gov:8080/snpworld/Search. Inserting locus
BG313769 into the map on the basis of synteny of 1AS1,
1BS9, and 1DS1 with Os5 placed it between recombination
block 36 (locus BE445121, which is at 56.85 cM on the Ae.
tauschii map and at nucleotide 1,679,201 in the Os5 pseudomolecule) and recombination block 37 (locus BF291549,
which is at 57.06 cM on the Ae. tauschii map and at
nucleotide 1,954,380 in the Os5 pseudomolecule). Locus
BG313769 and its diversity data were therefore placed
between loci BE445121 and BF291549. No cM value was
attached to the locus but its coordinates on Os5 were
given (Additional file 2, Table S1).
Loci corresponding to 484 ESTs were inserted into the
diversity maps on the basis of this process (Additional
file 2, Table S1), bringing the total number of loci on the
map to 1,354 (Table 3). Diversity was estimated from at
least one genome for 987 EST loci on the map. From
348,938 to 351,542 bp were sequenced and mapped on
the diversity maps for each genome × taxon combination
(Table 4). The numbers of discovered SNPs ranged from
377 in the T. aestivum D genome to 1,979 in the wild
emmer B genome. The highest average number of haplotypes per gene and highest average haplotype diversity
was in the D genome of synthetic wheats whereas the
lowest number of haplotypes per gene and lowest haplotype diversity was in the D genome of T. aestivum (Table
4). In wild emmer and T. aestivum, the average numbers
of haplotypes per gene and haplotype diversity did not
significantly differ between the A and B genomes (Table
4). However, both variables were significantly higher in
the genomes of wild emmer than in the corresponding
genomes of T. aestivum (Table 4).
Superimposition of diversity maps on the deletion-bin
maps

Wheat EST deletion-bin maps are an important resource for
the use of ESTs in wheat comparative mapping, map-based

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Table 2 Synthetic wheats used for SNP discovery
Name*

Database
code

Source of AB
genomes

Source of the D genome

ITMI synthetic

Sn24

T. t. durum ’Altar’

CIMMYT W7984, CIGM86.940, Ae. tauschii ssp. strangulata, (collected by H. Kihara near
Mazadaran, Iran)

RL5402(2)

Sn25

TetraCantach

RL5261, Ae. tauschii ssp. typica

RL5403(2)
RL5405(2)

Sn26
Sn27

TetraCantach
TetraCantach

RL5266, Ae. tauschii ssp. anathera
RL5288. Ae. tauschii ssp. strangulata, (originally supplied by M. Tanaka as KUSE 2144)

RL5406(2)

Sn28

TetraCantach

RL5289. Ae. tauschii ssp. meyeri,

62052_4(1)

Sn29

T. durum ’Croc_1’

CIMMYT 205, Ae. tauschii ssp. tauschii
(PI452130, Hunan, China)

62056_4(1)

Sn30

T. durum ’Croc_1’

CIMMYT 224 = CLAE 25, Ae. tauschii ssp.tauschii (collected by H. Kihara near Gilan, Iran)

161725_0(1)

Sn31

T. durum ’Ceta’

CIMMYT 372 = CIGM86.940, Ae. tauschii ssp. strangulata (collected by H. Kihara near Kabul,
Afghanistan)

Unknown

Unknown

(1)

Sear’s synthetic Sn32
(3)

* Accessions designated with (1) were developed and supplies by A. Mujeeb-Kazi, at CIMMYT, those designated with (2) were developed and supplied by E.R.
Kerber, Agriculture Canada, Winnipeg, Manitoba, and that designated with (3) was developed and supplied by E.R. Search, USDA-ARS, Columbia, Missouri

cloning of wheat genes, comparative genomics, and other
genetic and genomic applications. To facilitate crossreferencing of EST diversity data developed here with EST
deletion-bin maps, the wheat diversity maps were superimposed on the deletion bin maps (Additional file 2, Table S1).
The Ae. tauschii linkage map [30] and wheat deletionbin maps share large numbers of loci, which facilitated
comparison of the two sets of maps. Only loci mapped by
linkage were used for these comparisons. Totals of 534,
654, and 646 ESTs on the wheat A-, B- and D-genome
deletion-bin maps were compared, respectively. The bin
location of a locus was considered incongruent between
the genetic and deletion-bin maps if it disagreed with the
order of recombination blocks (Additional file 2, Table
S1); the order of loci within recombination blocks was
disregarded. The known translocation differences involving chromosome 4A and chromosome arms 5AL and
7BS [33,34] were not considered. Because the genetic
maps of Ae. tauschii chromosomes are highly colinear
with the rice pseudomolecules (Additional file 2,
Table S1) most of the disagreements between the linkage
maps and deletion-bin maps would have to be due to
structural differences between wheat and Ae. tauschii

chromosomes or due to incompleteness or inconsistencies in the deletion-bin maps.
The Ae. tauschii linkage map portion of the diversity
maps (Additional file 2, Table S1) is expected to be
more consistent with the D-genome deletion-bin map
than the A- and B-genome deletion-bin maps because
the Ae. tauschii chromosomes are phylogenetically more
closely related to those of the wheat D genome than to
those of the wheat A and B genomes, and this was
indeed observed. While the locations of only 8.8% of the
loci on the D-genome deletion-bin maps were incongruent with the linkage map, 10.8 and 12.4% of the A- and
B-genome loci were incongruent (Table 5). The greatest
discrepancies relative to gene order in Ae. tauschii and
rice were encountered in chromosome arms 1AL, 5AS,
7AL, 1BL, 5BS, 4DL, and 7DS, and none were found in
chromosome arms 2BS, 2DS, 2DL, 3DS, and 5DL (Table
5 and Additional file 2, Table S1).
Nucleotide diversity

From 609 to 704 genes with estimated diversity were
mapped in a genome × species combination (Table 6).
However, some of the loci were excluded from diversity

Table 3 Loci mapped on the basis of linkage and synteny and the total number of EST loci with estimated diversity
(Div. loci) on the map
Chromosome

Length (cM)

Total loci mapped

Linkage mapped loci

Synteny mapped loci

Div. loci

1

180.5

212

148

64

125

2
3

186.9
197.0

171
240

92
186

79
54

142
151

4

127.4

198

107

91

155

5

171.2

133

86

47

113

6

149.3

205

119

86

145

7

154.5

195

132

63

156

Total

1166.8

1354

870

484

987

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Table 4 Nucleotides sequenced, SNPs discovered,
average number of haplotypes (H), and haplotype
diversity (h)
Species

Genome

n

Nucl.

SNPs

Nucl./
SNP

H

h

T. aestivum

A

13 351542

966

364

T. aestivum

B

13 348938 1008

346

1.72c 0.21c

T. aestivum

D

13 349748

927

1.23d 0.06d

T. dicoccoides

A

10 351542 1516

232

2.05ab 0.31b

T. dicoccoides

B

10 348938 1979

176

2.22a 0.33b

Synthetic 6x
wheat

D

9 349029 1727

202

2.39a 0.47a

377

1.82c* 0.22c

* Means followed by the same letter are not significantly different at the 5%
probability level. Individual chromosome means (Table 9) were used as
variables in ANOVA.

analyses because of small sample size or because of
unreasonably high diversity indicating the possibility of
orthologous or paralogous sequences being included in
a diversity estimate. The numbers of loci used for analyses of diversity were therefore lower (Table 6). Of the
analyzed loci, 305 (52%) and 296 (51%) were polymorphic in the A and B genomes of T. aestivum,
respectively, and 316 (54%) and 338 (59%) were polymorphic in the A and B genomes of wild emmer,
respectively (Table 6). Only 138 (20%) loci of the 679
analyzed in the T. aestivum D genome were polymorphic (Table 6). Because the same GSPs resulted in
the discovery of 477 (74%) SNP-bearing loci in the D
genome of synthetic wheats (Table 6), the low number
of polymorphic loci in the wheat D genome must be an
attribute of wheat, not of Ae. tauschii, its diploid source.
Genome-wide θ w , and θ π were similar between the
T. aestivum A and B genomes (Table 7). Both estimates

were higher than those in the T. aestivum D genome
(Table 7). The estimates were also similar between the
A and B genomes in wild emmer, which showed higher
diversity than the corresponding genomes in T. aestivum (Table 7).
Tajima’s D contrasts θw, and θπ to detect differences
in the distribution of diversity relative to neutral expectations. The expectation for a neutral locus in a population is a Tajima’s D of zero. Positive values of Tajima’s
D indicate a paucity of rare alleles and a preponderance
of intermediate frequency alleles while negative values
indicate a preponderance of rare alleles and a paucity of
intermediate frequency alleles. Average Tajima’s D was
near zero in the A and B genomes of T. aestivum and
wild emmer but was negative in the T. aestivum D genome and positive in the Ae. tauschii genome present in
synthetic wheats (Table 7). The positive value of Tajima’s D in the D genome of synthetic wheats is very
likely due to strong subdivision of Ae. tauschii into two
major subpopulations. This subdivision has been
acknowledged taxonomically by elevating individuals of
the two subpopulations to subspecies, Ae. tauschii ssp.
strangulata and Ae. tauschii ssp. tauschii [35]. Estimates
of diversity at the replacement to silent codon sites in
the D genome were similar to those in Ae. tauschii and
differed in both genomes from those in the A and B
genomes of T. aestivum and wild emmer (Table 7).
Diversity among individual chromosomes

In the A genome of wild emmer and T. aestivum, diversity was lower in chromosome 4A than in the remaining
chromosomes (Table 8). This was true for diversity in
coding sequences and in replacement and silent codon

Table 5 Agreement between the locations of EST loci on the Ae. tauschii linkage map and wheat deletion-bin maps
A genome
Chrom. arm

B genome
Total loci

D genome

Total loci

% discordant loci

% discordant loci

Total loci

1S

62

6.5

60

20.0

73

% discordant loci
6.8

1L

38

31.6

49

20.4

48

16.7

2S
2L

24
39

8.3
2.6

36
36

0.0
5.6

26
37

0.0
0.0

3S

48

2.1

48

14.6

45

0.0

3L

82

1.2

92

8.7

97

9.3

4S

-*

-

27

14.8

29

10.3

4L

-*

-

55

1.8

59

27.1

5S

23

21.7

24

25.0

23

4.3

5L

39

12.8

37

10.8

33

0.0

6S
6L

29
53

3.4
9.4

26
59

19.2
11.9

24
48

12.5
8.3

7S

51

3.9

56

8.9

55

18.2

7L

46

23.9

49

12.2

49

10.2

Mean

10.8

12.4

8.8

* Chromosome 4A is extensively rearranged, and the congruence of the 4A deletion-bin map could not be meaningfully compared with the Ae. tauschii linkage
map.

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Table 6 Numbers of loci on the diversity maps harboring one or more SNPs, the total numbers of loci with estimated
diversity (nt), and the total numbers of loci used for analyses (na)
T. aestivum

T. dicoccoides

Synthetic wheat

Chromosome

A genome

B genome

D genome

A genome

B genome

D genome

1

43

49

23

53

50

66

2
3

42
27

45
36

30
11

49
30

49
40

66
54

4

55

28

20

54

62

75

5

45

35

6

37

31

58

6

39

47

25

42

50

87

7

54

56

23

51

56

71

Total *

305a

296a

138b

316a

338a

477c

nt

619

609

704

619

609

704

na

590

584

679

585

576

650

*Sums followed by the same letter are not statistically different at the 5% probability level.

positions (Additional file 1, Tables S1, S2). Because
chromosome 4A differs structurally from the Ae.
tauschii homoeologue, the distribution of diversity along
the chromosome was not investigated and it is not
included in Figure 2 and Additional file 1, Figure S2,
which illustrate the distribution of nucleotide diversity
and the number of haplotypes per gene among and
along the A-genome chromosomes. The distribution of
diversity on chromosome 4A relative to its rearrangements will be addressed separately. In wild emmer,
chromosome 5A also had lower diversity than the genome-wide average. Chromosome 5A of T. aestivum and
chromosomes 2A and 7A of wild emmer had higher
diversity than the genome-wide average. With the sole
exception of T. aestivum chromosome 2A, diversity was
low in genes in proximal chromosomal regions and high
in genes in distal chromosomal regions (Figure 2). In T.
aestivum chromosome 3A, most genes had only one or
two haplotypes (Additional file 1, Figure S2). Average
Tajima’s D was close to zero in most A-genome chromosomes in T. aestivum with the exception of chromosome 7A, which had a negative average value, and
chromosome 6A, which had a positive average value
(Table 8). In wild emmer, chromosome 4A had a

negative Tajima’s D and chromosomes 2A and 7A had
positive values.
In the B genome of T. aestivum, chromosome 2B had
higher diversity and chromosome 4B had lower diversity
than the rest of the chromosomes (Table 8). Diversity
was low across the entire chromosome 4B (Figure 3),
which also had the lowest number of haplotypes per
locus and lowest haplotype diversity (Table 9). Except
for genes in the distal region of the short arm of 4B, in
which three haplotypes were observed in several genes,
the proximal region of the short arm and the entire
long arm had only one or two haplotypes per gene
(Additional file 1, Figure S3). The sole exception to this
trend in the entire long arm was locus BF201102, which
had three haplotypes. However, the third haplotype
caused by a singleton SNP that was not observed in any
of the remaining 31 accessions could be a sequencing
error. No other B-genome chromosome showed a similar pattern either in T. aestivum or wild emmer (Figure
3 and Additional file 1, Figure S3). Wild emmer chromosome 5B had reduced diversity, lower number of
haplotypes per gene, and lower haplotype diversity compared to the genome mean (Tables 8 and 9). Diversity
was reduced only in the proximal regions of both arms

Table 7 Total nucleotide diversity, diversity in coding sequences, noncoding sequences (introns and UTRs),
replacement and silent codon positions
Genome

Coding
(× 10-3)

Total diversity
(× 10-3)

Noncoding
(× 10-3)

Replacement
(× 10-3)

Silent
(× 10-3)

Ratio replac./silent

θw

θπ

D

θw

θπ

θw

θπ

θw

θπ

θw

θπ

θw

θπ

T.a. A

0.59c

0.57c

-0.09b

0.54b

0.55b

0.76bc

0.72bc

0.30b

0.30b

1.38bc

1.46a

0.25a

0.24a

T.a. B

0.56c

0.57c

0.00b

0.55b

0.59b

0.89bc

0.89b

0.32b

0.36b

1.40bc

1.44a

0.22a

0.22a

T.a. D

0.22d

0.18e

-0.57c

0.22c

0.18c

0.36d

0.32d

0.06c

0.06c

0.79d

0.59b

0.10b

0.11b

T.d. A
T.d. B

0.68b
0.76b

0.70d
0.72cb

0.09b
-0.12b

0.58b
0.56b

0.60b
0.55b

0.91bc
1.21b

0.98b
1.17b

0.32b
0.31b

0.36b
0.30b

1.43bc
1.47b

1.47a
1.46a

0.23a
0.29a

0.26a
0.29a

Synth. D

1.31a

1.51a

0.56a

1.13a

1.25a

1.84a

2.09a

0.52a

0.53a

3.35a

3.87d

0.17ab

0.16b

* Means sharing the same letter are not significantly different at the 0.05 probability levels.

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Page 8 of 22

Table 8 Average nucleotide polymorphism (θw), nucleotide diversity (θπ), and Tajima’s D per chromosome
T. aestivum
A genome
(× 10-3)

Wild emmer

B genome
(× 10-3)

D genome
(× 10-3)

A genome
(× 10-3)

Synthetic wheats

B genome
(× 10-3)

D genome
(× 10-3)

Chromosome

θw

θπ

D

θw

θπ

D

θw

θπ

1

0.67

0.64

-0.14

0.66

0.71

0.07

0.31

0.32

-0.10*

0.69

0.73

0.09

0.82

0.90

0.37

1.18

1.28

D

θw

θπ

D

θw

θπ

D

θw

θπ

D
0.42

2

0.56

0.57

0.08

0.74* 0.87*

0.30

0.40

0.29

-0.65

0.83* 0.96*

0.50*

0.79

0.81

0.06

1.07*

1.23

0.57

3

0.49

0.46

-0.21

0.66

0.30

0.11* 0.08*

-0.79

0.60

0.65

0.21

0.81

0.76

-0.10

1.16

1.37

0.73

4

0.45*

0.42* -0.11 0.24* 0.16* -0.78*

0.15

0.10

-0.77

0.59

0.50* -0.39*

0.75

0.61

-0.56*

1.19

5

0.77*

0.76*

0.21

0.09* 0.06*

-0.84

0.54* 0.51*

6

0.60

7

0.63

Coef. variat.

0.18

0.72

1.41

0.61

-0.18

0.56* 0.47* -0.58* 1.65* 1.82*

0.39

0.80*

0.05

0.79

0.76

-0.07

1.67* 1.90*

0.44

0.69* 0.77*

0.38*

0.77

0.74

-0.05

1.34

1.63

0.78*

0.12

0.19

0.18

0.18

0.15

0.45

0.48

0.66

0.26

0.65

0.66

0.06

0.21

0.21

-0.27

0.77

0.51

-0.57

0.66

0.59

-0.28*

0.24

0.16

-0.86

0.30

0.38

0.52

0.59

0.21

0.15

0.23

* Means outside of the 99% bootstrap confidence interval of the genome mean

(Figure 3), and three or more haplotypes were observed
in many genes (Additional file 1, Figure S3). As in the A
genome, most chromosomes in both T. aestivum and
wild emmer showed low diversity in the proximal
regions (Figure 3). T. aestivum chromosome 4B and
wild emmer chromosome 5B had highly negative average Tajima’s D. In wild emmer, chromosome 4B also
had a negative Tajima’s D and a high ratio of silent to
replacement sites (Additional file 1, Table S1).
The D genome was the most uneven of the three
T. aestivum genomes in terms of average nucleotide
diversity per chromosome (Table 8). The coefficient of
variation among the D-genome chromosomes was three
times greater than in the D genome of synthetic wheats
(Table 8). Nucleotide polymorphism θw and nucleotide
diversity θπ, the number of haplotypes per gene H, and
haplotype diversity h were high in chromosomes 1 D
and 2 D compared to genome averages (Table 9) and
high values were distributed across the entire lengths of
the chromosomes (Figure 4). Diversity was low in chromosomes 3 D and 5 D and both chromosomes were
diversity impoverished across their entire lengths. Chromosome 4 D had low diversity across its length except
for the distal region of the long arm in which diversity
was high. A similar pattern was observed in chromosome 6 D, in which genes in both distal regions showed
relatively high diversity and those in the proximal
regions showed low diversity. In only a few genes were
there more than two haplotypes (Additional file 1, Figure S4). Genes with more than two haplotypes were
invariably in regions of elevated nucleotide diversity in
the D genome. Computation of ratios of silent and
replacement sites was greatly affected by the low levels
of diversity in the D genome and was of limited value
(Additional file 1, Table S2). All D-genome chromosomes had negative values of Tajima’s D but all seven
D-genome chromosomes in synthetic wheats had positive values of Tajima’s D (Table 8), which, like the

estimates of diversity, shows that negative Tajima’s D is
an attribute of the T. aestivum D genome but not of its
ancestor.
Wall’s B is a measure of intralocus linkage disequilibrium (LD). The higher the value of Wall’s B the greater
the proportion of neighboring sites in complete disequilibrium. In wild emmer and T. aestivum, the A and B
genomes showed similar values of Wall’s B, which ranged from 0.40 to 0.49 (Table 10). No significant differences were observed among the chromosomes. Triticum
aestivum chromosome 7B and the low diversity T. aestivum chromosome 4B had the highest Wall’s B values,
0.70 and 0.67, respectively, indicating that genes on
those chromosomes have on average the highest levels
of intralocus LD in the A and B genomes. The average
Wall’s B value of combined T. aestivum A and B genomes (0.49) was significantly higher (P = 0.024, paired
t-test) than that of wild emmer (0.41), indicating a
higher LD in T. aestivum than in wild emmer. In the D
genome, average Wall’s B (0.81) was significantly higher
than in the A and B genomes indicating stronger linkage
disequilibrium in the D-genome genes than in the
A- and B-genome genes.
Minor allele frequency in the populations of 10 wild
emmer chromosomes and 13 T. aestivum chromosomes
giving rise to the folded site frequency spectra were
computed for each polymorphic A, B, and D genome
locus (Figure 5). Folded site frequency spectrum measures the number of times a SNP is observed in a sample. In both species, most minor alleles were present
once. While the spectra were unimodal in T. aestivum
they were bimodal in wild emmer, with the sole exception of the B-genome silent site spectrum. The difference between the spectra of the two populations
suggests different demographic histories; the T. aestivum
spectra resemble those typical of an expanding population whereas wild emmer spectra resemble a spectrum
generated by a past bottleneck [36]. The folded spectra

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T. dicoccoides

T.aestivum
7.00

7.00

1A

6.00

6.00

5.00

5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79

2A

7.00
6.00

7.00
6.00
5.00

5.00
4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00

1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97

3A

7.00

Nucleotide diversity θπ (x 10-3)

1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97

7.00

6.00

6.00

5.00

5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00
1

4

7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64

1

7.00

5A

6.00
5.00

4

7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67

7.00
6.00
5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76

7.00

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76

6A

6.00

7.00
6.00

5.00

5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79

0.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79

7.00

7A

6.00

8.00
7.00

5.00

6.00

4.00

5.00
4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00
1

5

9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88

Sequence of loci along the chromosome
Figure 2 Nucleotide diversity θπ of individual A-genome genes. Gene diversity along the A-genome chromosomes in T. aestivum and wild
emmer (T. dicoccoides) in the Diyarbakir region in Turkey. Chromosome 4A is excluded because the order of genes does not conform to the Ae.
tauschii genetic map. Monomorphic loci are depicted with zero diversity. The gene order along the diversity maps in Additional file 2 Table S1 is
used on the X-axis, and the maps are oriented with the most distal gene in the short arm to the left. Centromere is indicated by a triangle.
Genetic distances between genes are not depicted.

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T.aestivum

T. dicoccoides

7.00

7.00

6.00

1B

5.00

6.00
5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79

9.00
8.00

2B

7.00

7.00

6.00

6.00

5.00

5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00
1

7.00

1

7

13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115

7.00

3B

6.00
5.00

Nucleotide diversity θπ (x 10-3)

0.00

5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89

6.00
5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00
1 4

7 10 13 16 19 22

25

0.00

28 31 34 37 40 43 46 49 52 55 58 61 64 67

1

4B

7.00
6.00

4

7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67

7.00
6.00

5.00

5.00

4.00

4.00

3.00

3.00

2.00

2.00
1.00

1.00

0.00

0.00
1

1

7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121

5B

7.00

7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115

7.00

6.00

6.00

5.00

5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70

6B

7.00

7.00

6.00

6.00

5.00

5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79

7B

7.00

7.00

6.00

6.00

5.00

5.00

4.00

4.00

3.00

3.00

2.00

2.00

1.00

1.00

0.00

0.00
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93

1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93

Sequence of loci along the chromosome
Figure 3 Nucleotide diversity θπ of individual B-genome genes. Gene diversity along the B-genome chromosomes in T. aestivum and wild
emmer (T. dicoccoides) in the Diyarbakir region in Turkey. See Figure 2 for details.

for the T. aestivum D genome declined faster than the
spectra for the T. aestivum A and B genomes, which is
consistent with higher numbers of rare polymorphisms
in the D genome than in the A and B genomes as
indicated by negative Tajima’s D for the T. aestivum Dgenome chromosomes.

Discussion
SNP discovery

A SNP discovery strategy based on the development of
GSPs and their deployment in the search for SNPs in
wheat is reported here. As a first step in SNP discovery,
a CP pipeline was built starting with 6,045 ESTs [37].

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Table 9 The average numbers of haplotypes per locus (H) and haplotype diversity (h)
T. aestivum

T. dicoccoides

Synthetic wheats

A genome

B genome

D genome

A genome

B genome

Chrom.

H

h

H

h

H

h

H

H

h

1

2.09*

0.29*

1.92*

0.26*

1.25

0.08*

2.13

0.30

2.17

0.36*

2.47

0.52

2

1.85

0.23

1.79

0.25

1.33*

0.11*

2.25*

0.33*

2.59*

0.34

2.40

0.47

3

1.65*

0.17*

1.78

0.24

1.13*

0.03*

2.03

0.30

2.11

0.32

2.26

0.41*

4

1.56*

0.17*

1.31*

0.07*

1.20

0.06

1.92

0.28

2.17

0.29*

2.24

0.43

5

2.04*

0.28*

1.68

0.21

1.11*

0.03*

2.01

0.31

1.99*

0.27*

2.43

0.50

6
7

1.71
1.84

0.22
0.18

1.86
1.74

0.25
0.20

1.27
1.31

0.08*
0.06

1.84*
2.14

0.27*
0.35*

2.22
2.33

0.35
0.35

2.63*
2.31

0.50
0.47

h

D genome
H

h

* Means outside of the 99% bootstrap confidence interval of the genome mean

For these ESTs and an additional 290 for which primers
were generated manually, only a small portion, about
17.4%, ultimately resulted in validated GSPs. Even
though the wheat genomes show low SNP levels, more
than half of the A- and B-genome GSPs yielded an SNP,
demonstrating that the development of GSPs is more
difficult in polyploid wheat than SNP discovery.
The rationale for GSP development is that they make
SNP markers versatile; any SNP detection method can
theoretically be used if GSPs are available for the site in
polyploid wheat. However, some SNP genotyping methods do not require prior PCR amplification of the SNPcontaining targets in polyploid wheat [38,39] making
GSPs superfluous. The cost/benefit ratio of GSP development should therefore be considered in the future
development of SNPs for wheat.
Another aspect of the SNP development strategy
employed here that needs consideration is the use of a
distantly related relative as a source of information about
the exon-splicing boundaries in ESTs for the design of
CPs [37]. The reliance on wheat-rice comparisons preferentially selected for the conserved gene repertoire, which
is concentrated in the proximal, low-recombination
regions of wheat chromosomes [5,30,40-42]. There is
also the potential that a focus on conserved loci could
result in a downward bias in diversity estimates.
A focus on single-copy loci may also affect the distribution of loci with SNPs along the chromosomes. In
wheat [40], as in other plants [43], single-copy genes are
preferentially located in the proximal, low-recombination regions whereas distal, high-recombination regions
are enriched for multigene families. Focusing on ESTs
from single-copy genes may cause preferential development of SNP markers for genes located in the proximal,
low-recombination regions of chromosomes.
For these multiple reasons, the SNP markers developed
here are more abundant in the proximal, low-recombination
regions of wheat chromosomes than in distal, high-recombination regions. This is particularly true for the distal 30 cM

of the short arms of chromosomes in homoeologous groups
1, 2, and 3, which are poorly populated with SNP markers.
Diversity maps

Comparative mapping based on RFLP markers showed
that gene order along the T. aestivum homoeologous
chromosomes is highly conserved and that any one
chromosome of a trio of homoeologous chromosomes
can be used to approximate gene order along the other
two [44] and, as a matter of fact, along homoeologous
chromosomes of other species throughout the tribe Triticeae [45]. Gene order is also surprisingly conserved
across the entire grass family. Approximately 64, 65, and
66% of the loci on the Ae. tauschii genetic map are
colinear with genes along the sorghum, B. distachyon,
and rice pseudomolecules, respectively [30].
The conservation of gene order among wheat homoeologous chromosome and across the grass family was
exploited here to summarize diversity in the wheat genomes using a single map. A comparative map of Ae.
tauschii [30] was selected for that purpose. The high
degree of gene synteny across grasses was exploited to
insert into that map additional genes that in wheat contain SNPs but could not be mapped in Ae. tauschii for
lack of polymorphism.
The utility of the Ae. tauschii linkage map as a representation of the linear order of genes in the wheat genomes depends on the extent to which the assumption of
colinearity of the Ae. tauschii and wheat chromosomes is
true. Known translocations exist among chromosomes
4A, 5A, and 7B, and chromosome 4A also acquired pericentric and paracentric inversions [33,34]. For chromosomes 4A and the translocated regions of 5A and 7B, the
diversity maps reported here are of limited relevance.
Since virtually all of the ESTs employed in SNP discovery here had been previously mapped on the wheat
deletion-bin maps, this is the first time it is therefore
possible to compare the wheat bin maps with a high
density genetic map of a closely related genome. The

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T.aestivum

Page 12 of 22

Table 10 Average values of Wall’s B in the N number of
genes
T. aestivum

1D

4.00

T. dicoccoides

Chromosome

N

Wall’s B

N

Wall’s B

1A

18

0.29c*

15

0.31ab

2A
3A

25
9

0.57ab
0.42bc

13
17

0.60a
0.36ab

4A

12

0.82a

16

0.59a

5A

24

0.40c

15

0.19b

6A

20

0.45bc

14

0.35ab

7A

3.50

23

0.50bc

14

0.42ab

3.00
2.50
2.00
1.50
1.00
0.50
0.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88

2D

3.50
3.00
2.50
2.00

Mean

1.50

0.49a

0.40a

1.00

3D

3.00

Nucleotide diversity θπ (x 10-3)

13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115

2.50
2.00

23
36

0.44ab
0.28a

3B

18

0.33bc

17

0.37ab

9

0.67a

29

0.49b

12

0.45ab

19

0.46ab

26

0.42b

24

0.38ab

7B

7

0.32bc
0.46ab

6B

1

3.50

25
24

5B

0.00

1B
2B
4B

0.50

27

0.70a

26

0.48ab

1.50
1.00
0.50
0.00
1

7

13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109

4D

3.50

Mean

0.48a

1.50
1.00
0.50
0.00
1

6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101

5D

3.50

2

1.00a

3

0.78a

5D

2.00

2

1.00a

6D

8

0.46b

7D

6

0.41a

0.88a
0.82a

4D

2.50

11
15

3D

3.00

1D
2D

0.75ab

3.00

Mean

2.50

0.81b

2.00

*Means followed by the same letter are not statistically different at the 5%
probability level.

1.50
1.00
0.50
0.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73

6D

3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
1

3.50

6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106

7D

3.00
2.50
2.00
1.50
1.00
0.50
0.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88

Sequence of loci along the chromosome
Figure 4 Nucleotide diversity θπ of individual D-genome genes.
Gene diversity along the D-genome chromosomes in T. aestivum.
See Figure 2 for details.

Ae. tauschii genetic map that formed the backbone of
the diversity maps was highly colinear with rice, B. distachyon, and sorghum genomic sequences [30,31].
There was a remarkably good agreement between the

deletion-bin maps and the Ae. tauschii genetic map for
most chromosome arms and discrepancies were found
for less than 10% of the loci. Some of these discrepancies were biological in nature. The greatest number of
discrepancies was in the B-genome deletion-bin map
and the smallest in the D-genome deletion-bin map.
The numbers of paralogous loci in the B genome outnumber those in the A or D genomes 2 to 1 [41]. The B
genome is also more prone to translocation [46-48] and
undoubtedly other structural changes. Both paralogous
gene duplications and changes in chromosome structure
manifest themselves as breaks in synteny between the
Ae. tauschii genetic map and wheat deletion-bin maps.
The poorest fit between the genetic map and the deletion-bin maps found here for the B genome is therefore
consistent with greater divergence of the B genome relative to the A and D genomes.
Although the wheat D-genome map was the most
similar to the Ae. tauschii map of the three wheat deletion-bin maps, it too showed discrepancies relative to
the Ae. tauschii map in several chromosome arms. The
largest number of loci showing a perturbed location on

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Page 13 of 22

1000

A

Number of SNPs

900

Td (A) silent

800

Td (A) replacement

600

Td (B) replacement

B

Td (B) silent

700

700
Td (A) silent

600

Td (B) silent

500

Td (D) silent
Td (A) replacement

400

Td (B) replacement

500
300

400
300

Td (D) replacement

200

200
100

100
0

0
1

2

3

4

5

1

2

3

4

5

6

Number of chromosomes
Figure 5 The folded site frequency spectra. Folded site frequency spectra of minor SNP alleles at silent and replacement positions in the A
and B genomes, Td (A) and Td (B) respectively, in a sample of 10 homozygous accessions of wild emmer (T. dicoccoides). Each homozygous
accession is equivalent to one chromosome. The plot depicts numbers of SNPs with the minor allele being observed in an indicated number of
sampled chromosomes. (B) The folded site frequency spectrum of minor SNP alleles at the silent and replacement positions in the A, B, and D
genomes, Ta (A), Ta (B), and Ta (D), respectively, in a sample of 13 homozygous accessions of T. aestivum. Each homozygous accession is
equivalent to one chromosome. The plot depicts numbers of SNPs with the minor allele being observed in an indicated number of sampled
chromosomes.

the deletion-bin map was observed in chromosome arm
4DL. Ordering of loci in the 4DL arm bins on the basis
of the Ae. tauschii genetic map resulted in interdigitation of loci mapped in the neighboring bins 4DL12 and
4DL13. The Ae. tauschii genetic map shows many rearrangements in that region compared to rice chromosome Os3 [30]. It is therefore possible that chromosome
4 D may contain a paracentric inversion spanning the
boundary of bins 4DL12 and 4DL13, which could
account for the difficulties encountered during an
attempt to recombine wheat homoeologous chromosome arms 4DL and 4BL in the KNA1 region [49].
A total of 36% of the loci on the diversity maps was
mapped on the basis of synteny with rice. Even though
mapping of these loci was based on several lines of corroborating information, it is nevertheless an inference
and must be treated with caution. The prerequisite corroborating information was not available for the remaining 209 (11.7%) of the A-, B-, and D-genome loci
harboring SNPs, and these markers were neither
included on the diversity maps nor used in computations of diversity estimates, although they were included
in the database http://probes.pw.usda.gov:8080/
snpworld/Search. The most frequent reason for the
inability to map a locus on the basis of synteny was the
failure to identify an orthologous region in rice. Synteny
is more rapidly lost in the distal regions of wheat chromosomes due to greater rates of gene deletions and
gene duplications in the distal regions than in the proximal regions [5,40,41]. This factor contributed to the
poor SNP marker coverage in the distal regions of some
of the chromosomes. For the same reasons, however,

ESTs harboring SNPs that could not be mapped on the
basis of synteny are preferentially located in distal chromosome regions. The project SNP database should
therefore be interrogated if additional SNPs are needed,
particularly those in the distal chromosome regions.
Genetic application of the diversity maps

The diversity maps reported in Additional file 2, Table S1
provide a convenient summary of SNPs http://probes.pw.
usda.gov:8080/snpworld/Search in genes that were
mapped on the Ae. tauschii map. A θw value of zero indicates no SNP was present and high values suggest several
SNPs at a locus in the respective population of T. aestivum and wild emmer lines. Negative Tajima’s D values
indicate low frequency SNPs and positive Tajima’s
D values indicate a predominance of intermediate
frequency SNPs at a locus.
Tetraploid wheats were parents of nine synthetic
wheats that were screened with data subsequently
reported in the SNP database. They included durum lines
(Sn24, Sn29, Sn30, and Sn31), the tetraploid component
of T. aestivum ’Canthatch’ (Sn25 to Sn28), and an emmer
line (Sn31). SNPs present in these lines are tabulated in
the database. Because they were not used in the computation of diversity measures, θw may be 0.00 for a gene in
Additional file 2, Table S1 but SNPs may exist in the A
and B genomes of synthetics wheats in the database. This
fact should be kept in mind when a specific locus is interrogated for a SNP on the diversity maps.
Although synthetic wheats RL5402, RL5403, RL5405,
and RL5406 share tetraploid Canthatch as the source of
their A and B genomes, they are occasionally polymorphic

Akhunov et al. BMC Genomics 2010, 11:702
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in the database. The tetraploid Canthatch was developed
by recurrent backcrossing of the pentaploid hybrid
T. durum ’Steward’ × T. aestivum ’Canthatch’ to T. aestivum Canthatch selecting tetraploid offspring in each generation [50]. SNPs occasionally observed among the four
synthetic wheats are presumably residual germplasm of
T. durum Steward present in the tetraploid Canthatch,
indicating that a complete extraction of hexaploid wheat
A and B genomes was not reached in tetraploid Canthatch
and that the tetraploid is heterozygous at some loci.
Sampling nucleotide diversity for SNP development

Nucleotide diversity measured as θπ was similar in the
T. aestivum A and B genomes and averaged 0.59 × 10-3,
which is close to an estimate of 0.8 × 10-3 reported earlier [16]. The agreement between these two independent
studies suggests that the sample of the T. aestivum lines
used here was representative of T. aestivum and was
adequate for SNP discovery in all three wheat genomes.
However, nucleotide diversity averaged across genomes
of wild emmer (θ π = 0.72 × 10-3 ) was lower than the
estimated θ π = 2.7 × 10 -3 for wild emmer as a whole
[16] indicating that the population in the Diyarbakir
region has low diversity relative to species-wide samples
of wild emmer. This is consistent with earlier RFLP
results [22], which indicated that the greatest diversity
in wild emmer exists in northern Israel, southern Lebanon, and southwestern Syria [22]. Because T. aestivum
originated in Transcaucasia [35], the failure to sample
wild emmer in those regions may have had a limited
effect on the discovery of SNPs relevant for hexaploid
wheat. However, it must have had a great effect on the
discovery of SNPs relevant for durum wheat, because
durum wheat originated in the eastern Mediterranean
[22]. Inclusion of only a few durum accessions in the
sample screened for SNPs here was inadequate to characterize durum diversity, and an additional SNP search
is needed for cultivated tetraploid wheat.
Wheat diversity architecture

In spite of the fact that the three T. aestivum genomes
have coexisted within a single nucleus since the origin
of T. aestivum, profound differences were found among
them. The A and B genomes are more diverse and show
more uniform distributions of diversity across the genome than does the D genome. Because of the short time
that has elapsed since the origin of T. aestivum, 8,500
years or less [10], it is unlikely that most SNPs observed
in T. aestivum originated there. It is much more likely
that SNPs were contributed by gene flow from the
ancestral species, tetraploid wheat and diploid Ae.
tauschii, or potentially polyploid species of Aegilops
having a D genome, such as Ae. cylindrica, that occasionally hybridize with wheat [51,52].

Page 14 of 22

This intuitive argument is supported by differences in
the ratio of replacement to silent polymorphisms in the
T. aestivum genomes. Evolution in young polyploids is
accompanied by relaxed purifying selection acting on
genes, which is shown by an order of magnitude greater
rate of fixation of deletions of single-copy genes in tetraploid wheat than in diploid Ae. tauschii and T. urartu
[5]. If SNPs observed in T. aestivum were contributed
by gene flow, genes in the A and B genomes should
show ratios of replacement to silent site variation shifted
towards 1.0 (indicating relaxed selection) compared to
those in the D genome, which was observed. Additionally, if the haplotypes present in T. aestivum were largely contributed by gene flow, this could increase the
effective population size Ne of the A and B genomes
relative to the D genome because haplotype recombination in the A and B genomes could have taken place
during the evolution of wild emmer. Hence LD in the A
and B genomes of T. aestivum is expected to be stronger than in the A and B genomes of wild emmer and
LD in the D genome of T. aestivum is expected to be
stronger than in the T. aestivum A and B genomes,
which is what was observed. We therefore conclude that
most of the differences in diversity between the A and B
genomes on the one hand and the D genome on the
other hand can be attributed to differences in gene flow.
The difference in gene flow among the genomes has a
material basis. It is well known that very little reproductive isolation exists between hexaploid and tetraploid
wheat because these species readily hybridize and the
resulting pentaploid hybrids are usually fertile [53]. In
contrast, hybridization between hexaploid wheat and Ae.
tauschii is difficult and hybrids are sterile [54]. Landraces of hexaploid and tetraploid wheat have often been
grown together, which has facilitated hybridization. In
contrast, sympatry between T. aestivum and Ae. tauschii
has been limited by the geographic distribution of Ae.
tauschii. Greater gene flow from the T. aestivum ancestors into the A and B genomes than into the D genome
is therefore expected.
This study substantiated a previous survey of modern
wheat varieties with SNPs developed here [55] and
showed that limited gene flow into the T. aestivum D
genome has enriched it for rare alleles. The preponderance of rare alleles in the D genome is indicated by the
negative average Tajima’s D observed in all seven Dgenome chromosomes. Site frequency spectra in the T.
aestivum genomes show a steeper decline in the D genome than in the A and B genomes, which is consistent
with more limited gene flow into the T. aestivum D
genome than into the A and B genomes. These observations agree with previous isozyme, RFLP, and SNP studies on the origin of hexaploid wheat, which suggested
that wheat originated via a very limited number of

Akhunov et al. BMC Genomics 2010, 11:702
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hybridization events [23,24,26,56-58]. SNP data generated here showed that 93% of the 138 polymorphic
genes in the D genome include only two haplotypes.
Diversity contributed by gene flow into wheat was
further shaped by several factors. One was reduced
effective recombination accompanying self-pollination,
the prevalent mating system in wheat. Self-pollination
can reduce the effective population size to half that
expected under cross-pollination [59] and enhance the
effects of genetic drift on diversity [60]. Self-pollination,
by greatly impacting effective recombination [59],
increases the sizes of chromosomal segments hitchhiking along with positively selected genes [61-64]. Low
effective recombination is likely one of the contributing
factors of the greatly uneven distribution of diversity in
the T. aestivum D genome compared to the A and B
genomes; the average θπ per chromosome was found to
be six-fold higher in the most-diverse D-genome chromosome compared to the least-diverse D-genome chromosome. Diversity is high along the entirety of
chromosomes 1 D and 2 D, the distal portion of the
long arm of chromosome 4 D, and both distal regions
of chromosome 6 D. In contrast, the entirety of chromosomes 3 D and 5 D, three-quarters of chromosome 4
D, and proximal regions of 6 D have very low levels of
diversity. This suggests that under limited gene flow and
self-pollination, genetic drift and selection may impact
diversity along large chromosomal regions in wheat.
Several A- and B-genome chromosomes show that
effects shaping the diversity of entire chromosomes may
occasionally take place even under the regime of moderate gene flow in polyploid genomes. Diversity in T. aestivum chromosome 4B mimics in all respects diversity
in the D genome. The entire chromosome is diversity
impoverished and the chromosome has a highly negative
Tajima’s D. As in the D-genome chromosomes, most of
the 4B genes have either one or two haplotypes. Chromosome 4B is polymorphic for a pericentric inversion in
T. aestivum [65], and homoeologous group 4 has a
lower number of genes than the remaining six Triticeae
homoeologous groups [29], presumably due to the
translocation of the gene-rich terminal region of the
short arm of chromosome 4 to the long arm of chromosome 5 [30]. Recombination takes place primarily in
genes. Low number of genes on chromosome 4B would
probably result in low crossover frequencies in this
chromosome, which was observed [66]. The net effects
of limited effective recombination may be that a large
portion of this chromosome has hitchhiked during episodes of positive selection during the evolution of T.
aestivum or was subjected to a reduction in effective
population size during episodes of background selection
[60]. A long-range loss of diversity may have also taken
place in wild emmer chromosome 5B, which also has a

Page 15 of 22

negative average Tajima’s D. Another chromosome in
which a chromosome-sized loss of diversity has taken
place is 4A. In this chromosome, the loss of diversity
was undoubtedly caused by the fixation of inversions
suppressing recombination in a heterozygous state.
Another factor that must have had a significant
impact on the architecture of diversity in wheat is the
expression of the Ph1 locus, which is unique to polyploid wheat. Its primary function is to preclude recombination between homoeologous chromosomes [67-69].
Importantly, Ph1 also negatively effects recombination
between heterozygous homologues [66]. The activity of
Ph1 therefore has similar effects on diversity as self-pollination. For an unknown reason, Ph1 negatively affects
recombination in the B genome more than in the A
genome [66]. The T. aestivum B genome shows greater
variation in diversity among chromosomes than the A
genome. The coefficients of variation were 0.18 and 0.21
for θw, and θπ among the T. aestivum A-genome chromosomes but were respectively 0.30 and 0.38 among the
T. aestivum B-genome chromosomes, which is consistent with more reduced recombination in the B genome
than in the A genome due to Ph1 effects. Recombination between the Ae. tauschii chromosomes and wheat
D-genome chromosomes is even more affected by Ph1
than recombination between wheat heterozygous homologues [70]. In agreement, T. aestivum D-genome chromosomes show the greatest variation in diversity among
the three genomes; coefficients of variation were respectively 0.52 and 0.59 for θw, and θπ among the D-genome
chromosomes. We suggest that the synergy of self-pollination and suppression of recombination due to Ph1
results in high levels of random drift, loss of diversity
from large chromosome regions, and relatively high variance in diversity among chromosomes.

Conclusions
Distinctly different diversity patterns were found in two
closely related polyploid species of differing age, the
recently evolved T. aestivum and the older wild emmer.
In wild emmer, diversity is uniform among genomes and
chromosomes but in T. aestivum, diversity is heterogeneous both among both genomes and chromosomes.
These observations suggest the following scenario of
polyploid evolution. In a nascent polyploid, diversity
almost entirely depends on gene flow from the ancestral
species. During that period, diversity is greatly affected by
stochastic and directional processes, particularly under
self-pollination that is wide spread in polyploids. Dependence on gene flow and the synergy of self-pollination
and action of Ph1-like genes results in low and heterogeneous diversity across genomes. If gene flow cannot keep
pace with the population expansion, diversity is dominated by rare alleles. Large chromosomal regions or

Akhunov et al. BMC Genomics 2010, 11:702
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whole chromosomes are subjected to genetic drift and
hitchhiking resulting in their low diversity. As time
passes, the accumulation of new mutations results in an
increased and more uniformly distributed diversity across
the genome, as is seen in wild emmer.

Methods
CP design

ESTs showing simple cDNA hybridization profiles with
T. aestivum genomic DNA in Southern blots http://
wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi were
selected from the wEST database [71] for CP design.
Only ESTs mapped on the wheat deletion bin maps
[32,72-77] were used. The wheat deletion-bin maps
were constructed by hybridization of random cDNA
clones with DNAs of 101 deletion stocks [78] and a set
of wheat telocentric stocks [79] that subdivided the 21
wheat chromosomes into 159 bins [80]. CPs located in
exons flanking one or more introns were designed on
the basis of comparison of wheat EST sequences with
rice genomic sequence. EST contigs or EST singletons
were extracted from the wEST database http://wheat.pw.
usda.gov/cgi-bin/westsql/map_locus.cgi and compared
with rice genomic sequences to identify exon/exon junctions. The Primer3 program [81] was modified for PCR
primer design in batch mode [82]. A pipeline for batch
homology search between wheat ESTs and rice genomic
sequence http://avena.pw.usda.gov/SNP/new/bioinformatics.shtml was built [37,82]. With this pipeline, PCR
primers were successfully designed for 2,223 EST unigenes, 1,958 EST contigs and 265 EST singletons. Of
these, primer pairs for 1,624 were from 5’ ESTs and 599
from 3’ ESTs. Since these primers were located in exons
and were designed on the basis of homology with the
rice exonic sequences, they were highly conserved in
grasses; hence their name. An additional 290 primers
for loci in the distal bins were designed manually using
the Primer3 program or the GeneTools primer design
program [83].
GSP design

Genomic DNAs of T. urartu accessions G1812 (PI
428198, Turkey) and ICTW600161 (Syria, supplied by J.
Valkoun, ICARDA, Syria), Ae. tauschii ssp. strangulata
accession AL8/78 (Armenia, supplied by V. Jaaska, Estonian University, Tartu), and Ae. tauschii ssp. tauschii
accession AS75 (Shaanxi, China) (Table 11) were used
as PCR templates. Pairs of accessions were selected
among 193 and 188 accessions representative of the
geographic distribution of T. urartu and Ae. tauschii¸
respectively, and genotyped with random restriction
fragment length polymorphism (RFLP) markers [23].
A pair of genetically distant accessions was selected
within each species. Target DNAs were PCR amplified

Page 16 of 22

using CPs and amplicons were directly sequenced, using
CPs as sequencing primers. Triticum uratu and Ae.
tauschii are self-pollinating species, and the four accessions were assumed to be homozygous at the targeted
loci but Ae. speltoides is cross-pollinating, and it was
expected to be heterozygous at many loci targeted for
sequencing. DNA of two randomly selected Ae. speltoides F4 plants from the cross 2-12-4 × PI 136909-12II/134-1 [84] were therefore used as PCR templates in
the hope that at least one was homozygous at a targeted
locus and the amplicon could be sequenced.
B-genome sequences were obtained from ‘Langdon’
durum wheat by PCR amplification of Langdon genomic
DNA using CPs. The amplicons were purified using the
Promega PCR amplicon purification kit and cloned to
the TA cloning site of the pGEM-T vector (Promega) following manufacturer’s recommendations, and after transformation, E. coli cells were plated on LB agar medium.
Twelve positive transformants were picked and plasmid
inserts were PCR amplified using M13-48 and T7 Universal Primers. The PCR reaction consisted of 1X Taq
polymerase buffer, 0.2 mM dNTPs mix, 50 pmols of primers, 1U of Taq polymerase, and sterile distilled water.
PCR conditions were 10 min at 94°C, 10 cycles of 94°C
for 20 sec, 58°C for 20 sec, and 72°C for 2 min, followed
by 35 cycles at 94°C for 20 sec, 55°C for 20 sec, and 72°C
for 2 min. PCR was terminated by final extension at 72°C
for 5 min. Success of PCR amplification was checked by
1% agarose electrophoresis. For sequencing, 5μl of amplified DNA was treated with exonuclease I (USB) and
shrimp alkaline phosphatase (USB) according to manufacturer’s recommendations in a 10μl reaction volume.
The reaction was diluted to 18μl with water before an aliquot was taken for subsequent sequencing. The clones
were sequenced as described below. The sequences of
the clones were compared with the T. urartu and Ae.
speltoides sequences and each Langdon clone was
assigned to either the A or B genome. Because CPs
annealed to both A- and B-genome templates during
PCR amplification of Langdon DNA, chimeric amplicons
could be generated during the amplification process [85].
A-genome/B-genome chimeric clones were occasionally
encountered and attention was paid to their presence
during the assignment of sequences to genomes.
The T. urartu, Ae. speltoides, Ae. tauschii, and Langdon sequences were assembled into contigs with the
Staden program [86]. Clean FASTA sequences were
aligned with ClustalW [87] or MUSCLE [88] programs.
Sequence alignments were visually checked using Bioedit (Tom Hall, Ibis Therapeutics, Carlsbad, CA). Genome-specific nucleotide substitutions were recorded and
GSPs were designed. The primers were limited to the
Tm range of 55 to 60°C, so that subsequent PCR amplifications could be performed in batches. Genome-

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Table 11 Diploid and tetraploid species used for the development of genome-specific primers
Species

Database code

Genome

Line

Origin

T. urartu

Tu01

A

PI428198 = G1812

Turkey

T. urartu

Tu02

A

ICTW600161 (ICARDA)

Syria

Ae. speltoides

As01

S

F4 /2-12-4//PI136909-12-II/134-1

Experimental line

Ae. speltoides

As02

S

F4 /2-12-4//PI136909-12-II/134-2

Experimental line

Ae. tauschii

At01

D

AL8/78

Armenia

Ae. tauschii

At02

D

AS75

China

T. turgidum ssp. durum

Lgd1 through 12

AB

Langdon, 12 clones

Cultivar

specific nucleotide substitution was used as the 3’ end of
each GSP [89]. In addition, the third nucleotide from
the 3’ end was occasionally purposely mismatch with
the template to increase the genome specificity of the
primer. These modifications are included in the GSPs
reported in the SNP database http://probes.pw.usda.
gov:8080/snpworld/Search. A GSP was combined with
one of the CPs to obtain a primer pair for genomespecific amplification. Most of the amplicons therefore
consisted of exonic and intronic sequences.
GSP validation

Because the bin location of each targeted gene was
known, only the chromosomes of the homoeologous
group in which the bins were located were used for
PCR validation of GSPs. DNA was PCR amplified from
the relevant N-T in the T. aestivum ’Chinese Spring’
genetic background [90]. If a GSP functioned properly,
DNA of the N-T line nullisomic for the chromosome in
the targeted genome produced no amplicon but DNA of
the N-Ts for the remaining two chromosomes of the
homoeologous group produced amplicons. Primers that
produced amplicons with DNA of all three N-Ts failed
the validation step. This could happen if the gene was
actually located in a different homoeologous group than
assumed. Primers that failed the N-T test were therefore
used in PCR with N-Ts for all 21 wheat chromosomes.
If amplification occurred in all but one N-T, it was
assumed that the targeted gene was located on the chromosome that was absent in the N-T line that failed to
produce an amplicon. Such primers were considered
validated. If none of the N-Ts consistently showed
absence of an amplicon in one of the N-T lines, the
putative GSP was discarded.
SNP discovery

To maximize the likelihood of the relevance of discovered SNPs to cultivated wheat while minimizing the
number of lines screened for SNPs, a resequencing
panel representative of lines of wild emmer from the
Diyarbakir region (10 lines) and T. aestivum (13 lines)
(Table 1) was used. Twelve of the 13 T. aestivum lines
were selected from representative branches of a

neighbor-joining tree constructed for 476 T. aestivum
lines genotyped at 153 RFLP loci [91] (Additional file 1,
Figure S1). One T. aestivum line (’Opata 85’) was added
because it was one of the parents of the International
Triticeae Mapping Initiative (ITMI) mapping population
(Table 1) [92]. The wild emmer lines were selected from
wild emmer populations in the Diyarbakir region so that
each represented a branch in a neighbor-joining tree
(Additional file 1, Figure S5) based on genetic distances
using 131 RFLP loci [22]. In addition, 9 synthetic hexaploid wheats produced by crossing tetraploid wheat with
Ae. tauschii and doubling the chromosome number [12]
were included in the screening population. Synthetic
wheat is used in wheat breeding as a source of new Dgenome variation [93-95]. Four synthetics (Sn25 through
Sn28, Table 2) selected for the project were supplied by
E.R. Kerber (Agriculture Canada, Winnipeg). They were
included because they were previously used as sources
of Ae. tauschii chromosomes in the development of
disomic substitution lines of single Ae. tauschii chromosomes in the Chinese Spring genetic background (J.
Dvorak, unpublished). The donor of the A and B genomes of these synthetic wheats was a tetraploid extraction of T. aestivum ’Canthatch’ [50]. Synthetic Sn24 was
the parent of the ITMI mapping population [92]. Synthetic wheats Sn29 through Sn31 were extensively used
in the CIMMYT wheat breeding program. Finally, synthetic wheat Sn32 was the parent of a RFLP mapping
population [44]. The tetraploid parents of Sn24, Sn29,
Sn30, and Sn31 were durum and that of Sn32 was
emmer. The Ae. tauschii parents of the synthetics, if
known, are indicated in Table 2.
Target DNAs of these 32 lines were amplified with
GSPs, the sequences were aligned and edited as
described above and SNPs were submitted to the central
database http://probes.pw.usda.gov:8080/snpworld/
Search. All wheat sequences were also submitted to
NCBI. Their accession numbers are HQ389550 to
HQ391340.
DNA sequencing

As explained above, a GSP primer pair consisted of a
CP and a GSP primer. PCR amplification was performed

Akhunov et al. BMC Genomics 2010, 11:702
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in seven different labs but sequencing of the amplicons
was performed centrally at the Western Regional
Research Center, USDA-ARS, Albany, California. Two
replicas containing 96 different processed amplicons
were made in semi-skirted 96-well PCR plates. To make
the first replica, 3 μl of a processed amplicon and 1μl of
the corresponding CP primer (3.2 pmol/ul) were placed
into a well of one plate. To make the second replica,
3 μl of a processed amplicon and 1μl of the corresponding GSP primer (3.2 pmol/ul) were placed into the corresponding well of a second plate. The plates were
frozen and shipped on dry ice to the sequencing lab
along with a directory of the amplicons in each well.
The plates were thawed in the sequencing lab and
1.5μl of 5X sequencing buffer, 1μl of 50% DMSO, 1μl of
Big Dye v.3.1, and 2.5μl of deionized water were added
to each well. The cycling conditions were: 5 min at 98°C
followed by 40 cycles at 10 sec at 96°C, 5 sec at 50°C,
and 4 min at 60°C. DNA was precipitated with ethanol
followed by a 70% ethanol rinse, dried, 12 μl of sequencing grade formamide was added, and the DNA was
sequenced on an ABI3730xl. Both strands of each
amplicon were sequenced; one was produced in the
plate containing CPs as sequencing primers (1 st DNA
strand) and the other was produced in the plate containing GSPs as sequencing primers (2nd DNA strand).
The Phred/Phrap [96] or Staden package http://staden.
sourceforge.net/ programs were used for base calling
and assembly of sequencing trace files. Assembled contigs were edited with the Staden package. Perl and Java
programs were written to manipulate the data. The
PolyPhred v. 5.0 program [97] and mutation detection
modules from the Staden package were utilized for SNP
detection.
Map construction

A genetic map based on segregation of markers in a
population of 572 F2 plants from the cross Ae. tauschii
AL8/78 × Ae. tauschii AS75 [30] was used as a backbone for the development of diversity maps. The backbone map contained 878 markers of which 863 were
ESTs; 12 of the remaining loci were random RFLP markers and three were microsatellite loci. ESTs were
mapped either on the basis of RFLP or SNP. The latter
were mapped with the SNaPshot™SNP assay (Applied
Biosystems, Foster City, California) or GoldenGate BeadArray SNP assay (Illumina Inc., SanDiego, California).
A total of 174 F2 plants was used for RFLP and SNaPshot mapping and 560 F2 were used for Illumina GoldenGate assays.
The EST markers were compared with the NCBI rice
genomic sequence to assess the wheat-rice macrosynteny
(henceforth synteny) [30]. Loci that cosegregated were
grouped into “recombination blocks” [30] within which

Page 18 of 22

they were arranged to parallel the order of orthologous
genes in rice. Genes that could not be mapped because of
the lack of polymorphism between the parents of the Ae.
tauschii mapping population were inserted into the Ae.
tauschii map at a location corresponding to that of a
putative rice orthologue, provided that the following conditions were met: (1) the allocation of the gene to wheat
chromosome by PCR using N-T lines and GSPs agreed
with the previous deletion-bin mapping, (2) the section
of the wheat chromosome in which the gene resided was
homoeologous with the rice chromosome on which the
rice orthologue resided, and (3) the bin location of the
gene http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi agreed with the location of the putative rice
orthologue. If any ambiguity was encountered, the location of the gene on the Brachypodium distachyon and
sorghum pseudomolecules [30,31] was taken into
account. The loci inserted into the diversity maps on the
basis of synteny with rice are indicated in Additional file
2, Table S1 by having no cM value assigned.
Comparisons of genetic maps with wheat deletion-bin
maps

The positions of genes on the Ae. tauschii genetic map
were compared with their locations on the wheat deletion-bin maps compiled in the GrainGenes database
http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi.
The deletion bins in Additional file 2, Table S1 were
named according to the proximal breakpoint delimiting
a bin. The proximal-most bin delimited by the centromeric break on the proximal side received the letter c
(for centromeric) following the chromosome arm name.
Bins within a chromosome were arbitrarily colored in
Additional file 2, Table S1. If a gene was previously
mapped into a wheat bin located in a different homoeologous group, the cell of the bin map was not colored. If
no bin information was available for a locus, the cells
were left blank in Additional file 2, Table S1. The bin
location of a locus was considered inconsistent with the
location of the locus on the deletion-bin map if it conflicted with the linear order of recombination blocks on
the genetic map.
Diversity estimation

Diversity was estimated only for mapped loci. The edited alignments of the 13 T. aestivum lines were compared. Nucleotide polymorphism θ w [98], nucleotide
diversity θ π [18], average number of haplotypes per
locus (H), haplotype diversity (h) [99], Tajima’s D [100],
and Wall’s B [101] were computed for each gene using
tools from the libsequence library [102]. The same
descriptive statistics were computed for 10 lines representative of wild emmer in the Diyarbakir region and 9
accessions of synthetic wheat.

Akhunov et al. BMC Genomics 2010, 11:702
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An unbiased estimation of diversity of a population
requires the alignment of homologous sequences. This
prerequisite is complicated in polyploid populations by
the potential for inadvertently incorporating homoeologous sequences into the alignments, which would
upwardly bias the estimates of average sequence diversity, sometimes dramatically. Coalescent simulations
were used to estimate diversity variance expected under
neutral coalescent histories. Simulations were performed
in ms [103] and results were summarized using the
msstats tool from the libsequence library [102]. For the
A and B genomes, simulations were based on estimates
of diversity in wild emmer, with the generative value of
θ based on mean θ for each chromosome and the average length of amplicons for the same chromosome. A
total of 10,000 simulations per chromosome was performed. The 99th percentile of θπ from the 10,000 simulations was taken as the upper bound of θπ expected for
each chromosome. Loci in both wild emmer and T. aestivum for which empirical estimates of θπ exceeded the
upper bound were excluded from estimation of
sequence diversity. Simulations for the D genome were
based on the mean chromosome-specific estimates of θπ
for the D genome of synthetic wheats. Loci for which
empirical estimates of θπ in synthetic wheats exceeded
the upper bound for the chromosome were excluded
from further analysis in both synthetic wheats and T.
aestivum. Loci excluded from computations are reported
in Additional file 2, Table S1 but are indicted by a yellow cell color. Data containing less than 75% of the
lines were also excluded from the analyses. They are
reported in Additional file, Table S1 but are indicted by
a red cell color.
The polydNdS program from libsequence [102] was
used to estimate polymorphism at replacement and
silent codon positions. The outputs estimated diversity
for the whole gene, exons only, introns plus flanking
sequences (UTRs) only, and replacement and silent
polymorphisms. Only codons that differed at one position or codons that differed at two positions where the
sites could be unambiguously assigned as synonymous
or nonsynonymous were used.
The frequency spectrum of the less frequent (minor)
allele was estimated in the sample of 10 homozygous
wild emmer lines (10 chromosomes) and 13 homozygous T. aestivum lines (13 chromosomes). The distribution of the minor allele frequency in a sample of size n
is described by the folded spectrum [36,104], which estimates the frequency of SNP sites with a minor allele in
the ith chromosome of n investigated chromosomes (i
ranges from 1 to ≤ n/2). The folded spectra were computed for silent and replacement codon positions for
individual genomes of wild emmer and T. aestivum.

Page 19 of 22

Statistical tests

Significance of differences among genomes was tested
with the GLM and LSD procedures (SAS), using mean
θ, the mean number of haplotypes (H) and mean haplotype diversity (h) per chromosome as variables.
Because θ, H, and h across loci are not normally distributed, the GLM procedure could not be used to
evaluate the significance of differences in these variables among chromosomes. The significance of differences between chromosome means was tested by
estimating a 99% confidence interval (CI) about the
genome mean of θ, H, and h from the distribution of
1000 means of random samples drawn with replacement from the population of θ, H, and h of genes
within a genome (A, B, D genomes) nested within a
species (T. aestivum and wild emmer). Chromosome
means outside of the 99% CI were declared significantly different from the genome mean.

Additional material
Additional file 1: Table S1 summarizes estimates of nucleotide
polymorphism θw and nucleotide diversity θπ at the replacement
(N) and silent (S) codon positions, and noncoding portions of genes
and the ratios of diversity at the replacement and silent codon
positions in genes in the individual chromosomes of the A and B
genomes of T. dicoccoides population from the Diyarbarkir region
in Turkey. Table S2 summarizes estimates of nucleotide polymorphism
θw and nucleotide diversity θπ at the replacement (N) and silent (S)
codon positions and in noncoding portions of genes and the ratios of
diversity at the replacement and silent codon positions in the A, B, and
D genomes of T. aestivum. Figure S1 is a neighbor joining unrooted tree
of 476 T. aestivum accessions constructed from Nei’s genetic distances
computed from RFLP at 131 loci. The tree depicts genetic relationships
among 13 T. aestivum lines used for resequencing and SNP discovery.
Figures S2 and S3 show the numbers of haplotypes per gene along the
A-genome and B-genome chromosomes, respectively, in T. aestivum and
wild emmer (T. dicoccoides) in the Diyarbakir region in Turkey. Figure S4
shows the numbers of haplotypes per gene along the D-genome
chromosomes in T. aestivum. Figure S5 is a neighbor joining unrooted
tree of 55 wild emmer (T. dicoccoides) accessions from the Diyarbakir
region in Turkey constructed from Nei’s genetic distances computed
from RFLP at 153 loci. The tree depicts genetic relationships among 10
wild emmer accessions used for resequencing and SNP discovery in wild
emmer.
Additional file 2: Table S1 is an Xcel table summarizing locus
diversity measures in the A, B, and D genomes of T. aestivum, the
A and B genomes of Diyarbakir population of wild emmer, and the
D genome of synthetic wheats. Table S1 further shows synteny of the
diversity map with the wheat deletion-bin maps and the rice 12
pseudomolecules.

Acknowledgements
We thank Professor Peter Langridge (Australian Centre for Plant Functional
Genomics, University of Adelaide, Adelaide, Australia) and Patrick S. Schnable
(Center for Plant Genomics, Department of Agronomy, Iowa State University,
Ames, Iowa) for advising us.
Author details
1
Department of Plant Sciences, University of California, Davis, CA 95616, USA.
2
Department of Plant Pathology, KSU, Manhattan, KS 66506, USA. 3Genomics

Akhunov et al. BMC Genomics 2010, 11:702
http://www.biomedcentral.com/1471-2164/11/702

and Gene Discovery Unit, USDA/ARS Western Regional Research Center,
Albany, CA 94710, USA. 4Department of Agronomy and Plant Genetics,
University of Minnesota, St. Paul, MN 55108, USA. 5Department of Plant
Sciences and Plant Pathology, Montana State University, Bozeman, MT
59717, USA. 6Department of Ecology and Evolutionary Biology, University of
California, Irvine, CA 92697, USA. 7Department of Plant Pathology, Kansas
State University, Manhattan KS 66506, USA. 8Department of Botany and Plant
Sciences, University of California, Riverside, CA 92521, USA. 9USDA-ARS,
Cornell University, Ithaca, NY 14853, USA. 10Philippine Rice Research Institute,
Maligaya, Nueva Ecija, Philippines. 11The International Maize and Wheat
Improvement Center (CIMMYT), 06600 Mexico, D.F., Mexico. 12Corn Host
Plant Research Resistance Unit, USDA/ARS MSU MS 39762, USA.
Authors’ contributions
EDA, ODA, JAA, MTC, JDu, BSG, YQG, MCL, PEM, COQ, LET, MLW, and JDv
designed research; EDA and JDv coordinated research activity among the
laboratories and PEM coordinated communication among the laboratories. CT,
MLW, and JDv isolated genomic DNAs. FMY and ODA designed CP primers,
ARA, NB, EJC, KRD, JH, HH, NH, DT, DMT, CT, and WZ performed PCR
amplification, purification of amplicons and their submission for sequencing,
alignment of sequences and design of GSPs; ODA, DCD, and CCC sequenced
PCR amplicons. EDA, ODA, DEM, GRL, FMY, and JDv designed the project
database and JR and FMY input data into the database. EDA, PLM, MTC, FMY,
and JDv analyzed data. JDv drafted the manuscript. All authors read and
approved the final version of the manuscript.
Received: 27 April 2010 Accepted: 14 December 2010
Published: 14 December 2010
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doi:10.1186/1471-2164-11-702
Cite this article as: Akhunov et al.: Nucleotide diversity maps reveal
variation in diversity among wheat genomes and chromosomes. BMC
Genomics 2010 11:702.

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