Independent Wheat B and G Genome Origins in Outcrossing Aegilops Progenitor Haplotypes

Abstract

The origin of modern wheats involved alloploidization among related genomes. To determine if Aegilops speltoides was the donor of the B and G genomes in AABB and AAGG tetraploids, we used a 3-tiered approach. Using 70 amplified fragment
length polymorphism (AFLP) loci, we sampled molecular diversity among 480 wheat lines from their natural habitats encompassing
all S genome Aegilops, the putative progenitors of wheat B and G genomes. Fifty-nine Aegilops representatives for S genome diversity were compared at 375 AFLP loci with diploid, tetraploid, and 11 nulli–tetrasomic Triticum aestivum wheat lines. B genome–specific markers allowed pinning the origin of the B genome to S chromosomes of A. speltoides, while excluding other lineages. The outbreeding nature of A. speltoides influences its molecular diversity and bears upon inferences of B and G genome origins. Haplotypes at nuclear and chloroplast
loci ACC1, G6PDH, GPT, PGK1, Q, VRN1, and ndhF for ∼70 Aegilops and Triticum lines (0.73 Mb sequenced) reveal both B and G genomes of polyploid wheats as unique samples of A. speltoides haplotype diversity. These have been sequestered by the AABB Triticum dicoccoides and AAGG Triticum araraticum lineages during their independent origins.

Full text

Independent Wheat B and G Genome Origins in Outcrossing Aegilops
Progenitor Haplotypes
B. Kilian,*H. O
¨zkan,àO. Deusch,S. Effgen,*A. Brandolini,§J. Kohl,kW. Martin,and
F. Salamini*{
*Max Planck Institute for Plant Breeding Research, Ko¨ln, Germany; Institute of Botany III, Heinrich-Heine-Universita¨t Du¨sseldorf,
Germany; àDepartment of Field Crops, Faculty of Agriculture, University of Cukurova, Adana, Turkey; §Istituto Sperimentale per la
Cerealicoltura—CRA, S. Angelo Lodigiano, Italy; kInstitute of Bioinformatics, Heinrich-Heine-Universita¨t Du¨sseldorf, Germany; and
{Fondazione Parco Tecnologico Padano, Via Einstein, Lodi, Italy
The origin of modern wheats involved alloploidization among related genomes. To determine if Aegilops speltoides was
the donor of the B and G genomes in AABB and AAGG tetraploids, we used a 3-tiered approach. Using 70 amplified
fragment length polymorphism (AFLP) loci, we sampled molecular diversity among 480 wheat lines from their natural
habitats encompassing all S genome Aegilops, the putative progenitors of wheat B and G genomes. Fifty-nine Aegilops
representatives for S genome diversity were compared at 375 AFLP loci with diploid, tetraploid, and 11 nulli–tetrasomic
Triticum aestivum wheat lines. B genome–specific markers allowed pinning the origin of the B genome to S chromosomes
of A. speltoides, while excluding other lineages. The outbreeding nature of A. speltoides influences its molecular diversity
and bears upon inferences of B and G genome origins. Haplotypes at nuclear and chloroplast loci ACC1,G6PDH,GPT,
PGK1,Q,VRN1, and ndhF for ;70 Aegilops and Triticum lines (0.73 Mb sequenced) reveal both B and G genomes of
polyploid wheats as unique samples of A. speltoides haplotype diversity. These have been sequestered by the AABB
Triticum dicoccoides and AAGG Triticum araraticum lineages during their independent origins.
Introduction
Bread wheat, Triticum aestivum, has no direct hexa-
ploid wild progenitor (Morris and Sears 1967; Kimber
and Feldman 1987). The species possesses 3 sets of homol-
ogous chromosomes, designated as AABBDD, whose
origins have differing degrees of certainty. The D chromo-
somes stem from the wild diploid Aegilops tauschii (Kihara
1944) through alloploidization with the wild AABB tetra-
ploid Triticum dicoccoides. The A and B chromosomes of
that tetraploid derive from an earlier hybridization between
the wild AA diploid Triticum urartu (Dvorak et al. 1993) and
a wild diploid B genome donor: the ultimate source of this B
genome is still discussed. A related conundrum is the origin
of AAGG Triticum araraticum, whose A genome also stems
from T. urartu, whereby the wild G progenitor is frequently
reported to be Aegilops speltoides (Rodriguez, Maestra et al.
2000). The B donor is traditionally sought in the Sitopsis sec-
tion of the genus Aegilops (Sarkar and Stebbins 1956; Kerby
and Kuspira 1988). Previous molecular analyses of single-
gene loci for a few accessions are not inconsistent with the
view that both the B genome of T. dicoccoides (AABB) and
the G genome of T. araraticum (AAGG) might trace to the
Sitopsis section, in genetic proximity to wild A. speltoides
(Blake et al. 1999; Rodriguez, Maestra et al. 2000; Zhang
et al. 2002). However, there are caveats.
First, nuclear and cytoplasmically inherited markers
yield contrasting results on the issue of B genome origin
(discussed in Wang et al. 1997). In addition, ancient allelic
diversity among wild ancestors, compounded by the possi-
bility of unrecognized hybridization events, renders infer-
ences of the B progenitor questionable (Huang et al. 2002)
in the absence of genome-wide surveys for many loci and
accessions. Furthermore, the outcrossing nature of A. spel-
toides (Kimber and Feldman 1987) renders introgression
for individual loci difficult to exclude in the absence of ex-
tensive lineage sampling. Importantly, cytogenetic evi-
dence does not support the view that A. speltoides was the
donor of B or G genome, even though such suggestions
can be found (Maestra and Naranjo 1998). When synthetic
SSAA genomes (S contributed by A. speltoides and A by
Triticum) are crossed to Triticum durum (AABB, the do-
mesticated form of T. dicoccoides), sterility is observed,
pointing to differences between S and B genomes; the same
is reported for S and G genomes (Dvorak 1972; Kimber and
Athwal 1972). Moreover, B–S pairing in wheat/A. spel-
toides hybrids is comparable to that noted for wheat/Aegi-
lops longissima and wheat/Aegilops sharonensis hybrids
(Fernandez-Calvin and Orellana 1994), suggesting that B
chromosomes of polyploid wheats do not pair preferentially
to those of A. speltoides.
Understanding hexaploid wheat origin would further
its genetic improvement (Salamini et al. 2002; Chantret
et al. 2005). Here, we report a comprehensive amplified
fragment length polymorphism (AFLP) survey of genomic
diversity among 1372 individuals from 480 wild B genome
progenitor candidates. Through the analysis of Sears’s
(1954) nulli–tetrasomic (AADDDD) lines, B genome–
specific AFLPs were identified. For ;70 domesticated
and progenitor lines representing the breadth of wild geno-
mic diversity, haplotypes at nuclear loci ACC1,G6PDH,
GPT,PGK1,Q,VRN1 and of the chloroplast locus ndhF
were determined. Comparisons to haplotypes from AA
Triticum boeoticum,Triticum monococcum, and T. urartu
identified haplotypes specific to the B genome to allow
comparison to Sitopsis accessions. The data circumscribe
molecular diversity among Sitopsis Aegilops species and
specify the nature of wheat B and G genome origins.
Methods
AFLP Analysis
The 480 Aegilops lines used in this study are listed in
Supplementary table S1 (Supplementary Material online).
Key words: molecular evolution, Triticum,Aegilops, hybridization,
alloploidization, AFLPs.
E-mail: francesco.salamini@tecnoparco.org.
Mol. Biol. Evol. 24(1):217–227. 2007
doi:10.1093/molbev/msl151
Advance Access publication October 19, 2006
ÓThe Author 2006. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: journals.permissions@oxfordjournals.org

DNA was isolated from freeze-dried or silica-dried leaves
of 1372 plants (Supplementary table S2, Supplementary
Material online), using the Qiagen (Hilden) DNeasy Kit,
and amplified as described by Zabeau and Vos (1993) using
the primer combinations E
ACC
/M
ACA
,E
ACC
/M
AGC
, and
E
ACC
/M
AGG
. The AFLP bands were scored as 1 or 0 (pres-
ent or absent). Jaccard (1908) similarities of the 850 indi-
viduals with different AFLP patterns (Supplementary table
S3, Supplementary Material online) were computed using
DistAFLP (Mougel et al. 2002), and Neighbor-Joining (NJ)
bootstrap trees were inferred with PHYLIP 3.6 (Felsenstein
2002). DNA from 94 selected Aegilops,T. boeoticum,T.
urartu,T. dicoccoides,T. araraticum, and T. aestivum ac-
cessions (Supplementary table S4, Supplementary Material
online), along with T. aestivum Chinese Spring aneuploids:
6 nulliB–tetraD (N1BT1D, N2BT2D, N3BT3D, N4BT4D,
N5BT5D, N6BT6D) and 5 nulliB–tetraA (N1BT1A,
N2BT2A, N3BT3A, N5BT5A, N7BT7A) (Sears 1954),
was amplified using primer combinations E
ACC
/M
AGC
,
E
ACC
/M
AGG
,E
ACG
/M
ACC
,E
ACG
/M
ACT
,E
ACG
/M
AGG
,
E
ACG
/M
AGT
,E
AGC
/M
AGC
, and E
AGC
/M
ATA
. NeighborNet
(NNet) planar graphs (Bryant and Moulton 2004) of AFLP
Hamming distances between individuals were constructed
with SplitsTree 4.1 (Huson and Bryant 2006).
Haplotype Analysis
Genes and accessions considered for haplotype anal-
ysis are recorded in Supplementary tables S5–S7 (Supple-
mentary Material online). Sixty-seven lines were common
to all loci—T. dicoccoides (34), T. dicoccum (5), T. durum
(1), T. araraticum (5), Triticum timopheevii (6), Aegilops
bicornis (2), A. longissima (2), Aegilops searsii (2), A. shar-
onensis (2), A. speltoides (7), and A. tauschii (1). Other se-
quences from additional lines of those same species, from
T. boeoticum,T. monococcum,T. urartu,T. araraticum,
and T. timopheevii (table 2), as well as available published
sequences were included. DNA was isolated as described
above. Primers (Supplementary table S8, Supplementary
Material online) were designed with Primer3 against
sequences for ACC1 (Huang et al. 2002), G6PDH (Nemoto
et al. 1999), GPT (GenBank AF548741), PGK1 (Huang
et al. 2002), Q(Faris et al. 2003), VRN1 (Sherman et al.
2004; Yan et al. 2004), and ndhF (Ogihara et al. 2002).
Some accessions of A. speltoides have 2 copies each of
the genes ACC1 and PGK1 (Huang et al. 2002); primers
for these 2 genes were used allowing the amplification
of the same gene in all A. speltoides. DNA amplifications
were performed in 25 ll containing ;100 ng of leaf DNA,
0.4 lM of each primer, 125 lM of each deoxynucleoside
triphosphate (AB gene, Surrey, UK), 3 mM MgCl
2,
4% di-
methyl sulfoxide, and 1 unit of Taq DNA polymerase in-
cubated in a PTC-225 Tetrad Thermal Cycler (MJ
Research) as follows: 94 °C for 3 min, 28–33 cycles of
30 s at 94 °C, 20–40 s at 59–65 °C, 50–95 s at 72 °C,
and a final extension step of 6 min at 72 °C. Polymerase
chain reaction products were sequenced on both strands
(Supplementary table S9, Supplementary Material online).
Sequences (Supplementary table S10, Supplementary Mate-
rial online) were processed with Applied Biosystems DNA
Sequencing Analysis Software 5.1.1 and manually in-
spected with BioEdit version 7.0.1 (Hall 1999). The align-
ments were generated with ClustalW, and the haplotypes
were scored manually and with DnaSP (Rozas et al.
2003). For homozygous loci, only one haplotype per line
was included in the alignment, both haplotypes for hetero-
zygous loci. Median-joining (MJ) networks (Bandelt et al.
1999) were constructed with the Network 4.1.1.2 program
(Fluxus Technology Ltd, Clare, Suffolk, UK). Total num-
ber of substitutions per site between populations, D
a
(equa-
tion 10.21; Nei 1987), using the Jukes–Cantor method was
calculated with DnaSP (Rozas et al. 2003).
To obtain A, B, and G genome–specific haplotype
sequences for polyploid wheats, three approaches were
used. When B genome–specific sequence differences were
available, 1) primer combinations were designed and used
for haplotype-specific amplification and sequencing; 2) am-
plification products from A, B, and G genomes were ob-
tained with the A genome primers, but sequenced using
genome-specific primers; and 3) in the remaining cases, am-
plification products for A, B, and G genomes were obtained
using nondiscriminating A genome primers, cloned in Es-
cherichia coli, and at least 3 sequences per clone obtained,
until both haplotypes were identified by comparison to
existing A and B genome data for the locus.
Results
Genomic Diversity within the Sitopsis Section of Aegilops
To survey the molecular diversity among candidate B
genome donors, we studied 2–3 plants each from a total of
501 accessions spanning all 5 Sitopsis Aegilops species—A.
searsii,A. bicornis,A. sharonensis,A. longissima, and A.
speltoides—collected along the Eastern Mediterranean
(fig. 1). (We follow Dorofeev et al. [1979] for Triticum
binomial nomenclature and Van Slageren [1994] for Aegi-
lops). Accessions were grown in 2003 and morphologically
reidentified, whereby 21 misassigned lines or interspecific
hybrids were discarded. DNA was collected from 1372
plants: A. bicornis (39 accessions, 105 individuals), A. long-
issima (81, 227), A. searsii (97, 285), A. sharonensis (112,
327), A. speltoides (149, 422) and from the D genome out-
group species A. tauschii (2, 6).
A screen using 3 AFLP primer combinations uncov-
ered a total of 70 polymorphic bands across all plants. This
revealed 850 individuals with different AFLP patterns: A.
bicornis (36 accessions, 44 individuals), A. longissima (80,
165), A. searsii (54, 77), A. sharonensis (101, 176), A. spel-
toides (147, 386), and A. tauschii (2, 2). The NJ tree of
Jaccard (1908) distances is shown in figure 2 and provides
an overview of Sitopsis genome diversity. The primary
screen revealed the breadth of divergence within each spe-
cies, helping to choose accessions for subsequent fine-scale
analyses. In addition, two notable results emerged. First, in
all intraspecific pairwise comparisons, AFLP-based genetic
similarity was lower in the outbreeder A. speltoides than in
any of the other members of the section which are in-
breeders (Kimber and Feldman 1987) (fig. 2). Second, fig-
ure 1 shows that the Sitopsis section (with one exception,
a plant of A. bicornis mapping outside but near the major
cluster of the species) does not include other genetically
distinct major groups, besides those represented by A.
218 Kilian et al.

FIG. 1.—Natural distribution of the different Aegilops section Sitopsis species and Aegilops tauschii.(A) Distribution in the Eastern Mediterranean
region and the Near East (Hall 1999) are indicated by colors, 387 collection sites are drawn with symbols. (B) Enlarged Israel region showing 253
collection sites. Numbers in red below the symbol indicate how many accessions were collected from the same site. Among the 480 accessions were
analyzed: Aegilops bicornis (39 accessions), Aegilops longissima (81), Aegilops searsii (97), Aegilops sharonensis (112), Aegilops speltoides (149), and
A. tauschii (2). Accessions sorted by country: China (1), Egypt (18), Iraq (4), Israel (253), Jordan (37), Libya (2), Portugal (2), Syria (29), Turkey (70), and
Ukraine (1), 63 analyzed accessions of unknown origin are not shown in the figure. Aegilops species sorted by country: China (1 SPE), Egypt (18 BIC),
Iraq (4 SPE), Israel (9 BIC, 69 LOG, 52 SEA, 104 SHA, 20 SPE), Jordan (4 BIC, 5 LOG, 28 SEA), Libya (2 BIC); Portugal (2 SPE), Syria (16 SEA, 13
SPE), Turkey (1 LOG, 2 SHA, 64 SPE, 2 TAU), Ukraine (1 SPE), and unknown (6 BIC, 6 LOG, 1 SEA, 6 SHA, 44 SPE). Country known, but exact
collection site unknown within the country 27 (Israel: 3 LOG, 3 SHA, 1 SPE; Jordan: 2 BIC, 2 LOG, 6 SEA; Syria: 2 SEA, 7 SPE; Turkey: 1 SPE). (C)
Spike morphologies for Aegilops species relevant to this study.
Origin of Wheat B and G Genomes 219

bicornis,A. searsii,A. speltoides, and the A. sharonensis–
A. longissima cluster (the two last species, however, map-
ped to some degree separately when using more AFLP
primer combinations, like in fig. 3Aand B). It is concluded
that the Sitopsis section does not contain cryptic species
molecularly distinct from those currently recognized
(Kimber and Feldman 1987), such that our sample appears
to span the full breadth and depth of molecular diversity
within the section. The inability to distinguish A. sharonen-
sis from A. longissima individuals in the coarse screen is
irrelevant here, as are the low bootstrap proportions for
branches separating species at these 70 loci.
Higher Resolution among Wild and Domesticated
Genomes
A reduced set of 59 Aegilops plants representative for
molecular diversity within the section was considered for
further AFLP studies, carried out with a higher number
of primer combinations. The selected plants were A. bicor-
nis (11 individuals), A. longissima (8), A. searsii (13), A.
sharonensis (13), and A. speltoides (14). Selection of plants
within species was carried out, maximizing the average ge-
netic distances among selected plants. Two plants from the
A. tauschii (D genome) outgroup were included, as were 5
T. aestivum cultivars (AABBDD), 3 wild T. dicoccoides
(AABB), 4 wild T. araraticum (AAGG, the progenitor
of the domesticated T. timopheevii), 9 wild T. boeoticum
(A
b
A
b
, the progenitor of the domesticated T. monococcum,
A
m
A
m
), and 12 wild T. urartu (AA). The choice of the 9 and
12 accessions, respectively, of T. boeoticum and T. urartu
considered the criterion specified above, based on pub-
lished and unpublished molecular data of the authors. Other
Triticum accessions were chosen as representative of mo-
lecular diversity among the species considered in experi-
ments previously published by the authors (references in
Salamini et al. 2002).
Eleven AFLP primer combinations amplified 375
polymorphic bands across these 94 lines, from which NNets
were constructed (fig. 3A). NNet splits graphs can be inter-
preted like trees in that they contain splits (branches) with
weights (lengths). Parallel lines identify the same split or
FIG. 3.—NNets of Hamming distances for AFLP polymorphisms among Sitopsis section, Aegilops species, and Aegilops tauschii (outgroup) with
polyploidy wheats. (A) NNet for 375 polymorphic loci (11 AFLP primer combinations) and 94 lines. AES, Triticum aestivum; ARA, Triticum araraticum;
BIC, Aegilops bicornis; BOE, Triticum boeoticum; DIC, Triticum dicoccoides; LOG, Aegilops longissima; SEA, Aegilops searsii; SHA, Aegilops shar-
onensis; SPE, Aegilops speltoides; TAU, A. tauschii; and URA, Triticum urartu. The number of individuals considered was 5 (AES), 4 (ARA), 11 (BIC), 9
(BOE), 3 (DIC), 8 (LOG), 13 (SEA), 13 (SHA), 14 (SPE), 2 (TAU), and 12 (URA). Genome assignments are shown. Relevant splits are highlighted (see
text). (B) NNets based on 65 AFLP polymorphic loci (11 primer combinations) assigned specifically to B chromosomes of T. aestivum using
Chinese Spring nulli–tetrasomic lines for the same 94 lines. Other details as in (A). (C) Jaccard (1908) genetic similarity index (average proportion
of identical B genome–specific AFLP bands) among T. dicoccoides (DIC)/T. araraticum (ARA) lines and Aegilops species based on the 65 AFLP
B bands in (B).
!
FIG. 2.—Unrooted NJ tree of Jaccard (1908) distances based on AFLP markers describing genetic relationships among 850 individuals of the genus
Aegilops, section Sitopsis, and Aegilops tauschii. The 850 genotypes—Aegilops bicornis (36 accessions, 44 individuals), Aegilops longissima (80, 165),
Aegilops searsii (54, 77), Aegilops sharonensis (101, 176), Aegilops speltoides (147, 386), and A. tauschii (2, 2)—were selected as unique pattern out of
a total of 1372 one-plant samples. A total of 70 AFLP polymorphic loci were generated with primer combinations E36M35, E36M40, and E36M41.
Bootstrap proportions for the main internal edges are shown. Numbers within boxes report the average intraspecific value of Jaccard (1908) genetic
similarity (6standard error).
220 Kilian et al.

Origin of Wheat B and G Genomes 221

branch. Boxes indicate support for 2 competing patterns of
taxon relationship. NNet splits graphs highlight the pre-
dominant phylogenetic signals in the data and the extent
to which these signals may or may not be tree-like (Huson
and Bryant 2006). In cases of reticulate evolutionary his-
tory, hybrid taxa are suggested by the occurrence of incom-
patible splits (which appear as boxes), often with hybrid
taxa being linked by splits to their potential parents. NNet
split graphs only display the contradictory splits that can
be visualized in a single plane and should not be considered
an explicit model of reticulate evolutionary history. Never-
theless, they provide an implicit representation of evolu-
tionary history (Huson and Bryant 2006) and one that is
useful for identifying and exploring different signals and
their meaning. For allopolyploid species, NNet has an ad-
vantage for data visualization over tree-building methods,
which assume that the data have evolved on a single bifur-
cating tree. At this level of genome-wide comparison, the
only Sitopsis member that shared a split with the AABB,
AAGG, and AABBDD polyploids was A. speltoides. That
split reflects a higher proportion of shared AFLP bands be-
tween polyploid wheats and A. speltoides as compared with
other SS genomes. A second split divides T. urartu (AA)
from all diploids sampled but excludes T. boeticum (A
b
A
b
),
and indeed T. urartu is the A genome donor (Dvorak et al.
1993). No split links A. tauschii (DD) to the hexaploid
AABBDD genome. However, this might be expected be-
cause the NNet method can only represent incompatible
splits projected onto 2 dimensions (Huson and Bryant
2006). With the D genome absent in the 7 tetraploids sam-
pled, signals from A, B, G, and S genomes will override the
weaker signal linking AABBDD and DD genomes. A
strong split linking the AA diploids with T. araraticum
(AAGG) to the exclusion of T. dicoccoides (AABB) is also
observed, indicating that the AABB and AAGG genomes,
both involving T. urartu, are the result of independent poly-
ploidization events.
The B Genome
Six nulliB–tetraD and 5 nulliB–tetraA lines of the hexa-
ploid cultivar Chinese Spring (Sears 1954) were included in
the AFLP analysis. They identified 65 bands that reside spe-
cifically on the B genome. If the split that links A. speltoides
to polyploids is a historical component of genome similarity,
it should become more prominent in the NNet based on the
65 B genome–specific bands. This is observed in figure 3B,
where the split linking A. speltoides to polyploid wheats is
highlighted in blue. These B bands were selected by virtue of
their occurrence in hexaploid wheat, not by virtue of their
character state among tetraploids or diploids. Hence, they
do not skew the locus sample systematically toward any po-
tential B genome donor. They represent B genome–specific
signals with regard to polyploid origins; competing A- and
D-specific signals are diminished, but not abolished, be-
cause A, B, G, and D genomes are still related at these loci.
Figure 3Breveals that the A. speltoides genome is most
similar to the B and G genomes of polyploid wheats. And
because we have extensively sampled genome diversity
across the Sitopsis (fig. 2), this indicates that the A. spel-
toides S genome is the extant version of B and G genomes
of polyploid wheats. Identical B genome–specific AFLP
bands shared between tetraploid wheats and the species
of the Sitopsis section using the Jaccard (1908) similarity
(fig. 3C) further support that conclusion.
The NNet shows a strong split linking hexaploid wheat
with T. dicoccoides to the exclusion of T. araraticum, high-
lighted in green in figure 3B. This corresponds to the well-
known participation of T. dicoccoides in bread wheat origin
(Dvorak et al. 1993). Evidence for additional hybridization
events was uncovered, namely the strong component of
similarity linking few T. urartu (AA) accessions to the
T. boeticum complex (A
b
A
b
), probably reflecting interspe-
cific crosses. However, even in the B genome–specific data,
no A. speltoides genome sampled was specifically more
similar to all polyploids sampled. Nonetheless, if the B
and G genomes stem from within A. speltoides, then
genome-specific haplotypes from polyploids should pro-
vide more detailed evidence for that origin.
Congruent Evidence from Haplotypes
Haplotypes recognized in DNA fragments for the nu-
clear genes ACC1,G6PDH,GPT,PGK1,Q,andVRN1 and
in a 719-bp region of the chloroplast gene ndhF were de-
termined for tetraploids, Sitopsis members, and AA dip-
loids. In total, 0.73 Mb of sequence data were obtained
and combined with 80 000 bp from previous studies (Sup-
plementary tables S5–S7, Supplementary Material online)
for analysis. At all nuclear loci investigated, the number of
net nucleotide substitutions per site between populations
(Nei 1987), D
a
, revealed that T. dicoccoides B genome hap-
lotypes were always more similar to those in A. speltoides
than those in any other species. The same was true for com-
parisons of the T. araraticum G genome haplotypes (table
1), providing additional evidence for an origin of both B
and G genomes from a A. speltoides donor. The same
was evident for the cytoplasmically inherited ndhF gene
(table 1).
MJ networks for these loci (fig. 4) reveal higher levels
of haplotype diversity within the outbreeder A. speltoides
than in other wheats. Furthermore, B and G genome hap-
lotypes of the tetraploids were consistently more closely re-
lated to A. speltoides than to other sources. At G6PDH,8A.
speltoides haplotypes were observed: SPE-I is the closest
relative of B and G haplotypes, which are monomor-
phic for T. dicoccoides and dimorphic for T. araraticum,
whereas other Sitopsis or A genome haplotypes are distinct
by 20 substitutions. At ACC1,A. speltoides revealed 7
haplotypes: SPE-I and -II are identical to those found in
G genome, SPE-III is the closest relative of the major T.
dicoccoides B haplotype; no ACC1 haplotypes are shared
between A. speltoides and remaining Sitopsis species. A ge-
nome haplotypes at ACC1 were much less diverse than B
genome homologues. At GPT, only one A. speltoides hap-
lotype was observed, which is identical to the major T. ara-
raticum haplotype and shows only 2 nucleotide differences
to the main T. dicoccoides B haplotype; other Sitopsis or A
genome haplotypes were clearly distinct. Qwas by far the
most polymorphic locus sampled: the closest progenitor to
the main T. dicoccoides B genome haplotype was SPE-I,
different by 7 nucleotides to the T. araraticum haplotype
222 Kilian et al.

and by 17 nucleotides from the major T. dicoccoides B hap-
lotype; the other haplotypes were more distant to B and G
genomes. At PGK1,13A. speltoides haplotypes were
found: SPE-I differed by 2 nucleotides from the main B
haplotype of T. dicoccoides; the rare SPE-II is not more
closely related to the single T. araraticum G haplotype than
haplotypes found among other Sitopsis; for this gene,
a greater diversity of A. speltoides haplotypes relative to
other Sitopsis was particularly evident. VRN1 (Supplemen-
tary fig. S1, Supplementary Material online) did not reveal
a closer relationship for either A. speltoides or other Sitopsis
to B or G genome. For this gene, the simplest interpretation
is that our present lineage sample at VRN1 did not uncover
A. speltoides B genome progenitor haplotypes: only a clear
distinction between A and B/G genome–specific haplo-
types was evident.
The main A. speltoides ndhF haplotype was identical
with that of tetraploids and hexaploids (fig. 4). The net-
work, while excluding the progenitors of A. bicornis,A.
longissima,A. sharonensis, and A. searsii as the B female
recipients in the cross with the A genome, provides evi-
dence that female gametes of A. speltoides generated the
AABB and AAGG genomes.
In summary, the MJ networks uncover no Sitopsis
haplotypes that are more similar to B or G genome than
A. speltoides haplotypes are, indicating that the A. spel-
toides gene pool participated in the synthesis of AABB
and AAGG genomes. Furthermore, loci that are highly
polymorphic in A. speltoides, such as PGK1, underscore
the need to sample many lineages to uncover B genome
progenitor alleles.
The 2 distinct G genome haplotypes at ACC1 differing
by 4 substitutions, each identical to haplotypes occurring in
A. speltoides, could, at face value, suggest 2 independent
origins for T. araraticum. However, for all loci at which
the B genome donor was heterozygous—for instance, in
unreduced gametes—both alleles should persist in modern
tetraploids. This problem is related to the outcrossing nature
of A. speltoides (table 2): of the 39 loci investigated, 76%
were heterozygous as compared with 7.4% for remaining
Aegilops species, all predominantly inbreeders (Kimber
and Feldman 1987). The distinctness of B and G genome
haplotypes at all nuclear loci, and the proximity of A. spel-
toides progenitors in most cases, clearly indicates indepen-
dent alloploidization events underlying T. araraticum and
T. dicoccoides origins, consistent with their divergent
positions in the analysis of B genome–specific AFLPs
(fig. 3B). The results of table 3, which underscore the close
relationship of B and G genomes relative to A genome,
support this conclusion.
Discussion
Domestication of wheats commenced about 10 000
years ago (Salamini et al. 2002), but the events that gave
rise to wild polyploids are older. Estimates for the age of
T. dicoccoides origin range from .0.5 MYA (Huang
et al. 2002), 0.25–1.3 MYA (Mori et al. 1995; Huang
Table 1
Sequence divergence between tetraploid wheat and Sitopsis section haplotypes (boldface: divergence between S and
B or G genomes)
Gene (L)
b
Genome (n)
D
a
6SE 310
3
(n)
a
in comparison with
Aegilops bicornis Aegilops longissima Aegilops searsii Aegilops sharonensis Aegilops speltoides
ACC1 (366) dicA
c
(37) 14 62 (3) 12 62 (5) 17 62 (6) 14 62 (4) 5 61 (23)
dicB
d
(37) 14 62 (3) 12 62 (5) 17 62 (6) 14 63 (4) 561 (23)
araA
e
(5) 14 65 (3) 12 64 (5) 17 64 (6) 14 65 (4) 5 61 (23)
araG
f
(6) 10 65 (3) 8 65 (5) 13 65 (6) 10 65 (4) 162 (23)
G6PDH (537) dicA (34) 54 68 (2) 53 68 (2) 51 68 (2) 53 68 (2) 43 64 (15)
dicB (34) 52 68 (2) 52 68 (2) 50 68 (2) 52 68 (2) 12 63 (15)
araA (5) 52 619 (2) 52 619 (2) 50 618 (2) 52 619 (2) 39 68 (15)
araG (6) 48 618 (2) 48 618 (2) 46 617 (2) 48 618 (2) 10 64 (15)
GPT (673) dicA (34) 9 61 (2) 14 62 (2) 9 61 (2) 14 62 (2) 21 62 (7)
dicB (34) 15 62 (2) 17 63 (2) 15 62 (2) 17 63 (2) 360.3 (7)
araA (5) 9 63 (2) 13 65 (2) 9 63 (2) 14 65 (2) 20 64 (7)
araG (5) 15 66 (2) 17 66 (2) 15 66 (2) 17 66 (2) 060.4 (7)
PGK1 (665) dicA (35) 24 65 (2) 21 63 (2) 26 64 (2) 21 63 (2) 12 62 (16)
dicB (35) 24 65 (2) 19 63 (2) 24 64 (2) 21 63 (2) 15 64 (16)
araA (5) 24 610 (2) 21 67 (2) 26 610 (2) 21 67 (2) 12 64 (16)
araG (5) 19 69 (2) 16 66 (2) 19 67 (2) 16 66 (2) 15 65 (16)
Q(917) dicA (36) 111 616 (2) 81 614 (3) 100 615 (2) 94 616 (3) 68 66 (13)
dicB (36) 77 611 (2) 48 611 (3) 85 612 (2) 55 613 (3) 20 63 (13)
araA (5) 113 641 (2) 83 632 (3) 102 637 (2) 96 635 (3) 70 614 (13)
araG (5) 65 624 (2) 35 618 (3) 75 628 (2) 43 620 (3) 12 65 (13)
VRN1 (304) dicA (34) 53 68 (2) 53 68 (2) 56 68 (2) 53 68 (2) 58 65 (11)
dicB (34) 28 64 (2) 28 64 (2) 35 65 (2) 28 64 (2) 26 63 (11)
araA (5) 53 619 (2) 54 619 (2) 57 621 (2) 53 619 (2) 58 611 (11)
araG (5) 20 67 (2) 21 67 (2) 28 610 (2) 20 67 (2) 19 65 (11)
ndhF (719) dic-cp
g
(34) 6 61 (2) 6 61 (2) 6 61 (2) 6 61 (2) 060.2 (7)
ara-cp (9) 6 62 (2) 6 62 (2) 6 61 (2) 6 61 (2) 060.3 7)
a
Total number of substitutions per site between populations 6standard error (SE) (Nei 1987) calculated with DnaSP (Rozas et al. 2003); n, number of loci sequenced
for each species.
b
Number of sites compared, all gapped sites excluded.
c
Triticum dicoccoides A genome.
d
T. dicoccoides B genome.
e
Triticum araraticum A genome.
f
T. araraticum G genome.
g
Chloroplast genome.
Origin of Wheat B and G Genomes 223

FIG. 4.—MJ networks derived from DNA sequence haplotypes among accessions, involving in all loci Triticum dicoccoides (34), T. dicoccum (5), Triticum durum (1), Triticum araraticum (5), Triticum
timopheevii (6), Aegilops bicornis (2), Aegilops longissima (2), Aegilops searsii (2), Aegilops sharonensis (2), Aegilops speltoides (7), and Aegilops tauschii (1) plus additional haplotype data for each locus as
available. Distance between 2 black dots is one nucleotide substitution. AES, Triticum aestivum; ARA, T. araraticum; BIC, A. bicornis; BOE, Triticum boeoticum; DIC, T. dicoccoides; DIM, T. dicoccum; DUR, T.
durum; LOG, A. longissima; MONO, Triticum monococcum; SEA, A. searsii; SHA, A. sharonensis; SPE, A. speltoides; TAU, A. tauschii; TIM, T. timopheevii; and URA, Triticum urartu. Species names according
to Dorofeev et al. (1979) and Van Slageren (1994). G6PDH—For this MJ network, we used 72 lines from this project,for a total of 132 sequences. ACC1—116 lines: 79 from this project; 37 from published results of
other lines; total of 176 sequences: 139 from this project; 37from published sequences. GPT—72: 71; 1; 124: 123; 1. PGK1—86: 76; 10; 144: 134; 10. Q: 93: 81; 12; 157: 145; 12. ndhF—79: 78; 1; 79: 78; 1.
224 Kilian et al.

et al. 2002), or 0.36 MYA (Dvorak and Akhunov 2005) but
are heavily subject to haplotype sampling variance, as our
data underscore, for which reason the B genome donor
has remained in issue. Though the source of the B genome
has been sought among Sitopsis (Sarkar and Stebbins 1956;
Kerby and Kuspira 1988), with a focus on A. speltoides
(Dvorak and Zhang 1990; Huang et al. 2002), genetic data
have been equivocal (Dvorak 1972; Kimber and Athwal
1972; Fernandez-Calvin and Orellana 1994) in the absence
of studies sampling many loci and accessions.
Our data indicate an origin of the B genome from
within A. speltoides. First, the analysis of 375 AFLP loci
links specifically polyploid wheats with A. speltoides to
the exclusion of other Sitopsis species (fig. 3A). In addition,
65 AFLPs specific to the T. aestivum B genome link A. spel-
toides even more closely to the B and G genomes (fig. 3B).
Second, with the exception of VRN1, haplotypes from chlo-
roplast and nuclear loci show that A. speltoides shares the
highest average sequence identity with the B and G ge-
nomes (table 3) and reveals specific progenitor-descendant
relationships in the MJ networks (fig. 4). These findings can
be incorporated into a broader scheme of wheat genome
evolution (Supplementary fig. S2, Supplementary Material
online) with resolved positions of the B genome relative to
S progenitors and G sisters.
AABB and AAGG genome origins have been attrib-
uted to the same single hybridization event (Wagenaar
1961; Tanaka et al. 1979; Gill and Chen 1987; Provan
et al. 2004) or to separate alloploidization events (Mori
et al. 1995; Brown-Guedira et al. 1996; Rodriguez,
Perera et al. 2000; Huang et al. 2002). In support of the
former view, T. dicoccoides and T. araraticum have al-
most identical morphology, but they have F1 hybrids
showing 100% sterility (Tanaka et al. 1979) (with normal
chromosome pairing, Rao and Smith 1968; Rawal and
Harlan 1975; Tanaka et al. 1979). In addition, some lines
of T. araraticum produce hybrids with a significant level
of fertility when crossed to T. dicoccoides (Rao and Smith
1968; Rawal and Harlan 1975). Our data resolve this issue.
The hybridization events leading to AABB and AAGG
genomes occurred independently as evidenced 1) by their
distinct positions in AFLP analyses, 2) by the finding that
each has sequestered different samples of A. speltoides
haplotype diversity, and 3) from the comparison of diver-
gence within and among A, B, and G genome haplotypes
(table 3). The B and G genomes are clearly distinct, incom-
patible with the view of a single-hybrid origin (Rodriguez,
Maestra et al. 2002).
Wheat is no exception to the rule that specific poly-
ploids arose recurrently during flowering plant evolution
(Soltis 2005), accompanied by extensive and rapid genome
restructuring (Leicht and Bennett 1997). Alloploidization
often involves intergenomic recombination (McGrath
et al. 1990; Jang and Gill 1994; Song et al. 1995; Soltis
2005) and rapid loss of DNA (O
¨zkan et al. 2001), whereby
subsequent diploidization restores disomic genetics (Levy
and Feldman 2002). The genetic control of chromosome
pairing provides insights on wheat alloploid evolution.
Aegilops speltoides forms are known that suppress pair-
ing among homologous chromosomes (Ph1 activity)
(Aghaee-Sarbarzeh et al. 2000). If Ph1 genotypes participate
in polyploidization events, interspecific hybrids acquire a
Table 2
Heterozygosity among Aegilops accessions at loci sampled
ACC1 G6PDH GPT PGK1 Q VRN1
Species n
a
H
b
h
c
nHhnHhnH h nH h nHh
Aegilops speltoides 8 7 587870186118510746
Aegilops bicornis 2 0 120220120 220 2201
Aegilops longissima 2 1 3 20120120 121 3201
Aegilops searsii 2 0 120120120 220 2201
Aegilops sharonensis 2 0 120120120 121 2201
Aegilops tauschii 1 0 110110110 111 2101
a
Number of individuals sampled.
b
Number of heterozygous individuals found.
c
Number of haplotypes found (including publically available sequences).
Table 3
Average between- and within-genome haplotype sequence divergence (boldface: significant divergence between B to A and B to
G comparisons)
D
a
6SE 310
3
(n)
a
;Triticum
dicoccoides B versus
Average sequence divergence within genomes
Gene (L)
d
A genome
b
G genome
c
T. dicoccoides B Other B
e
Triticum araraticum GTriticum timopheevii G
ACC1 (366) 5.8 60.4 (72) 7.7 61.1 (15) 1.7 61.9 (37) 1.5 61.8 (43) 5.9 65.7 (6) 4.2 65.3 (15)
G6PDH (537) 49.7 61.7 (56) 19.3 61.6 (11) 0.0 60.0 (34) 0.0 60.0 (41) 1.7 62.1 (5) 2.1 62.1 (11)
GPT (673) 20.2 60.8 (57) 3.6 60.4 (11) 0.2 60.6 (34) 0.2 60.5 (40) 1.5 61.0 (5) 1.2 61.2 (11)
PGK1 (665) 23.1 61.4 (63) 20.6 61.5 (11) 0.8 61.3 (35) 0.7 61.2 (41) 0.0 60.0 (5) 0.0 60.0 (11)
Q(917) 81.3 62.3 (73) 21.7 61.5 (11) 0.5 60.8 (36) 0.4 60.7 (43) 0.0 60.0 (5) 0.2 60.5 (11)
VRN1 (304) 55.9 61.8 (64) 6.8 60.6 (11) 2.6 62.2 (34) 2.5 62.2 (41) 0.0 60.0 (5) 0.0 60.0 (11)
a
Number of net nucleotide substitutions per site between populations 6standard error (SE) (Nei 1987) calculated with DnaSP (Rozas et al. 2003) using the Jukes–Cantor
method; n, number of loci sequenced for each species.
b
A genome of Triticum monococcum,Triticum boeoticum,Triticum urartu,T. dicoccoides,Triticum durum,T. di-
coccum,T. araraticum, and T. timopheevii.
c
T. araraticum and T. timopheevii.
d
Number of sites compared, all gapped sites excluded. Alignments available as Supplementary
Material online.
e
T. durum,T. dicoccum.
Origin of Wheat B and G Genomes 225

bivalent type of chromosome pairing, the case of T. dicoc-
coides (Sears 1976). Other lines of A. speltoides do not show
Ph1-like activity (Kimber and Feldman 1987), having loci
that suppress Ph1, thus allowing homologous pairing in
interspecific crosses (Sears 1976). The absence of Ph1 or
Ph1-like activity favors tetravalent formation and, possibly,
intergenomic translocations. Triticum araraticum has ex-
tensive DNA loss (O
¨zkan et al. 2001) and 6 chromosomal
rearrangements relative to T. dicoccoides (Rodriguez,
Maestra et al. 2000; Rodriguez, Perera et al. 2000), 4 of
which are intergenomic G–A translocations. A possibility
is that in the AAGG genome synthesis, the Ph1 allele of
A. speltoides was suppressed and later on restored via genetic
segregation (today T. araraticum has an active Ph1 allele).
Similar evolutionary mechanisms may underlie the cytoge-
netic distinctness of S, B, and G genomes, whose evolution-
ary relationships are nonetheless revealed by AFLP and
haplotype data.
Previous studies suggested that T. araraticum in-
herited a A. speltoides cytoplasm (Mori et al. 1997; Wang
et al. 2000; Provan et al. 2004) but were conflicting for T.
dicoccoides (Wang et al. 2000; Provan et al. 2004). Our
ndhF data assign the A. speltoides cytoplasm to both T.
dicoccoides and T. araraticum. This cytoplasm is distinct
from that of other Sitopsis species.
We identified no A. speltoides line that shares greater
similarity to all polyploids sampled than any other A. spel-
toides line. Furthermore, the A. speltoides haplotypes most
similar or identical to B and G genome haplotypes are dis-
persed across different individual lines. Further sampling
within A. speltoides might uncover lines that carry the same
combination of haplotypes as the B genome donor con-
tained. However, because the species is an outbreeder, it
is more likely that no modern A. speltoides lines have pre-
served the B donor genotype in its contiguous ancestral
state.
Supplementary Material
Supplementary material mentioned in the text, com-
prising 10 supplementary tables and 2 supplementary
figures are available online at http://en.tecnoparco.org/
Default.aspx?tabid5118 and at Molecular Biology and
Evolution online (http://www.mbe.oxfordjournals.org). Se-
quence data from this article are deposited in GenBank
Data library under accession no. provided in Supplemen-
tary table S10.
Acknowledgments
We thank Charlotte Bulich and Isabelle Fuchs for ex-
cellent technical assistance, the Deutsche Forschungsge-
meinschaft for financial support, and the Alexander von
Humboldt Foundation for a stipend to H.O
¨.
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Accepted October 5, 2006
Origin of Wheat B and G Genomes 227