Plant Syst Evol (2011) 291:117–131 DOI 10.1007/s00606-010-0375-1
ORIGINAL ARTICLE
Phylogeny and genetic diversity of D-genome species of Aegilops and Triticum (Triticeae, Poaceae) from Iran based on microsatellites, ITS, and trnL-F Firouzeh Bordbar • Mohammad Reza Rahiminejad Hojjatollah Saeidi • Frank R. Blattner
•
Received: 12 March 2010 / Accepted: 4 October 2010 / Published online: 30 October 2010 Ó Springer-Verlag 2010
Abstract Cereal species of the grass tribe Triticeae are economically important and provide staple food for large parts of the human population. The Fertile Crescent of Southwest Asia harbors high genetic and morphological diversity of these species. In this study, we analyzed genetic diversity and phylogenetic relationships among D genomebearing species of the wheat relatives of the genus Aegilops from Iran and adjacent areas using allelic diversity at 25 nuclear microsatellite loci, nuclear rDNA ITS, and chloroplast trnL-F sequences. Our analyses revealed high microsatellite diversity in Aegilops tauschii and the D genomes of Triticum aestivum and Ae. ventricosa, low genetic diversity in Ae. cylindrica, two different Ae. tauschii gene pools, and a close relationship among Ae. crassa, Ae. juvenalis, and Ae. vavilovii. In the latter species group, cloned sequences revealed high diversity at the ITS region, while in most other polyploids, homogenization of the ITS region towards one parental type seems to have taken place. The chloroplast genealogy of the trnL-F haplotypes showed close relationships within the D genome Aegilops species and T. aestivum, the presence of shared haplotypes in up to three species, and up to three different haplotypes within single species, and indicates chloroplast capture from an
Electronic supplementary material The online version of this article (doi:10.1007/s00606-010-0375-1) contains supplementary material, which is available to authorized users. F. Bordbar M. R. Rahiminejad H. Saeidi Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran F. Bordbar F. R. Blattner (&) Institute of Plant Genetics and Crop Research (IPK), 06466 Gatersleben, Germany e-mail:
[email protected]
unidentified species in Ae. markgrafii. The ITS phylogeny revealed Triticum as monophyletic and Aegilops as monophyletic when Amblyopyrum muticum is included. Keywords Chloroplast trnL-F D genome Evolution Internal transcribed spacer (ITS) Microsatellite (SSR) Systematics
Introduction Iran, as part of the Fertile Crescent, is within the center of diversity of many species of the grass tribe Triticeae. Thus, phylogenetic studies of the taxa of the tribe in this area provide information about the closest relatives of crops such as bread wheat (Triticum aestivum L., 2n = 6x = 42, haploid genome composition ABD) (Saeidi et al. 2006). As the D genome was involved in the formation of bread wheat (Dvorak et al. 1998; Petersen et al. 2006), the genetic diversity present within Aegilops L. species bearing the D genome is of considerable interest. They are potential sources of genetic variation and useful alleles for breeding purposes of T. aestivum and can serve as a secondary gene pool of this species (Feldman and Sears 1981; Kilian et al. 2011). The genus Aegilops contains 22 species comprising both diploids and polyploids (van Slageren 1994) composed of C, D, M, N, S, and U genomes at the diploid level. Kihara (1954) identified for the first time the D genome as a pivotal genome in some species of Aegilops by chromosome pairing in interspecific hybrids. The D genomebearing species of the genus have been classified into Aegilops sections Cylindropyrum and Vertebrata, both of which possess a D genome component derived from their diploid progenitor Ae. tauschii Coss. (2n = 2x = 14, D)
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(Kimber and Zhao 1983; Wang et al. 2000). The D genome exists at three ploidy levels: diploid (Ae. tauschii); tetraploid (2n = 4x = 28; Ae. cylindrica Host., CD; Ae. ventricosa Tausch, DN; and the tetraploid cytotype of Ae. crassa Boiss., XD); and hexaploid [2n = 6x = 42; Ae. vavilovii (Zhuk.) Chennav., XDS; Ae. juvenalis (Thell.) Eig, XDU; and the hexaploid cytotype of Ae. crassa, XDD] (van Slageren 1994). These species are distributed in Iran with the exception of Ae. ventricosa, Ae. vavilovii, and the hexaploid cytotype of Ae. crassa. Cytogenetic investigations have been carried out to establish genome differentiations and phylogenetic insights into the D genome-bearing species. On the basis of meiotic pairing studies in interspecific hybrids involving D genome-bearing species, Kimber and Zhao (1983) divided the species into three groups. The first group included bread wheat, Ae. cylindrica, and Ae. ventricosa with a slight modification in comparison to the D genome of Ae. tauschii. The D genomes of tetraploid and hexaploid cytotypes of Ae. crassa were found to be slightly different and formed the second group. The D genomes of the two hexaploid species Ae. juvenalis and Ae. vavilovii possess substantial modifications and were therefore placed into a third group. Rayburn and Gill (1987) studied genome differentiation of tetraploid species that contain the D genome by using in situ hybridization. They differentiated two patterns in the D genome species. The first included T. aestivum, Ae. cylindrica, and Ae. tauschii and the second Ae. crassa and Ae. ventricosa. These patterns confirmed the groups described by Kimber and Zhao (1983), however the D genome of Ae. crassa was found to be more similar to that of Ae. ventricosa. Badaeva et al. (2002) provided compatible results by using C-banding and in situ hybridization in D genome-bearing species. They showed that the D genome of Ae. ventricosa was more similar to the D genome of Ae. crassa than to that of Ae. cylindrica. In addition they found two different C-banding patterns for Ae. tauschii, one similar to the D genome of T. aestivum, the other to the D genomes of polyploid species of Aegilops. Up to now, there are few molecular studies that have investigated genetic diversity and phylogenetic relationships of all members of the D genome-bearing species. Using nuclear ribosomal DNA internal transcribed spacers (ITS) of polyploid species of Aegilops, Wang et al. (2000) found that the D genome-derived sequences of allopolyploid species were slightly different from those derived from Ae. tauschii and assumed that they might be largely homogenized by concerted evolution toward one of their other ancestors during the process of hybridization and polyploidization, i.e., Ae. cylindrica towards Ae. markgrafii (Greuter) Hammer (synonym Ae. caudata L., 2x = 14, C) and Ae. juvenalis towards Ae. crassa. Using random
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amplified polymorphic DNA markers, Goryunova et al. (2004) studied the diversity and relationships of the D genome-bearing species of Aegilops. The authors found the highest genetic diversity of all D genome species in Ae. tauschii, while relationships among taxa were mainly unresolved. In the present study, we investigate genetic diversity at the inter- and intraspecific level and phylogenetic relationships of the D genome-bearing species of AegilopsTriticum complex using microsatellite (simple sequence repeats, SSRs) markers, and nuclear rDNA internal transcribed spacer (ITS) and chloroplast trnL-F sequences. Microsatellites provide valuable genetic markers due to their high abundance, co-dominant nature, locus specificity, and efficiency in detecting variation among and within plant species (Powell et al. 1996; Ro¨der et al. 1998; Pestsova et al. 2000b; Gandhi et al. 2005). Numerous studies have demonstrated the utility of the ITS region (Hsiao et al. 1995; Wang et al. 2000; Blattner 2004; Jakob and Blattner 2010) and the trnL (UAA) intron and the trnLtrnF (GAA) intergenic spacer (Jakob and Blattner 2006; Liu et al. 2008) for resolving relationships among closely related species in Triticeae. The objectives of this study are (1) to estimate the genetic diversity within the D genome of T. aestivum in comparison to the other polyploids carrying this genome, (2) to present a comprehensive phylogenetic reconstruction of the D genome-bearing species of Aegilops, and (3) to investigate the relationships between T. aestivum and Aegilops D genome-bearing species in Iran by using nuclear and chloroplast DNA markers.
Materials and methods Plant material and DNA isolation We used 122 accessions of different D genome-bearing species in this study. Accessions (Table 1) were selected to represent different geographical distributions and morphological characters of Aegilops and Triticum species in Iran. Seventy-seven accessions were provided by the herbarium of the University of Isfahan, Iran. Seven accessions of Ae. juvenalis, Ae. ventricosa, and Ae. vavilovii were obtained from the collections of the John Innes Centre (Norwich, UK), and 38 accessions from the gene bank of the Leibniz Institute of Plant Genetics and Crop Research (IPK Gatersleben, Germany). For a better representation of the relevant taxa we also added seven accessions of Ae. markgrafii. Total genomic DNA was extracted from young leaves according to Gawel and Jarret (1991) or by using the DNeasy Plant Kit (Qiagen, Hilden, Germany). DNAs of up to 20 individuals per accession were pooled,
4x
4x
4x
6x
6x
2x
6x
A. crassa (cra)
A. cylindrica (cyl)
A. ventricosa (ven)
A. juvenalis (juv)
A. vavilovii (vav)
A. markgrafii (mark or mk)
Triticum aestivum (Trc)
d
c
b
ABD
C
XDS
XDU
DN
CD
XD
D
Genome (Badaeva et al. 2002)
Accession used in trnL-F analysis
Accession used in ITS analysis
Accession provided by John Innes Centre
Accession provided by IPK
2x
A. tauschii (tau)
a
Ploidy level
Taxon (abbrev.)
Table 1 List of the accessions used in this study
Unknown Algeria Greece Romania Germany France
2270001b,c,d (701),2270002b,c,d (702), 2270003b,c,d (703), 2270004b,c,d (704) 645a 10a 20a,c,d 304a 897a,c,d
Germany
1522a
c
a
97 , 107, 70, 57 , 47 , 74, 28, 100 , 51 , 90, 5980 , 5727 , 5581 , 5721a, 5479a, 5550a, 5898a, 6095a, 5956a, 6064a
c
Iran
Unknown c
108176a, 109176a, 1379a,c,d, 1380a,c,d, 1381a c
Iraq
743a,c,d c
Iran
15650c,d
Unknown Syria
(vav601)
2260002b,c,d (vav602)
2260001
England
91a b,c,d
Iran Unknown
182a, 555a, 578a, 1495a, 537a, 582a, 2280001b (801)
UK
15649c,d, 68c,d, 261c,d, 457c,d
1511a
Romania
Morocco
1568a 1515
Germany
29a
a,d
Libya Italy
842a,d 986a
689
a
Iran
508, 513c,d, 479, 489c,d, 490c, 477c,d, 488, 484, 474, 470c,d, 491, 494, 519, 482c, 516, 529c, 520c,d
a
Iran
201, 2, 80, 56, 18c,d, 28, 442, 11c,d, 9, 183, 92, 90, 266, 41, 7, 1c,d, 464c,d, 3, 6, 73d, 12, 82, 4, 261, 361a, 327a
Spain
Iran
537, 26, 20, 413, 18, 28, 411, 412, 9, 1c,d,4, 415, 540, 24c,d, 535, 533c,d, 10c,d, 407c,d, 21, 17c,d, 536c,d
a
Origin
Accession numbers
6.00
3.28
2.08
3.68
4.56
2.24
5.36
11.1
Average allele number
139
121
127
125
135
131
128
138
Average allele length (bp)
0.58
0.46
0.32
0.43
0.42
0.24
0.54
0.81
Polymorphism information content
Phylogeny and genetic diversity of D-genome species 119
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F. Bordbar et al.
and DNA concentration and purity were estimated by spectrophotometry. All of these accessions were included in SSR analysis. For ITS and chloroplast DNA analyses, two to eight accessions of each species were included to represent various geographical regions and morphologically different types to address intraspecific variation. Microsatellite analysis Fifteen Xgwm (Gatersleben wheat microsatellites) and 11 Xgdm (Gatersleben D genome microsatellites) microsatellite loci were selected in order to cover all chromosomes and different arms of the D genome chromosomes (Ro¨der et al. 1998; Pestsova et al. 2000a) and allow reliable amplification of polymorphic fragments. PCR was carried out as described in Ro¨der et al. (1998). The amplification protocol consisted of 94°C for 4 min; 35 cycles of 1 min at 94°C, 1 min at the respective annealing temperature of 50–65°C (see Table 2), 72°C for 1 min; followed by a final extension step of 72°C for 10 min (Saeidi et al. 2006). One of each set of primers was labeled with a
fluorescent dye (6-FAM, HEX, NED) to allow fragment profiling on an automatic DNA sequencer (MegaBACE 1000) using ET400-R size standard (Amersham). The wheat cultivar Chinese Spring (CS) was included as a control in each run to ensure size accuracy. Alleles were scored (Karp et al. 1998; Bredemeijer et al. 1998) using Fragment Profiler version 1.2 (Amersham). In the case of weak or no fragment products, PCR amplifications were repeated to exclude failed PCR reactions as the cause of any null allele. Population genetic analysis Two estimates of genetic diversity for different loci and different species studied were measured using the Microsatellite Toolkit (Park 2001), namely, allelic diversity, which is the mean number of alleles per locus, and allelic polymorphism information content (PIC) values according to the formula of Nei (1973): X PIC ¼ 1 P2ij
Table 2 Microsatellite markers used in this study Microsatellite
Chromosomal location
Motif
Annealing temperature (°C)
Fragment size in CS (bp)
Allele size range
Alleles (n)
PIC
Xgdm111 Xgwm337
1DL 1DS
(CA) 13 (CT) 5 (CACT)6(CA) 43
55 60
193 183
Null, 82–206 Null, 131–211
16 23
0.86 0.87
Xgwm458
1DL
(CA) 13
60
113
91–135
15
0.89
Xgdm6
2DL
(GT) 27
55
152
Null, 82–236
28
0.90
Xgwm102
2DS
(CT) 15
65
143
Null, 121–193
30
0.90
Xgwm539
2DL
(GA) 27
60
147
Null, 103–163
24
0.86
Xgwm261
2DS
(CT) 21
55
192
Null, 130–226
21
0.87
Xgdm8
3DL
(CT) 19
60
137
Null, 121–191
22
0.89
Xgdm72
3DS
(CT) 25imp
60
115
Null, 107–167
19
0.91
Xgwm314
3DL
(CT) 25imp
55
170
98–186
23
0.88
Xgwm3
3DL
(CA) 18
55
84
Null, 64–134
17
0.81
Xgdm61
4DL
(GT) 12
60
115
93–139
17
0.86
Xgwm194
4DL
(CT) 32imp
55
131
Null, 93–137
18
0.88
Xgdm43
5DL
(GA) 24
55
142
Null, 101–181
16
0.89
Xgdm63
5DL
(CT) 20
60
150
Null, 108–178
21
0.86
Xgwm190 Xgwm358
5DS 5DS
(CT) 22 (GA) 18 (G)2(GA) 4
60 55
201 164
Null, 132–246 Null, 132–180
26 18
0.91 0.84
Xgwm212
5DL
(CT) 20
60
104
76–134
19
0.85
Xgwm325
6DS
(CT) 16
60
131
Null, 105–147
13
0.87
Xgdm98
6DL
(GT) 21
60
146
Null, 121–171
14
0.86
Xgdm36
6DS
(GT) 21
60
148
117–219
14
0.71
Xgdm67
7DL
(GT) 20
60
106
Null, 76–172
28
0.92
Xgdm130
7DS
(CT) 27
60
112
Null, 75–131
18
0.86
Xgwm437
7DL
(CT) 24
50
109
Null, 73–129
23
0.91
Xgwm44
7DS
(GA) 28
60
182
94–190
19
0.84
L Long arm, S short arm
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where P2ij is the frequency of the jth allele for the ith locus summed across all alleles for the locus. Binary data were used to compute a pairwise similarity matrix using the Dice similarity index. Pairwise similarities were used to construct a dendrogram by using UPGMA (unweighted pair group method with arithmetic averages) employing SAHN (sequential, agglomerative, hierarchical and nested) clustering in NTSYS-pc version 2.11a (Rohlf 2002). ITS amplification, cloning, and sequencing For amplification of the ITS region, we used primers ITS-A and ITS-B (Blattner 1999). Polymerase chain reactions were performed according to the following cycling program: initial denaturation at 95°C for 3 min; 38 cycles of 95°C for 30 s, 53°C for 45 s, 68°C for 1 min; followed by a final elongation period at 70°C for 8 min. A final volume of 50 ll for each PCR reaction was prepared, containing 20 ng of total DNA, 50 pmol of each primer, 3 ll of 25 lM MgCl2, 5 ll of 109 PCR-buffer, 10 ll of Q-Solution (Qiagen), 0.2 lM of each dNTP, and 1.5 U Taq DNA polymerase (Qiagen). PCR products (amplicons) were purified using Nucleofast 96 PCR plates (Macherey-Nagel) and resuspended in 35 ll TE buffer. PCR products were directly sequenced on MegaBACE 1000 automatic DNA sequencer using the respective dye-terminator sequencing technology (Amersham Biosciences, Freiburg, Germany) and the amplification primers. Sequence heterogeneity was found within ITS sequences of Ae. crassa, Ae. juvenalis, and Ae. vavilovii. Therefore, a cloning step was included to obtain clean sequences for this region. Cloning was performed using Promega’s T-Easy Vector System (Promega), and then the ITS region was reamplified from the transformed bacterial colonies by using a small portion of a colony as the PCR template. Amplicons were purified and directly sequenced as described before. ITS sequence alignment and data analysis Forward and reverse sequences obtained for all samples were inspected and, where necessary, manually edited and combined into unique consensus sequences. When sequences from the same accession were identical, only one was included in the data set. We found some putative pseudogenes, characterized by several point mutations in the otherwise conserved 5.8S rDNA, which were excluded from the analyses. Sequences were stored in the EMBL nucleotide database (accession numbers FR716067-FR716138). Additional sequences of Aegilops species and Triticeae were obtained from EMBL nucleotide database and included in the analyses. The best-fitted model of sequence evolution was estimated to be GTR?G?I with the Akaike information
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criterion in MODELTEST (Posada and Crandall 1998). Phylogenetic trees were calculated in PAUP* 4.0b10 (Swofford 2002) using neighbor-joining cluster analysis (NJ) of pair-wise maximum-likelihood distances and maximum parsimony (MP) with a two-step procedure (Blattner 2004) with the heuristic search algorithm. Initially 1,000 random sequence additions were conducted with TBR branch-swapping and restricting the number of equally most parsimonious trees retained to 10 per replicate. The best trees found in this analysis were used as starting trees in a second heuristic analysis restricting the number of saved trees to 80,000. Hordeum and Psathyrostachys species were defined as outgroup according to previous studies (Hsiao et al. 1995; Blattner 2004). Bootstrap support of branches was calculated for the NJ analysis with 1,000 data resamples. Bayesian analysis (BI) was performed with MrBayes 3.1 (Ronquist and Huelsenbeck 2003) under the GTR ? I ? G model of sequence evolution, running two analyses for 9 million generations, sampling a tree every 200 generations. The initial 30% of the trees were discarded as burn-in, and posterior probabilities were calculated on the basis of the remaining trees. TrnL-F amplification and sequencing To analyze the chloroplast trnL-F region, the locus was amplified according to the protocol given by Jakob and Blattner (2006). Purification and sequencing were performed as described before for ITS sequencing. TrnL-F sequence alignment and data analysis Forward and reverse sequences obtained for all samples were manually edited and combined into unique consensus sequences. These were stored in the EMBL nucleotide sequence database (accession numbers FR716025FR716066). Sequences were aligned using ClustalX (Thompson et al. 1997) and manually improved in Se-Al V2.0a11 (Rambaut 1996). Additional sequences of other Aegilops and Triticeae species obtained from the EMBL nucleotide database were included in the alignment. MODELTEST (Posada and Crandall 1998) estimated GTR?I?G as model of sequence evolution. A phylogenetic tree was calculated in PAUP* 4.0b10 (Swofford 2002) using NJ analysis with maximum-likelihood distances. Sequences of Hordeum and Secale species were included as outgroups. Bootstrap support of branches was calculated with 500 data resamples. Identical trnL-F sequences were combined into haplotypes and analyzed by a statistical parsimony network approach with the program TCS 1.13 (Clement et al. 2000) to infer a chloroplast allele genealogy. In this analysis each insertion/deletion (indel) was considered as a single mutation event, and all indels
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were therefore coded as single positions in the final alignment. Three mononucleotide repeat stretches (A/T) within the sequences were excluded from the analysis due to uncertain homology of the sequence positions, and only Aegilops, Triticum, and Secale species were included, as genetic distances to the Hordeum species were quite high, which makes statistical parsimony networks perform badly.
Results Genetic diversity using microsatellites Twenty-five microsatellite markers that proved to be highly polymorphic (Pestsova et al. 2000b; Saeidi et al. 2006) were used to characterize and evaluate the genetic diversity of 122 accessions of the D genome-bearing species of diverse origins and phenotypes. Microsatellite markers resulted in one or two peaks depending on whether or not they were homozygous or heterozygous, but as bulk DNAs of about 20 individuals were used, it was not possible to distinguish between a DNA mixture of heterogeneous varieties or heterozygosity of single individuals. Six microsatellites of both Xgdm and Xgwm marker sets of different chromosomes gave no amplification products in some accessions: Xgwm337 and Xgwm190 for all accessions of Ae. crassa, and in Ae. markgrafii no alleles were observed for three loci and amplification products were generated only in one out of seven individuals for four loci. As Ae. markgrafii is a diploid bearing the C genome, and D genome-specific primers were employed in PCR, this result was predictable. Absence of amplification was confirmed in repeated experiments and finally scored as null alleles (Table 2). A total of 500 alleles were detected. The number of alleles per locus ranged from 13 for Xgwm325-6D to 30 for Xgwm102-2D with an average number of 20.1 per locus (Table 2). For most of the loci targeted, one or two alleles were fixed in Ae. cylindrica. All microsatellites showed a high level of genetic diversity (PIC), which ranged from 0.71 for Xgdm36-6D to 0.92 for Xgdm67-7D (Table 2). Among the seven D genome-bearing species, Ae. tauschii showed the highest average number of alleles per locus (11.1) and PIC (0.81) followed by T. aestivum with an average of 6 alleles per locus and a PIC value of 0.58. The minimum number of average alleles was found in Ae. vavilovii (2.08) (attributed to the low number of accessions), and the smallest PIC value (0.24) was obtained in Ae. cylindrica (Table 1). Average allele lengths for all loci and species were 132 base pairs (bp) and 131 bp when excluding C genome Ae. markgrafii (ranging from 139 bp for T. aestivum and 138 bp for Ae. tauschii to 127 bp for Ae. vavilovii and 125 bp for Ae. juvenalis; Table 1).
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Variation in ITS and trnL-F sequences The total length of the ITS regions within the D genomebearing species of Aegilops-Triticum and Ae. markgrafii ranged from 600 to 602 bp. The lengths of ITS1 and 5.8S rDNA were identical in all species, 222 and 162 bp, respectively. The only difference observed occurred in the length of ITS2, which ranged from 214 to 216 bp. This difference was due to one dinucleotide (AA/TT) deletion at positions 185–186 of ITS2 in some sequences of Ae. crassa, Ae. vavilovii, and Ae. juvenalis. The sequenced trnL-F region of the D genome-bearing species and Ae. markgrafii ranged between 889 and 951 bp. The length variation was mostly the result of large duplications or deletions within the intergenic spacer. The accessions of Ae. markgrafii were different from the remaining taxa and shared a 25 bp duplication, once in mark1379, 1380, and 15650 and twice in mark743, and a duplication (5 bp) in mark743. Phylogenetic relationships Microsatellite data The UPGMA dendrogram (Fig. 1) based on D genomespecific SSRs divided the species in two main groups, one including T. aestivum, Ae. tauschii, Ae. cylindrica, and Ae. markgrafii, and the second comprising Ae. crassa, Ae. vavilovii, Ae. juvenalis, and Ae. ventricosa. All accessions of T. aestivum grouped together, although internally separated by quite high genetic distances. The accessions of Ae. tauschii showed a different pattern. Most of the accessions clustered together and were sister to T. aestivum, while accessions tau1, 4, 9, 26, and 28 formed the sister group of Ae. cylindrica plus mark108, and tau536 was sister to both groups. This differentiation within Ae. tauschii was not related to geographical distribution or the intraspecific classification of the taxon. The accessions of Ae. markgrafii formed a group with the exception of mark108, which was sister of Ae. cylindrica. In the second group of the dendrogram, Ae. ventricosa (all accessions) was grouped as sister species of Ae. vavilovii, Ae. crassa, and Ae. juvenalis. ITS analyses All analysis approaches resulted in similar species groups with sequences derived from diploid Triticum species and Aegilops plus Amblyopyrum being each monophyletic. The latter group also contains sequences derived from T. aestivum. Relationships among the Aegilops/Amblyopyrum species groups were partly different among the different analysis algorithms or not very well resolved. However,
Phylogeny and genetic diversity of D-genome species Fig. 1 Phenogram of a UPGMA cluster analysis of pairwise distances derived from allelic diversity at 25 D genome-specific microsatellite loci
123 CS -6x (ABD) trc97-6x (ABD) trc107-6x (ABD) trc100-6x (ABD) trc51-6x (ABD) trc57-6x (ABD) trc90-6x (ABD) trc47-6x (ABD) trc28-6x (ABD) trc5980-6x (ABD) trc5727-6x (ABD) trc5581-6x (ABD) trc5479-6x (ABD) trc5550-6x (ABD) trc5721-6x (ABD) trc5956-6x (ABD) trc74-6x (ABD) trc5898-6x (ABD) trc6095-6x (ABD) trc70-6x (ABD) trc6064-6x (ABD) tau537-2x (D) tau535-2x (D) tau533-2x (D) tau18-2x (D) tau10-2x (D) tau17-2x (D) tau540-2x (D) tau24-2x (D) tau20-2x (D) tau413-2x (D) tau411-2x (D) tau412-2x (D) tau21-2x (D) tau415-2x (D) tau407-2x (D) tau26-2x (D) tau28-2x (D) tau9-2x (D) tau1-2x (D) tau4-2x (D) cy508-4x (DC) cy516-4x (DC) cy488-4x (DC) cy484-4x (DC) cy519-4x (DC) cy529-4x (DC) cy482-4x (DC) cy479-4x (DC) cy477-4x (DC) cy494-4x (DC) cy474-4x (DC) cy520-4x (DC) cy489-4x (DC) cy491-4x (DC) cy490-4x (DC) cy513-4x (DC) cy470-4x (DC) mark108-2x (C) tau536-2x (D) mark109-2x (C) mark15650-2x (C) mark1381-2x (C) mark743-2x (C) mark1379-2x (C) mark1380-2x (C) cr201-4x (XD) cr4-4x (XD) cr7-4x (XD) cr80-4x (XD) cr361-4x (XD) cr18-4x (XD) cr28-4x (XD) cr9-4x (XD) cr464-4x (XD) cr41-4x (XD) cr183-4x (XD) cr11-4x (XD) cr82-4x (XD) cr92-4x (XD) cr327-4x (XD) cr90-4x (XD) cr266-4x (XD) cr442-4x (XD) cr12-4x (XD) cr2-4x (XD) cr73-4x (XD) cr56-4x (XD) cr261-4x (XD) cr1-4x (XD) cr6-4x (XD) cr3-4x (XD) juv801-6x (XDU) juv578-6x (XDU) juv537-6x (XDU) juv91-6x (XDU) juv582-6x (XDU) juv1495-6x (XDU) juv555-6x (XDU) juv182-6x (XDU) juv261-6x (XDU) juv68-6x (XDU) juv457-6x (XDU) juv15649-6x (XDU) vav601-6x (XDS) vav602-6x (XDU) ven1522-4x (DN) ven1515-4x (DN) ven20-4x (DN) ven10-4x (DN) ven29-4x (DN) ven689-4x (DN) ven1511-4x (DN) ven304-4x (DN) ven701-4x (DN) ven704-4x (DN) ven702-4x (DN) ven703-4x (DN) ven1568-4x (DN) ven645-4x (DN) ven897-4x (DN) ven842-4x (DN) ven986-4x (DN)
T. aestivum
Ae. tauschii
Ae. cylindrica
Ae. markgrafii Ae. tauschii Ae. markgrafii
Ae. crassa
Ae. juvenalis
Ae. vavilovii
Ae. ventricosa
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Ae. biuncialis AF157003 4x (UM) Ae. neglecta AF157004 4x (UM) Ae. kotschyi AF1577002 4x (SU) Amblyopyrum muticum 2x (T) 0.69 Ae. peregrina AF156996 4x (SU) 0.6 Ae. columnaris AF156997 4x (UM) Ae. geniculata AF156998 4x (UM) Ae. umbellulata AF149197 2x (U) Ae. comosa AF149198 2x (M) Ae. uniaristata AY775272 2x (N) 1 Ae. uniaristata AY775273 2x (N) 0.73 Ae. uniaristata AF149200 2x (N) Ae. ventricosa 701 4x (DN) Ae. ventricosa 702 4x (DN) 0.68 Ae. ventricosa 703 4x (DN) Ae. ventricosa 704 4x (DN) Ae. ventricosa 20 4x (DN) 0.51 Ae. ventricosa 897 4x (DN) Ae. speltoides AJ301804 2x (S) 0.97 Ae. comosa subventricosa AY 775264 2x (M) Ae. comosa subventricosa AY 775265 2x (M) Ae. juvenalis 261 6x (XDU) 0.8 Ae. juvenalis 261a 6x (XDU) Ae. juvenalis 261g 6x (XDU) 0.5 Ae. juvenalis 68d 6x (XDU) Ae. juvenalis 457e 6x (XDU) 0.9 Ae. juvenalis 457b 6x (XDU) Ae. juvenalis 457g 6x (XDU) 0.63 Ae. juvenalis 15649d 6x (XDU) 0.59 Ae. juvenalis 68 6x (XDU) Ae. juvenalis 15649 6x (XDU) Ae. juvenalis 457 6x (XDU) Ae. juvenalis 261c 6x (XDU) Ae. juvenalis 15649e 6x (XDU) Ae. juvenalis 15649f 6x (XDU) Ae. cylindrica 477 4x (CD) Ae. cylindrica 513 4x (CD) Ae. cylindrica 489 4x (CD) Ae. cylindrica 470 4x (CD) 0.59 Ae. cylindrica 520 4x (CD) Ae. cylindrica 529 4x (CD) Ae. cylindrica 482 4x (CD) Ae. cylindrica 490 4x (CD) Ae. triuncialis AF156994 4x (CU) Ae. markgrafii 1379 2x (C) 0.81 Ae. markgrafii 1380 2x (C) Ae. markgrafii 7431 2x (C) Ae. markgrafii 15650 2x (C) Ae. markgrafii AY775263 2x (C) 1 Ae. vavilovii 602b 6x (XDS) 0.86 Ae. vavilovii 602c 6x (XDS) Ae. vavilovii 602a 6x (XDS) Ae. crassa 464c 4x (XD) Ae. crassa 464a 4x (XD) 0.61 Ae. crassa 18c 4x (XD) Ae. crassa 18a 4x (XD) 0.52 Ae. crassa 11c 4x (XD) Ae. vavilovii 602d 6x (XDS) Ae. crassa 18f 4x (XD) 0.56 Ae. crassa 1f 4x (XD) Ae. crassa 464b 4x (XD) Ae. crassa 18e 4x (XD) Ae. crassa 11a 4x (XD) 0.94 Ae. crassa 11e 4x (XD) 0.73 Ae. vavilovii 602e 6x (XDS) Ae. juvenalis 68g 6x (XDU) 0.6 0.79 Ae. vavilovii 601b 6x (XDS) 0.93 Ae. vavilovii 601g 6x (XDS) 0.55 Ae. vavilovii 601c 6x (XDS) Ae. juvenalis 15649c 6x (XDU) Ae. crassa 1d 4x (XD) Ae. juvenalis 68f 6x (XDU) Ae. tauschii 1 2x (D) 1 Ae. tauschii 407 2x (D) 0.86 Ae. tauschii 536 2x (D) Ae. bicornis AF149192 2x (Sb) Ae. tauschii 533 2x (D) 0.99 1 Ae. tauschii 24 2x (D) Ae. tauschii 17 2x (D) Ae. tauschii 10 2x (D) T. aestivum 97 6x (ABD) T. aestivum 47 6x (ABD) 1 T. aestivum 100 6x (ABD) T. aestivum 51 6x (ABD) 1 T. aestivum 57 6x (ABD) 1 Ae. speltoides AY450267 2x (S) 0.94 Ae. speltoides AY450268 2x (S) Ae. sharonensis AF149195 2x (Sl) Ae. vavilovii 601a 6x (XDS) 1 Ae. vavilovii 601e 6x (XDS) 1 Ae. vavilovii 601d 6x (XDS) 0.75 Ae. vavilovii 601f 6x (XDS) 0.81 Ae. searsii AF149194 2x (Ss) Ae. longissima AF149196 2x (Sl) Ae. crassa 1c 4x (XD) 0.81 T. monococcum AJ301800 2x (Am) 1 T. urartu AJ301803 2x (Au) T. urartu AY450265 2x (Au) 1 Secale vavilovii AJ608152 2x (R) Secale cereale AF303400 2x (R) Taeniatherum caput-medusae AJ608153 2x (T) Agropyrum cristatum AJ608149 2x (P) Dasypyrum villosum AJ608150 2x (V) Hordeum vulgare AF438189 2x (H) Hordeum murinum glaucum AJ607986 2x (Xa) Hordeum bogdanii AJ607819 2x (I) Psathyrostachys juncea AJ608151 2x (Ns) 0.53
U
M+U
N
M
X?
C
D?
D D
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1
S
0.93
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Chloroplast analyses
b Fig. 2 Phylogenetic tree derived from nuclear rDNA ITS sequences
inferred by Bayesian phylogenetics. Numbers along branches give posterior probabilities, bold branches indicate groups also present in the strict consensus tree of a maximum parsimony analysis. To the right probable genome affiliations of sequence groups are given. Sequences obtained from the nucleotide database are depicted by sequence accession numbers after taxon names. Hordeum and Psathyrostachys sequences were defined as outgroups in the analyses
The NJ analysis of the trnL-F region revealed Triticum/ Aegilops as clearly separate from Secale and the Hordeum species used as outgroups. The analysis resolved several species and species groups, although few branches within Aegilops got bootstrap support [50%. A specific feature of the NJ tree is the occurrence of many zero-length branches, typical for analyses where taxa not only group at the tips of a tree but also at internal branching points (Fig. S1, available as supplemental online material). Therefore, this tree essentially represents a network (Jakob and Blattner 2006) and was analyzed by a statistical parsimony network approach. TCS calculated a 95% parsimony connection limit of 12 steps for the trnL-F alignment consisting of 20 haplotypes, 8 of them exclusively found in the D genome-bearing species (Fig. 3). Twenty haplotypes were inferred but not found in the analyzed individuals (missing intermediates), mostly separating S. cereale from Triticum/Aegilops. Five positions in the network revealed closed loops caused by homoplastic alignment positions, of which one could not be resolved unambiguously. The D genome-bearing species possess eight closely related chloroplast haplotypes, mostly one or two mutational steps away from the central haplotype 1 (HT1). Eight chloroplast haplotypes are shared between two and four species. The majority of individuals in our analysis possess HT1, occurring in Ae. tauschii, Ae. cylindrica, and Ae. ventricosa. The highest allele numbers were found in Ae. tauschii and Ae. ventricosa, each species with three haplotypes, while T. aestivum, Ae. juvenalis, Ae.
none of the relationships among these groupings got convincing statistical support in NJ, MP, or BI. Therefore, we show in Fig. 2 the BI tree, where the Aegilops groups occur along a large polytomy, and indicate groups also present in the strict consensus of 80,000 MP trees (length 408 steps, CI = 0.59, RI = 0.77). This tree might be seen as a conservative estimation of phylogenetic relationships among the ITS sequences. It shows, however, a peculiar position of the Ae. bicornis sequence within the Ae. tauschii clade that none of the other analyses provided, as in MP and NJ Ae. tauschii was monophyletic, although Ae. bicornis was sister to Ae. tauschii in MP. In all analysis approaches, sequences derived from the cloned PCR products of Ae. crassa, Ae. vavilovii, and Ae. juvenalis occurred in a mixed clade consisting of two subclades, with additional sequences of Ae. juvenalis forming a separate clade. Sequences of the other D genome species, where we included several individuals per taxon, group according to their species affiliation. For the Triticum and Aegilops clades, we tried to correlate sequence groups with their putative genomic origin (Fig. 2), which was not unambiguously possible for the two sequence groups derived from Ae. crassa, Ae. vavilovii, and Ae.juvenalis. 16 T.mon
8
ven 842
7 ven703, 897
3 Trc47,51,57, 97,100,107
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18 A.biu, A.gen A.umb Am.mut
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cra1; vav601, 602
5 tau24, 407
15 mk743
10 A.per
9
A.kot A.bic A.sea
11 A.sha
17
S.cer
Fig. 3 Genealogical network of haplotype relationships derived from sequences of the chloroplast trnL-F region. Haplotypes were arbitrarily numbered. Sequences of D genome species generated in this study are indicated by the accession numbers of individuals, and sequences obtained from the nucleotide database are indicated by
abbreviated species names (for EMBL database accession numbers, see Fig. S1). Black dots indicate inferred haplotypes not present in the analyzed individuals. The probable root of the network within Aegilops/Triticum, defined by the inclusion of Secale cereale, is depicted by an asterisk
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cylindrica, and Ae. vavilovii possess only a single haplotype each, one of them shared between Ae. crassa and Ae. vavilovii (HT4). The haplotype found in T. aestivum (HT3) is closest to one of the Ae. tauschii haplotypes (HT6). The other species of Aegilops fall outside of this central group within the network. The accessions of Ae. markgrafii comprise three haplotypes, two with close relationships to Ae. triuncialis and a quite distant one closely related to alleles of Ae. columnaris and Ae. neglecta.
Discussion Genetic diversity based on SSR, ITS, and trnL-F data Genetic diversity of the D genome-bearing species, a comparison of nuclear and chloroplast data Based on SSR analysis, the accessions belonging to Ae. tauschii showed a wide range of alleles. The maximum PIC value indicates higher genetic differentiation in this species than in any of the studied D genome polyploids. High levels of genetic diversity in this species in Iran were reported by Saeidi et al. (2006) and Naghavi et al. (2009) using SSR and AFLP markers, and Naghavi et al. (2008) using SSRs. Saeidi et al. (2006) suggested the possibility of Iran as the ancestral center of origin of Ae. tauschii. Based on variation at 10 chloroplast SSR loci in Ae. tauschii, Matsuoka et al. (2005) found the Caspian region of northern Iran to be the area with highest haplotype diversity. In our analysis, genetic diversity of Ae. tauschii was also revealed using chloroplast sequences, as we found three trnL-F haplotypes in this species, although we excluded the variation of three chloroplast microsatellite loci present within the trnL-F spacer due to uncertain character homology. Also ITS sequencing resolved three different ribotypes for this species. Thus, we conclude that Ae. tauschii populations in Iran contain high levels of genetic diversity. The accessions of Ae. cylindrica showed only one allele in most SSR loci, and it is evident from the low PIC value and small genetic distances that individuals are genetically quite similar throughout the study area. Thus, Iran might harbor only marginal populations with reduced genetic diverstiy of this more westerly distributed species. Badaeva et al. (2002), based on C-banding and FISH analysis, mentioned that the low amount of intraspecific polymorphism of Ae. cylindrica might reflect a recent origin of this species. The single chloroplast haplotype of the trnL-F region can support such a view. However, in the ITS region we found no indications of the presence of a second ITS type that is often present in young allopolyploids depending on the tempo of intergenomic rDNA
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homogenization (Blattner 2004). Thus, Ae. cylindrica seems to be old enough to have allowed locus homogenization between the two genomes of this allopolyploid (but see below). In the accessions of T. aestivum, only one chloroplast haplotype was found, and also with regard to the ITS region, the accessions were completely uniform (Figs. 2, 3). In contrast, SSR markers revealed the second highest genetic diversity (PIC = 0.58) among the D genomes screened. This result is quite surprising, as the formation of hexaploid wheat happened only about 8,000 years ago (Nesbitt and Samuel 1996) by hybridization of diploid Ae. tauschii with the already domesticated tetraploid emmer wheat, and the D genome of bread wheat is thought to be the least diverse within this species (Kilian et al. 2011). If T. aestivum originated via a single hybridization event (Petersen et al. 2006), all allelic diversity in its D genome should have evolved since then. Assuming mainly equal mutation rates for homologous SSR loci among D genome polyploids would mean that they are all quite young (\8,000 years), as none of their D genomes is genetically as diverse as in T. aestivum. This seems a highly unlikely assumption to us for the naturally occurring polyploids. Therefore, we have to assume either multiple origins of the hexaploid, prolonged gene flow from wild Ae. tauschii into T. aestivum, elevated SSR mutation rates after hexaploid formation, or a technical bias of the used marker regions. Multiple origins of bread wheat are in contradiction with the archeological record indicating only the Caspian region as area of origin of hexaploid wheat (Nesbitt and Samuel 1996), although it does not exclude the possibility that hexaploid formation happened several times in this area (Kilian et al. 2011). The possibility that different genomes within this species carry different amounts of genetic diversity was already discussed by Petersen et al. (2006), although they could not arrive at a convincing explanation when taking into account a single origin of the polyploid. A bias of detected genetic diversity has been reported several times when SSR loci were transferred to other species. The reason for this is most probably the selection of SSR loci for a high number of repeat units during SSR development to ensure high allelic diversity. In other species, however, a lower number of repeat units might be present at homologous loci, resulting in lower mutation rates and accordingly allele numbers in comparison to the species for which the SSR loci were initially developed (Pleines et al. 2009). As the SSR loci of our study were developed for the D genomes of bread wheat and Ae. tauschii, we cannot exclude such a technical bias as a reason for the exceptionally high SSR diversity found in T. aestivum, particularly as highest average allele lengths per species in our dataset occur in Ae. tauschii (138 bp) and T. aestivum (139 bp). However, Ae. cylindrica (131 bp) meets the
Phylogeny and genetic diversity of D-genome species
average allele length over all species and loci screened and possesses quite low SSR diversity (PIC = 0.24), while in Ae. juvenalis average allele length is only 125 bp, although genetic diversity of this species is comparatively high (PIC = 0.43). Thus, although highest SSR allele numbers correlate with longer loci on average, shorter loci can also be quite diverse. As we did not resequence the SSR loci in the analyzed species, we cannot exclude that indels outside the repeat motifs contribute to allele length differences without influencing SSR repeat numbers themselves. Gene flow from diploid to polyploid Triticeae species has been reported (Jakob and Blattner 2006) and seems possible in low-intensity farming in early agriculture in the Fertile Crescent, where domesticated and wild grasses grew in close proximity or even mixed stands. It is, however, assumed to be rare in Aegilops (Meimberg et al. 2009). Also from our data, it seems unlikely that extended gene flow from diploid Ae. tauschii into bread wheat resulted in genome-specific recombination in the nucleus, enriching SSR diversity in the hexaploid, without leaving traces in its ITS region. To clarify reasons for the exceptionally high SSR diversity in the cultivated species in comparison to its wild relatives, we think it will be necessary to (1) screen genome-specific diversity of the A and B genomes of bread wheat in comparison to wild species with these genomes (Petersen et al. 2006), (2) sequence a diverse array of SSR alleles in the D genome species, and (3) analyze species histories to gain insights into the influence of demographic parameters on genetic diversity (Jakob et al. 2009, 2010). A reversed pattern of genetic diversity was found in Ae. crassa, where D genome-specific SSRs resulted in modest genetic distances among individuals in comparison to the other species included here (Fig. 1), but two chloroplast haplotypes were found, and a high number of ITS ribotypes occur within single individuals as well as among different individuals. This pattern might also be explained with a biased repeat number in SSR loci, resulting in lower allelic diversity in this species. Both chloroplast haplotypes are one mutation step apart, thus it is possible that this variation originated within the species and is no indication of multiple origins of this tetraploid. While only one chloroplast haplotype was detected in Ae. juvenalis, SSR and ITS diversity is high in ITS sequences of this species occurring in two major groups in Fig. 2, and in the informally named D0 clade, ITS sequences from this species can be found in both subgroups, always together with sequences derived from Ae. crassa and Ae. vavilovii. For all three species, we cannot exclude that some of the ITS diversity was introduced by PCR errors, which are easily visible in cloned PCR amplicons. However, even direct sequencing produced different ITS sequences, indicating that at least to some extent the detected ITS diversity is real.
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In the SSR analysis (Fig. 1), Ae. ventricosa contains a group of geographically widespread but genetically rather narrow accessions embedded within lineages with higher genetic diversity mostly from Iran. In the chloroplast, three haplotypes could be identified, two of them occurring exclusively in this species. In contrast, all individuals sequenced possess the same ITS type indicating homogenization of the ITS region towards the N genome progenitor of this tetraploid. Also in Ae. markgrafii chloroplast diversity is relatively high with three very diverse trnL-F haplotypes (Fig. 3), probably indicating hybridization and chloroplast capture. While just one type of ITS sequence was found, SSR diversity was high, at least for the loci amplified in this species. Phylogenetic relationships Homology of diploid Ae. tauschii to other D genome-bearing species Based on the cluster analysis of SSR markers, all of the accessions tended to be clustered according to their attributed species classification with the exception of Ae. tauschii and Ae. markgrafii. This indicates that the D genomes present in the different polyploid species have sufficiently diverged during evolution to be distinguished by the used SSR markers. Aegilops tauschii, however, was subdivided in two groups (Fig. 1), as some of the accessions grouped with T. aestivum while others were clustered with the accessions of Ae. cylindrica. Isozyme analysis (Jaaska 1981) as well as FISH and C-banding pattern (Badaeva et al. 1996, 2002) already revealed intraspecific diversity within Ae. tauschii, and different D genomes of Ae. tauschii in bread wheat versus other polyploid Aegilops species were postulated. Also Gandhi et al. (2005) resolved two different groups within Ae. tauschii based on nuclear SSR markers. Our analysis of Iranian materials revealed that both types of Ae. tauschii are present in Iran. Three of the accessions grouped with Ae. cylindrica (tau1, 4, and 9) are from northwestern Iran. Based on retroelement polymorphisms and phenotypic characters, Saeidi et al. (2008a) found a clearly different gene pool near the Iranian-Turkish border in comparison to populations from other areas of this species’ distribution. However, the D genome type of Ae. tauschii found to be close to Ae. cylindrica occurs not only in this area but also in the northeastern part of Iran (tau26, 28, 36), whereas it is seemingly absent in the Iranian inland populations. Aegilops tauschii is traditionally classified into two morphologically and geographically defined subspecies, strangulata (Eig) Tzvelev and tauschii (Hammer 1980), which differ mainly by traits related to spike shape
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(Takumi et al. 2009). Among the accessions studied, tau407, 535, and 537 were classified as belonging to subsp. strangulata. We found, however, no correlation between groups within Ae. tauschii in our SSR analysis and the described subspecies of this taxon, confirming earlier results of molecular studies (Dvorak et al. 1998; Knaggs et al. 2000; Lelley et al. 2000; Dudnikov and Kawahara 2006; Saeidi et al. 2008a, b; Naghavi et al. 2008). Also van Slageren (1994), who reported a high number of morphologically intermediate forms within Ae. tauschii subspecies, concluded that the intraspecific classification of this species is not adequate. ITS data resolve Ae. tauschii sequences as a separate unit within the large polytomy of Aegilops/Triticum species (Fig. 2). Neither ITS sequences of T. aestivum or Ae. cylindrica nor any other polyploid assumed to contain a D genome fall in a clade with this species. This suggests that (1) Ae. tauschii was not the D genome donor (it might have been a now-extinct close relative; see e.g., Jakob and Blattner 2010), (2) ITS sequences in all polyploid D genome-bearing species have already substantially diverged from their putative progenitor sequences, or (3) they were homogenized towards the ITS types provided by their non-D genome donor. This result is in agreement with the results of Wang et al. (2000) who mostly found only single ITS types within polyploid Aegilops species (but see below). Moreover, ITS sequences are not able to safely resolve species and genome relationships within Aegilops/ Triticum, as species and species groups all occur along a polytomy in the phylogenetic tree, although ITS has proved suitable in other genera of Triticeae. This could be an indication for a relatively fast radiation of these groups initially during the evolution of Aegilops/Triticum about 2–3 million years ago (Chalupska et al. 2008) or indicates that some of the ITS types originated from recombinant molecules, as the inclusion of hybrid sequences often results in a loss of resolution in phylogenetic analyses. Analysis of chloroplast trnL-F sequences found an identical haplotype in Ae. tauschii, Ae. cylindrica, and Ae. ventricosa, and closely related alleles in all other D genome species, which might indicate different Ae. tauschii types as maternal progenitors of these polyploids. The haplotype of T. aestivum was different, although closely related to the types found in D genome Aegilops species, supporting a paternal contribution of Ae. tauschii in the evolution of bread wheat (Petersen et al. 2006). Phylogeny of polyploid D-genome species: nuclear and chloroplast inference In our analysis of D genome Aegilops species, we initially used direct sequencing of amplicons of the rDNA ITS region to infer presence or absence of different ITS copies
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in polyploid species. This approach resulted in the detection of the presence of multiple ITS copies only for Ae. crassa, Ae. juvenalis, and Ae. vavilovii. Sequencing of cloned PCR products confirmed the presence of quite diverse ITS types in these cases, indicating the persistence of different parental rDNA clusters in the polyploids. No such types could be inferred in the tetraploids Ae. cylindrica and Ae. ventricosa and hexaploid T. aestivum. The sensitivity of the initial detection of different ITS types depends, however, on the contribution of approximately equal amounts of these DNAs during the initial cycles of PCR. As this cannot be guaranteed, finding no indications of different ITS types in these species does not mean that all rDNA clusters in the genome are already homogenized towards one parental type. Therefore, sequencing a high number of cloned ITS amplicons might provide evidence that traces of these parental types are still present in the taxa where we or Wang et al. (2000) did not find them. As discussed below, the complex mode of evolution of the nuclear rDNA renders the ITS region less suitable for polyploid analysis in comparison with nuclear single-copy loci (Alvarez and Wendel 2003; Small et al. 2004), as intergenome homogenization is much less likely for lowcopy loci. They should therefore carry the information of parental genomes incorporated in allopolyploids for a much longer time. There are, however, trade-offs regarding evolution rates and availability of universal PCR primers for low-copy loci, making them often less suitable in narrow taxonomic groups in comparison with ITS sequencing (Jakob and Blattner 2010). Aegilops cylindrica (DC) originated from hybridization between Ae. markgrafii (C) and Ae. tauschii (D) (Kimber and Zhao 1983). Based on protein electrophoretic studies, Jaaska (1978) hypothesized that the origin of Ae. cylindrica was in the eastern part of Turkey where the distribution areas of the parental diploids overlap. In our chloroplast analysis, Ae. tauschii and Ae. cylindrica haplotypes revealed a high homology and are only distantly related to the chloroplast types occurring in Ae. markgrafii, indicating that Ae. tauschii was the maternal progenitor of Ae. cylindrica. In the ITS analysis, a close relationship of the sequences from Ae. markgrafii and Ae. cylindrica was found. Therefore, we assume that the rDNA clusters of Ae. cylindrica were homogenized towards Ae. markgrafii. Using chloroplast DNA data Yamane and Kawahara (2005) found Ae. markgrafii (Ae. caudata in their publication) to be polyphyletic with individuals from the western part of the distribution area grouping with Ae. umbelullata and Amblyopyron muticum, while other individuals were resolved as sister to members of Aegilops section Comopyrum. Also in our study sequence, data support two different groups of chloroplasts present in Ae. markgrafii (Fig. 3). However, nuclear data do not reflect
Phylogeny and genetic diversity of D-genome species
this split. Thus, neither ITS nor SSR analysis places accession mk1379 (with chloroplast HT20) outside the bulk of Ae. markgrafii individuals possessing HT13 and 15. This indicates ancient introgression resulting in chloroplast capture and, thus, the occurrence of diverse chloroplast types in Ae. markgrafii, without leaving traces in the nucleus, at least not in the parts screened by the markers we used. The nuclear data therefore do not support the existence of two cryptic species within this taxon as implied by Yamane and Kawahara (2005). Aegilops markgrafii groups in the SSR dataset (Fig. 1) with the non-Triticum part of the Ae. tauschii individuals and Ae. cylindrica, and in the ITS tree with the C genomes of Ae. cylindrica and Ae. triuncialis in a clade with M, N, U, and putative X genome diploids (Fig. 2). From our data we are not able to deduce if this means that its C genome is closely related to the D genomes, as implied by partial amplification of our D genome-specific SSRs in Ae. markgrafii, or if the C genome is more closely related to the M/N/U/X group, as indicated by the ITS dataset, particularly as statistic support for this latter hypothesis is very low. Aegilops crassa occurs as tetraploid and hexaploid cytotypes. Cytogenetic analyses on Ae. crassa individuals from all over Iran showed that the hexaploid form does not exist in this area (data not shown). The D genome of tetraploid Ae. crassa (XD) is thought to be derived from the D genome of Ae. tauschii, while the origin of the X genome is not clear (Badaeva et al. 2002). The D genomes of Ae. vavilovii (6x) and Ae. juvenalis (6x) were inherited from tetraploid Ae. crassa (Kihara 1957). The S genome of Ae. vavilovii stems from Ae. searsii and the U genome of Ae. juvenalis from Ae. umbellulata (Badaeva et al. 2002). In our analysis, Ae. crassa and Ae. juvenalis share chloroplast HT2, and Ae. crassa and Ae. vavilovii HT4. Both haplotypes are closely related, indicating the close maternal relationship within this species group. Also in the ITS analyses, sequences derived from these three species occur together in one group (D0 ), with additional sequences from Ae. juvenalis forming a separate clade (X). As the D0 group is subdivided in two clades both containing sequences from all three species, we conclude that two ITS types occur in tetraploid Ae. crassa and hexaploid Ae. vavilovii, while Ae. juvenalis, which is also hexaploid, contains three ITS types. However, none of the detected types groups with good statistical support with ITS sequences generated from diploid species with the basic genomes occurring in Aegilops. Our results are in contrast to Wang et al. (2000) who assumed homogenization of the rDNA sequences towards only one of the parental species in all polyploid Aegilops. This is not the case here because cloned ITS amplicons indicate the persistence of different rDNA types within these polyploids. What we can conclude from our ITS data is a close relationship among Ae. crassa, Ae. juvenalis, and
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Ae. vavilovii, which is supported by the results from SSR analysis that also groups these species together in a clade. Based on the analysis of ITS and trnL-F sequences, Am. muticum falls within the group of Aegilops species included in our analysis, supporting the view that this species belongs to Aegilops (e.g., Petersen et al. 2006; Baum et al. 2009) and should be treated as Ae. mutica (Yamane and Kawahara 2005). Sequences generated from Aegilops plus Amblyopyrum form a monophyletic group in our ITS analyses separate from sequences of the two Triticum diploids included here, which are also monophyletic. Although the Aegilops/Amblyopyrum clade includes ITS sequences of T. aestivum, this does not change its monophyletic status, as these ITS copies are most likely derived from the Ae. speltoides-like progenitor of bread wheat contributing the B genome. Due to the restriction of our analysis to few diploid Triticeae species outside Aegilops and Triticum, the monophyly of both genera in ITS analyses might, however, not be completely resolved. Acknowledgments We thank S. Reader (John Innes Center, Norwich, UK), the gene bank of the IPK, and the herbarium of the University of Isfahan for providing plant materials. Thanks to P. Oswald for assistance with laboratory work, C. Koch for greenhouse sample management, K. Zeynali Nejad for technical help with SSR markers, and M. Gurushidze, E. Achigan-Dako, and S. Jakob for help in data analysis. We acknowledge helpful remarks on the manuscript by N. Haider. The authors wish to thank the Office of Graduate Studies of the University of Isfahan for their support.
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