Chromosome and Molecular Analyses Reveal Significant Karyotype Diversity and Provide New Evidence on the Origin of Aegilops columnaris

Aegilops columnaris Zhuk. is tetraploid grass species (2n = 4x = 28, UcUcXcXc) closely related to Ae. neglecta and growing in Western Asia and a western part of the Fertile Crescent. Genetic diversity of Ae. columnaris was assessed using C-banding, FISH, nuclear and chloroplast (cp) DNA analyses, and gliadin electrophoresis. Cytogenetically Ae. columnaris was subdivided into two groups, C-I and C-II, showing different karyotype structure, C-banding, and FISH patterns. C-I group was more similar to Ae. neglecta. All types of markers revealed significant heterogeneity in C-II group, although group C-I was also polymorphic. Two chromosomal groups were consistent with plastogroups identified in a current study based on sequencing of three chloroplast intergenic spacer regions. The similarity of group C-I of Ae. columnaris with Ae. neglecta and their distinctness from C-II indicate that divergence of the C-I group was associated with minor genome modifications. Group C-II could emerge from C-I relatively recently, probably due to introgression from another Aegilops species followed by a reorganization of the parental genomes. Most C-II accessions were collected from a very narrow geographic region, and they might originate from a common ancestor. We suggest that the C-II group is at the initial stage of species divergence and undergoing an extensive speciation process.


Introduction
Aegilops columnaris Zhuk. is annual tetraploid (2n = 4x = 28) grass species naturally growing in Western Asia, mainly in Turkey, Armenia, and in a western part of the Fertile Crescent [1][2][3]. It is also native to Crete, Iraq, Lebanon, Azerbaijan, and Iran but found as adventive species in France, near Marseille [1]. Despite a relatively broad distribution area, Ae. columnaris is uncommon throughout its range. Biodiversity Collecting Mission Database included 816 Ae. columnaris site records (https://www.gbif.org/), and according to Genesys, 763 accessions are currently maintained in gene banks worldwide (https: //www.genesys-pgr.org/). This number, however, may be overestimated owing to a large number of potentially duplicated and incorrectly classified accessions. From the other The aim of the present study was the analysis of intraspecific diversity of Ae. columnaris on a broader sample of accessions using cytogenetic (C-banding, FISH with various DNA probes), biochemical (seed storage proteins-gliadins), and molecular (comparative sequence analysis of nuclear and chloroplast DNA fragments) markers.

C-Banding Analysis of Ae. columnaris
We showed that most of Ae. columnaris accessions were karyotypically uniform, but two, K−4224 and a sample provided by Drs. E.A. Nazarova and A.G. Gukasyan in 1998, consisted each of three distinct biotypes, while PI 554187-of two biotypes (Table 1). Three accessions (PI 564182 from Turkey and K−4553 and K−4233 from Armenia) maintained in gene banks under the name Ae. columnaris were found to be taxonomically misclassified and were in fact Ae. neglecta. Accession IG 49067 was the mix of Ae. columnaris and Ae. biuncialis, whereas accessions K−2344 from Armenia and AE 1607 of unknown origin-the mix of Ae. columnaris and Ae. triuncialis. One plant in K−4224 accession was found to be the F 1 hybrid between Ae. columnaris and Ae. triuncialis (Figure 1c).   The C-banding analysis revealed that Ae. columnaris gene pool consists of two distinct karyotypic groups that differed from each other and from Ae. neglecta ( Figure  1a,b,d) in karyotype structure, the number of satellite chromosomes, and C-banding patterns. The larger group was designated C-I, whereas the smaller one-C-II. Group C-I triuncialis (genotype unknown) carrying reciprocal translocations 1U c :5U c derived from Ae. columnaris and 1U t :7C t derived from Ae. triuncialis; (d)-Ae. neglecta (K−4553). Chromosomes are designated according to genetic nomenclature; the U c /U tr chromosomes are labeled in dark green, the X c /X tr chromosomes in dark blue, the C t of Ae. triuncialis-in red, and the U t in grey color). Red arrowheads point to satellite chromosomes. Blue arrows show translocated 5U c :6X c chromosomes (a).
The C-banding analysis revealed that Ae. columnaris gene pool consists of two distinct karyotypic groups that differed from each other and from Ae. neglecta (Figure 1a,b,d) in karyotype structure, the number of satellite chromosomes, and C-banding patterns. The larger group was designated C-I, whereas the smaller one-C-II. Group C-I included 62 accessions collected from an entire distribution range, while C-II comprised only seven accessions, six from the southeast coastal part of Turkey and one from Iraq ( Figure 2). Both Ae. columnaris groups demonstrated high diversity in the C-banding patterns and, in the case of C-I, broad translocation polymorphism (Figures 3-7). Thus, karyotypes of 31 C-I accessions (50%) differed from each other only in the presence/absence or the size of Giemsa C-bands in particular positions; this karyotypic variant was considered basic or "normal (N)." Karyotypes of 31 accessions derived from normal as a result of one or more structural chromosome rearrangements. Accessions collected from geographically closer regions usually had more similar banding patterns than accessions from distant locations, and this trend was also observed in genotypes with chromosomal rearrangements. The highest C-banding and translocation polymorphisms were observed in Turkey. Ae. neglecta was similar to Ae. columnaris in the amount and distribution of C-bands on most chromosomes, but differed in the morphology of 6X c , which was more metacentric (arm ratio L/S = 1.173 vs. 1.924). In contrast to the C-II group, Ae. neglecta carried three pairs of satellite chromosomes as the C-I accessions (Figure 1a  Ae. neglecta was similar to Ae. columnaris in the amount and distribution of C-bands on most chromosomes, but differed in the morphology of 6X c , which was more metacentric (arm ratio L/S = 1.173 vs. 1.924). In contrast to the C-II group, Ae. neglecta carried three pairs of satellite chromosomes as the C-I accessions (Figure 1a,d). Three accessions of Ae. neglecta had similar C-banding patterns (Figure 3t-v) and did not possess chromosomal rearrangements.
Twenty-six C-I accessions were collected in different regions of Turkey (Table 1; Figure 4). Nearly half of them (12 accessions) had normal karyotypes (N), and 14 (including segregating accession AE 1607) carried 11 variants of chromosomal rearrangements ( Table 2 (Table 2).  Twenty-six C-I accessions were collected in different regions of Turkey (Table 1; Figure 4). Nearly half of them (12 accessions) had normal karyotypes (N), and 14 (including segregating accession AE 1607) carried 11 variants of chromosomal rearrangements ( Table 2  Transcaucasia was represented by 19 Armenian and one Azerbaijani accession (Table 1). Nine accessions had normal karyotypes, and five variants of translocations were identified in the remaining ten accessions (Table 2; Figure 5). Translocation T1U c :5U c -T10 (Figure 6f,g) was present in five Armenian accessions and in PI 488258 of unknown origin. This translocation gave rise to two double translocations: T1U c :5U c + T3U c :5X c (T13) T1U c :5U c + T4U c :6U c (T12) found in one accession each and one triple translocation T1U c :5U c + T7U c :3X c + T3U c :4U c -T17 (Figure 6e,i-k) detected in two accessions (Table 2). Interestingly, another complex translocation, the derivative of T1U c :5U c -T13, was found in Turkey ( Figure 4b). The only translocation not related to T1U c :5U c was T3U c :4X c (T3) identified in two Armenian accessions (Figure 6o,p).
Two of the four Iranian accessions analyzed in a current study carried chromosomal rearrangements (Figure 6s-v). These were a single translocation T5U c :6X c (T5) and double cyclic translocation T2X c :4X c :6X c (T15).  Five accessions were from Syria. Three of them had normal karyotypes, and two different complex translocations were identified in the remaining two accessions (Figure 3a,b). K−4372 and K−4362 carried T2U c :4X c + T4U c :6X c (T18) and T2U c :5X c + T4U c :2X c (T20), respectively. In both cases, the original single translocations were not found. Transcaucasia was represented by 19 Armenian and one Azerbaijani accessi (Table 1). Nine accessions had normal karyotypes, and five variants of translocatio were identified in the remaining ten accessions (Table 2; Figure 5). Translocati T1U c :5U c -T10 (Figure 6f,g) was present in five Armenian accessions and in PI 488258 unknown origin. This translocation gave rise to two double translocations: T1U c :5U T3U c :5X c (T13) T1U c :5U c + T4U c :6U c (T12) found in one accession each and one tri translocation T1U c :5U c + T7U c :3X c + T3U c :4U c -T17 (Figure 6e,i-k) detected in t accessions (Table 2). Interestingly, another complex translocation, the derivative T1U c :5U c -T13, was found in Turkey ( Figure 4b). The only translocation not related T1U c :5U c was T3U c :4X c (T3) identified in two Armenian accessions (Figure 6o,p). The origin of four accessions, AE 1512, AE1607, TX01, and CIae 34, was unknown. We found that AE 1607 consisted of two biotypes differing in chromosomal rearrangements (inv2X c /inv2X c −2 + T6U c :7X c ) and the C-banding patterns. This accession also contained Ae. triuncialis seeds. Three accessions, CIae34, TX01, and AE 1521, carried pericentric inversion of 7U c (Figure 3p-r). This rearrangement was recorded only in Ae. columnaris collected from Central Anatolian in Turkey ( Figure 2, outlined with green dotted lines); therefore, we suggested that these three accessions may originate from the same region.
Seven Ae. columnaris accessions, six from Turkey and one from Iraq, were karyotypically distinct from all other accessions of the species and exhibited significant variation in the C-banding patterns (Figure 7). They were assigned to the C-II group. Accession TA2084 carried at least two whole-arm reciprocal translocations; unidentifiable minor translocations may present in other accessions causing variation in the C-banding patterns. Despite heterogeneity, karyotypes of all C-II accessions shared some distinct features discriminating them from the C-I group and Ae. neglecta: (1) They had only two pairs of the satellite (SAT) chromosomes; (2) Chromosome 1U c was more heterochromatic; (3) Chromosome 4U c of C-II contained less heterochromatin compared to C-I ( Figure 7); (4) Chromosome 7U c did not possess a prominent C-band complex in a proximal part of the long arm, which was found in the orthologous chromosomes of all C-I or Ae. neglecta accessions.

FISH Analysis of Ae. columnaris
In order to get a deeper insight into genetic differences between groups C-I and C-II of Ae. columnaris and to assess their relationship with Ae. neglecta, we applied FISH with ribosomal DNA probes pTa71 (45S rDNA), pTa794 (5S rDNA), three microsatellite sequences (GAA)n, (GTT)n, (ACT)n, and three families of the Triticeae-specific satellite DNA sequences pSc119.2, pAs1, and pTa−713. The pTa−535 probe was not considered because it produced signals only on a few chromosomes ( Figure S1, o; green signals), uninformative for our analyses.
Hybridization of pTa71 and pTa794 probes revealed three pairs of major, nearly equal pTa71 signals on chromosomes of C-I and Ae. neglecta, but only two pairs of major NORs in the C-II accessions (Figure 8; Figure S1a,b; Figure S2a,c). Instead, all C-II accessions possessed faint pTa71 signals on a chromosome pair carrying a clear distal 5S rDNA locus. This chromosome was classified as 1X c based on results of sequential FISH with 5S + 45S rDNAs followed by (GAA)n + (GTT)n/pTa−713 probes ( Figure S2b,d). An additional minor NORs were found in the middle of 6U*L ( Figure S3) of all Ae. columnaris (g)-TA2084 (all from Turkey). C-I accession i−570045 (=PI 554184) from Turkey is shown for comparison. Rearranged chromosomes are indicated with black arrows. The red arrow indicates a chromosome, which was presumably introgressed from the C-I group; green arrow shows the chromosome, which could be introgressed from Ae. neglecta.
Morphology and the C-banding pattern of chromosome 5U c in both C-I and C-II accessions were similar; however, 1U c of C-II was more heterochromatic than the 1U c in C-I (Figures 7 and 8). Significant differences existed in C-banding patterns of other C-I and C-II chromosomes, although some polymorphisms could result from introgression. Thus, chromosome 3U c of PI 554182 (Figure 7d) had the C-banding pattern typical for Turkish and Transcaucasian C-I accessions (i.e., PI 554186, PI 554187 on Figure 4e,i) and may originate via introgression between C-I and C-II groups. A C-banding pattern of chromosome 4X c of PI 564180 was more similar to 4X t of Ae. neglecta (Figure 3t-v) than other C-II or C-I accessions.

FISH Analysis of Ae. columnaris
In order to get a deeper insight into genetic differences between groups C-I and C-II of Ae. columnaris and to assess their relationship with Ae. neglecta, we applied FISH with ribosomal DNA probes pTa71 (45S rDNA), pTa794 (5S rDNA), three microsatellite sequences (GAA) n , (GTT) n , (ACT) n , and three families of the Triticeae-specific satellite DNA sequences pSc119.2, pAs1, and pTa−713. The pTa−535 probe was not considered because it produced signals only on a few chromosomes ( Figure S1, o; green signals), uninformative for our analyses.  Hybridization of pTa71 and pTa794 probes revealed three pairs of major, nearly equal pTa71 signals on chromosomes of C-I and Ae. neglecta, but only two pairs of major NORs in the C-II accessions (Figure 8; Figure S1a,b; Figure S2a,c). Instead, all C-II accessions possessed faint pTa71 signals on a chromosome pair carrying a clear distal 5S rDNA locus. This chromosome was classified as 1X c based on results of sequential FISH with 5S + 45S rDNAs followed by (GAA) n + (GTT) n /pTa−713 probes ( Figure S2b,d). An additional minor NORs were found in the middle of 6U*L ( Figure S3) of all Ae. columnaris and Ae. neglecta accessions. Ae. neglecta differed from Ae. columnaris in the presence of a minor 45S rDNA site in a distal part of an arm of a pair of large metacentric X*-genome chromosome tentatively designated as 6X t (Figure 8; Figure S1c, arrowed; Figure 2e; Figure S3a). The application of FAM-labeled oligo-probes allowed us to detect very weak minor pTa71signals at the terminus of 5X*L, a distal quarter of 1U*L, and in a proximal part of 3X c S ( Figure S3). Similar signals were obtained on chromosomes of the C-II accession PI 564181 (data not shown). However, these minor sites never appeared when the plasmid DNA was used as a probe, and they were not considered in the analysis.
Apparent differences between C-I, C-II groups, and Ae. neglecta existed in the pattern of 5S rDNA probe. All Ae. columnaris C-I and Ae. neglecta accessions contained ten 5S rDNA signals distributed among four chromosome pairs ( Figure S2a,e; Figure S3). The chromosome 1X * possessed two pTa794 sites: one located distally to the NOR, while the second-proximally to it ( Figure 8; Figure S1a,c; Figure S2a,e). By contrast, four chromosome pairs in all C-II accessions carried a single 5S rDNA signal each.
In Ae. columnaris and Ae. neglecta labeling patterns of (GAA) n probe were largely consistent with the C-banding patterns, while the (GTT) n hybridized predominantly on the X c chromosomes ( Figure S1g-i). Only 2U*, 4U*, and 5U* contained small (GTT) n sites in pericentromeric/ proximal regions, and a faint signal was present in the middle of the 7U c L arm (Figure 8) in four of the five C-II accessions. By contrast, all X c genome chromosomes demonstrated prominent (GTT) n signals located predominantly in the proximal chromosome regions. Positions of the (GTT) n clusters on the X c chromosomes only partially overlapped with the (GAA) n locations; some chromosomes (e.g., 5X c or 7X c ) that were poorly labeled with (GAA) n , showed extremely heavy labeling with (GTT) n ( Figure 8). Hybridization patterns of (ACT) n probe were almost identical to that of (GTT) n ( Figure S1m,n).
The pSc119.2 probe hybridized to subtelomeric regions of one or both arms of most Ae. columnaris chromosomes except for 7X c , which lacks pSc119.2 signals in all C-I and most C-II accessions ( Figure 8; Figure S1j,k,n,o). Intercalary sites appeared only on the long arm of 7U c and rarely on 6U c L, as in a diploid Ae. umbellulata. Labeling patterns of the pSc119.2 probe were polymorphic between and among C-I and C-II accessions ( Figure 8). Four of the five C-II accessions studied by FISH possessed intercalary pSc119.2 site also on the chromosome 2U c L (Figure 8c), but this site was never observed in C-I or Ae. neglecta. On the other side, the C-II accession PI 564181 did not possess any pSc119.2 signals on chromosome 2U c (Figure 8i).
The D-genome specific probes pAs1 and especially pTa−535 were not very informative for chromosome identification in Ae. columnaris and Ae. neglecta. Distinct pAs1 sites were observed in the pericentromeric region of 6U * and 4X* chromosomes of all studied species, whereas 2-3 weak signals were present on 4X*S and 7X * L arms. The chromosome 5X c of C-I also contained a single, small pAs1 site in the distal half of the short arm. Hybridization sites of the pTa−535 probe emerged on the 6U c L arm, but only in a few accessions studied ( Figure S1o; Figure S3b).
The pTa−713 probe hybridized to most Ae. columnaris (C-I and C-II) and Ae. neglecta chromosomes, while 3U c , 4X c , and 5X c (in Ae. neglecta-also 7X t ) lacked the signals completely. In most cases, the distribution of pTa−713 sites on chromosomes of all three groups was similar; however, some differences between them were observed (Figure 8; Figure S4). In particular, a large pTa−713 signal was present on the short arm of 1U c of all C-I and Ae. neglecta accessions, but it was absent in the C-II. Most C-I and one Ae. neglecta (K−4233) possessed a distinct site in the proximal half of 2U c L, which was not found in C-II and two Ae. neglecta accessions ( Figure S4g,h). We did not observe proximal pTa−713 sites in the short arm of 2X c L and 3X c L in the C-II group, but they were present in all C-I and Ae. neglecta accessions. A large pTa−713 signal present on the 1X c L arm of all C-II accessions was never observed in the C-I group or Ae. neglecta ( Figure S4). Position of hybridization sites on 5U c , 6U c , and 7U c , was similar in all three groups, but Ae. columnaris differed from Ae. neglecta in morphology and/ or labeling patterns of chromosomes 6X * and 7X* (Figure 8).

Analysis of Gliadin Spectra of Ae. columnaris
Electrophoretic analysis revealed a high diversity of gliadin spectra in 25 Ae. columnaris accessions and their distinctness from the spectra of Ae. neglecta ( Figure S5). Only two of 25 Ae. columnaris accessions, K−4413 and K−4418 from Iran, shared similar gliadin spectra, whereas four C-II accessions included in our analysis were highly diverse. However, all contained electrophoretic (EP) components, whose position did not match the overall pattern specific for Ae. columnaris C-I accessions (Figure 9). diverse. However, all contained electrophoretic (EP) components, whose position did not match the overall pattern specific for Ae. columnaris C-I accessions ( Figure 9). Thus, electrophoretic profiles of PI 564180 and PI 542191 were characterized by low-intense, virtually invisible ("minor") components in the α-zone; their intensities and position were distinct from other accessions of Ae. columnaris (Figure 9c,d; Figure S5). Based on comparison with the K−1193 spectrum, we proposed that these components can be encoded by both the X c and U c genomes ( Figure 9).  [27] are shown schematically at the right side of the electrophoretic spectrum; (b) EP spectrum of the accession TA2084 in comparison with wheat cultivar Bezostaya−1; (c) EP spectrum of PI 564180; (d) EP spectra of Ae. columnaris accessions illustrating protein components presumably encoded by the X c (red dots) and U c (yellow dots) chromosomes. The unique components, which were not found in any other Ae. columnaris accessions, are shown schematically (parts (b-d)).
Protein components located in the ω-zone of the spectra of all Ae. columnaris C-I accessions were similar in intensity and position ( Figure S5). Among them, components designated as "2" and "3" (Figure 9, indicated with red dots) corresponded to components detected in the spectrum of K−1193, which were coded by chromosome 1X c . In contrast to other materials, accession PI 564181 contained the unique double band instead of "component 3" (Figure 9d). In addition, it displayed a distinct distribution of components located in the β-γ zone, which, by comparison with the K−1193 spectrum, can be coded by group-6 chromosomes of the U c and X c genomes. Such distribution was more typical for common wheat, and the respective zone was controlled by wheat chromosomes 6B and 6D [35].  [27] are shown schematically at the right side of the electrophoretic spectrum; (b) EP spectrum of the accession TA2084 in comparison with wheat cultivar Bezostaya−1; (c) EP spectrum of PI 564180; (d) EP spectra of Ae. columnaris accessions illustrating protein components presumably encoded by the X c (red dots) and U c (yellow dots) chromosomes. The unique components, which were not found in any other Ae. columnaris accessions, are shown schematically (parts (b-d)).
Thus, electrophoretic profiles of PI 564180 and PI 542191 were characterized by lowintense, virtually invisible ("minor") components in the α-zone; their intensities and position were distinct from other accessions of Ae. columnaris (Figure 9c,d; Figure S5).
Based on comparison with the K−1193 spectrum, we proposed that these components can be encoded by both the X c and U c genomes (Figure 9).
Protein components located in the ω-zone of the spectra of all Ae. columnaris C-I accessions were similar in intensity and position ( Figure S5). Among them, components designated as "2" and "3" (Figure 9, indicated with red dots) corresponded to components detected in the spectrum of K−1193, which were coded by chromosome 1X c . In contrast to other materials, accession PI 564181 contained the unique double band instead of "component 3" (Figure 9d). In addition, it displayed a distinct distribution of components located in the β-γ zone, which, by comparison with the K−1193 spectrum, can be coded by group-6 chromosomes of the U c and X c genomes. Such distribution was more typical for common wheat, and the respective zone was controlled by wheat chromosomes 6B and 6D [35].
Protein components encoded by chromosome 1U c were characterized by low intensities (Figure 9a,c; indicated by yellow dots). By contrast, the spectra of TA 2084 and PI 564180 possessed several intense components in the upper part of ω-zone designated 1 , 2 , and 3 . By comparing with the spectrum of K−1193, we hypothesized that they could be controlled by the chromosome 1U c (Figure 9b,c). TA 2084 and PI 564180 spectra shared components 2 and 3 with similar mobility and intensity, but they differed in the presence of additional minor component 1 , which showed slower mobility in TA2084.

Variability of the U-Genome Specific U31 Nuclear Fragment in Ae. columnaris and Ae. neglecta
Amplification and further sequencing of the U-genome-specific U31 nuclear fragment was performed with primers U31a and U31b in 15 accessions, including ten Ae. columnaris (K−4225, K−4228, K−4409, K−4413, and PI554186 from different countries and representing chromosomal group C-I, and PI542191, PI564179, PI 564180, PI564181, and TA2084 all from Turkey representing group C-II), two Ae. neglecta from Algeria and Turkey (PI 170209 and AE 646) in comparison with three accessions of their diploid parental species Ae. umbellulata (AE 1339, AE 155, and AE 820) of different geographic origin (Table 1). All accessions analyzed generated 363 bp fragments, except for Ae. columnaris PI 554186. In this accession, the fragment length was reduced to 270 bp due to a 123 bp deletion ( Figure 10; Figure S6). 1′, 2′, and 3′. By comparing with the spectrum of K−1193, we hypothesized that they could be controlled by the chromosome 1U c (Figure 9b,c). TA 2084 and PI 564180 spectra shared components 2′ and 3′with similar mobility and intensity, but they differed in the presence of additional minor component 1′, which showed slower mobility in TA2084.

Variability of the U-Genome Specific U31 Nuclear Fragment in Ae. columnaris and Ae. neglecta
Amplification and further sequencing of the U-genome-specific U31 nuclear fragment was performed with primers U31a and U31b in 15 accessions, including ten Ae. columnaris (K−4225, K−4228, K−4409, K−4413, and PI554186 from different countries and representing chromosomal group C-I, and PI542191, PI564179, PI 564180, PI564181, and TA2084 all from Turkey representing group C-II), two Ae. neglecta from Algeria and Turkey (PI 170209 and AE 646) in comparison with three accessions of their diploid parental species Ae. umbellulata (AE 1339, AE 155, and AE 820) of different geographic origin (Table 1). All accessions analyzed generated 363 bp fragments, except for Ae. columnaris PI 554186. In this accession, the fragment length was reduced to 270 bp due to a 123 bp deletion ( Figure 10; Figure S6).
The sequence of the U31 fragment obtained from Ae. columnaris accessions fall into three types, which corresponded to designations proposed earlier by Kadosumi et al. [23] based on fragment length and the presence of MspI restriction site (CCGG). Type-I having the full-length U31 fragment and an intact MspI site was found in seven Ae. columnaris accessions as well as in all analyzed Ae. neglecta and Ae. umbellulata accessions ( Figure S6; Figure 10).  15 Ae. columnaris (U c U c X c X c ), Ae. neglecta (U t U t X t X t ), and Ae umbellulata (UU) sequences. Dots correspond to nucleotides identical to consensus sequences. The MspI restriction site is highlighted in red.
The type-II U31 fragment was identified in two Ae. columnaris accessions, both from the C-II chromosomal group (Figure 10). It emerged as a result of sequence changes at the MspI restriction site: a mononucleotide deletion in position 292 was found in TA2084, while C/T290 substitution in PI 564181. Accession PI 554186 (C-I) possessed the type-III U31 fragment with a 123 bp deletion ( Figure S6). All U31-alleles assigned to type-II corresponded to those reported by Kadosumi et al. [23] in Ae. columnaris or Ae. neglecta. Among U31 type-I accessions of Ae. columnaris, four allelic variants were found, three of which were novel alleles ( Figure 10). Two of them were identified in C-II and one in C-I accession.
The U31 sequences of Ae. umbellulata accessions AE 155 and AE 820 and both Ae. neglecta accessions (PI 170209 and AE 646) belonged to type-I and showed just a few (1-2) Figure 10. Nucleotide substitutions in the U31 region in 15 Ae. columnaris (U c U c X c X c ), Ae. neglecta (U t U t X t X t ), and Ae. umbellulata (UU) sequences. Dots correspond to nucleotides identical to consensus sequences. The MspI restriction site is highlighted in red.
The sequence of the U31 fragment obtained from Ae. columnaris accessions fall into three types, which corresponded to designations proposed earlier by Kadosumi et al. [23] based on fragment length and the presence of MspI restriction site (CCGG). Type-I having the full-length U31 fragment and an intact MspI site was found in seven Ae. columnaris accessions as well as in all analyzed Ae. neglecta and Ae. umbellulata accessions ( Figure S6; Figure 10).
The type-II U31 fragment was identified in two Ae. columnaris accessions, both from the C-II chromosomal group (Figure 10). It emerged as a result of sequence changes at the MspI restriction site: a mononucleotide deletion in position 292 was found in TA2084, while C/T 290 substitution in PI 564181. Accession PI 554186 (C-I) possessed the type-III U31 fragment with a 123 bp deletion ( Figure S6). All U31-alleles assigned to type-II corresponded to those reported by Kadosumi et al. [23] in Ae. columnaris or Ae. neglecta. Among U31 type-I accessions of Ae. columnaris, four allelic variants were found, three of which were novel alleles ( Figure 10). Two of them were identified in C-II and one in C-I accession.
The U31 sequences of Ae. umbellulata accessions AE 155 and AE 820 and both Ae. neglecta accessions (PI 170209 and AE 646) belonged to type-I and showed just a few (1-2) nucleotide substitutions, while almost 12 SNPs were detected in the U31 sequence of Ae. umbellulata, AE 1339 from Greece, which was also assigned to type-I ( Figure S6). Most of the U31 alleles of Ae. umbellulata or Ae. neglecta discovered in this study ( Figure 10) were not identified earlier, and only Ae. neglecta accession PI 170209 carried the same allele as Ae. columnaris (KU−2953A) from Armenia, described earlier by Kadosumi et al. [23].
An ML tree ( Figure 11) shows the possible evolutionary relationship between accessions and species based on comparative sequencing of the U31 alleles. All Aegilops accessions except AE 1339 (Ae. umbellulata) formed one common cluster on the tree obtained. No species-specific or ploidy-specific clusters have been observed. Three Ae. columnaris accessions including two of type-II U31 alleles (PI 564179 and PI 564180) and one type-III accession (PI 554186) formed a separate sub-cluster with 79% bootstrap support. Other accessions representing different species (Ae. columnaris and Ae. neglecta) and different U31 allele types (I and II) fall into one common sub-cluster with Ae. umbellulata (AE 115 and AE 820) showing a closer relationship. Ae. neglecta accession (AE 646) form an individual branch.

Variability of Three Plastome Intergenic Spacers in Ae. columnaris and Ae. neglecta
Variability of three plastome fragments, trnH(gtg)-psbA, trnT(ugu)-trnL(uaa), and rpL32-trnL(tag) DNA, were assessed on the same set of 10 Ae. columnaris accessions as for nuclear U31 fragment. The total length of plastome sequences obtained corresponded to 1825 bp (trnH-psbA-558 bp, trnT-trnL-577 bp, and rpL32-trnL-690 bp). Polymorphism levels differed between the analyzed fragments: only three SNPs were found in the trnT-trnL spacer, while rpL32-trnL and trnH-psbA sequences were much more polymorphic. In contrast to Ae. columnaris, spacer sequences of two Ae. umbellulata accessions (AE 155 and AE 1339) were invariable ( Figure 12). According to the analysis of all three plastome regions, 10 Ae. columnaris accessions split into two groups (plastogroups). Four C-I accessions (K−4225, K−4228, K−4409, and PI 554186) had identical sequences of the plastome spacers, while K−4413 (C-I, Iran) differed at a single site: substitution of the hexamer sequence CCTCAT by ATGAGG at position 470−475 of the rpl32-trnL spacer ( Figure 12). Accessions in group C-II, PI542191, PI564179, PI 564180, PI564181, and TA2084, showed significantly higher sequence polymorphisms at all three plastome regions. Nevertheless, they all shared the same deletion of one of the two AAGAA 5-bp repeats, as well as the deletion of the mononucleotide T at position 446. (Figure 12). In addition, they all carried G/T substitution at position 468 of trnT-trnL, as Ae neglecta and Ae. umbellulata accessions.
C-II accession PI 564179 possessed the highest number of mutations, especially in the rpL32-trnL sequence. Together with C-II accession PI 564181 and Iranian C-I K−4413, it carried ATGAGG/CCTCAT sequence substitution. The same substitution was also identified in Ae. umbellulata and Ae. neglecta ( Figure 12). Comparison of the observed plastogroups with groups discriminated based on C-banding and FISH analyses showed that all Ae. columnaris accessions characterized by an increased variability (PI542191, PI564179, PI 564180, PI564181, and TA2084) belonged to group C-II, while low polymorphic accessions (K−4225, K−4228, K−4409, K−4413, PI 554186) fall to C-I.
According to the analysis of all three plastome regions, 10 Ae. columnaris accessions split into two groups (plastogroups). Four C-I accessions (K−4225, K−4228, K−4409, and PI 554186) had identical sequences of the plastome spacers, while K−4413 (C-I, Iran) differed at a single site: substitution of the hexamer sequence CCTCAT by ATGAGG at position 470−475 of the rpl32-trnL spacer ( Figure 12). Accessions in group C-II, PI542191, PI564179, PI 564180, PI564181, and TA2084, showed significantly higher sequence polymorphisms at all three plastome regions. Nevertheless, they all shared the same deletion of one of the two AAGAA 5-bp repeats, as well as the deletion of the mononucleotide T at position 446. (Figure 12). In addition, they all carried G/T substitution at position 468 of trnT-trnL, as Ae. neglecta and Ae. umbellulata accessions.
C-II accession PI 564179 possessed the highest number of mutations, especially in the rpL32-trnL sequence. Together with C-II accession PI 564181 and Iranian C-I K−4413, it carried ATGAGG/CCTCAT sequence substitution. The same substitution was also identified in Ae. umbellulata and Ae. neglecta (Figure 12). Comparison of the observed plastogroups with groups discriminated based on C-banding and FISH analyses showed that all Ae. columnaris accessions characterized by an increased variability (PI542191, PI564179, PI 564180, PI564181, and TA2084) belonged to group C-II, while low polymorphic accessions (K−4225, K−4228, K−4409, K−4413, PI 554186) fall to C-I.
On the ML tree ( Figure S7), all Ae. columnaris accessions with invariable plastome sequences clustered together, whereas K−4413 formed a separate branch in a common sub-cluster with two Ae. umbellulata accessions (bootstrap = 67). Five genetically variable group was also shown by gliadin analysis. All four C-II accessions had different gliadin profiles, which did not possess any common components. The spectra of each of the C-II accessions (PI 564180, PI 564181, TA2084, and PI 542191), however, carried a number of features (band loss or gain; bands that differed in intensity or position) which were not observed in the C-I group.
The comparative sequence analysis of the U31 nuclear fragment and three plastome intergenic spacer regions also revealed the highly heterogeneous composition of the C-II groups. Thus, the U31 fragment of type-I was found in three C-II accessions (Figure 10), but two of them carried mutant alleles. All accessions with type-II U31 fragment belonged to the C-II group. It was an interesting observation because, according to Kadosumi et al. [23], type-II U31 fragment occurred extremely rare in Ae. columnaris, although frequently in Ae. umbellulata. Both type-II alleles identified here in the C-II accessions corresponded to those described earlier by these authors, but they found one allele in Ae. columnaris from Syria, while the second-in Ae. neglecta. Kadosumi et al. [23] also identified an additional type-II U31 allele, not found in this work, in Ae. columnaris from Iran; however, the karyotypic group of this accession was not determined.
In contrast to the relatively conservative C-I group, from three to 27 SNPs covering all three intergenic spacer regions of cpDNA were identified among accessions of the C-II group.
An interesting fact uncovered by molecular analysis of the U31 nuclear fragment was an unexpectedly high number of SNPs (12) identified in Ae. umbellulata accession AE 1339 from Greece ( Figure S6), which showed no changes in the cpDNA ( Figure 12). According to FISH [40], this accession was karyotypically normal and similar to other Ae. umbellulata genotypes in the distribution of repetitive DNA probes [4,[41][42][43][44][45][46]. All these indicated that the observed mutations in AE 1339 were not caused by chromosomal rearrangement. From the other hand, Kawahara [47] has already uncovered the distinctness of Ae. umbellulata population from Greek Islands based on morphological and isozyme markers.
Summarizing our results, we can conclude that Ae. columnaris is phylogenetically very close to Ae. neglecta, and probably derived from this species (or their common ancestor). It is supported by the following observations.
Owing to a species-specific inversion in chromosome 6X c , the karyotype of Ae. columnaris becomes more "asymmetric" compared to Ae. neglecta. According to Stebbins [48], an increase in karyotype asymmetry is a trend of evolution in plant species and, therefore, Ae. neglecta karyotype should be considered "more primitive", while Ae. columnaris-"more advanced";

1.
The chromosome 6X t of Ae. neglecta possesses a minor 45S rDNA locus, which probably pre-existed in the progenitor Aegilops species; however, this locus is absent in Ae. columnaris.

2.
Ae. columnaris is characterized by chromosome instability expressed in a higher proportion and broader diversity of chromosomal rearrangements (20 variants in more than 55% of accessions). Chromosome instability is an essential factor of speciation [49,50] and is usually more expressed in phylogenetically new species. In addition, we found significant intraspecific polymorphism in Ae. columnaris plastome, although the only low variation of the chloroplast DNA sequences was recorded in Triticum and Aegilops species [51,52].
The similarity of rDNA and repetitive DNA patterns of chromosomes of Ae. neglecta and group C-I of Ae. columnaris and their distinctness from chromosomes of the C-II accessions indicate that the C-I group diverged from Ae. neglecta or their common ancestor as a result of minor genome modifications. Group C-II could derive from a progenitor presumably belonging to group C-I of Ae. columnaris relatively recently, probably due to introgression from another Aegilops species, accompanied by significant reorganization of the parental genomes. As most C-II accessions with known collection sites originated from a very narrow geographic region of the southeastern coastal part of Turkey (Figure 2, red boxes), they might originate from one common ancestor. Significant heterogeneity of the C-II accessions in karyotype structure, C-banding, and FISH patterns, gliadin composition, and nuclear and chloroplast DNA sequences may indicate that they are currently at the initial stage of species divergence; most likely, this group is undergoing an extensive speciation process.

Giemsa C-Banding Method
The C-banding procedure was carried out as described in Badaeva et al. [60]. Chromosomes of Ae. columnaris were classified according to genetic nomenclature developed earlier by Badaeva et al. [20] based on analysis of introgressive lines. Chromosomes of Ae. neglecta were classified according to similarity with Ae. columnaris chromosomes. Designation of Ae. umbellulata chromosomes followed the nomenclature suggested by Friebe et al. [41].

Fluorescence In Situ Hybridization
FISH was carried out according to the protocol described in Badaeva et al. [61]. The probes labeled with fluorescein were detected using anti-fluorescein/Oregon green ® , rabbit IgG fraction, Alexa Fluor ® 488 conjugate (Molecular Probes, Eugene, OR, USA). The slides were counter-stained with DAPI (4 ,6-diamidino-2-phenylindole) in Vectashield mounting media (Vector Laboratories, Peterborough, UK) and examined on a Zeiss Imager D−1 microscope. Selected metaphase cells were captured with AxioCam MRm digital camera using software AxioVision, version 4.6. Images were processed in Adobe Photoshop R , version CS5 (Adobe Systems, Edinburgh, UK).

Seed Storage Protein (Gliadin) Analysis
Electrophoresis (EP) in polyacrylamide gel (PAG) according to the previously published protocol [62] was employed to obtain gliadin spectra of the 25 Ae. columnaris and one Ae. neglecta accessions. The spectra of wheat cultivar Bezostaya−1 (a standard for gliadin spectra of common wheat) and Ae. columnaris K−1193 with the known genetic control of gliadin components [27] were used to compare gliadin profiles of other Aegilops accessions (Figure 9a).

DNA Extraction, PCR Amplification, and DNA Sequencing
Ten accessions of Ae. columnaris (five C-I representing five countries and five C-II from Turkey), Ae. umbellulata (3 accessions) and Ae. neglecta (2 accessions) were selected for analyses by molecular methods. Genomic DNA was extracted from 10-day-old seedlings using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany). DNA quantitative and qualitative evaluation was performed using NanoDrop 2000c spectrophotometer (ThermoFisher-Scientific, Madison, WI, USA).
Amplification of the U-genome-specific U31 nuclear fragment was performed using primers U31a and U31b [23] with PCR conditions: an initial denaturation step of 95 • C for 5 min followed by 30 cycles of 94 • C for 1 min, 55 • C for 1 min, and 72 • C for 1 min with a final extension step at 72 • C for 3 min. The amplified fragments were sequenced directly from both ends with the same U31a and U31b primers.
Amplification of the three intergenic spacers regions (trnH(ugu)-psbA, rpl32-trnL(tag), trnT(ugu)-trnL(uaa)) of the plastome DNA of Aegilops accessions was performed using primer sets listed in Table S1. PCR amplification was performed in a 15 µL reaction mixture containing approximately 50 ng genomic DNA, 1.5 µL of 10× PCR buffer, 1.5 mM MgCl 2 , 0.2 mM of dNTPs, 0.3 µM of each primer, and 0.5 unit of Taq DNA polymerase. The PCR conditions were as follows: an initial denaturation step of 95 • C for 5 min, followed by 30 cycles of 94 • C for 1 min, annealing at the appropriate Tm for 1 min, and 72 • C for 1 min with a final extension step at 72 • C for 5 min. Annealing temperatures for trnH-psbA was 58 • C; trnL-rpl32-56 • C; and trnT-trnL-55 • C. The same primers were used to sequence the obtained chloroplast DNA fragments; PCR products were cleaned before sequencing using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany). PCR products were sequenced using standard protocols with the ABI Prism Big Dye Terminator cycle sequencing kit v. 3.1. Sequences were resolved on an ABI Prism 3100 automated sequencer.
A phylogenetic tree was constructed based on U31 data and combined chloroplast sequence data using MEGA 7 [63] based on ML (maximum likelihood) method. Kimura 2-parameter model was used for U31 and Tamura−3 parameter model for cpDNA, which was selected using Modeltest; 1000 bootstrap replicates were applied for the branch support evaluation. The SNP data from 10 Ae. columnaris genotypes were taken for subsequent analyses. The SNP position was determined from the first nucleotide of U31 or of each of the analyzed chloroplast spacers. IPK, Gatersleben, Germany. Nine samples were collected by H. Özkan in Turkey, and one sample was kindly provided by E.A. Nazarova and A.G. Gukasyan, Institute of Botany after A. Takhtajyan, Academy of Sciences of the Republic of Armenia, Erevan

Conflicts of Interest:
The authors declare no conflict of interest.