Special Issue: Breeding Towards Agricultural Sustainability - Invited

Wheat yields are stagnating around the world and new sources of genes for resistance or tolerances to abiotic traits are required. In this context, the tetraploid wheat wild relatives are among the key candidates for wheat improvement. Despite of its potential huge value for wheat breeding, the tetraploid GGA t A t genepool is largely neglected. Understanding the population structure, native distribution range, intraspecic variation of the entire tetraploid GGA t A t genepool and its domestication history would further its use for wheat improvement. We report the rst comprehensive survey of genomic and cytogenetic diversity sampling the full breadth and depth of the tetraploid GGA t A t genepool. We show that the extant GGA t A t genepool consists of three distinct lineages. We provide detailed insights into the cytogenetic composition of GGA t A t wheats, revealed group-, and population-specic markers and show that chromosomal rearrangements play an important role in intraspecic diversity of T. araraticum. We discuss the origin and domestication history of the GGA t A t lineages in the context of state-of-the-art archaeobotanical nds. We shed new light on the complex evolutionary history of the GGA t A t wheat genepool. We provide the basis for an increased use of the GGA t A t wheat genepool for wheat improvement. The ndings have implications for our understanding of the origins of agriculture in southwest Asia. comprehensive survey of cytogenetic and genomic diversity of the GGA t A t genepool of wheat, thereby unlocking these plant genetic resources for wheat improvement.


Introduction
The domestication of plants since the Neolithic Age resulted in the crops that feed the world today. However, successive rounds of selection during the history of domestication led to a reduction in genetic diversity, which now limits the ability of the crops to further evolve ( However, in nature, no wild hexaploid wheat has ever been found. Only two wild tetraploid wheat species (2n = 4x = 28) were discovered, namely, (i) wild emmer wheat T. dicoccoides (Körn. ex Asch. et Graebn.) of the cytoplasmic genomes of Ae. speltoides and T. araraticum indicated that Ae. speltoides was the maternal parent of T. araraticum (Tsunewaki 1996). Hybrids between T. araraticum × T. dicoccoides were reported as sterile because of meiotic disturbances and gene interactions (Makushina 1938;Svetozarova 1939; Tanaka and Ishii 1973;Wagenaar 1961), although a few authors (Noda and Ge 1989;Sachs 1953; Tanaka and Ichikawa 1972;Tanaka and Kawahara 1976) reported relatively good chromosome pairing in the F 1 hybrids in some T. araraticum × T. dicoccoides combinations.
Triticum dicoccoides is considered to be the older species than T. araraticum (Gornicki et al. 2014;Huang et al. 2002), which is supported by higher similarity of the S-G genomes compared to the S-B genomes (Jiang and Gill 1993; Kilian et al. 2007; Rodríguez et al. 2000a). Cytogenetic and molecular data showed that the speciation of T. araraticum was accompanied by complex species-speci c translocations involving chromosomes 1G-6A t -4G and 3A t -4A t (Chen and Gill 1984;Jiang and Gill 1993;Rodríguez et al. 2000b; Salina et al. 2006) as well as with mutations of the primary DNA structure causing the divergence of homoeologous chromosomes (e.g., chromosomes 3A-3A t ) (Dobrovolskaya et al. 2009). These changes of karyotype structure are speci c for the whole section Timopheevii as compared to the emmer wheat lineage (BBAA, BBAADD) (Badaeva et al. 1986;Hutchinson et al. 1982;Zhang et al. 2013).
Most recent phylogenetic studies based on whole chloroplast genome sequences, genome-wide sequence information and enlarged taxon sampling provided increased resolution of the evolutionary history within the Triticeae tribe, thereby shedding also new light on the GGA t A t wheat genepool (Bernhardt et al. 2017; Gornicki et al. 2014).
Research and pre-breeding activities have focused on T. dicoccoides because it gave rise to the economically most important wheats, T. durum Desf. and T. aestivum L. (Avni et al. 2017;El Haddad et al. 2021). However, T. araraticum and T. timopheevii have also contributed to bread wheat improvement. Important genes were transferred such as genes controlling resistance against stem rust, leaf rust, powdery mildew or wheat leaf blotch (Allard and Shands 1954;Brown-Guedira et al. 1996, 2003Dyck 1992;McIntosh and Gyarfas 1971). Cytoplasmic male sterility (CMS) induced by T. timopheevii cytoplasm showed great potential for heterotic hybrid technology (Maan and Lucken 1972;Mikó et al. 2011;Würschum et al. 2017). However, despite of its potential huge value for bread and durum wheat improvement, only a comparatively small number of genes was transferred from T. timopheevii (even less from T. araraticum). Most of the gene contributions originate from only one line (D-357-1) bred by R. Allard at the University of Wisconsin in 1948 (Martynov et al. 2018).
Understanding the population structure and the intraspeci c variation of the entire tetraploid GGA t A t genepool would further its use for wheat improvement. In this study, we report the rst comprehensive survey of cytogenetic and genomic diversity sampling the full breadth and depth of the tetraploid GGA t A t genepool. We provide new insights into the genetic relationships among GGA t A t wheats and its domestication, taking into account the state-of-the-art archaeobotanical nds. A subset of 787 tetraploid wheat genotypes representing 765 genebank accessions was examined using Sequence-Speci c Ampli cation Polymorphism (SSAP) markers. The subset included 360 genotypes of T. araraticum, 76 T. timopheevii (including two T. militinae), while 351 genotypes of T. dicoccoides were considered as an outgroup. Of them, 243 (67%) T. araraticum, 139 (39.6%), and nine T. timopheevii genotypes (including one T. zhukovsky) were shared with C-banding analysis. The whole collection was single-seed descended (SSD) at least twice under eld conditions (2009-2012) and taxonomically reidenti ed in the eld at IPK, Gatersleben in 2011 (Supplementary Table S2).

Materials And Methods
Based on the results of the SSAP analysis, a subset of 103 genotypes, including 37 T. araraticum collected from different geographic regions and representing all genetic groups, one T. militinae, 14 T. timopheevii, 38 T. dicoccoides, 9 T. dicoccon, and four T. durum was selected for a complementary analysis using Ampli ed Fragment Length Polymorphism (AFLP) markers to infer the population structure of GGA t A t wheats (Supplementary Table S2). Forty-seven T. araraticum and T. timopheevii accessions (88.7%) were shared with SSAP and 39 (73.6%) with C-banding analyses.
Karyotype diversity of 370 T. araraticum accessions was assessed by C-banding and Fluorescence in situ hybridization (FISH) in comparison with 17 T. timopheevii and one T. zhukovskyi genotypes (Supplementary Table S2). According to the C-banding analysis, most T. araraticum accessions (353 of 370) were karyotypically uniform and were treated as single genotype each. Seventeen accessions were heterogeneous and consisted of two (13 accessions) or even three (four accessions) cytogenetically distinct genotypes, which were treated as different entities (genotypes). In order to infer the population structure of GGA t A t wheats, 265 typical genotypes of T. araraticum were selected representing all karyotypic variants, seven T. timopheevii, one T. zhukovskyi. A total of 87 T. araraticum genotypes representing all geographic regions and chromosomal groups were selected for Fluorescence in situ hybridization (FISH) analysis.

Molecular analysis SSAP analysis
Evolutionary relationships among the comprehensive collection of wild and domesticated tetraploid wheat taxa were rst inferred based on polymorphic retrotransposon insertions. For this, the highly multiplex genome ngerprinting method SSAP was implemented based on polymorphic insertions of retrotransposon families BARE-1 and Jeli spread across the wheat chromosomes. DNA was isolated from freeze-dried leaves of 787 SSD plants, using the Qiagen DNeasy Kit (Hilden, Germany). The SSAP protocol was based on Konovalov et al. (2010) with further optimizations for capillary-based fragment detection (Supplementary Material S3). In total, 656 polymorphic markers were generated for BBAA-and GGA t A t -genome wheats by ampli cation of multiple retrotransposon insertion sites. Data analysis was performed in SplitsTree 4.15.1 (Huson and Bryant 2006). NeighborNet planar graphs of Dice distances (Dice 1945) were constructed based on presence/absence of SSAP bands in the samples. AFLP analysis The AFLP protocol, as described by Zabeau and Vos (1993), was performed with minor modi cations according to Altıntaş et al. (2008) and Alsaleh et al. (2015). In total, six AFLP primer combinations were used to screen the collection of 103 lines (Supplementary Material S3). Neighbor-Joining (NJ) trees were computed based on Jaccard distances (Jaccard 1908;Perrier et al. 2003). NeighborNet planar graphs were generated based on Hamming distances (Huson and Bryant 2006). Genetic diversity parameters and genetic distances were calculated using Genalex 6.5 (Peakall and Smouse 2012). Cytogenetic analysis

C-banding
Chromosomal preparation and C-banding procedure followed the protocol published by Badaeva et al. (1994). The A t -and G-genome chromosomes were classi ed according to the nomenclature proposed by Badaeva et al. (1991) except for chromosomes 3A t and 4A t . Based on meiotic analysis of the F 1 T. timopheevii × T. turgidum hybrids (Rodríguez et al. 2000b) and considering karyotype structure of the synthetic wheat T. × soveticum (Zhebrak) (Mitrofanova et al. 2016), the chromosomes 3A t and 4A t were exchanged. Population structure of T. araraticum and the phylogenetic relationship with T. timopheevii were inferred based on chromosomal passports compiled for 265 genotypes representing all geographic regions and chromosomal groups (247 T. araraticum, 17 T. timopheevii, one T. zhukovskyi). Chromosomal passports were constructed by comparing the karyotype of the particular accession with the generalized idiogram of A t -and G-genome chromosomes ( genotypes considered for FISH analysis. Additionally, 26 of the 95 genotypes were analyzed using the probe pAesp_SAT86 (Badaeva et al. 2015a), which also showed differences between the accessions. The probes pSc119.2 (Bedbrook et al. 1980), GAA n (Pedersen et al. 1996), and pTa-535 (Komuro et al. 2013) were subsequently hybridized to the same chromosomal spread to allow chromosome identi cation. Classi cation of pSc119.2-and pTa-535-labeled chromosomes followed the nomenclature of Jiang and Gill 1993). Chromosomes hybridized with the GAA 10 microsatellite sequence were classi ed according to nomenclature suggested for C-banded chromosomes.

Results
Genetic diversity and population structure of the GGA t A t genepool Simultaneous ampli cation of multiple retrotransposon insertion sites using eight Long Terminal Repeat (LTR) primer combinations generated 656 polymorphic Sequence-Speci c Ampli cation Polymorphism (SSAP) markers: 255 markers were obtained for Jeli insertions and 401 markers for BARE-1 insertions. According to our previous study (Konovalov et al. 2010), Jeli targets mostly the A genome, while BARE-1 is distributed between A-and B/G-genome chromosomes. Altogether, 787 wheat genotypes representing 753 genebank accessions were considered for data analysis, after excluding apparently misidenti ed taxa and several cases of low-yield DNA extraction (Supplementary Table S2 Table S6); (v) differences between A t -and A t +G genome diversity patterns based on Jeli and BARE-1 were discovered, respectively (Supplementary Table S6); and (vi) Nei's genetic distance between lineages based on all 656 SSAP markers or considering only the BARE-1 markers revealed that ARA-0 is phylogenetically more closely related to TIM than ARA-1. However, considering only the Jeli markers, ARA-1 was more closely related to TIM. Triticum dicoccoides (DIC) was genetically related most closely to the ARA-1 lineage (Supplementary Table S6). Importantly, the two lineages of T. araraticum and their distribution ranges were veri ed; (ii) the summary statistics highlight that, in contrast to the SSAP analysis, ARA-1 was more diverse than TIM for all parameters (Supplementary Table S8); (iii) ARA-1 was found to be genetically most closely related to TIM (Supplementary Table S8); (iv) ARA-1 was related most closely to DIC Supplementary Table S8) Fluorescence in situ hybridization (FISH) using six DNA probes was carried out for 95 genotypes (Supplementary Table S2, column 13), which, in turn, corroborates the ndings obtained using Jeli markers, AFLP and C-banding markers (Supplementary Table S11). Intraspeci c genetic diversity of T. araraticum based on karyotype structure and C-banding patterns Cytogenetic analysis of 391 T. araraticum genotypes in comparison with 17 T. timopheevii genotypes provided detailed insights into the genetic composition of GGA t A t wheats. First, cytogenetic analysis highlighted signi cant differences between wild emmer T. dicoccoides and T. araraticum/T. timopheevii in karyotype structure and C-banding patterns (Fig. 4). Second, we revealed high diversity of the Cbanding patterns and broad translocation polymorphisms within T. araraticum (Figs. 4,5). The karyotype lacking chromosomal rearrangements was de nd as 'normal' (N) and was the most frequent karyotype variant shared by T. timopheevii and T. araraticum. The 'normal' karyotype was found in 175 of 391 T. araraticum genotypes (44.6%) (more speci cally: 155 of 342 ARA-0 = 45.32%; 20 of 49 ARA-1 = 40.81%). These 175 genotypes differed from each other only in the presence/absence or size of one to several Cbands. The ratio of karyotypically normal genotypes decreased from 86.7% in Azerbaijan (excluding Nakhichevan), to 60.0% in Turkey, 46.5% in Iraq, 30.6% in Armenia, 20.0% in Nakhichevan, Azerbaijan, 16.7% in Syria, to 9.1% in Iran (Supplementary Figure S12). Local populations differed in the ratio of normal/rearranged genotypes. For example, some populations from Dahuk, Iraq, possessed only karyotypically normal genotypes, while in others all genotypes possessed chromosomal rearrangements. Similarly, in Turkey the frequency of karyotypically normal genotypes varied from 100% (Mardin) to 0% (Kilis) (Supplementary Table S13).
C-banding patterns of T. araraticum were highly polymorphic. Based on the presence or absence of particular C-bands (Figs. 1, 4), all genotypes were divided into two groups. The rst, larger group (ARA-0) comprised of 342 genotypes (Supplementary Figures S14 -S19). The second group (ARA-1) included 49 genotypes (Supplementary Figure S18 h1-h5; h6-h9; Supplementary Figure S20). Interestingly, genebank accession TA1900 presumably collected 32 km S of Denizli, Dulkadiroğlu district, Kahramanmaraş province in the Taurus Mountain Range of Turkey in 1959 shared karyotypic features of T. timopheevii and ARA-0 ( Fig. 4) and, probably, it is a natural or arti cially produced hybrid of T. timopheevii with an unknown genotype of T. araraticum.
Only few C-bands were characteristic for the ARA-0 lineage (Figs. 1, 4). Three C-bands appeared with a frequency of over 95%, and two of them, 6A t L3 and 5GL15, occurred only in the ARA-0 group. The third Cband, 2GL13, was detected in 97% of ARA-0, but also in few ARA-1 genotypes.
'Region-speci c' C-bands (in terms of highest frequency) (Badaeva et al. 1994) were detected for ARA-0.  Figure S16). The speci city of C-banding patterns in genotypes originating from the same geographic region was not only determined by single bands, but usually by a particular combination of C-bands on several chromosomes. These region-speci c banding patterns were observed for both, normal and translocated forms. For example, the unique banding pattern of chromosome 7G, lacking the marker C-band 7GS11, but carrying large bands for 7GS13, 7GS17 and 7GS21 on the short arm, and 7GL13 and 7GL15 on the long arm was common in Transcaucasia (Supplementary Figure S14). The chromosome 6G lacking telomeric C-bands on the long arm was frequent in genotypes from Dahuk and Sulaymaniyah (both Iraq), and also occurred in ARA-1 genotypes from Kahramanmaraş, Turkey (Supplementary Figure S20). Chromosomal rearrangements play an important role in intraspeci c diversity of T. araraticum In total, 216 out of 391 (55.4%) T. araraticum accessions carried a translocated karyotype. Seventy-six variants of chromosomal rearrangements including single and multiple translocations, paracentric and pericentric inversions were identi ed (Figs. 5, 6). Novel rearrangements were represented by 44 variants, while 32 variants were described earlier (Badaeva et al. 1990(Badaeva et al. , 1994Kawahara et al. 1996; Tanaka 1977, 1981). One-hundred-forty-seven genotypes differed from the 'normal' karyotype by one, 45 genotypes by two (double translocations), 21 genotypes by three (triple translocations) and three genotypes -by four chromosomal rearrangements (quadruple translocations) (Supplementary Table  S13).
Altogether, we revealed 52 (33 novel) variants of single chromosomal rearrangements (Figs. 5, 6). They included paracentric (one variant) and pericentric inversions (seven variants) and 44 single translocations involving A t -A t , A t -G, or G-G-genome chromosomes. Double rearrangements were represented by 16 independent and three cyclic translocations; among them 10 were novel. Triple translocations were represented by three variants, two of which -T2A t :7G + T6A t :5G:6G and T3G:7G:7A t + T6A t :6G -were found here for the rst time. Both variants of quadruple translocations have been identi ed earlier in Transcaucasia (Badaeva et al. 1990(Badaeva et al. , 1994. A translocation between two chromosomes could give rise to different products depending on the breakpoint position and arm combination in rearranged chromosomes. For example, a centromeric translocation between 1G and 2G resulted in two translocation variants which were distinct in arm combinations (S:S vs. S:L). Three translocation variants involving chromosomes 3G and 4G differed from each other in arm combination and breakpoint position. To discriminate different translocation variants involving same chromosomes, they were designated as T1G:2G-1 and T1G:2G-2, etc. (Supplementary   Table S13).
Most variants of chromosomal rearrangements were unique and identi ed in one or few genotypes, and only four variants were relatively frequent. These were a triple translocation T2A t :4G:7G + T4A t :7A t , perInv7A t -1, T6G:7G, and T2G:4G:6G (21, 16, 12, and 13 genotypes, respectively). Taken together, these four variants accounted for approximately 16% of the whole materials we studied. Genotypes carrying the same rearrangement usually had similar C-banding patterns and were collected from the same, or closely located geographic regions. For example, (i) all genotypes with T2A t :4G:7G + T4A t :7A t originated from Nakhichevan, Azerbaijan; (ii) T6G:7G and T2G:4G:6G were found in Erbil, Iraq; and (iii) perInv7A t -1 was collected in Iran and in the neighboring region of Sulaymaniyah, Iraq.
Other frequent translocations had a more restricted distribution and usually occurred in a single population. Only few genotypes carrying the same translocations were identi ed in spatially separated populations. These genotypes differed in their C-banding patterns, for example: (i) T2G:4G was found in four genotypes from Erbil, Iraq and in two genotypes from Siirt, Turkey; (ii) T1G:3G was identi ed in ve ARA-0 genotypes from Dahuk, Iraq, one ARA-1 from Turkey and one ARA-1 genotype of a mixed accession IG 117895 collected in Syria; and (iii) T1G:5G was identi ed not only in three cytogenetically distinct T. araraticum ARA-0 genotypes from Armenia and Azerbaijan and ARA-1 from Turkey ( Cytogenetic diversity of GGA t A t wheats assessed using FISH markers To further investigate the intraspeci c diversity of T. araraticum and to assess their phylogenetic relationships with T. timopheevii, we carried out FISH using six DNA probes. The probes pTa-535, pSc119.2 and GAA n ensured chromosome identi cation  whereas Spelt-1, Spelt-52 and pAesp_SAT86 were used to estimate intra-and interspeci c variation (Fig. 7).
The variability of pAesp_SAT86 hybridization patterns was analyzed in four T. timopheevii and 26 T. araraticum genotypes, of them seven were from ARA-1 and 19 from ARA-0 lineages (Supplementary Table S2). Distribution of the pAesp_SAT86 probe in all T. timopheevii genotypes was similar except for chromosome 7A t in genotype K-38555, which was modi ed due to a paracentric inversion or insertion of an unknown chromosomal fragment. Labeling patterns, however, were highly polymorphic for T. araraticum ( Fig. 7; Supplementary Figure S22). Large pAesp_SAT86 sites were found only on some Ggenome chromosomes. The A t -genome chromosomes possessed several small, but genetically informative polymorphic sites. Some of these sites were lineage-speci c. Most obvious differences were observed for 3A t , 4G and 7G chromosomes (Fig. 1). Thus, all TIM and ARA-1 genotypes carried the pAesp_SAT86 signal in the middle of 3A t S, while for ARA-0 it was located sub-terminally on the long arm. One large pAesp_SAT86 cluster was present on the long arm of 4G in ARA-0, but on the short arm in ARA-  Table S26).

Discussion
We present the most comprehensive survey of cytogenetic and genomic diversity of GGA t A t wheats. We describe the composition, distribution and characteristics of the GGA t A t genepool. Building on our results, the latest published complementary genomics studies and state-of-the-art archaeobotanical evidence we revisit the domestication history of the GGA t A t wheats. We arrived at the following four key ndings:

The GGA t A t genepool consists of three distinct lineages
We sampled the full breadth and depth of GGA t A t wheat diversity and discovered a clear genetic and geographic differentiation among extant GGA t A t wheats. Surprisingly, and supported by all marker types, three clearly distinct lineages were identi ed. The rst lineage is comprised of all T. timopheevii genotypes (and the derived T. militinae and T. zhukovskyi; note that all T. militinae and all T. zhukovskyi accessions maintained ex situ in genebanks are each derived from only one original genotype). Interestingly, T. araraticum consists of two lineages that we preliminarily describe as 'ARA-0' and 'ARA-1'. This nding is in contrast to Kimber and Feldman (1987) who concluded that T. araraticum does not contain cryptic species, molecularly distinct from those currently recognized. Based on passport data, ARA-0 was found across the whole predicted area of species distribution. ARA-1 was only detected in south-eastern Turkey and in neighboring north-western Syria. It is interesting to note that only in this part of the Fertile Crescent, the two wild tetraploid wheat species, T. dicoccoides and T. araraticum, grow in abundance in mixed stands.  Table  S2, column 17), provided increased resolution of the chloroplast genome phylogeny and showed that the T. timopheevii lineage possibly originated in northern Iraq (and thus according to our data, belong to the ARA-0 lineage as no ARA-1 occurs in Iraq). This was supported by Bernhardt et al. (2017), who, based on re-sequencing 194 individuals at the chloroplast locus ndhF (2232 bp) and on whole genome chloroplast sequences of 183 individuals representing 15 Triticeae genera, showed that some ARA-0 and TIM genotypes are most closely related. All GGA t A t wheats re-sequenced by Bernhardt et al. (2017) were considered in our study (Supplementary Table S2, column 16). Haplotype analysis of the Brittle rachis 1 (BTR1-A) gene in a set of 32 T. araraticum in comparison with two T. timopheevii accessions (Nave et al. 2021) also showed closer relationships of domesticated T. timopheevii to wild T. araraticum from Iraq. That is more, one of these accessions, TA102 (PI 538461, 1 km NE of Salahaddin) shared the same haplotype with T. timopheevii and it was assigned to ARA-0 group by our study (Supplementary Table S2, column 18).
It is important to note that our results (i.e., the characteristics, composition and geographical distribution of ARA-0 and ARA-1 lineages) are not in agreement with the latest comprehensive taxonomical  Table S2, column 2). We propose to re-classify the GGA t A t genepool taxonomically in the future. The karyotype 'similar' to those in 'normal' T. timopheevii was found in 44.6% of all T. araraticum genotypes. This is the group of candidates, in which the closest wild relative(s) to T. timopheevii is (are) expected. The frequency of the normal karyotype varied among countries and between populations (Supplementary Figure S12). It is interesting to note that the Samaxi-Akhsu population in Azerbaijan and some populations near Dahuk (Iraq) possessed mostly karyotypically normal genotypes. Diagnostic Cbands for the ARA-1 lineage, both with normal and rearranged karyotypes, were 1A t L3, 4A t S7, 5A t L3, 1GL5, 2GL7, 3GL7 + L11 (Fig. 1). As expected, the number of C-bands characteristic for ARA-0 was smaller (due to the wide geographical distribution) and only two C-bands were lineage-speci c and found in normal as well as translocated genotypes: 6A t L3 and 5GL15.

The karyotypic composition of GGA t A t wheats is as complex as the phylogenetic history of the
However, some FISH patterns suggested that T. timopheevii probably originated in Turkey and probably from ARA-1 (or, ARA-1 and TIM may have originated from a common ancestor, but then diverged). This is supported by the following observations: (i) TIM and ARA-1 carry the pSc119.2 signal in the middle of 1A t long arm, while this site was absent from ARA-0; (ii) all ARA-0 and most ARA-1 possessed the Spelt-52 signal on 6GL, but it is absent in all TIM and ve ARA-1 genotypes from Gaziantep-Kilis, Turkey. The distribution of Spelt-1 and Spelt-52 probes on chromosomes of these ve genotypes was similar to, and in accession IG 116165 (ARA-1 from Gaziantep) almost identical with TIM; (iii) the pAesp_SAT86 patterns on chromosomes 3A t , 4G, and 7G are similar in TIM and ARA-1 but differed from ARA-0. Differences between ARA-1 and TIM based on FISH patterns of some other chromosomes as well as the results of Cbanding and molecular analyses suggest that extant ARA-1 genotypes are not the direct progenitors of TIM but that the ARA-1 lineage is most closely related to it.
Based on AFLP, C-banding, FISH and Jeli retrotransposon markers, TIM was genetically most closely related to ARA-1. Additional evidence for the close relationship between TIM and ARA-1 lineages comes from allelic variation at the VRN-1 locus of genome A t (Shcherban et al. 2016). This analysis revealed a 2.7 kb deletion in intron 1 of VRN-A1 in three T. timopheevii and four T. araraticum accessions, which, according to our data, belong to the ARA-1 lineage. However, at Vrn-G1, TIM from Kastamonu in Turkey (PI 119442) shared the same haplotype (Vrn1Ga) with ARA-1 samples, while TIM from Georgia harbored haplotype VRN-G1 as found in ARA-0. These results suggest multiple introgression events and incomplete lineage sorting as suggested by Bernhardt et al. (2017Bernhardt et al. ( , 2020. Regular chromosome pairing observed in the F 1 hybrids of lines with 'normal' karyotypes (Kawahara et al. 1996), identi ed in our study as ARA-0 × ARA-1 (Supplementary Table S2, column 14), suggested that karyotypic differences between ARA-0 and ARA-1 lineages are not associated with structural chromosomal rearrangements such as large translocations or inversions.
The emergence or loss of most lineage-speci c Giemsa C-bands (Fig. 3) or FISH loci ( Supplementary   Fig. 22, Supplementary Fig. 23) could be due to heterochromatin re-pattering: ampli cation, elimination or transposition of repetitive DNA sequences. Wide hybridization can also induce changes in C-banding and FISH patterns of T. araraticum chromosomes. Changes in pAesp_SAT86 hybridization patterns on 4G and 7G chromosomes, however, are likely to be caused by pericentric inversions, which are also frequent in common wheat (Qi et al. 2006). The role of inversions in inter-and intraspeci c divergence is probably underestimated. In our case, it seems possible that divergence between ARA-1/TIM (two inversions) from ARA-0 (no inversion) was associated with at least two pericentric inversions.
We did not nd any genotype harboring both ARA-0 and ARA-1 speci c FISH sites, although ARA-0 and ARA-1 genotypes co-existed in two populations in Turkey and Syria. However, based on FISH (Spelt-1 site on chromosome 6GL and 7GS, respectively), hybridization between certain ARA-1 and ARA-0 lines can be predicted.
Iran occupies a marginal part of the distribution range of T. araraticum. An abundance of the pericentric inversion of the 7A t chromosome in the Iranian group indicates that it is derived from Iraq. The karyotypically 'normal' genotype was probably introduced to Transcaucasia via Western Azerbaijan (Iran).
The low diversity of FISH patterns and the low C-banding polymorphism of T. araraticum from Transcaucasia indicate that T. araraticum was introduced as a single event. Interestingly, the AFLP data suggested some similarity between ARA-0 from Armenia and Azerbaijan and T. timopheevii.
We hypothesize that homoploid hybrid speciation (HHS) (Abbott et al. 2010;Nieto Feliner et al. 2017;Soltis and Soltis 2009) and incomplete lineage sorting may be the possible mechanisms explaining the origin of the ARA-1 lineage. Although this assumption was not experimentally supported, it is favored by some indirect evidence. ARA-1 grows in sympatry and in mixed populations with T. dicoccoides (Fig. 3) and is phylogenetically most closely related to T. dicoccoides (Supplementary Table S6; Supplementary  Table S8). ARA-1 is morphologically more similar to T. dicoccoides. Thus, ve of 10 misclassi ed T. araraticum accessions belonged to ARA-1 group (Supplementary Table S2) Much less is known about the domestication history of T. timopheevii. It is believed that T. timopheevii is the domesticated form of T. araraticum (Dorofeev et al. 1979;Jakubziner 1932). In contrast to T. dicoccon, T. timopheevii was, since its discovery, considered as a 'monomorphous narrowly endemic species' (Dekaprelevich and Menabde 1932)  Based on intensive archaeobotanical investigations at Çatalhöyük in central Anatolia, for example, NGW was the predominant hulled wheat, overtaking emmer wheat around 6500 cal BC and remaining dominant until the site's abandonment c. 5500 cal BC. The nds suggested that this wheat was a distinct crop, processed, stored, and presumably grown, separately from other glume wheats. NGW formed part of a diverse plant food assemblage at Neolithic Çatalhöyük, including six cereals, ve pulses and a range of fruits, nuts and other plants, which enabled this early farming community to persist for 1500 years (c. 7100 to 5500 cal BC) (Bogaard et al. 2013(Bogaard et al. , 2017. Recently, polymerase chain reactions speci c for the wheat B and G genomes, and extraction procedures optimized for retrieval of DNA fragments from heat-damaged charred material, have been used to identify archaeological nds of NGW ). DNA sequences from the G genome were detected in two of these samples, the rst comprising grain from the mid 7th millennium BC at Çatalhöyük in Turkey, and the second made up of glume bases from the later 5th millennium BC site of Miechowice 4 in Poland. These results provide evidence that NGW is indeed a cultivated member of the GGA t A t genepool ). As NGW is a recognized wheat type across a broad geographic area in prehistory, dating back to the 9th millennium BC in SW Asia, this indicates that T. timopheevii (sensu lato, s.l. = domesticated GGA t A t wheat in general), was domesticated from T. araraticum during early agriculture, and was widely cultivated in the prehistoric past (Czajkowska et al. 2020).
This raises the question of whether the few populations of T. timopheevii (sensu stricto, s.str.) found in western Georgia were the last remnants of a wider GGA t A t wheat cultivation or whether the T. timopheevii of Georgia was a local domestication independent of the domestication of T. araraticum in SW Asia. To answer this question, sequence information for NGW, the Georgian T. timopheevii, and the two lineages of T. araraticum (ARA-0, ARA-1) need to be compared. Vavilov (1935) suggested that T. timopheevii of western Georgia was probably originally introduced from north-eastern Turkey. As cited by Dorofeev et al. (1979), Menabde and Ericzjan (1942) associated the origin of T. timopheevii with the region of the ancient kingdom of Urartu, whence immigrant ancestors of modern-day Georgians introduced it into western Georgia. Certainly, the possibility of introduction of Timofeev's wheat into Georgia from the south should not be rejected (Dorofeev et al. 1979 We screened all available passport data and found three cases, which could potentially help to reconstruct the recent past cultivation range of T. timopheevii s.str: Interestingly, two T. timopheevii accessions maintained in two ex situ genebanks are reported to originate from Turkey (Supplementary Table S2 Figure S19, i31). However, we are not fully convinced that this line is a true natural hybrid. Based on the passport data, this accession could potentially have escaped from an experimental eld or a breeding station, or received introgression(s) during ex situ maintenance (Zencirci et al. 2018). Assuming that the passport data is correct, we could speculate that T. timopheevii may have been cultivated in Turkey during the rst half of the 20th century. However, is this realistic option?
We believe not. As reported by Stoletova (1924-25), Menabde (1929, 1932), Menabde (1948), Dekaprelevich (1954), T. timopheevii s.str. was part of the spring landrace Zanduri (mixture of T. timopheevii s.str. and T. monococcum) and well adapted to the historical provinces Lechkhumi and Racha of Georgia (Supplementary Material S28). The Zanduri landrace was cultivated in the 'humid and moderately cool climate zone 400-800' m above the sea level (Dorofeev et al. 1979). Martynov et al. (2018) reported that T. timopheevii potentially has a 'low potential for plasticity' and is not drought tolerant. Climate at origin based on bioclimatic variables (Fick et al. 2017; R Core Team 2017) clearly differs between the regions of Western Georgia where the Zanduri landrace grew till the recent past, and both Kastamonu and Kahramanmaraş regions in Turkey (Supplementary Material S28). We speculate that the three T. timopheevii accessions which were collected in Turkey were probably introduced from Transcaucasia or elsewhere and may have been left over from unsuccessful cultivation or breeding experiments of T. timopheevii s.str. in the recent historical past. Also, based on botanical records, T. timopheevii (s.str.) has only been identi ed in Georgia, but not in Turkey or elsewhere (Davis 1965(Davis -1988Hanelt 2001). From this we conclude that the cultivation range of T. timopheevii (s str.) was not wider in the recent past.

Conclusions
The evolutionary history of the GGA t A t wheat genepool is complex. However, some pieces of the puzzle are clearly recognizable: the region around Dahuk in northern Iraq can be considered the center of origin, but also the center of diversity of T. araraticum.
The origin of T. timopheevii s.str. remains unclear, but we speculate that it was probably introduced from Turkey, on the grounds that wild T. araraticum does not grow in Georgia and that T. timopheevii s.str. is more closely related to T. araraticum from Turkey or northern Iraq than to the Transcaucasian types.
Based on bioclimatic variables, we predict that T. timopheevii s.str. is maladapted to the climate outside Western Georgia. If this speculation is correct, it suggests a sister-group relationship between (i) the Georgian T. timopheevii (s.str.) and both T. araraticum lineages (ARA-0, ARA-1), but also between (ii) T. timopheevii s.str. and the prehistoric SW Asian T. timopheevii s.l. The distribution area of the ARA-1 lineage requires our attention. It is interesting to note that the ARA-1 lineage (GGA t A t ) and T. dicoccoides (BBAA) grow in sympatry and in mixed populations only in a peculiar geographical area in the Northern Levant. This speci c area is located between Kahramanmaraş in the north, Gaziantep in the east, Aleppo in the south and the eastern foothills of the Nur Dağlari mountains in the west. It is interesting that the closest extant wild relatives of domesticated einkorn, barley and emmer were also collected here: (i) the beta race of wild einkorn (Kilian et al. 2007) and the closest wild relatives to einkorn btr1 type (Pourkheirandish et al. 2018); (ii) the closest wild barley to btr2 barley (Pourkheirandish et al. 2015) and to btr1b barley (Civáň and Brown 2017). This speci c region was among the areas predicted with high probability as potential refugia for wild barley during the Last Glacial Declarations and support. We thank two anonymous reviewers, who helped us to improve an earlier version of this manuscript.

Con icts of interest/ Competing interests
The authors declare that they have no con ict of interest.

Availability of data and material
All data sets supporting the conclusions of this article are available in the Electronic supplementary material and from the corresponding author Ekaterina D. Badaeva (katerinabadaeva@gmail.com HK veri ed genebank information concerning the origin of the material, translated texts from Russian into English, and contributed to the discussions and editing of the manuscript. AF, supported the molecular data analysis.
AR contributed to the FISH analysis.
ZK conducted the simulation of regional agro-ecological variation based on bioclimatic variables. SZ contributed to the work on mapping of Spelt-1 and Spelt-52 probes on T. timopheevii and T. araraticum genotypes.
SS designed and synthesized oligo-probes for FISH analyses.
KN contributed to data analysis, supported the eld trials and edited the manuscript.
AG contributed to the discussions and editing of the manuscript.
AF phenotyped and taxonomically re-identi ed the collection, contributed to the discussions and edited the manuscript.
KH phenotyped and taxonomically re-identi ed the collection, contributed to the discussions and edited the manuscript.
ABcontributed to the discussions and editing of the manuscript.
GJ contributed to the discussions and editing of the manuscript.
HÖ conducted the AFLP experiments, analyzed the data,contributed to the discussions and edited the manuscript.
BK conceived the project, established the germplasm collection, analyzed data, interpreted results, wrote and edited the manuscript.

Ethics approval
The authors declare that the experiments comply with the current laws of Germany.

Consent to participate
Not applicable Consent for publication Not applicable Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

Figure 1
Generalized idiogram and nomenclature of the At-and G-genome chromosomes. The C-banding pattern is shown on the left, the pSc119.2 (red) and pAesp_SAT86 (green) pattern on the right side of each chromosome. 1-7 -homoeologous groups; S -short arm, L -long arm. The numerals on the left-hand side designate putative positions of C-bands/FISH sites that can be detected on the chromosome arm; Cbands speci c for the ARA-1 group are shown with pink numerals, C-bands speci c for the ARA-0 group are indicated by green numerals. Red asterisks on the right-hand side indicate C-bands that were considered for the "chromosomal passport".  Natural geographical distribution of wild tetraploid T. araraticum and T. dicoccoides. Green dots correspond to collection sites of ARA-0 accessions, pink dots to ARA-1, and dark blue dots to T. dicoccoides (DIC). The collection sites of T. timopheevii and T. zhukovskyi are shown with turquoise and yellow dots, respectively. Key excavation sites in Turkey where NGW was identi ed are indicated with red triangles. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.   Intraspeci c divergence of T. araraticum and T. timopheevii. Combinations of chromosome arms in rearranged chromosomes are designated. Line colors mark the different groups: ARA-0 (green), ARA-1 (pink) and T. timopheevii (black). Solid arrows designate novel rearrangements; arrows with asterisk designate previously described rearrangements (Badaeva et al. 1990(Badaeva et al. , 1994. The numerals above/next to the arrows indicate the number of accessions carrying the respective translocation.

Figure 7
Distribution of different families of tandem repeats on chromosomes of T. timopheevii and T. araraticum.

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