Traits to Differentiate Lineages and Subspecies of Aegilops tauschii , the D Genome Progenitor Species of Bread Wheat

: Aegilops tauschii Coss. , the D genome donor of hexaploid wheat ( Triticum aestivum L.), is the most promising resource used to broaden the genetic diversity of wheat. Taxonomical studies have classified Ae. tauschii into two subspecies, ssp. tauschii and ssp. strangulata . However, molecular analysis revealed three distantly related lineages, TauL1, TauL2 and TauL3. TauL1 and TauL3 in ‐ cludes the only ssp. tauschii , whereas TauL2 includes both subspecies. This study aimed to clarify the phylogeny of Ae. tauschii and to find the traits that can differentiate between TauL1, TauL2 and TauL3, or between ssp. tauschii and ssp. strangulata . We studied the genetic and morpho ‐ physiolog ‐ ical diversity in 293 accessions of Ae. tauschii, covering the entire range of the species. A total of 5880 high ‐ quality SNPs derived from DArTseq were used for phylogenetic cluster analyses. As a result, we observed wide morpho ‐ physiological variation in each lineage and subspecies. Despite this var ‐ iation, no key traits can discriminate lineages or subspecies though some traits were significantly different. Of 124 accessions previously lacking the passport data, 66 were allocated to TauL1, 57 to TauL2, and one to TauL3. markers. Origin of accessions: SYR, Syria; TUR, Turkey; GEO, Georgia; ARM, Armenia; AZE, Azerbaijan; DAG, Dagestan; IRN, Iran; TKM, Turkmenistan; AFG, Afghanistan; PAK, Pakistan; TAJ, Tajikistan; UZB, Uzbekistan; KGZ, Kyrgyzstan; KAS, Kazakhstan; CHN, China and UN, un ‐ known country.


Plant Materials
We used 293 Ae. tauschii accessions collected from the entire range of the natural distribution of this species (Table 1, Figure 1). Of these accessions, 201 have full passport data, including geographical coordinates, lineages and subspecies classification [14] (Figure 1). Five of the 201 accessions (AT 55, AT 60, AT 76, PI 499262 and PI 508262) represent adventive populations in the Shaanxi and Henan provinces of China. Among the 201 accessions, 132 belong to TauL1, 64 to TauL2 and 5 to TauL3 [14]. Based on sensu stricto criteria for subspecies classification, only accessions with distinctly moniliform spikes were classified to Ae. tauschii ssp. strangulata. In contrast, accessions having mildly moniliform and cylindrical spikes were classified to Ae. tauschii ssp. tauschii [14]. Of 293 accessions used in this study, 169 were previously studied by Matsuoka et al. (2009) [14] who classified 110, 55 and 4 to TauL1, TauL2 and TauL3, respectively.  [14] and confirmed by cluster analysis in this study ( Figure S1).

Genomic Analysis and Statistaical Analysis of Molecular Data
Genomic DNA was extracted using the CTAB method [28]. The DNA samples (30 μL; 50-100 ng μL −1 ) were sent to Diversity Arrays Technology Pty. Ltd, Canberra, Australia (http://www.diversityarrays.com, accessed on 29 January 2018) for a whole-genome scan using the DArTseq platform. Sequencing-based DArT genotyping applies two complexity-reduction methods optimized for several plant species i.e., PstI/HpaII and PstI/HhaI were used to select a subset of the corresponding fragments [29]. At the DArT facility, the DArT soft marker extraction pipeline was used to filter and identify the informative markers. We performed the hierarchical clustering analysis in the statistical software R with the pvclust package [30]. The DArTseq SNPs data of 5880 markers without any missing data for 293 accessions of Ae. tauschii from 16 countries (some accessions are from unknown origin) were used for the analysis. Pvclust package computes the AU (approximately unbiased) P-value and BP (bootstrap probability) value via multiscale bootstrap resampling. These values can show how strong the clustering result is supported by the data. The dendrogram was generated by using the Euclidean distance matrix and complete method. The summary of SNP data sequences used for constructing phylogenetic tree was provided in Table S1.

Morpho-Physiological Evaluation
The morphological and physiological traits of all the accessions were measured at the research field of the Arid Land Research Center, Tottori University (Tottori, Japan; 35°32′N 134°13′E) during the winter and spring seasons of 2016/17 and 2017/18 by using an augmented complete block design with three randomly selected accessions as checks (GE12-14-O-1, GE12-28-O-2 and KU-20-2), and five plants were grown per accession. To estimate the phenotypic variation, we measured two leaf parameters (flag leaf length, FLL; flag leaf width, FLW), four spike parameters (spike length, SPL; spike width, SPW; seed number per spike, SN/SP; spike weight, SPWg), days to heading (DH), biomass weight (Bio) and three physiological traits (Normalized Difference Vegetative Index, NDVI; canopy temperature, CT; and chlorophyll content, SPAD). To measure SPWg, we covered the spikes with a transparent envelope before physiological maturity to avoid shattering. The measurement methods are summarized in Table 2.

Chlorophyll content SPAD
Measured at the flowering stage from the middle of the flag leaf of three tillers using a Minolta brand chlorophyll meter (Model SPAD-502; Spectrum Technologies Inc., Plainfield, IL, USA).

Statistical Analysis of Morpho-Physiological Data
Analyses of the phenotypic data, including mean, standard deviation, range distribution and analysis of variance (F and P-values in one-way ANOVA) for the morphophysiological variations were calculated using Plant Breeding Tools (PBTools) version 1.4 (International Rice Research Institute, http://bbi.irri.org/products, 15 February 2020). Due to the significant genotype × season interaction, best linear unbiased predictions (BLUPs) were estimated for each trait.

Phylogenetical Allocation of Uncertain Accessions by Molecular Markers
Following Matsouka et al. (2009) [14], we carefully observed the key morphological traits of the 124 accessions that lacked taxonomical information and identified 7 accessions as ssp. strangulata and the remaining 117 as ssp. tauschii. Among the seven accessions identified as ssp. strangulata, AE 525 was collected from Iran, AE 692 from Uzbekistan and AE 426, AE 428, AE 429, AE 430 and AE 434 from unknown regions. To know the lineages (TauL1, TauL2 or TauL3) of all 124 accessions, we conducted cluster analysis using 5,880 DArTseq markers. As a result, 66, 57 and 1 were clustered in TauL1, TauL2 and TauL3, respectively (Figure 2, Figure S1). All the accessions in TauL1 were ssp. tauschii, whereas in TauL2, 50 were ssp. tauschii and 7 were ssp. strangulata. The accessions in the TauL3 were ssp. tauschii. These findings supported previous results that the ssp. strangulata is present only in TauL2.
From these studies, we found that all 293 accessions of Ae. tauschii were classified as 175 TauL1, 113 TauL2 and 5 TauL3. In TauL2, 15 accessions were ssp. strangulata and others including accessions in TauL1 and TauL3 were ssp. tauschii.
The TauL1 cluster contained accessions from Syria, Turkey, Georgia, Armenia, Azerbaijan, Dagestan, Iran, Turkmenistan, Afghanistan, Pakistan, Tajikistan, Uzbekistan, Kyrgyzstan, Kazakhstan, China and unknown countries. The TauL2 cluster contained accessions from Syria, Turkey, Georgia, Armenia, Azerbaijan, Dagestan, Iran, Turkmenistan, Uzbekistan and unknown countries (Table 1, Figure 2, Figure S1). The ssp. strangulata accessions were clustered in one clade in TauL2, and most of the accessions were from Iran.

Morpho-Physiological Differences between TauL1 and TauL2
A large variation was observed for all the morpho-physiological traits in TauL1 and TauL2 (Table 3). Statistical analyses showed a significant difference between these two lineages in SPW, SPWg, DH and Bio. The means in these traits were larger in TauL2 than in TauL1, indicating that the accessions in TauL2 tend to be higher than TauL1. On the other hand, the means of the physiological traits (NDVI, CT and SPAD), and leaf traits (FLL and FLW) were not significantly different between them. The ranges of these traits overlapped between the two lineages, and thus we cannot discriminate the two groups with these traits (Table 3).

Morpho-Physiological Variation between ssp. tauschii Belonging to TauL1 and TauL2
We designated ssp. tauschii in TauL1 and TauL2 as 'TauL1T' and 'TauL2T', respectively, and compared accessions in these groups. A large variation was observed for all the morpho-physiological traits in TauL1T and TauL2T (Table 4). Statistical analyses showed significant differences between the two groups in FLL, DH and Bio. The mean of FLL was higher in TauL1T, whereas those of DH and Bio were higher in TauL2T. On the other hand, the means of the physiological traits (NDVI, CT and SPAD), and spike traits (SPL, SPW, SN/SP and SPWg) were not significantly different between them. The ranges of these traits overlapped between TauL1T and TauL2T, and thus we cannot discriminate the two groups with these traits (Table 4).

Morpho-Physiological Variation between ssp. tauschii and ssp. strangulata
A large variation was observed for all the morpho-physiological traits in ssp. tauschii and ssp. strangulata (Table 5). Statistical analyses showed significant difference between these two subspecies in SPL, SN/SP, SPWg and DH. The means of SPL and SN/SP were higher in ssp. tauschii than in ssp. strangulata, whereas those of SPWg and DH were higher in ssp. strangulata than in ssp. tauschii. On the other hand, the means of the leaf traits (FLL and FLW), SPW and physiological traits (NDVI, CT and SPAD) were not significantly different between them. The ranges of these traits overlapped between the two subspecies ( Table 5).

Morpho-Physiological Variation of Accessions in TauL3
In this study, only five accessions (AE 454, AE 929, AE 929a, KU-2829A and KU-2832) belong to TauL3. Therefore, we did not compare them with TauL1 and TauL2. All the accessions originated from Georgia and showed a similar plant morphology to ssp. tauschii with an intermediate spike shape between TauL1 and TauL2. Genomic analysis revealed that these accessions are clearly differentiated from both TauL1 and TauL2.

Geographical Clines of Morphological Variation in Subspecies and Lineage Classification
The main putative area of origin of Ae. tauschii is the Transcaucasus, from which it has spread to the east and south [10] (Figure 1). While ssp. tauschii has cylindrical spike forms and ssp. strangulata moniliform spike forms, some Ae. tauschii accessions have mildly moniliform spike forms (TauL3) which suggest a hybrid origin. Overall, spikelet morphology is the main trait not only for discriminating the two subspecies but also for intraspecific diversification in Ae. tauschii, even though the genetic basis of spikelet morphology divergence has not yet been studied. Nishijima et al. (2017) [31] divided Ae. tauschii into two main lineages TauL1 and TauL2, and a minor lineage (TauL3) by Bayesian population structure analysis with genome-wide marker genotyping. Using DArTseq genotyping of a large number of accessions, we confirmed their results ( Figure  2, Figure S1). The TauL1 accessions are spread from the western geographical range (Transcaucasus, northern regions of Iran) to the eastern geographical range (Pakistan and Afghanistan), whereas TauL2 is limited only to the western range, and ssp. strangulata is included only in TauL2.
This result is consistent with Mizuno et al. (2010) [23] using AFLPs. Thus, the differentiation of the ssp. strangulata is believed to have occurred in TauL2. Furthermore, we found that the most probable origin of ssp. strangulata is Iran and that this subspecies clusters in one clade within TauL2 (Figure 2, Figure S1). This finding strongly indicates that speciation had occurred in the ssp. tauschii included in TauL2, resulting in appearance of ssp. strangulata-type spike morphology. The D genome of ssp. strangulata is involved in the D genome of bread wheat. This was revealed by sequencing [32], single nucleotide polymorphisms [33], variation in the AP2 homoeologs, the genes underlying lodicule development [34], SSR markers [35], NADP-dependent aromatic alcohol dehydrogenase [36] and aspartate aminotransferase and alcohol dehydrogenase isoenzymes [37]. Overall, using the DArTseq genotyping platform, we have allocated 124 accessions with no previous lineage description into TauL1, TauL2 or TauL3. Furthermore, based on this data, we have reclassified 5 accessions: 2 accessions from Iran (KU-2109 and KU-2158) formerly classified in TauL2 by chloroplast DNA [14] were now placed in TauL1, and 3 accessions (PI 486274 from Turkey, IG 127015 from Armenia and IG 120735 from Turkmenistan) formerly classified in TauL1 were now placed in TauL2. The inconsistency of the nucleus and cytoplasmic genomes may be attributable to the cytoplasmic substitution origin by hybrids between the two lineages and the backcrossing in the evolution of these accessions. Furthermore, previous studies reported that accessions in TauL2 were distributed in the regions near the Caspian Sea. However, here we found that five accessions (AE 192, AE 213, AE 250, CGN10733 and IG 120735) which originated from Turkmenistan and AE 692 from Uzbekistan were clustered in TauL2 (Table 1). These accessions may have been transferred to the regions naturally or by human activity.

Potential for Adaptive Convergence in Ae. tauschii Evolution
Molecular evolutionary studies have explained the origin of crops more clearly than before [38,39], especially for the main crops that were domesticated without ploidy modification. Phylogeographic analyses based on nuclear and chloroplast DNA sequences have shown multiple evolutionary origins of cultivated rice in East Asia [40] and barley in the Fertile Crescent and Central Asia [41,42], whereas phylogenetic analysis based on multilocus microsatellite genotyping has shown a single domestication event for maize ca. 9000 years ago [43]. One of the fundamental problems in understanding the evolution of Ae. tauschii is the relationship between the different lineages and subspecies. In the current study, although some traits examined differed significantly between the lineages and subspecies, the range of the diversity was overlapped (Tables 3-5). The phenotypes convergence may have originated through either divergent genetic solutions [44,45] or the same pathways, genes or even nucleotide positions in independent lineages [46,47]. Convergence at the genetic level can in turn result from (i) mutations arising independently in separate populations or organisms (parallel genetic evolution); (ii) evolution of a polymorphic allele in a common ancestral population or species (trans-specific polymorphism); and (iii) evolution of an allele introduced by hybridization (introgression) from one population to another (e.g., TauL1 and TauL2). Another possibility that can explain the phenotypic similarities between the different Ae. tauschii lineages is the occurrence of genetic differentiation after the geographical isolation under similar environmental condition without morphological or physiological differentiation. Local standing genetic diversity combined with spatial population structure restricting dispersal in an ecologically patchy area promotes rapid convergence [48].

Implications of Ae. tauschii Diversity in Wheat Breeding
Among the species in genus Aegilops, only Ae. tauschii can be used efficiently for wheat improvement owing to the mostly regular pairing of its chromosomes with the D genome chromosomes of bread wheat [49]. It is believed that Ae. tauschii is an excellent source to widen the narrow genetic base of bread wheat. Currently, with the new advances in plant science and the rapid development of sequencing and genome-editing tools, identification and characterization of genes of interest in wheat are in progress and can be expected to become easier and more straightforward in the coming decades. Once the gene in question is identified and characterized, it is easy to transfer and utilize the gene in breeding programs. This will pave the way to utilize the genes from Ae. tauschii as it will help to overcome the limitations related to the irregular chromosome pairing.  Data Availability Statement: This study did not report any data.