Wild Emmer (Triticum turgidum ssp. dicoccoides) Diversity in Southern Turkey: Evaluation of SSR and Morphological Variations

1. Introduction

Crop wild relatives are the primary sources of novel genetic diversity that is needed for crop improvement in all aspects [1]. A comprehensive evaluation of In-situ and Ex-situ resources as well as common cultivars to mine new alleles is a continuously needed process for crop improvement and agricultural sustainability [2]. The utilization of genetic resources not only requires a clear understanding of the allelic constitution of such species [3] but also requires the successful introduction of this diversity into the domesticated gene pool [4].

Wild emmer wheat (Triticum turgidum ssp. dicoccoides), as one of the closest known wild relatives of the domesticated tetraploid wheat, holds significant potential for wheat improvement [5,6,7]. It is the most likely ancestral species of cultivated tetraploid wheat with a genome constitution of BBAA. The geographical distribution area of wild emmer covers the Fertile Crescent in South-west Asia from Israel, Jordan, Lebanon, Syria, southern Turkey, northern Iraq to south/southwest Iran [8,9]. Due to its full interfertility with domesticated emmer wheat (T. turgidum subsp. dicoccum (Schrank ex Schübl.) Thell.), it can serve as one of the most important genetic resources to improve durum (Triticum turgidum L. ssp. durum (Desf.) as well as bread wheat (Triticum aestivum L.). Wild emmer has been used for the allele mining for many needs of wheat breeding, including but not limited to, drought tolerance [10,11], salinity stress tolerance [5,12], and for the biotic stress factors, fusarium head blight [13,14], stripe rust [15,16], root-lesion nematodes [17], and powdery mildew [18,19,20,21,22].

Due to its possible domestication in or near the Levant [6,9], there has been a high level of reports from Israel and vicinities in the Jordan Valley [23,24,25]. There were only a few studies on the accessions of wild emmer wheat from some locations out of Turkey [9,26,27,28], but there is not any comprehensive evaluation of In-situ or Ex-situ gene-pools from Southeast Anatolia. On the other hand, since the early 20th century, there has been a significant number of reports on the diversity and distribution of wild emmer wheat from the Levant and Jordan Valley, mostly Israel and Lebanon [23,24,29,30,31,32,33,34,35,36,37,38,39]. This concentrated evaluation of a specific region for wild emmer did continue in the following years [7,11,20,40,41,42,43,44,45,46,47,48,49,50,51,52,53]. Evaluation of the natural populations in the central Fertile Crescent, especially the Karacadağ region of Southeast Anatolia, is neglected. Even though this region is recently emphasized for its role in wheat domestication [9,54], there is no comprehensive report of the natural wild emmer diversity in this region and Turkey. Therefore, the purpose of this study was to document the phenotypic and genotypic diversity between and within 169 accessions of 38 In-situ populations of wild emmer wheat collected from the Karacadağ region of Southeast Anatolia.

2. Materials and Methods

In this study, 169 accessions of 38 wild emmer wheat populations recently collected from Southeast Anatolia were used (Table 1). The study was conducted in Adana, Turkey during 2020-2021 growing season. The seeds from each population were grown at the experimental area of Çukurova University, Adana, Turkey, for studying genotypic and phenotypic variation. Initially, ten seeds from each genotype were germinated in Petri dishes. Once the seedlings developed, they were transferred to small pots. Each seedling was then transplanted into a separate row, maintaining a spacing of 30 cm between and within the rows. To prevent damage from spike breakage and to ensure complete self-pollination while preserving seed purity, the main spikes of all wild emmer plants (Triticum turgidum subsp. dicoccoides) were bagged for self-pollination.

Total genomic DNA was isolated from young leaves according to cetyltrimethylammonium bromide (CTAB) protocol [55] with some modification of Ozkan, et al. [56]. The extracted DNA was evaluated qualitatively in addition to quantity, measured by 0.8% agarose gel electrophoresis. Before using DNA for molecular analysis, the DNA diluted to the required concentration for 10 ng/ml for SSR applications. Initially, 100 SSR primers mapped to the A and B genomes were first screened on eight wild emmer genotypes not only to detect their polymorphism level but also PCR amplification. After screening, 16 SSR primer was select for further work (Table 2). M13 tailed-primer PCR amplification of SSRs according to [57] was performed in a 12 µL PCR mix containing 1X buffer, 0.125 mM dNTPs, 0.4 pmol M13 sequences tailed forward primer, 0.3 pmol reverse primers, 3.0 pmol universal M13 primer labeled with one of four (6-FAM, VIC, NED or PET) fluorescent dyes, 0.12U Taq DNA polymerase, and approximately 50ng genomic DNA. PCR amplification was performed with an initial denaturation at 94°C for 5 min; 30 cycles of 94 °C for 1 min, 55 to 67 °C (annealing temperature depending on primers) for 1 min, 72 °C for 1 min; followed by 8 cycles of 94°C for 30 s, 53°C for 45 s, and 72°C for 45 s; and a final extension at 72°C for 10 min. A set of four PCR products (1 μl each) labeled with a different dye was combined with 0.25 μL GeneScan-400 LIZ^® size standards (Applied Biosystems) and 9.86µL Hi-Di™ Formamide (Applied Biosystems), denatured at 94°C for 5min, chilled on ice, and separated on an ABI 3130xl Genetic Analyzer (Applied Biosystems). The SSR fragments were scored and checked twice using the Gene Mapper software v3.7 (Applied Biosystems) as described in the user manual.

The SSR was scored as binary data (1/0), indicating the presence or absence of a marker in the genomic representation of each sample. Several genetic diversity parameters were calculated for each locus and population using the GENALEX6.5 program [58]. Principal coordinate analysis (PCoA) used to explore multivariate relationships among inter-individual genetic distances within and among populations was also performed with the GENALEX6.5 program.

3. Results

3.1. Agro-Morphological Variation in Wild Emmer Populations

A total of 169 accessions, collected as 38 populations from the East and West Karacadağ region of Southeast Anatolia (Figure 1), were evaluated for agro-morphological and genetic diversity-related traits. According to the variance analysis (ANOVA), there were significant (p<0.05) differences between the evaluated panel of accessions for plant height, peduncle length, flag leaf length and area, length of the uppermost awn in the spikelet, length of the lowermost awn in the spikelet, width and height of the glume, length and width of the anther and 1000 grain weight (Data not shown). Out of 23 agro-morphological parameters evaluated, populations from Karacadag/EAST had the highest values in spike length (9.28 cm), spikelet number (20.69), glume hull thickness (0.26 mm), anther width (0.59 mm). On the other hand, Karadag-1/WEST populations had the highest agro-morphological trait values in heading date (170.38 days), flag leaf area (17.76 cm²), auriculas width (5.68 mm), length of the uppermost awn in the spikelet (68.41 mm), spikelet length (16.13 mm), lemma length (13.95 mm), palea length (12.88 mm). Finally, populations from Karadag-2/WEST had the highest values in plant height (128.31 cm), heading date (167.49 days), peduncle length (41.48 cm), auriculas length (4.65 mm), lemma width (2.62 mm), palea width (2.03 mm), glume length (12.61 mm), glume height (2.55 mm), anther length (4.10 mm) and maturation day (199.86 days). According to overall data Karadag-2/WEST was the region with the maximum agro-morphological diversity and the highest values in phenotypical traits (Table 3).

3.2. Genetic Diversity Within and Between the Populations and Sub-Regions

Genetic diversity of the evaluated accessions was assessed with analysis of molecular variance (AMOVA), and several diversity parameters including Nei genetic distance and identity values (Table 4 and Table 5). AMOVA was calculated to assess the variance within and between 38 populations of wild emmer wheat collected from three sub-regions of Karacadağ mountains (Table 4). There was a significant level of variation within the populations (84%), while variation among the populations was 16%. Nei genetic distance and identity values for the three sub-regions are given in Table 5. According to the results, the regions with the most distance were Karacadag/EAST and Karadag-2/WEST (0.539), while the ones with the closest identity values were Karadag-1/WEST and Karadag-2/WEST (0.788). The lowest distance was seen between two Karacadağ west sub-regions (0.214) and the lowest identity was between Karacadag/EAST and Karadag-2/WEST (0.463).

We have also calculated several genetic diversity parameters to estimate the variation within and between the populations. These parameters were the number of alleles per locus (Na), the number of effective alleles per locus (Ne), Shannon’s information index (I), expected heterozygosity (He), and unbiased heterozygosity (uHe). The highest number of alleles per locus was in Karacadag/EAST populations, while the lowest was in Karadag-1/WEST. Similarly, Karacadag/EAST populations had the highest values in Ne, I, He, and uHe compared to Karadag-1/WEST and Karadag-2/WEST populations (Table 6). In contrast, the lowest values in all parameters were obtained in Karadag-1/WEST populations. According to the above results, maximum and minimum genetic diversity was obtained in the populations from Karacadag/EAST and Karadag-1/WEST populations, respectively.

3.3. PCoA and Neighbor-Joining Grouping Patterns in Wild Emmer Populations

The PCoA analysis was applied to define the population interactions, and 169 accessions from 38 populations were separated into 2 major clusters, without any mixture between Karacadag/EAST and both Karadag-1/WEST and Karadag-2/WEST. The distinction between East and West was quite sharp and there was no mixture (Figure 1) between these regions. Two sub-sets of Karadağ/WEST (1 and 2) were almost entirely mixed. So, it was not possible to separate them further into smaller sub-sets. Of the sub-cluster in Karacadag/EAST, there were several accessions located far away from the rest of the accessions.

The relationships within and between the three regional groups were also evaluated using Neighbor-Joining analysis (Figure 2). Neighbor-Joining tree dendrograms produced a similar distribution to PCoA clustering. Two main branches were formed with accessions from WEST and EAST. These two main clusters did not have any admixture. There were several sub-clusters on the main clusters EAST and WEST. There were several sub-clusters in the main cluster WEST, which are almost entirely constituted from the mixed accessions of WEST-1 and WEST-2. There were only a few sub-clusters (WEST-uppermost branch) with a clear divergence from the rest of the group. Even though there were mixtures over the entire main cluster WEST, each small sub-set was formed with at least several accession from the same sub-region.

4. Discussion

Crop wild relatives are one of the main sources of allelic diversity, and so the hub for climate-resilient crop breeding. To “feed the billions” [59] and meet the pace of climate change and population increase, there is a continuous need for allele mining and germplasm screening [60,61,62,63,64]. Wild emmer wheat, one of the closest relatives of durum wheat [65], is a possible shortcut to the wide wild allelic gene pool in Triticum sp. [8,66,67,68]. As highlighted, there is a need for a “walk on the wild side” [61]. With this objective in mind, we characterized a set of In-situ germplasm accessions from the Southeast Anatolia, Karacadağ mountains, the home of Göbeklitepe, the oldest known temple, for genetic diversity assessment [69,70].

4.1. Agro-Morphological Diversity

As a result of germplasm collection from the three different sub-regions around Karacadağ mountains in Southeast Anatolia, 169 accessions within 38 populations were characterized. Agro-morphological characterization and genetic variation assessment through SSR markers were utilized to estimate the natural population diversity of this region. Wild emmer is thought to be domesticated near the southern Levant [6,9], and there has been intensity in the number of reports from the possible domestication center. However, it has a much wider species distribution [6,8,9] and the number of reports from other regions (Turkey and Iran) is quite limited [26,27,71]. In addition, none of the previous reports from Turkey built an in-depth agro-morphological and/or genetic diversity evaluation among natural (In-situ) populations.

Here we obtained significant agro-morphological and genetic diversity among and within the subsets of wild emmer wheat collected (Table 3 and Table 4). The main component of the traits we evaluated were spike, plant growth, and phenological traits.

Even though there were a vast number of studies in relation to biotic stress tolerance, such as powdery mildew [20,72], fusarium head blight [13], and stripe rust [15] in wild emmer, we did not find any reports concerning spike and phenology traits within this region. The results highlight the potential of In-situ populations and hidden gems in the wild [73] reports that several spike-related traits such as purple coleoptile, purple auricle, purple culm, hairy auricle, hairy rachilla, and fragility of spike were controlled by single dominant genes, making the transfer of such traits much straight-forward compared to quantitatively inherited traits. Further studies should try to utilize germplasm resources with novel allelic diversity into the common crops as candidates for abiotic and biotic stress tolerance as well as yield and growth-related traits [13,20,72,74,75].

4.2. Genetic Diversity of In-Situ Populations

To evaluate the genetic diversity within and between the populations, 16 specific SSR markers were applied (Table 2). According to the results of AMOVA and other genetic diversity parameters, there was significant diversity among the evaluated panel of In-situ accession. There were 84% within and 16% between populations diversity. The results were in a similar range with previous reports in tetraploid wheat. In similar studies, [76] reported 19 and 81% within and between population diversity, while Teklu, et al. [77] reported wider levels of diversity among 73 wild emmer accessions from 11 different geographic regions. Nei genetic distance values in this study ranged between 0.214 (between Karadag-1/WEST and Karadag-2/WEST) and 0.539 (between Karacadag/EAST and Karadag-2/WEST). The results showed a clear-cut separation between the EAST and WEST populations. Mountainous landscape of the region seems to reduce genetic drift and mixture even within a relatively close distance of about 200 km. The distance between WEST-1 and WEST-2 populations were closer, about 55 km and their genetic distance was quite small, compared to WEST-EAST distance (Table 5). According to Harlan and Zohary [78], Zohary and Hopf [79] and Ozkan, Willcox, Graner, Salamini and Kilian [9] wild emmer has a wide distribution from the Levant to Turkey and south/southwest Iran. The level of observed diversity within the Karacadağ region demonstrates the potential of this unexplored habitat for crop improvement [6,11,80].

The number of alleles per locus (Na) is an indicator of the diversity at the gene level [81]. Here, we observed a three-fold difference between three regional groups from 3.93 in Karadag-1/WEST to 9.93 in Karacadag/EAST. Since all accessions were from the same species within the same wider region, this level of difference may be due to unequal population size distribution among sub-sets or some other unknown reasons. Li, Roder, Fahima, Kirzhner, Beiles, Korol and Nevo [43] evaluated the number of microsatellites among 105 individuals from the Yehudiyya region of Israel, the Na values they obtained were lower than the current study. In their follow-up study, the same group obtained 1.88, 3.86, and 5.89 Na values at chromosome, genome, and genome x chromosome levels. In a similar study, on a different region, Li, Fahima, Peng, Roder, Kirzhner, Beiles, Korol and Nevo [39] reported an average of 7.1 Na value using 28 microsatellite markers among 155 individuals from two sub-regions of Tabigha, Israel. In our study, the number of effective alleles (Ne) followed the same trend with the Na values, which ranged between 2.75 and 5.50 per locus. The Ne values were higher than Arystanbekkyzy, Nadeem, Aktas, Yeken, Zencirci, Nawaz, Ali, Haider, Tunc, Chung and Baloch [26], who reported an average 1.962 Ne value among 29 wild and 4 cultivated emmer wheat collected from different regions of Turkey.

We obtained a Shannon index (I) between 1.030 (Karadag-1/WEST) and 1.692 (Karacadag/EAST). Expected heterozygosity (He) was between 0.561 and 0.725. The I and He values we obtained was significantly higher compared to Dong, Wei, Chen, Li, Wang, Nevo and Zheng [7] and Fahima, Sun, Beharav, Krugman, Beiles and Nevo [37], while it was in similar ranges with Fahima, Roder, Wendehake, Kirzhner and Nevo [42]. A similar study [28], compared accessions from Israel and Turkey (Diyarbakır region) for genetic diversity using AFLP markers. The He values they reported were much lower compared to the current study. Ozbek, et al. [82] evaluated a set of accessions (120) from Israel and also reported lower He values compared to our results. Here, Na, Ne, I, He, and uHe values we obtained, shows the genetic diversity of the natural wild emmer populations in the Karacadağ region.

4.3. PCoA and Neighbor-Joining Analysis

PCoA and Neighbor-Joining trees followed the same trend (Figure 1 and Figure 2). Both methods separated WEST and EAST populations and did not create any mixture zones. On the other hand, two sub-sets of WEST (1 &2) were almost entirely mixed and it was not possible to make a distinction between these two. Neighbor-Joining Dendrograms and PCoA clustering were similar to our previous report with AFLP markers [9], which distributed EAST and WEST populations in two separate clusters. When we closely looked at the Neighbor-Joining dendrogram for branching and genotypes positions (Figure 3), it was seen that most of the individuals from the specific populations were located closely on the same branch, with some exceptions that did not follow any trend.

5. Conclusions

Wild and domesticated emmer wheat are well-characterized and excessively utilized species for crop improvement, especially in biotic stress tolerance. Here, a panel of wild emmer wheat was characterized for agro-morphological traits and genetic diversity. According to genetic diversity values obtained, PCoA and Neighbor-Joining dendrograms, two regional groups with approximately 200 km distance had significantly different characteristics in terms of allelic distribution and some phenotypic traits. The screening and utilization of In-situ germplasm sources from this region would help widen the genetic diversity in durum as well as common wheat breeding.