Genetic Diversity of Grasspea and Its Relative Species Revealed by SSR Markers

The study of genetic diversity between Lathyrus sativus L. and its relative species may yield fundamental insights into evolutionary history and provide options to meet the challenge of climate changes. 30 SSR loci were employed to assess the genetic diversity and population structure of 283 individuals from wild and domesticated populations from Africa, Europe, Asia and ICARDA. The allele number per loci ranged from 3 to 14. The average gene diversity index and average polymorphism information content (PIC) was 0.5340 and 0.4817, respectively. A model based population structure analysis divided the germplasm resources into three subgroups: the relative species, the grasspea from Asia, and the grasspea from Europe and Africa. The UPGMA dendrogram and PCA cluster also demonstrated that Asian group was convincingly separated from the other group. The AMOVA result showed that the cultivated species was quite distinct from its relative species, however a low level of differentiation was revealed among their geographic origins. In all, these results provided a molecular basis for understanding genetic diversity of L. sativus and its relatives.


Introduction
The genus Lathyrus L. includes as many as 187 species [1,2]. These are distributed throughout temperate regions of the Northern Hemisphere and extend into tropical East Africa and South America. However, the main centers of diversity include the Mediterranean and Irano-Turanian regions [3]. Grasspea (Lathyrus sativus L.) is the only species widely cultivated as a food crop in the genus Lathyrus, whereas other species (Lathyrus cicera L. and Lathyrus ochrus L.) are cultivated to a lesser extent [4]. Moreover, grasspea has great agronomic potential as a grain and forage legume in the fragile agro-ecosystems, because of its ability to survive under extreme climatic conditions such as drought, flood and salinity [5].
There have been recent studies of genetic diversity in Lathyrus sativus. PCR-based molecular markers utilized so far in L. sativus and its relative species include random amplification of polymorphic DNA (RAPD) [6,7], restriction fragment length polymorphism (RFLP) [8] which was indicated the highly similarity between L. sylvestris L. and L. latifolius L., amplified fragment length polymorphism (AFLP) [9] clarified that 20 central Italy grasspea accessions were divided into the Household populations and the Commercial populations which was useful for the grasspea bereeding in central Italy, and inter-simple sequence repeat (ISSR) was used for exploring the genetic diversity among L. sativus, L. cicera, and L. ochrus [10].
Up to now, there was little study of genetic diversity in Lathyrus sativus using simple repeat sequence (SSR) markers [11][12][13] (Table 1). Lioi et al. searched for EST sequences of L. sativus with the European Molecular Biology Laboratory (EMBL) nucleotide sequence database. Amplification was successful only in 10 out of 20 of the SSR primers, with only 6 of these exhibiting size polymorphism and subsequently used in genetic diversity analysis for 13 Italian landraces [11]. Shiferaw et al. used 11 EST-SSRs developed from L. sativus. EST-SSRs derived from Medicago truncatula L. to investigate the genetic diversity among 20 grasspea accessions from Ethiopia [12].
Using the 454 FLX Titanium pyrosequencing technique, a large-scale microsatellite approach was developed in Lathyrus sativus [13]. Potentially these SSR primers can make a significant contribution to genomics enabled improvement of grasspea. To broaden the genetic variation of cultivated grasspea in the future for China, it is necessary to perform a more comprehensive analysis of genetic diversity and population structure in the national genebank. We used 30 polymorphic genomic-SSR markers developed by Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China (ICS/CAAS) [13], to study the genetic diversity among 266 accessions from L. sativus and 17 accessions from its cultivated and wild relatives ( Fig. 1).

Plant materials
A total of 266 grasspea accessions (Table 2) and 17 relative accessions (Table 3) were collected and tested in the protected field of experimental farm within CAAS campus (39°57' 38" N, 116°19' 27" E). For Lathyrus sativus, European germplasm comprised 100 accessions from 14 countries, while Asian germplasm contained 20 accessions from China and 98 non-Chinese accessions. African germplasm included 33 accessions and ICARDA comprised 15 accessions (Fig. 1). The 17 accessions of 9 relative species are from Europe, Asia, and Africa (Fig. 1). Seed supplies direct from collected samples were sourced from ICS/CAAS, as well as from N. I. Vavilov Research Institute of Plant Industry, St. Petersburg, Russia. Maps of the genus Lathyrus collection sites were conducted with DIVA-GIS based on latitude and longitude coordinates [14].

DNA extraction
Genomic DNA was extracted from pooled ten random young seedlings of each accession using the CTAB method [15,16] with 1% PVP added.

Data analysis
The genetic diversity parameters and polymorphism information content (PIC) of each primer pair were calculated by Powermarker v3.25 [17] using the following formulas: Gene diversity: Þ=ð1−ð1 þ f Þ=nÞ; PIC = ∑(1-pi 2 )/n, where pi is the frequency of the ith allele, n is the total number of genotypes [18]. POPGENE version 1.32 [19] was used to calculate Nei's genetic distance [20]. The program STRUCTURE V2.3.3 [21,22] was used to examine population structure and differentiation. The simulations were run with a burn-in of 100,000 iterations and from K = 1 through 10. Runs for each K were replicated 160 times and the true K was determined according to the method described by Evanno et al. [23]. The number of subgroups (K) was identified based on maximum likelihood and delta K (ΔK) values. The cluster analysis of different geographical groups was carried out using unweighted pair-group method with arithmetic average (UPGMA), and the dendrogram was drawn by MEGA 5.02 [24]. Analysis of molecular variance (AMOVA) was used to assess the variance among and within populations from different geographical origin with GenAlEx 6.41 software [25]. Principal component analysis (PCA) was applied to show the distribution of individual accessions in scatter diagram and two-dimension PCA graph was drawn using the NTSYSpc 2.2 statistical package [26].

Results
SSRs polymorphic testing 120 SSR markers were randomly selected to validate polymorphism at first. 25% of them were polymorphic (Table 4). 30 SSR makers amplified 258 polymorphic bands with an average of 8.6, ranged from 3 to 14 per primer pair (Table 5). Gene diversity was from 0.0708 to 0.8505, and the average was 0.5340. Meanwhile, polymorphism information content (PIC) of each primer pair ranged from 0.0688 to 0.8338 with an average of 0.4817. These results demonstrated polymorphic SSR markers which we used were good enough for further genetic diversity analysis.
Genetic diversity and classification analysis among populations of Lathyrus sativus and its relative species The population structure of Lathyrus sativus and its relatives was inferred by using STRUC-TURE V2.3.3 based on 30 SSR markers. At K = 2, all the germplasm were divided into L. sativus and its relatives. But, according to the method described by Evanno et al. [23], three populations should be identified theoretically based on delta K (ΔK) values (Fig. 2), therefore the genetic structure of 283 accessions can be described with greatest probability and no gain in discrimination. At K = 3, the related accessions were in one subgroup and the L. sativus also divided into 2 subgroups (Fig. 3). One subgroup contained 79 accessions mainly from Asia. The other subgroup contained 187 accessions and most of them came from European and African countries.

Genetic relationships analysis
The Lathyrus sativus relatives as a group were marginally more similar to the Asian than to the African and European sources of L. sativus, whereas the African and European sources of L. sativus were more closely related than either to the Asian source (Table 6, Fig. 4). All Lathyrus accessions were clustered according to Nei's genetic distance [20] (Fig. 4). The largest genetic distance (0.6360) was between Lathyrus sativus relatives and European grasspea, and the smallest genetic distance (0.0038) was between African and European grasspea. Based on the origin of L. sativus accessions, the genetic distance between Africa and Asia (0.0141) was larger than it between Europe and Asia (0.0118). These result matched with structure analysis above. There were 17 accessions from nine different relative Lathyrus sativus species used in this study. Nei's genetic distance of 0.7247 between L. sativus and L. cicera was the smallest in our study among L. sativus and its nine relative species (Table 7). This result matched morphological [27] and cytogenetical [28] researches which suggest that L. cicera is the most probable progenitor of L. sativus. Among L. sativus relative species, the relationship between L. latifolius and L. sylvestris was the closest (Table 7). Meanwhile, the closer phylogenetic relationship between L. latifolius and L. sylvestris revealed in our research was also detected by Ceccarelli et al. [29] using satellite DNA and Asmussen and Liston [30] using chloroplast DNA study.
Clustering analysis based on Nei's genetic distance divided all the 10 species under genus Lathyrus accessions into two major groups (Fig. 5). One group included L. sativus, L. cicera L., Table 4. Characteristics of 30 polymorphic microsatellite loci used in this study (FP = forward primer, RP = reverse primer, Ta = annealing temperature).

Primer
Repeat motif Primer sequence (

Classification and PCA analysis of all the accessions used in this study
The genetic relationship of individual accessions was analyzed using principal component analysis (PCA); all the cultivated accessions were labeled according to their geographical origin. Within cultivated species, accessions from Asia were somewhat associated with their geographical origin and were different from other accessions (Fig. 6), especially, eight accessions from Bangladesh were quite apart from African and European accessions. The first two principal components explained 43.42% and 29.17% of the molecular variance, respectively.

Analysis of molecular variance
First of all, we evaluated genetic differentiation between Lathyrus sativus and its relatives by analysis of molecular variance (AMOVA). The results showed that the cultivated species was significantly distinct from its relatives at P-value of 0.0001 (Table 8). Among population variance explained 40% and within population explained 60% of genetic diversity. Secondly, significant genetic differentiation among the three population structure classified subgroups was detected by AMOVA at P-value of 0.0001 (Table 9). The results of AMOVA also indicated that the majority of the genetic variation among all the 283 accessions was due to within population variation (84%). Finally, we evaluated the genetic differentiation among accessions of grasspea (Table 10). The results show a low level of differentiation (3%) among Asia, Europe, and Africa.

Use of genetic diversity
Grasspea, as a neglected and underutilized species, is very popular among the resource poor farmers in marginal areas due to the ease with which it can be grown successfully under adverse agro-climatic conditions without much production inputs [5]. Genetic diversity is a source of traits for increased agricultural production and resistance to biotic and abiotic stresses [31]. Knowledge of genetic diversity will assist germplasm utilization in Lathyrus sativus breeding, and more climate-resilient varieties would be bred in the near future. There may be opportunities to exploit wiser genetic diversity in grasspea by combining germplasm between Asia and Africa/Europe, especially taking note of eco-geographical origins for complementation of extreme stress traits for drought tolerance, reproductive heat stress and salinity, for the breeding demands of specific target environments. Further such exploration of diversity could include the more closely related L. sativus relatives which have more limited geographic range in cultivation, and attention to sources of low toxin to reduce the risk of poisoning in situations where grasspea is a major component of human diet.

Comparison of grasspea genetic diversity
EST-SSRs have been used to detect the variability in grasspea accessions and to evaluate genetic diversity [11,12]. These markers were developed from Lathyrus sativus and transferable EST-SSRs from L. japonicus L. and Medicago truncatula respectively and the number was limited. In this study, the SSRs were developed by NGS sequencing of L. sativus genomic DNA [13]. Compared with the previous study [11,12], the genetic diversity of 283 accessions was higher, as the average allele number per locus was 8.6, and the average PIC value was 0.4817. In comparison with the L. sativus relatives, the cultivated germplasm, which came from Africa, Europe, Asia, and ICARDA, had much wider diversity than local germplasm, such as Ethiopia [12] and Italy [11], respectively. The level of polymorphism detected with genomic-SSRs was higher than that of EST-SSRs matching with the previous reports [32,33].

Possibility of Genetic Flow
All the Lathyrus accessions were divided into three subgroups, under cultivated subgroups the accessions were classified according to geographical origins. The Lathyrus sativus relatives were separated from L. sativus clearly. Within the cultivated species, European and African accessions were aggregated together, and partially overlapped with some Asian accessions due to possibility of flow between the two subgroups. For example, Island Cyprus and ICARDA located in Asia, but 21 and 13 accessions were divided into European and African subgroup and only 1 and 2 accessions consisted to Asian subgroup, respectively [34,35].

Richness of genetic diversity
The PCA of cultivated accessions by geographical distribution indicates that the first two principal components explained over 72% of the total genetic variation. Although most European and African materials flowed together, Asian accessions dispersed in much more extensive scope, as the PCA indicated (Fig. 6). More interestingly, the eight accessions from Bangladesh were relatively separated from others, as showed in Fig. 6. It means that the genetic diversity of cultivated accessions of grasspea originated from Asia is much richer than that from Europe and Africa.

Genetic relationship and origin of Lathyrus sativus
Our accessions used in this study occupied vast territories of Southwestern, Western and Eastern Asia. It also occurred on isolated sites in Africa (Ethiopia and Eritrea). The European accessions were widespread throughout Southern and partly Central Europe, penetrating to the northern coast of Africa (Algeria, Morocco and Tunisia). The result of structure demonstrated that Lathyrus sativus divided into 2 population (Fig. 3). One contained 79 accessions and most of them distributed in Asia. The other included 187 accessions and most of them came from European and African countries. The UPGMA dendrogram (Fig. 4) also supported this hypothesis that there was a smaller genetic distance between African and European accessions than that of Asian accessions. AMOVA based on geographic origins (cultivated species divided into Asian, African, and European accessions) revealed that, in the total genetic variance, geographic-related variance was very limited (Table 10). Although Vavilov described Central Asia and Abyssinia as the centers of origin for L. sativus [36], our research results based on genotyping method partially supported the hypothesis that India together with adjacent areas was the primary centre of origin [37] which based on traditional phenotyping method. In conclusion, the natural distribution of L. sativus was obscured by cultivation, making it difficult to precisely locate its center of origin as described by Singh et al [38].