Combination of multiple molecular markers can more accurately determine the genetic relationship among species. For example, RAPD, ISSR, IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon microsatellite amplified polymorphism) markers revealed genetic diversity and relationship among the Citrus species (Biswas et al., 2010). Genetic relationships and modes of Robinsonia species were illustrated based on AFLP and SSR markers (Takayama et al., 2015). Combining SSR and ISSR markers could better reflect genetic relationship of D. trifida than single marker (Nascimento et al., 2013). At the molecular level, the percentage of polymorphic band, Nei’s genetic diversity (H), the effective number of alleles (Ne), Shannon’s information index (I), total heterozygosity (Ht) and the average heterozygosity within the species (Hs) are essential for evaluating the genetic diversity of germplasm (Mignouna et al., 2005; Narzary et al., 2009). In this study, ISSR, ISAP and SRAP molecular markers were used to analyse the genetic relationship between D. polystachya and its related species, and high levels of genetic diversity among Dioscorea species were observed with the above parameters. Our results also indicted that combining the three markers detected a higher degree of variation among Dioscorea species compared with single marker as reported previously (Shiwachi et al., 2000; Malapa et al., 2005; Zhou et al., 2008).
It is interesting that there was a relatively high gene flow (Nm = 0.3293) among the 6 species involved in this study, which is much higher than previous studies (Nm = 0.1081) (Wu et al., 2014). Meanwhile, Gene differentiation coefficient (Gst = 0.6029) among species was lower than previously reported (Gst = 0.8222) (Wu et al., 2014). An important reason for this phenomenon might be that the experimental materials involved in this study including many wild individuals. Some studies have indicated that there was more genepool exchange in wild Dioscorea species than cultivated crops (Mengesha et al., 2013a; Mengesha et al., 2013b). In previous studies, the samples of Dioscorea species were almost cultivated crops. In fact, the main reproductive mode of cultivated Dioscorea crops is asexual reproduction, which limits the genepool exchange between species (Takayama et al., 2015). This might be the reason why there was a low gene flow in some Dioscorea genetic diversity studies. Furthermore, we also conducted genetic structuring analysis, which is typically assessed using the so-called F-statistics (Foll et al., 2008). The lowest intra-species similarity coefficient (Fis) was observed in D. japonica, which means that there were the highest degree of intraspecific differentiation and variation in this species. Therefore, it might be suitable for potential breeding materials of Dioscorea. The Fis value of D. persimilis was the highest, indicating that the gene purity in the species was high, and the species may rarely have sexual reproduction. On the other hand, the lowest inter-species variation (Fst) was observed in D. polystachya. This indicated that there was less reproductive isolation between D. polystachya with other Dioscorea species, and it was a good parent material for Dioscorea. The highest Fst value was obtained in D. fordii, which indicating the highest differentiation between D. fordii and other five species. Combined with the results of phylogenetic tree, it could be induced that D. fordii was the ancestor of those Dioscorea species.
The genetic relationship of some edible species in Sect. Enantiophyllum Uline has always been controversial. For example, based on18S sequence analysis, it was speculated that D. persimilis might be a cultivar of D. polystachya and should not be classified as an independent species (Liu et al., 2000). However, our result showed that all individuals of D. persimilis had a distant relationship with D. polystachya. At the same time, phylogenetic tree also showed all the individuals of the two species were located in different branches. The evidence in this study indicated that D. persimilis should be an independent species. Another taxonomic problem in Dioscorea is whether D. alata is a true species or a putative cultigen. Some studies indicated that D. alata might be a cultigen of D. persimilis (D. hamiltonii synon.) due to the narrow genetic base (Hahn, 1991; Wilkin et al., 2007; Mengesha et al., 2013a). In fact, D. alata originates in North and East of Bay of Bengal, and it’s found to be naturalized in China (Hang and Sun, 2020). Our results showed all the individuals of D. alata clustered together in the phylogenetic tree. It is indicated that D. alata might be a true species and not a putative cultigen. This phenomenon was also found in previous studies using isozymes analysis (Lebot et al., 1998). Another interesting phenomenon in this study was the genetic relationship between D. exalata and D. persimilis. The closest genetic relationship was observed between them. Furthermore, PCoA analysis could not separate the individuals of the two species. Therefore, further work will be required to establish the genetic relationship of the two species.
In this study, the phylogenetic tree and genetic similarity results in this study showed the genetic relationship between D. japonica and D. polystachya was the closest. Since Song Dynasty, D. japonica have been called ‘Wild Yam’ (Hang and Sun, 2020). The morphological characteristics of D. japonica is very similar to D. polystachya, and there was no reproductive isolation between them (Araki et al., 1983; Mizuki et al., 2010). Moreover, in pharmacological experiments, unlike other Dioscorea species, D. japonica and D. polystachya had the same efficacy (Hang, 1995). For subsequent breeding programs or conservation actions, we suggested they might be referred to as D. polystachya-D. japonica complex according to the classification of Guinea yam (D. cayenensis-D. rotundata complex) (Martin and Rhodes, 1978; Mengesha et al., 2013a; Mengesha et al., 2013b; Loko et al., 2015;)
Identification with SCAR makers.
Nowadays, the specie-identification of the yam with medicinal properties is still obscure. Therefore, there is an urgent need for the development of a fast and robust identification method to distinguish D. polystachya from its relative species. In this study, a specie-specific band was obtained from SRAP and developed to a SCAR marker. In order to examine its accuracy and sensitivity, the specie-specific SCAR marker was conducted by Double-Blind Test. The result showed that all the individuals of D. polystachya amplified the specie-specific band, which was absent in the other species, confirming the specificity of the SCAR marker for specie-specific identification. In short, a converted-SCAR technique proved to be a helpful tool in identifying Dioscorea species. The results in this study indicated that PCR products can be used to covert SCAR marker and then used as a DNA barcoding for specie identification at a lower cost.