Cross-species Amplification and Molecular Diversity Analysis in Greengram [Vigna radiata (L.) Wilczek] and Ricebean [Vigna umbellata Thunb.]

Legumes belongs to the third largest flowering plant family (Leguminosae - 650 genera and 20,000 species) (Harouna et al., 2019). The most widely consumed legumes fit in to the genera Phaseolus and Vigna. Of which, the genus Vigna entail a huge set of important legumes namely, mung bean [V. radiata (L.) Wilczek], urd bean [V. mungo (L.) Hepper], cowpea [V. unguiculata (L.) Walp.], azuki bean [V. angularis (Willd.) Ohwi and Ohashi], bambara groundnut [V. subterranea (L.) Verdc.], moth bean [V. aconitifolia (Jacq.) Marechal] and rice bean [V. umbellata (Thunb.) Ohwi and Ohashi] (Tomooka et al., 2014).
 
Legumes rank third in global crop production and India is the largest producer, consumer and importer in the world. Greengram is the fourth dominant pulse crop grown in India because of its wide flexibility to adaptation, short duration, drought hardiness and low input requirement. According to the data available at Ministry of Agriculture and Social Welfare, Govt. of India there is a fluctuation in area, production and productivity of greengram (www.indiastat.com). In order to cope up with the fluctuations occurring each and every year there is a need to develop versatile greengram varieties. The major cause for these fluctuation is its high susceptibility to YMD and bruchids (Zhang et al., 2002). In order to improve productivity of the crop i) look for diversity available in the germplasm and make effective crosses ii) transfer resistance from related species to the crop.
        
The first step in any breeding programme is to identify and utilize the variability available in the germplasm. In order to maintain, evaluate and utilize germplasm effectively for breeding, it is important to investigate the extent of genetic diversity available. Morphological characterisation is cumbersome and is highly influenced by environment. Hence, molecular characterisation based on differences in DNA sequences was employed to detect polymorphisms between individuals which are highly stable than morphological and protein-based markers (Mignouna et al., 1998; Tanksley et al., 1989).
        
Ricebean (Vigna umbellata Thumb.) belongs to the tertiary genepool of greengram, is highly responsive to inputs with high harvest index and high degree of resistance to YMD and bruchids (Singh et al., 2013). Being a minor pulse crop, the availability of SSR markers is limited. Development of highly informative sequence based molecular markers is expensive. To overcome the limitation of molecular markers available to minor crops, cross species transferability studies were conducted employing molecular markers available in related species (Srimathy and Jayamani, 2010; Azevedo et al., 2012; Srimathy et al., 2013; Jena et al., 2015; Shivakumar and Ramesh, 2015). The microsatellite markers reported a cross species amplification rate of 50 to 100% (Peakall et al., 1998). Hence, the present study was conducted to test the cross-species amplification of SSR markers derived from different species of the genus Vigna in greengram and ricebean genotypes and to study the molecular diversity existing in it.

The molecular diversity analysis on 25 greengram and five ricebean genotypes using 20 SSR markers derived from adzukibean, commonbean and greengram were carried out in the Marker-Assisted Selection Laboratory, Department of Pulses, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore in 2018. The list of genotypes used in molecular diversity analysis is presented in Table 1. Primary leaves from cup-sown genotypes were collected and the DNA was extracted by CTAB (mini-prep) method. DNA was quantified by using Nano Drop spectrophotometer. The quality of DNA was also checked using 0.8 per cent agarose gel electrophoresis using 1X TBE buffer and ethidium bromide (5 µl/100 ml agarose). The DNA was then diluted to appropriate concentration for use in molecular analysis.
 

        
DNA from 25 greengram and five ricebean genotypes were amplified using 20 SSR markers derived from adzukibean (Srimathy et al., 2013; Tian et al., 2013), commonbean (Sathya and Jayamani, 2013) and greengram (Kumar et al., 2002; Sathya and Jayamani 2013; Somta et al., 2008). The details of SSR markers used in the study along with their annealing temperature is presented in Table 2. Amplification reaction was performed in a volume of 15 µl containing 50 ng of genomic DNA and amplification was performed in BIORAD - My cycler. PCR reaction mixture (15 µl) consisted of 7.75 µl of sterile water, 1.5 µl of buffer (10 X), 0.3 µl of dNTP’s (10 mM), 0.15 µl of MgCl₂ (50 mM), 3.0 µl of marker (5 µM), 0.3 µl of Taq DNA Polymerase and 2.0 µl of DNA.
        
The thermal profile consisted of initial denaturation was held at 94^oC for 3 minutes. Followed by 35 cycles of denaturation at 94^oC for 45 seconds, annealing at 47^o-60^oC for 1 minute and extension at 72^oC for 1 minute. The final extension at 72^oC was allowed for 10 minutes and held at 4^oC for infinite time. The PCR products were resolved on 3 per cent agarose gel in 1 X TBE and ethidium bromide (5 µl /100 ml of agarose) at 80 V for 3 hours using gel electrophoresis unit. The gel was documented in BIORAD documentation system.
        
The gels were scored and represented by their allelic sizes as allelic data. Image Lab software was used to determine the allele size. Using the DARwin -5.0 software package (Perrier and Jaqeuemond-Collet, 2005) a simple matching dissimilarity index was calculated from the allele-size data set with 100 bootstraps and this matrix was then subjected to UPGMA and Neighbour-Joining analysis. The PIC value are calculated using the formula given by Smith et al., (1997).
Where
pi = Frequency of the i^th allele.

The availability of microsatellite markers are less in number for greengram and not available for ricebean. Hence, microsatellite markers from related species were utilized for testing their transferability to greengram and ricebean. Cross-species amplification for adzukibean derived SSR markers were reported in prior by Srimathy and Jayamani (2010), Sathya and Jayamani (2013) and Jena et al., (2015). Jayamani and Sathya (2012) reported cross species amplification for adzukibean and cowpea derived markers. All the markers used in the present study shown amplification. In greengram, the per cent of polymorphism observed were 62.50, 100 and 54.54 per cent, respectively for the SSR markers derived from adzukibean, commonbean and greengram. The markers that exhibited polymorphism in greengram genotypes were CEDAAG 001, CEDG 015, CEDG 026, CEDG 154, CEDG 198, BM 170, MB 14, MB 17, MB 87, MB 91, DMBSSR 167 and VJ 3120 B.  For ricebean, the SSR markers derived from adzuki bean and greengram exhibited 62.50 and 36.36 per cent polymorphism, respectively. The markers shown polymorphism for ricebean genotypes were CEDAAG 001, CEDG 015, CEDG 026, CEDG 043, CEDG 198, MB 17, MB 87, MB 91 and DMBSSR 167.
        
The number of alleles detected in different SSR markers are presented in Table 2. The number of alleles ranged from one to four with an average of 2.55 alleles per locus. The average number of alleles per locus was low when compared to the results reported by Gwag et al., (2006) with 3 alleles per locus, Gwag et al., (2010), Sathya and Jayamani (2013) with 2.96 alleles per locus, Shrivastava et al., (2014) with 4.85 alleles per locus and Chen et al., (2015) with 2.66 alleles per locus in greengram. The lesser number of alleles per locus might be due to the existence of lower level of DNA polymorphism exhibited by the genotypes for the particular marker used and the conservation of that segment of DNA over years. The maximum number of alleles generated by adzukibean derived SSR marker was 4 (CEDAAG 001), commonbean derived SSR marker was 3 (BM 170) and greengram derived SSR marker was 4 (MB 91). Allele size varied from 100-290 bp for the SSR markers used.
 

        
Polymorphic information content (PIC) is a measure of informativeness of a genetic marker for linkage studies. The PIC value of the present study varied from 0 to 0.611 which represents the presence of highly conserved region (zero) in the two species which are not subjected to recombination, leading to lack of polymorphism for the marker and a moderate polymorphic nature indicates how ricebean have diverged from the greengram (Table 2). Li-Xia et al., (2009) and Chen et al., (2015) recorded an average PIC value of 0.360 in greengram, Somta et al., (2008) recorded a PIC value of 0.256 which was found to be lower than the average PIC value observed in the current study. The lower PIC value observed in the presence study indicates that the analysis was performed with lesser number of genotypes.
        
With respect to the markers used, the adzukibean derived marker CEDG 43 and greengram derived marker VJ 3144 A were found to be biallelic only in greengram indicating the heterozygosity for the particular locus whereas in ricebean it was found to be monoallelic. The adzukibean derived marker CEDG 198 expressed biallelic nature in both the species. In ricebean, the commonbean derived marker did not show polymorphism indicating the genetic conservation. The differential polymorphism exhibited by the markers in greengram and ricebean helps in fingerprinting studies and construction of linkage map to tag agronomically important traits.
        
The dissimilarity value between species was found to be higher than within species indicating higher diversity between species which is the natures rule. The maximum inter-specific dissimilarity value was observed between the greengram (PLS 274 and PLA 334) and the ricebean genotype (RB 559), paving their way to inter-specific hybridization programme. The genotypes LM 103/1 and ML 131 from greengram, the genotypes LRB 324 and RB 559 from ricebean, were identified for their deployment in intra-specific hybridization based upon their high dissimilarity value (Table 3).
 

        
In the present study, UPGMA grouped the 30 genotypes into five clusters (Fig 1). By using this method, Jayamani and Sathya (2012) reported six clusters from 20 greengram and one ricebean genotype, Sathya and Jayamani (2013) reported five clusters from 36 greengram genotypes, Chen et al., (2015) reported nine clusters from 157 cultivated and wild greengram accessions. The largest cluster formed by UPGMA was cluster I with 19 greengram genotypes. The cluster II (AC 241) and III (ML 131) had solitary genotype. The rice bean genotypes were grouped in a single cluster V. Based on weighted average for dissimilarity matrix, the Neighbour-Joining tree developed five groups from 30 genotypes (Fig 2). By using Neighbour-Joining tree, Tian et al., (2013) reported four clusters from 472 cultivated and wild accessions of ricebean.

 

        
The comparative study between the clusters and groups formed in UPGMA and Neighbour-Joining tree revealed that, the ricebean genotypes were grouped into a single cluster using both the methods. The results obtained confirmed the genetic divergence between the two Vigna species at the molecular level by distinct cluster formation. The result is supported by the findings of Jayamani and Sathya (2012) who also reported separate cluster formation for greengram and ricebean genotypes. The cluster V of dendrograms generated from UPGMA and group V of Neighbour-Joining tree method had same genotypes. These observations indicated the distinctness and stability of the cluster that remained unvaried with the method of analysis.

To conclude the SSR markers used in the present study displayed i) successful cross species amplification, ii) marker transferability to other crop species of the same genera, iii) existence of maximum interspecies diversity than intraspecies diversity and iv) clear cut cluster separation of the two species. The outcome of this study paves the way for the deployment of these molecular markers in species differentiation, intraspecies molecular characterization, identification of diverse genotypes for inter- and intraspecific hybridization, molecular identification and characterization of interspecific hybrids.