ISSR based diversity analysis in beachpea [Vigna marina (Burm.) Merr.] accessions of Andaman and Nicobar Islands, India 

Among Vigna species, beachpea [Vigna marina (Burm.) Merr.], is a wild relative of cultivated Vigna species distributed throughout the tropics (Sanjeewani et al., 2012). The habitats of beachpea [Vigna marina (Berm.) Merr.]  associated with seashore where it may occur from immediately above watermark to low coastal dunes and scrub, particularly over sandstone and sandy soils in tropical and subtropical regions (Chankaew, 2014 and Singh et al., 2014). As a species, beachpea is prostrate, creeping vine, perennial underutilized edible pulse crop which is also known as the nanea, wild mung and notched cowpea. It usually grows closest to the seashore. Vigna marina is determinate type, dune creeper having salt tolerant capacity (Elanchezian et al., 2009 and Sanjeewani et al., 2012). This vine is great for open, sunny areas as a ground cover and especially good for beach front properties and has a distinct advantage of being drought tolerant and can be grow as a feed and fodder crop in wide range of soils and environments. Beachpea possess succulent stems and leaflets, the later being ovate, its seeds are medium to bold, more glabrous, seeds are oval and brown to radish brown in colour.
       
Since, a very few studies on the analysis of the genetic diversity among wild species of Vigna has been done (Ajibade et al., 2000, Sonnate et al., 1997, Samarjeewa et al., 2002, Singh et al., 2015, Yoon et al., 2007) the local holdings of beachpea (Vigna marina) at the ICAR-Central Island Agricultural Research Institute, Port Blair were used for their molecular characterization.

Plant material
 
The experiment material for the present study comprised of collected 16 accessions of beachpea (Vigna marina) from South Andaman and Car Nicobar of Andaman & Nicobar Islands. Details of the accessions along with their morphological features are given in Table 1.
 

 
DNA extraction and PCR amplification
 
The plant genomic DNA was isolated from fresh 30 days old leaves of 16 accessions grows in the germplasm block.  Fresh leaves from young plants were collected and frozen in liquid nitrogen. DNA was isolated according to the cetyltrimethyl ammonium bromide (CTAB) protocol described (Murray and Thomson, 1980). DNA integrity was tested, using 1.5% agarose gel electrophoresis and its concentration was determined with a UV spectrophotometer. DNA was then diluted to 25 ng/µl for PCR amplification.
       
ISSR amplification reactions were carried out in 15 µl volume containing 25 ng template DNA, 0.5 units of Taq DNA polymerase, 0.1 mM dNTP each, 10mM primer, 1X reaction buffer and distilled de- ionized water. The PCR amplification was done using the Thermolcycler (Biorad, USA) with an initial denaturation step of 5 min at 94°C, followed by 45 cycles at 94°C for 1 min, 48°C- 53°C (depending upon the primer pair) for 2 min, 72°C for 1 min and final extension cycle of 72°C for 10 minutes. The details of primers used is given in Table 2. PCR amplified products were subjected to electrophoresis in a 3% agarose gel in 1x TBE buffer at 80 v for 3 hours. Ethidium bromide stained gels were documented using gel documentation system (Alpha Imager TM 1200).
 

 
Analysis of molecular data
 
The binary data of marker genotype matrix was used for analysis using NTSYS pc (Numerical taxonomy system, version 2.1, Rohlf, 2000). The SIMQUAL programme was used to calculate the Dice coefficient. The similarity coefficients were subjected to UPGMA (Unweighted Pair-group Method of Arithmetic average) method of clustering to generate the dendrogram. The polymorphism information content (PIC) values were calculated following Botstein et al., (1980) as follows:

                                        n
                                PIC =    1 - 𝚺    Pij²
                                          j=1

 

where P_ij is the frequency of the j^th allele for the i^th marker and summed over n alleles.

A total of randomly selected 17 ISSR primers specific to Vigna species were used to analyze the genetic diversity of 16 beachpea (Vigna marina) accessions. ISSR primers produced varying number of DNA fragments, depending on their ISSR patterns. Out of 17 ISSR primers studied 16 were polymorphic. A total of 861 amplicons were generated, 588 were polymorphic among the 16 accessions. Among the ISSR primers, HB 13 was found to amplify the highest number of ISSR fragments followed by UBC-824, UBC 826, UBC 810 and HB 15, while, the least number of ISSR fragments were produced by UBC-818. ISSR markers profiles produced by the 17 ISSR primers produced 861 clear and distinct bands across 16 accessions, of which 588 were polymorphic with an average of 34.59 / primer with 0.387 PIC value. The total number of amplified bands produced by each primer varied from 4 (UBC 818) to 110 (HB 13). The maximum number of polymorphic bands was produced by ISSR primer HB 13 (94 polymorphic bands) while, minimum polymorphic bands were produced by UBC 809 (0 number). The size of amplified bands also varied with different primers and it ranged from 125 to 3000 bp. Similarity coefficients values for the 16 beachpea accessions based on ISSR markers ranged from 0.382 to 1.000 (Table 3).
 

               
The ISSR genotyping data were used to generate UPGMA dendrogram (Fig 1). Cluster analysis revealed that the genotypes are grouped into two distinct clusters at 32% similarity coefficient. The present study has shown that the genotypes of beachpea (Vigna marina) ANBp-15-10 and ANBp-15-03 collected from South Andaman (Havelock Islands) and Car Nicobar showing more than 81 % similarity. The results indicated that ISSR individually can be effectively used in determination of genetic diversity among Vigna species. Alleles produced by different primers ranged from 4 to 110 with an average of 50.65 per primer and the level of polymorphism was found to be 34.59 percent. The maximum amount of polymorphism was produced by ISSR markers HB 13, HB 15, UBC 811 and UBC 824. The PIC value of primer varied from 0.000 (UBC 809) to 0.499 (UBC 810) with an average value of 0.387, indicating high resolving power of the ISSR markers. In the present study, ISSR markers detected 34.59% polymorphism. The results indicated that ISSR markers have been successfully utilized for assessing genetic diversity and revealed remarkable molecular discrimination between the 16 beachpea accessions. Similar results were reported by Hady et al., 2010, where they reported that RAPD and ISSR markers individually or in combination can be effectively used in determination of genetic relationship among Vigna species and Ajibade et al., 2000, where they found that the ability of ISSR technique to be effectively distinguish species in the genus Vigna.  
 

       
The studies on genetic diversity analysis and relationship studies in wild relatives and cultivated species of Vigna involving ISSR marker are very limited (Hady et al., 2010, Kaga et al., 2005, Padulosi and Ng, 1993, Pardhe and Satpute, 2011, Singh et al., 2014). The present study also shows that ISSR markers can be useful to supplement the morphological traits in the analysis of genetic diversity. The genetic diversity between the accessions is not necessarily reflecting the variability for agronomic traits as molecular markers based on repeat sequences can be found in non-genic portion of genome. However, it could be concluded that, the information of genetic diversity among beachpea accessions can be useful from the point of view of characterization and conservation of wild Vigna accessions for their use in Vigna breeding programmes.

Authors are grateful to Indian Council of Agricultural Research, Ministry of Agriculture & Farmers Welfare, Government of India, New Delhi for providing financial support. We are also grateful to the Director, ICAR - Central Island Agricultural Research Institute, Port Blair (Andaman & Nicobar Islands) for all kinds of facilities towards research and development activities carried out. The aouthors are also grateful to the Director, ICAR-Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India for providing technical support and able guidance.