ESTIMATION OF MORPHOLOGICAL AND MOLECULAR GENETIC DIVERSITY IN BLACKGRAM [ VIGNA MUNGO ( L . ) HEPPER ] UNDER YMV HOTSPOT REGIME

A phenotypic and molecular diversity study was conducted using seven traits and 19 SSR markers in a collection of 26 black gram genotypes. Phenotypic characterization was based on seven yield and yield related variable. The field experiment was laid out at Panboli village (YMV hotspot) of Tirunelveli District in Tamilnadu during summer 2017. Genetic divergence was estimated on the basis of D2values and 26 genotypes under study were grouped into six clusters by Tocher’s method. Seed yield per plant followed by Plant height and number of pods per plant contributed to the genetic divergence. The genetic distance announced using DICE dissimilarity co-efficient indicated highest divergence of 1.0 between VBN 8 and AUBG 17 and between VBN 8 and AUBG 19. The dendogram constructed using the DICE dissimilarity co-efficient between genotypes showed four apparent clusters based on marker allele distribution. Divergence was noted between the dissimilarity matrices based on the molecular and phenotypic diversity based on agronomic data.


INTRODUCTION
Pulses are one among the important crops in the world.They are grown on 21-22 million hectares of area globally with an annual production of 12-16 million tonnes [1].Among the pulses, pigeon pea, blackgram and greengram are the major contributors of the total pulses production.Blackgram or urad bean belongs to the family Leguminaceae and the genus Vigna.Consideration of genetic diversity existing in a population is the basic requirement for effective improvement programme [2].Hence an attempt was made to assess the phenotypic diversity using morphological traits and to estimate genetic diversity of blackgram accessions using molecular markers.

Molecular markers
A total of 19 microsatellite markers proved to be polymorphic and linked with YMV resistance in black gram and mung bean were selected from previously published literatures [3][4][5]

Sample preparation
The genomic DNA was isolated from leaf tissue of 20 d old seedlings following the standard protocol [6].

PCR amplification
PCR was conducted in a thermocycler (Mastercycler Personal, Eppendorf, USA).).The amplification reaction consisted of an initial denaturation at 94 °C for 4 min followed by 40 cycles of 1 min at 94 °C, 1 min at 50-62 °C (depending on marker type), and 1 min at 72 °C and finally terminated with an extension of 7 min at 72 °C.List  Email: agriman2001@yahoo.commarkers and their annealing temperatures are given in table 2. The amplified PCR products (10 μl) were resolved on 1.5% (w/v) agarose gels in 1X TBE buffer.The gel was stained with ethidium bromide as previously described.The size of the fragments was estimated using a 100 bp ladder (Genei, Bangalore) as a size marker.The gel was run till the dye reached the end of the gel, then the gel images were photographed using Gel Documentation system (Vilber Lourmat, France).All the PCR reactions were repeated thrice to ensure reproducibility and reliability of the results.

Data analysis
The amplified DNA fragments in gel images were transformed to binary data (0, 1), where 1 and 0 were scored for presence and absence of alleles, respectively.Percent polymorphism for each marker was generated by the formula: (Number of polymorphic alleles/Total number of alleles) x 100.The clustering was done by using the dissimilarity coefficient matrix for the twenty six genotypes by employing the unweighted neighbour joining method bootsraped over 5000 times.The clads showing more than 70% of the bootstrap value were considered arbitrarily as a strong cluster.Cluster analysis was performed using the neighbor-joining (NJ) method with the DARwin v. 5.0.157software [7].

Field experiment for phenotypic variability
Twenty six genotypes of black gram were evaluated for seven yield and yield attributing traits viz., Plant height, number of branches per plant, number of clusters per plant, number of pods per plant, pod length, number of seeds per pod, seed yield per plant.The genotypes were grown in the YMV hotspot, Panboli village of Tirunelveli District, Tamil Nadu during summer, 2017.Thee genotypes were sown in single row with spacing of 30 X 10 cm in two replications.The quantum of genetic divergence was also assessed by cluster analysis using Mahalanobis's euclidean squares distances which grouped the entire material into more precise clusters and estimates the average distance between them.
The quantum of genetic divergence was also assessed by cluster analysis using Mahalanobis's euclidean squares distances which grouped the entire material into more precise clusters and estimates the average distance between them.Genetic divergence was estimated on the basis of D2 value and 26 genotypes under study were grouped into six clusters by Tocher's method as presented in table 1 and fig. 2. Cluster VI was the largest with 12 genotypes, AUBG7, AUBG8, AUBG9, AUBG12, AUBG13, AUBG14, AUBG15, AUBG17, AUBG18, VBN3, VBN5 and VBN8.Cluster I with 5 AUBG1, AUBG2, AUBG3, AUBG4 and AUBG16 was the second largest cluster.Cluster IV was the third largest cluster with 3 genotypes namely AUBG5, AUBG10, VBN4.The remaining clusters II, III and IV had two genotypes each.
The average intra and inter cluster D 2 values are presented in table 2. Intra cluster values ranged from 12.86 (Cluster II) to 1188.15 (Cluster V).From the inter cluster values of six clusters, it can be seen that the highest divergence occured between cluster IV and cluster V (1591.36)followed by cluster V and cluster VI (1578.54),cluster II and cluster V (1554.80),cluster 1 and cluster V (1186.67) in that order of magnitude.The minimum inter cluster distance was noticed between cluster III and cluster IV (130.24))followed by cluster II and cluster IV (104.92) and cluster II and cluster III (51.12).
In the present study there was a divergence in phenotypic and molecular genotypic diversity.The phenotypic diversity analysis produced six clusters while the molecular diversity analysis produced three clusters.All the VBN genotypes (developed in National Pulses Research Centre,Vamban, Tamilnadu) were in same cluster (cluster 3) by molecular diversity while they were in three different clusters by phenotypic diversity.Similarly the genotypes AUBG 5 and AUBG 10, AUBG 19 AND AUBG 11 which was grouped in the same cluster by phenotypic analysis were in different clusters by genotypic analysis.There were instances of correlation between both analysis.The genetic distance announced by DICE dissimilarity co-efficient indicated the lowest value of 0.007 between AUBG12 with AUBG13 and AUBG 16, and VBN5 with VBN6.This was in correlation with phenotypic diversity analysis were AUBG 12 and AUBG 13 were grouped in the same cluster.The highest divergence of 1.0 was between VBN8 and AUBG17 and between VBN8 and AUBG19.In the phenotypic analysis too VBN 8 and VBN 19 were grouped in different clusters.
Although the genotypes were subjected to natural hotspot condition for Yellow mosaic virus resistance the phenotypic clustering pattern didn't correlate with the genotypic clustering with SSR primers specific for YMV resistance in most of the occasions.Many studies have found less relationships between molecular genotypic analysis and genetic distances estimated from morphological data [8][9][10][11][12][13][14].Although the neighbor joining procedure was bootstrapped 10000 times none of the clusters or clads showed bootstrap value more than 70%.The highest bootstrap value of 69% was shown by two genotypes VBN5 and VBN6 followed by their grouping with VBN7 (54%).