Received: 7 December, 2011. Accepted: 26 April, 2012. Original Research Paper International Journal of Plant Breeding ©2012 Global Science Books Genetic Diversity Analyses of Mungbean (Vigna radiata [L]. Wilczek) by ISSR Sunita Singh 1,2 • Srinivasulu K. Reddy 3 • Narendra Jawali 1* 1 Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India 2 Department of Biotechnology and Bioinformatics, Padmashree. Dr. D. Y. Patil University, Navi Mumbai-400614, India 3 Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India Corresponding author: *enjay@barc.gov.in ABSTRACT In the present study 3 anchored ISSR primers were designed, and after optimizing each primer annealing temperature for obtaining a higher number of markers per primer, they were used to analyze genetic diversity among 87 mungbean genotypes from India and neighboring countries. Following screening the 24 ISSR primers, 8 primers that yielded a clear banding pattern were chosen. Out of a total of 145 bands, 135 were polymorphic. An average of 18.12 bands and 16.87 polymorphic bands per primer was obtained. A dendogram revealed clustering of genotypes into 5 clusters. The first two clusters individually included the genotypes developed at two of the agricultural research institutes in India. Cluster three included genotypes developed in and around India, and cluster 4 included the genotypes that were local collections of mungbean from India and the fifth cluster included exotic and old selections of mungbean. Analyses showed that both resolving power of primer and number of polymorphic bands showed a positive correlation with the number of genotypes identified. _____________________________________________________________________________________________________________ Keywords: exotic varieties, genetic diversity, PCR optimization Abbreviations: ISSR, inter simple sequence repeat; Rp, resolving power; PCR, polymerase chain reaction; UPGMA, unweighted pair group method with arithmetic average INTRODUCTION Mungbean (Vigna radiata [L]. Wilczek), also known as green gram or moong, is grown in the Asian subcontinent. In India, it is one of the major pulse crops grown on about 3 million ha in arid, semi-arid and sub-humid regions. Mung- bean is native to India (Paroda and Thomas 1987). It is the third important legume crop in India after chickpea and pigeon pea. In India, mungbean is cultivated on 3.77 million ha and production is 1.56 million tonnes with an average yield of 413 Kg/ha during 2007-2008 (Singh 2009). The productivity varies year to year and among states (Singh 2009). In general, there is scope for the improve- ment of yield as productivity up to 800 tonnes/ha is repor- ted from the state of Punjab (Singh 2009). Although the germplasm collections from India are very large, much diversity has not been reported on the basis of morpholo- gical characters. There is an urgent need to identify genetic diversity based on a molecular basis for utilization in breed- ing programmes. Conventional morphological characters, isozyme and other biochemical markers have been used to examine genetic diversity in mungbean (Mohanty et al. 2001). But most of the breeding programs would require markers that can be conveniently used for tagging multiple genes for a phenotype, for identification of a variety, for genetic map- ping studies and to determine the genetic diversity in the germplasm collection (Kumar 1999). In the recent past DNA-based molecular markers have been applied for vari- ous purposes including assessment of genetic diversity, mapping of the genome and studying the species relation- ship within the genus Vigna. Genetic diversity among mungbean genotypes has been reported using different DNA markers (Lakhanpaul et al. 2000; Kumar et al. 2003; Afzal et al. 2004; Saini et al. 2004; Bhat et al. 2005; See- halak et al. 2006; Sangiri et al. 2007; Reddy et al. 2008; Chattopadhyay et al. 2008; Lavanya et al. 2008; Saini et al. 2008; Dikshit et al. 2009; Saini et al. 2010; Ullah et al. 2010; Tantasawat et al. 2010; Vir et al. 2010; Narasimhan et al. 2010; Raturi et al. 2011; Taunk et al. 2012). RAPD analysis has been used to study relationship among the species belonging to Ceratotropis (Kaga et al. 1996). Other marker such as AFLP was used for genetic diversity analy- ses of mungbean (Bhat et al. 2005) and other species of Vigna (Tomooka et al. 2002). The presence of two types of internal transcribed spacer (ITS) among mungbean geno- types based on the methylation status of a BamHI site pre- sent in the ITS region, were reported by Saini et al. (2000) and further analyses indicated the presence of multiple intragenomic ITS variants showing a high genetic diversity in mungbean (Saini et al. 2008). Recently the development of sequence tagged microsatellite sites and their use in analyzing a set of 87 genotypes has been reported (Singh and Jawali 2009), which included 48 genotypes that were used in genetic diversity analyses by AP-PCR (Saini et al. 2004). Among a large number of PCR-based methods available for assessing genetic diversity in plants (Kumar 1999), ISSR, which is based on microsatellite sequences and does not need flanking sequence information, is easy to develop. ISSRs are highly reproducible compared to RAPD. This reproducibility is attributed to the length of the primers and higher annealing temperature used in the ISSR analysis. ISSR has been used extensively in studying a variety of as- pects of plant biology (Rakoczy-Trojanowskam and Boli- bok 2004). The objective of the present study was to (a) optimize annealing temperature for each primer in PCR to achieve a larger number of markers per primer, (b) to use these for assessing the genetic diversity among the 87 mungbean ®