ABSTRACT
Aims: Medicago is known as the Queen of forage with potential economic importance to our society. The present study aimed at the use of RAPD-PCR DNA marker to identify the genetic fingerprints affinities of six species of Alfalfa. Place and Duration of Study: The study was conducted at the Department of Genetics, Garden Campus, Hazara University, Mansehra Pakistan during February, 2011 to August, 2013. Methodology: In this study, six species of Medicago namely TWAL (Tetraploid Wisconsin Alfalfa Line), Medicago arborea, Medicago falcata, Medicago sativa, Medicago lupulina and Medicago polymorpha were used to explore the diversity of alfalfa. Seven out of 120 decamers produced 34 polymorphic loci with 100% polymorphism to identify the different species of Medicago crop. The range of polymorphic loci was observed from 300 to 700 bp. Eleven species specific loci were generated by seven decamers. Primer B-18 generated single specific locus 700 bp against genomic DNA of M. lupulina and it is important to identify particular species of Alfalfa. The bivariate data were recorded as the presence of locus 1 and absence 0 and then this data was transferred into A and C respectively to make it suitable for DNAMAN software (version 5.2.2.0; Applied Biostatistics Inc). Moreover, cluster analysis was performed using sequence alignment and divergence function of the DNAMAN against the bivariate data collected from the products of decamers. All members clustered in a unique pattern except M. falcata and M. lupulina those shared 86% homology. Three distinct groups were observed during UPGMA (Unweighted pair Group Method with Arithmetic Mean). During the phylogenetic study, TWAL was observed to have genetic diversity from other five species of Alfalfa. Conclusion: So, the present study is enabling us to discriminate different species of Alfalfa and it could be useful to identify and authenticate different species of the same genus of medicinal important plant from the Flora of Pakistan.