Copyright © 2016 IJRAS, All right reserved 168 International Journal of Research in Agricultural Sciences Volume 3, Issue 3, ISSN (Online): 2348 – 3997 Study of Genetic Diversity of Berseem Accessions (Trifolium Alexandrinum L. SPP) With RAPD Markers S. ROUZ* Ecole Supérieure d’Agriculture de Mograne, 1121-El Magren, Tunisie F. BEN JEDDI Ecole Supérieure d’Agriculture de Mograne, 1121-El Magren, Tunisie M. ZOUAGHI Institut National Agronomique de Tunisie, 43 Avenue Charles Nicolle 1082 Tunis-Mahrajène, Tunisie Abstract – This study aimed to screening genetic diversity and ancestral relationship between Tunisian berseem accessions. The analysis of the genetic diversity of 29 accessions of berseem kept and recorded in the fodder index of Tunisian National Institute of Agronomy has been conducted via RAPD markers. Several prompts have been tested, but 5 only were selected for their reproductive potential and their high polymorphism rates. A total of 388 reproducible bands were obtained during PCR-based amplifications. The bands have a size ranging between 200 and 2000 pb; though, most of the bands are in the range of 200 to 600 pb. The correlations between the accessions have been evaluated by means of analyses according to the UPGMA method. The extent of dissimilarity between the accessions varies from 16 to 100%, which represents a high polymorphism level. The dendrogram, worked out via the Treecon software, reveals seven sets of accessions at the 60% threshold. T. berytheum, ancestor of berseem, clearly emerged from the other accessions. It is of interest for intensive crop-growing systems and may be used as parent in selection programs. T. meironense is genetically very close to the T. alexandrinum var serotinum. Three sets are generated by T. alexandrinum and T. berytheum crosses. The present study showed a high genetic diversity of studied berseem accession. Showed results can be used as a pathway for preservation and productivity improvement under different agricultural systems. Keywords – Trifolium Alexandrinum, Berseem, Genetic Diversity, T. Berytheum. I. INTRODUCTION Zohary and Heller, 1984; Lange and Schifino- Wittmann, 2000; Le Floc’h et al., 2010 reported that Tunisia is characterized by a great genetic diversity of the fodder and pastoral legumes of the genus Trifolium, having 32 species among a total of 237. According to Morris & Greene, 2001The berseem (Trifolium alexandrinum L.) is one of the most important crops in the Mediterranean agricultural system. The geographical area of the berseem is the Mediterranean Basin, the ranging from the Middle East to Irak and Central Asia, like Pakistan and India (Knight, 1985; Sardana and Narwal, 2000; Badr et al., 2008). The intensive use of a limited number of improved varieties of berseem reduces the genetic base of this species and contributes to genetic vulnerability of its biodiversity. The ex-situ conservation and the evaluation of genetic resources of berseem has begun since the 1980s in the Tunisian National Institute of Agronomy (INAT). The morphological identification of the genetic variability in this collection through morphological traits poses a major issue due to possible inter-crossings amongst species close to berseem and belonging to the subsection Alexandrina (Putiyevsky et al., 1975). The RAPD (Random Amplified Polymorphic DNA) had been used by Bullita (1995) to characterize varieties and populations of Trifolium. It demonstrated an efficiency to reveal the genetic polymorphisms between species and populations within the species. This method can be used for the improvement of berseem and for the management of its genetic resources. In this work we analyze and try to determine 29 entries of the genetic diversity of Egyptian clover available in the collection of the INAT by the RAPD method using primers which already gave successful results for the other species of clover (Bullita, 1995). II. MATERIAL & METHODS In this study, twenty nine entries of berseem are studied including a commercial cultivar Miscawi, two local cultivars, two naturalized cultivars and the rest are natural populations obtained by exchange with diverse research institutions, recorded in the fodder index of INAT. The Origin, the lot number, the name of the genotype and the code are summarized in table 1. Table 1. Biologic material, number and origin Species Number Origin T. alexandrinum L. 02 TUNISIA-(Local) T. alexandrinum L. 01 USA T. alexandrinum L. 02 EGYPT T. alexandrinum L. 24 INAT These entries of berseem have been cultivated in 2007/08 in the experimental station of the Agricultural College of Mograne; situated at a height of 156 m, a longitude of 10°04’ and a latitude of 36°26’, in a homogeneous clay-limestone soil. The sowing has been done in the same date, and the morphological and agronomic parameters have also been determined (height of plants, number of stems by plant, date of bloom, color of flowers, number of cutting, yield dry matter for each cut,…). Extraction and Quantification of the DNA To extract and purify the total DNA from berseem leaves, we adopted the protocol described by Ben Naceur (1998) which is a combination of three methods of extractions (Murray and Thomson, 1980; Saghai-Maroof et al., 1984; Webb and Knapp, 1990) bringing CTAB. A representative sample of 1g of leaves from every entry has been taken from 10 plants of berseem and crushed in the mortar, in the presence of liquid nitrogen, until obtaining of a very fine powder. Each mix has been transferred to a 10 ml tube, adding 7.2 ml of extraction