International Journal of Latest Technology in Engineering, Management & Applied Science (IJLTEMAS) Volume XI, Issue XI, November 2022|ISSN 2278-2540 www.ijltemas.in Page 7 Detected molecular markers for Alfalfa (Medicago sativa) using ISSR and SSR under Egyptian conditions. Samy A.A. Heiba 1 , Rania Tawfick Ali 1 , Hamdy M. Abdel-Rahman* 1 and Shimaa E. Rashad 2 1 Genetics and Cytology Department, Biotechnology Research Institute, National Research Centre, 33 El Buhouth ST., 12622, Dokki, Giza, Egypt 2 Microbial Genetics Department, Biotechnology Research Institute, National Research Center, Giza, Egypt. *Corresponding Author Abstract: Alfalfa (Medicago sativa L.) is worldwide forage and grown in different environmental conditions. This high geographical adaptation stimulates the genetic variation and gives breeders the possibility of using highly diverse gene pools. In this work five of Egyptian alfalfa (Medicago sativa L) cultivars of different geographic origin (Ismailia 1, Ismailia 2, Nubaria 1, Ramah and Sewi) in addition to the Australian cultivar (Medicago truncatula ) to complete a set of six cultivars represent six genotypes were evaluated for their responses to water regime on the level of some morphological characters such as flag leaf, stem diameter, plant height, leaf length and weight of 1000 grains then undergone a test for their genetic diversity with two selected molecular markers SSR and ISSR . The ISSR marker is more discriminating, provides more informative data than SSR marker. The five used ISSR primers succeeded in identifying four positive markers, the A1 & A2 primers generated positive bands at molecular size 650 & 510 bp in Nubaria 1 & Australian respectively which positively mark the width of flag leaf, stem diameter plant height and the pods productivity when correlates the obtained ISSR results with some of yield traits. The Sewi, Ramah and Australian cultivars which scored the highest plant and larger leaves under water regime are close relatives and genes can flow smoothly among them to produce new Alfalfa genotype with high tolerance to water deficit. Also, Ismailia 1 & 2 and Nubaria 1 are close relatives can exchange genes in between smoothly. Key words: Alfalfa morphological traits, Water regime, ISSR and SSR markers. I. INTRODUCTION aximum efforts from almost all countries to improve their nature gift resources to maximize benefits from it, plants are the first ring in the energy chain and so undergoes many scientific researches worldwide to develop new techniques and methods to increase plant production. The classical breeding methods depend on the breeder’s innovative experiences from mass selection to the use of a small set of selected families to obtain high yielding modified varieties, and to hybrid production by the use of a cytoplasmic male sterility system (Desprez et al. 1994). Although the success of this traditional way, it still has the undeniable disadvantage of the try and error method which is very time consuming. In alternative to classical methods, plant breeding based on their genome is very effective to get enough quantity production with high quality in shorter time and less effort to cover the growing population needs. Enlargement plants production accompanied with successful and advanced breeding for more cultivars in well harmony accommodation to the environment; depend mainly on the well understanding the genetic diversity among the already existing cultivar ancestors. This understanding assures the gene easy exchange and flow between relatives, save huge efforts, money and time. More recently, molecular tools such as QTL analysis (Paterson et al. 1988) and the molecular marker such as: ISSR and SSR markers for analysis of genetic diversity in the seagrass Posidonia oceanica(Serra et al., 2007), ISSR and SCoT markers for assessment the biodiversity among some Fennel genotypes (Ramadan et al., 2019), ISSR and Srap Markers for assessment of biodiversity among some sesame Genotypes (Aboelnaga et al., 2020) assisted selection have facilitated these endeavors. Forage quality traits such as digestibility, nutritional quality and palatability present the molecular biologist with interesting new targets for gene discovery. Genetic modification of these traits should enhance economics and animal health. Medicago sativa L. (alfalfa) one of the most important legume forage crops, consist of crud protein 16-25%), fibers (20-30%), vitamins (A, B, D, K and E) in addition to minerals (Patra & Paul, 2019). It is characterized by wide range of soil types can grow on despite its texture or alkalinity (El-Ramady et al., 2020). Alfalfa plant exists in Egypt in many forms of germplasm under different cultivar names (ex. Ismailia1&2, Nubaria1, Ramah and Sewi). Best irrigation interval 7-10 days, irrigation water in needs is about 7500m 3 /ha per season. The annual production of Alfalfa in Egypt reaches 400 tons and about 90%of this amount is exported to the gulf countries in exchange for hard currency. Egyptian alfalfa is cultivated in autumn season (late September). M