Microsatellite marker-based genetic diversity analysis and developing synthetic varieties in Alfalfa (Medicago sativa L.)
Abstract: Cultivated alfalfa (Medicago sativa L.) is an allogamous autotetraploid species with 2n = 4x = 32 chromosomes. Alfalfa is the most cultivated forage legume in the world mainly due to its wide adaptation to diverse environmental conditions and efficient nitrogen fixation ability. Kyrgyzstan is among the major alfalfa producing countries. The analysis of alfalfa genetic diversity helps to assess the future risk of genetic erosion and helps in the development of sustainable conservation strategies and wise use of the genetic variation in breeding programs. The aim of this Master thesis was to study the genetic diversity of alfalfa grown in Kyrgyzstan and in other countries and determine the genetic relationship using simple sequence repeat (SSR, microsatellite) molecular markers as well as to contribute to the development of synthetic alfalfa varieties for use as forage in Kyrgyzstan. A total of 78 cultivated alfalfa genotypes from 60 accessions representing 24 countries were used for the SSR analysis. Medicago lupulina and M. scutellata were represented by one sample each as outgroups. Ten SSR loci were analyzed in this study, nine of which were polymorphic. The locus AGGT-004 was monomorphic across the 78 cultivated alfalfa individuals analyzed. A total of 53 alleles were recorded across the ten loci of cultivated alfalfa accessions, with an average of 5.3 alleles per locus. The number of alleles (Na) for the nine polymorphic loci varied from two (locus ACT-026) to nine (locus AGAT-012). Of the ten SSR loci used, seven are highly recommended for alfalfa genetic diversity analysis, as high genetic variation was detected at these loci. The locus AGAT-012 is highly interesting in that it had several alleles at low frequencies which might be linked to desirable traits. At this locus, 98.6% of the individuals investigated were heterozygotes. Analysis of variance (AMOVA) revealed that 95% of the total genetic variation was found within countries and the remaining 5% differentiated the accessions according to their countries of origin (FST = 0.13; P = 0.005). AMOVA revealed no significant differentiation between accessions from the Western and Eastern countries (P = 0.14). The Jaccard similarity coefficient and cluster analysis revealed a significant genetic variation between Kyrgyz alfalfa genotypes, which is significant enough for alfalfa improvement, including the development of synthetic varieties. The use of Kyrgyz accessions, such as BM-5 and BM-9/4 for the development of synthetic varieties through crossing with genotypes from other Kyrgyz accessions is recommended, as the genotypes from these accessions were the most differentiated as compared to other genotypes originated from other Kyrgyz alfalfa accessions. The introduction of new alfalfa genetic material from countries such as Argentina, Oman, Australia, Iran, Iraq and Norway into Kyrgyzstan is a great opportunity to increase genetic variation in the alfalfa gene pool in the country.
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