| Medicago sativa L. is one of the most extensive and important forage legume crops in the world. It is the preferred forage plant in improving degraded grassland and developing artificial grassland. In recent years, with the rapid development of Medicago sativa’ industry in china, it is urgent need to cultivate high yield, good quality, strong resistance and wide adaptability of alfalfa cultivars. Therefore, molecular-assisted breeding is very important to accelerate the progress of alfalfa breeding.Forty-five Medicago materials were used in this study supplied by Institute of Animal Sciences, Chinese Academy of Agricultural Sciences. The work mainly included:1) to develop and characterize the polymorphic EST-SSR primers for Medicago sativa and their transferability in other Medicago species; 2) to evaluate the genetic diversity of alfalfa cultivars in China using SSR markers. The results were as followed:1 A total of 12,371 ESTs belonging to M. sativa were collected from NCBI genebank and 3,284 SSR containing ESTs were identified. A diverse range of SSR motifs was obtained which varied widely with mononucleotide repeats (1,285,39.1%) being the most abundant followed by tri-(1,278,38.9%). Nine main Repeat motifs were obtained. The most frequent occurring mononucleotide motifs were the A/T repeat (38.6%), followed by AG/CT(10.3%) and AAC/GTT(10.3%).2 One hundred primer pairs were successful designed among 216 SSR loci, and were first screened on a panel of 11 alfalfa materials.56%(56) of the 100 primer pairs produced SSR bands of expected size length in all accessions. Among them,25 primer pairs can produce polymorphic amplification profiles. The PIC values of these 25 EST-SSR markers ranged from 0.62 to 0.94 with the average value of 0.86.3 Fifty-six alfalfa EST-SSRs markers were tested across four different spccies for their transferability. The transferability rates of these EST-SSRs ranged from 100% in M. rigidula and M. truncatula, followed by 92.9% in M.polymorpya, and 91.1% in M.minama, with an average of 94.2%.4 The SSR markers showed the discriminatory powers to clearly separate the 15 Medicago genotypes into three main clusters (Ⅰ,ⅡandⅢ) based on 25 polymorphic SSR primer pairs. Eleven M.sativa accessions including eight M.sativa ssp sativa and each of M.sativa.ssp.falcata, M.sativa.ssp.varia,and M.sativa.ssp.coerulea clustered together (clusterⅠ), and M. polymorpha, M. truncatula, M.rigidula formed into clusterⅡ. M.minima appeared to be distinct from others and formed clusterⅢ.5 Eight polymorphic SSR primer pairs from eight alfalfa linkage groups were used to evaluate the genetic diversity of 34 alfalfa cultivars registrated in China. There were 99 alleles detected, with an average of 12.4. The mean heterozygosity ranged from 0.119 to 0.179. Higher PIC values were observed, and values ranged from 0.79～0.89, with a mean of 0.86.7 Thirty-four M.sativa species was seperated into three different groups (Ⅰ,ⅡandⅢ) with the method of UPGMA. Beijiang, Xinjiangdaye, Xinmu no.2 and Zhaodong were clustered into groupⅠ. Gongnong no.1, Gongnong no.2, Gannong no.1, and Gannong no.2 were formed into groupⅡ. The remain 26 materials were clustered into groupⅢ.The genetic similarity coefficient (GS) values ranged from 0.000～0.788, with an average of 0.274.The result of principal components analysis was agree with the result of UPGMA, and 34 M.sativa species was clustered into four different groups by principal components analysis. |
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