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Title: DEVELOPMENT AND CHARACTERIZATION OF SSR MARKERS AND THEIR USE TO ASSESS GENETIC RELATIONSHIPS AMONG ALFALFA GERMPLASM.

Author
item He, Chunlin
item Xia, Zheng-Lian
item Campbell, Travis
item Bauchan, Gary

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2005
Publication Date: N/A
Citation: N/A

Interpretive Summary: Molecular genetic markers are tools used by geneticists and plant breeders for a number of purposes including cultivar identification and the determination of the position of genes. In alfalfa, there are very few molecular markers available which can be utilized for these purposes. We developed 81 new molecular markers called simple sequence repeats (SSRs) from alfalfa. These molecular markers were used to study the historically distinct alfalfa germplasms which are important progenitors of current U.S. alfalfa cultivars. These new molecular markers are capable of distinguishing some of the historically distinct germplasms from each other and can be used to develop a map of alfalfa which can eventually be used to discover important genes. Alfalfa scientists both in the seed industry and the public sector can utilize these new molecular markers to identify alfalfa cultivars and ultimately identify genes important for the improvement of alfalfa.

Technical Abstract: Simple sequence repeat (SSR) or microsatellite markers are codominant, abundant and hypervariable molecular markers from eukaryotic genomes that are being widely used in genetic mapping, phylogenetic studies and marker-assisted selection. Currently, the number of SSR markers available from alfalfa (Medicago sativa L.) genomic libraries is very limited. This study was conducted to identify additional SSR markers in the alfalfa genome and to evaluate their ability to separate the nine progenitors of cultivated alfalfa (African, Chilean, Falcata, Flemish, Indian, Ladak, Peruvian, Turkistan and Varia) as well as wild tetraploid M. falcata, two very non dormant M. sativa accession, diploid M. coerulea and M. falcata and M. truncatula. Genomic DNA was extracted from the autotetraploid alfalfa population W10, digested with Sma I, Alue I, Rsa I, Nac I, Hinc II and Xmn I, and fragments were cloned into pUC19. Colonies were probed with the repeats of AC, AT, CT, CTT, GAT and GGT motifs. A total of 158 colonies with positive signals were identified and primers could be designed to amplify 81 SSRs. The majority of the primer pairs were functional. Sixty-one of the primer pairs generated polymorphisms with 2-11 alleles. The polymorphism information content ranged from 0.12 to 0.92 and was significantly correlated with the number of alleles amplified by each primer pair (r=0.77, p<0.001), but not significantly correlated with the number of repeats of the SSR motif (r=0.07, p=0.59). A dendrogram was constructed representing the genetic relationships which produced three main clusters: diploid ssp. falcata, M. truncatula, and all the remaining entries. However, additional entries could be effectively separated using multiple correspondence analysis including M. truncatula (Jemalong) , Ladak (Ladak), Very Non Dormant 11 (UC-1887), Indian (Sirsa Type 9), Flemish (Dupuit), Peruvian (Hairy Peruvian), African 2 (Moapa) and Turkistan (Kayseri) (marginally). However, the remaining germplasms within the ssp. sativa species could not be separated due to a limited number of SSR markers used in this study or due to the change of genetic constitution during the long term preservation of these historic germplasms.