Marmota Himalayan (marmota Himalayana), Microsatellite Loci Screening Applications

Relative species' microsatellite primers were used to find out microsatellite loci in Himalayan Marmot. The 18 microsatellite sequences of relative species areAF259372,AF259373,AF259374,AF259375,AF259376,AY197780,AY197781,AY197782,AY197783,AY197784,AY197785,AY702707,AY702708,AY702709,AY702710,AY702711,AY702712 and SS-Bible. After gene application, connection and translation, hundreds of clones were screened with PCR technique for recombinant positive clones containing microsatellite sequences. The primers used in PCR were M13 Primers M3/M13 Primers RV which located at both sides of multiple clone site of pBS-T and microsatellite primers (CA)8 designed according to core repeats motifs of microsatellite. 21 microsatellite loci were found. GenBank accession numbers of these microsatellite sequences are DQ888321-DQ888327, EF125660-EF125666, EF520117-EF520122. Among these microsatellite loci there are 14 perfect ones in 67%; 3 imperfect ones in 14%; 4 compound ones in 19%.Repeat number of most microsatellites varies from 3 to 20, few microsatellites exceed 20.Four microsatellite sequences were selected for designing oligonucleotide primers,using Oligo 6.0 and Primer 5.0 software.After extensive optimization, 4 pairs of primers gave satisfactory results and were polymorphism. Genetic polymorphism of Himalayan Marmot in Tuotuo River , Haixizhou, Wulan and Anduo were studied with these primers .The result indicate that genetic variation of Haixizhou population were highest, then were Wulan population, Tuotuo River and Anduo population. Cluster analysis showed that there were differences between four population, but differences between four populations were small. This experiment has made the foundation for further reserch of Himalayan Marmot in molecular ecology.