Genetic Diversity Of Different Geographical Populations Of Marmota Himalayana By SSR Analysis

Marmota himalayana belongs to Rodentia,Sciuridae,Marmota.Plague plots virus usually are carried and diffused by Marmota himalayana.However,no researches of Marmota himalayana in the field of molecular ecoloy have been found so far.Meanwhile,the construction of a genomic library is essential to researches on genome structure and function.A comprehensive genomie library makes it possible to screen and isolate any DNA fragments from it.In this paper,part of the genomie library of Marmota himalayana in four different geographical populations was constructed.The sampling localities include Delingha,Wulan, Tuotuohe,and Anduo,all were alongside the Qinghai-Tibet railway.Genomic DNA extracted from Marmota himalayana by Improved Method of Phenol-Chloroform.Genomic DNA digested by restriction endonuclease Sau3AI.Recovery 200-1000bp fragments from low melting point agarose gel.Plasmid pUC19 was extracted by alkaline lysis method and digested by restriction endonuclease BamH I to come into being the carriers.The fragments and the carriers were linked and transformed into the E.coli DH5αfeel competent cells which were prepared by CaCl₂ method.The positive clones were screened by two methods.One is the blue and white plots screening.The other is screening the positive clones by PCR with oligonucleotide primer(CA) ₈ and pUC 19 multiple cloning sites bilateral M13 primers(M3 and RV).4000 clones were screened,61 recombinants were obtained.The sequences of the recombinants were identified. Then nine colones including thirteen high various microsatellite loci were submitted to the GenBank and admitted,recorded.Genbank accession numbers were EF555518～EF555519, EF676084～EF676090.High homologous microsatellite sequences have not been found in Genbank.Indicated the thirteen microsatellite loci were first found.The corresponding primers were designed and synthesized as molecular markers to detect genetic diversity of the Marmota himalayana in the four populations.The analysis shows that the numbers of the alleles and the effective alleles of all loci are 2～6 and 1.166～3.073.The polymorphism information content(PIC)of all loci show that six loci are high polymorphism (PIC＞0.5)and six loci are middle polymorphism(0.5＞PIC＞0.25)and only one locus is low polymorphism(PIC＜0.25).Average heterozygosity(Het)and Sharmon index(â… )reveal the same results.PIC and I and Her also show that all of the four populations are high polymorphic populations.The Cluster Analysis based unweighted pair-group method using arithmetic averages show the theorical phylogenetic tree is consistent to the actuátl geographical situation. The genetic distance between Delingha populations and Wulan populations is the smallest value (0.1504).Then the genetic distance between Tuotuohe population and the two is nearer than that of the Anduo and the two.The genetic distance between Anduo population and Tuotuohe population is the biggest value(0.5104).Therefore twelve micro satallite lo ci(except SSR6)and the four populations may be used to the researches of genetic diversity ofMarmota himalayana.