Genetic diversity of microsatallite loci in Marmota himalayana in different geographical populations
【摘要】：正Marmota himalayana belongs to Rodentia,Sciuridae,Marmota.Plague plots virus usually were carried and diffused by Marmota himalayana.However,no research of Marmota himalayana in the field of molecular ecology has been found so far.Meanwhile,the construction of a genomic library was essential to researches on genome structure and function.A comprehensive genomic library made it possible to screen and isolate DNA fragments from it.In this paper,part of the genomic library of Marmota himalayana in four different geographical populations were constructed.The sampling localities in China include Delingha,Wulan,Tuotuohe,and Anduo, whose habitat were all alongside the Qinghai-Tibet railway.Genomic DNA extracted from Marmota himalayana by Improved Method of Phenol-Chloroform.Genomic DNA digested by restriction endonuclease Sau3AI.Fragments of 200-1000bp were extracted from low melting point agarose gel. Carriers were obtained from Plasmid pUC19,which was extracted by alkaline lysis method and digested by restriction endonuclease BamH I.After ligation,the plasmid containing the fragments was transformed into the E.coli DH5α competent cells,which were prepared by CaCl2 method. After growing overninght,the positive clones were screened by two methods.One was the blue and white plots screening.The other was screening the positive clones by PCR with oligonucleotide primer(CA)_8 and pUC19 multiple cloning sites bilateral M13 primers(M3 and RV). The sequences of the 61 recombinants,which were obtained from 4000 screened clones were identified.Then nine clones including thirteen high various microsatellite loci were submitted to the GenBank and admitted,recorded.Genbank accession rumbers of them were EF555518～ EF555519,EF676084～EF676090 respectively.High homologous microsatellite sequences were not found in Genbank,which indicated it is the first time that the thirteen microsatellite loci were found. The corresponding primers were designed and synthesized as molecular markers to detectgenetic diversity of the Marmota himalayana in all four populations mentioned before.The analysis showed 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 showed that six loci were in high polymorphism (PIC0.5);six loci were in middle polymorphism(0.5FIC0.25),but the PIC of one of the six locus was only 0.265;only one locus was in low polymcrphism(PIC0.25).The results were supported by the data from Average heterozygosity(Het)and Shannon index(Ⅰ).PIC and I and Het also revealed that all of the four populations were in high polymorphic populations.The Cluster Analysis based on unweighted pair-group method using ar thmetic averages showed the theorical phylogenetic tree was consistent to the actual geographical situation.The value of genetic distance between Delingha populations and Wulan populations was the smallest(0.1504).Then the genetic distance between Tuotuohe population and those two were nearer than that between the Anduo and those two.While the genetic distance between Anduo population and Tuotuohe population gave the biggest value(0.5104).In conclusion,all of the eleven microsatallite loci(except SS1 and SSR6) could be applied in the researches of genetic diversity of Marmota himalayana.All of the four populations of Marmota himalayana have high degree genetic polymorphism.The genetic distance of four populations Marmota himalayana are not always in accordance with the geographical distance,which means that geographical isolation may influence the population form and evolution of Marmota himalayana.