Development Of Microsatellite Loci And Population Genetic Analysis

Sable?Martes zibellina?,a kind of typically holarctic mammal,has been once widely distributed.There are 4 subspecies which are mainly distributed in the northeast and northwest of China.Aciculiailvae,as the habitation of sables,is sparsely populated and freezing.Because of the high value of the fur,sables are often caught and killed by poachers.Furthermore,habitat fragmentation caused by human activity has made a huge challenge for the living of sable.22 polymorphic microsatellite loci were isolated and developed for the sable through traditional method?magnetic beads enrichment?.Meanwhile,we also used the high-throughput sequencing data to develop microsatellites and got 16 tetranucleotide loci.Afterward,we analyzed the genetic diversity and population structure of three sable populations?Mu-shang,Mohe and Genhe;A total of 49 individuals?from northeast of China based on the 16 tetranucleotide loci.The following is the main results of the research:1.A total of 2252 clones were screened by construction of genomic library.After selecting and sequencing positive clone?200 out of 343 positive clones were sequenced?,and detection of primer?100 primer pairs?,26 pairs of primer could yield specific PCR products which were consistent with the expected size.22 Martes zibellina samples were used to detect the primers through capillary electrophoresis.At last,we got 22 polymorphic loci.The number of alleles varied from 3 to 11 for these loci.All loci showed high polymorphism?PIC>0.5?except the loci Mz24 and Mz26?0.25<PIC<0.5?.2.124 primer pairs were designed after searching genomic sequence?high-throughput sequencing?of sable.30 primer pairs,randomly selected,were detected by PCR.16 primer pairs could yield specific PCR products which were consistent with the expected size.The 16 pairs of primer were used to do genotyping and individual identification for 49 sable samples.The results showed all 16 loci were polymorphic,and no sample was from the same individual.?1?Genetic diversity.For the three sable populations,105 alleles were observed at 16 loci,with an average of 6.563 alleles.The average observed heterozygosity and PIC are 0.5893 and 0.672,which suggested all 16 microsatellite loci showed high polymorphism and the genetic diversity of sable is not low.?2?Genetic divergence.Using the F-statistics to analyze the three populations based on 16 loci.Results showed that the average of genetic differentiation(F_ST)was 0.060 and the inbreeding coefficient?FIS?was 0.103 in whole population,which suggested there were the phenomenons of inbreeding and heterozygote deficiency.Pairwise F_ST values of populations showed that the genetic differentiation between Mu-shang and Mohe was more significant(F_ST=0.056),and the genetic differentiation level between Mohe and Genhe was lower,which suggested there were different levels of genetic differentiation between every two populations.?3?Population structure.Based on the Nei's genetic distance of microsatellite data,a phylogenetic tree was constructed with the UPGMA method.The results showed populations of Mohe and Genhe,all from Daxinganling,formed one cluster,and Mu-shang from the region of Xiaoxinganling and Zhangguangcailing became the other one cluster alone.Meanwhile,based on the genetic distance between every two individuals,we constructed a phylogenetic tree of Neighbour-Joining.The tree topology was consistent with the results of phylogenetic tree of UPGMA,Principal coordinate analysis and STRUCTURE analysis.All the results,added with genetic differentiation coefficient(F_ST>0.05),suggested that there was a moderate level of genetic differentiation between populations from Daxinganling and region of Xiaoxinganling and Zhangguangcailing.