| The Blue-crowned Laughingthrush(Garrulax courtoisi)is an extremely endangered species,which is rare in population size and very narrow in distribution range.The breeding population can only be found in Wuyuan of Jiangxi Province.Genetic diversity level can determine the evolutionary potential of one species and be related to its further destiny.In order to explore the endangered status and evolutionary potential of the blue-crowned Laughingthrush on the molecular level,and better protect the Blue-crowned Laughingthrush,we developed novel SSRs molecular markers to analyze the genetic diversity and paternity testing for the blue-crowned laughingthrush populations.1.Using microsatellite searching and primers designing software,110 microsatellite primers were designed based on the partial genome sequences of the Blue-crowned Laughingthrush.69 blue-crowned Laughingthrush individuals were amplified by PCR,and,a total of 15 microsatellite loci with polymorphism were developed.12 out of 15 loci were highly polymorphic and 3 loci were moderate polymorphic.Moreover,the DNA template concentration,primer concentration and annealing temperature of each primer in the SSR-PCR reaction system were optimized to establish the optimal SSR-PCR reaction system.2.Seven pairs of microsatellite primers with high polymorphism were selected to analyze the genetic diversity of 69 Blue-crowned Laughingthrush samples.40 alleles were detected in 69 samples,of which 14 were rare alleles,accounting for 33.3%,and the average allele was 5.714.The heterozygosity ho = 0.773 ± 0.061(0.580 – 1),the expected heterozygosity he = 0.668 ± 0.033(0.583 – 0.791),and the polymorphism pic = 0.631 ±0.039(0.507 – 0.752).Compared with the related species and the artificial population,the genetic diversity of wild Blue-crowned Laughingthrush is higher.3.Through the analysis of F-statistic genetic differentiation of five breeding subpopulations of Blue-crowned Laughingthrush,it was found that the inbreeding coefficient of Blue-crowned Laughingthrush was-0.244 ± 0.066(-0.510-0.023),the inbreeding coefficient of the total population was-0.225 ± 0.063(-0.487-0.038),and there was a certain degree of inbreeding within the population.The genetic differentiation coefficient(FST)was 0.016 ± 0.007(-0.012-0.044),which indicated that the inbreeding level and genetic differentiation degree were low.The mean value of gene flow(nm)was8.430,indicating that there was a certain gene exchange between subgroups4.Population genetic structure analysis found that 69 Blue-crowned Laughingthrush can be divided into three ancestral groups;the Dexing Haikou(DXHAK)population and the Wuyuan Shimen(WYSM)population cluster into one branch first,and then cluster into with Wuyuan Hexi(and WYHX),Wuyuan Huokou(WYHOK),and Wuyuan Taibai(WYTB);Nei's genetic distance and genetic similarity index were performed by Popgene software,which found that WYSM and DXHAK had the closest genetic relationship,and WYHX and WYTB had the farthest genetic relationship.5.Primers P2 / P8 were used to carry outthe sex identification on 56 Blue-crowned Laughingthrush individuals.There were 16 females and 40 males with a sex ratio of 2/5,which significantly occupied more than the females.This is the first discoverythat the male Blue-crowned Laughingthrush is more than the females,and it is likely to be the most important constraint that causes its population increasing slowly.Paternity test were conducted within 56 Blue-crowned Laughingthrush individuals with known genders,and a pair of parent-child relationship and three pairs of parent-child relationship were identified.It showed that the Blue-crowned Laughingthrush marriage system may be the social breeding system monogamy. |
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