| Branchiostoma has a very high scientific value which has been regarded as a model animal for study of the origin and evolution of vertebrates.In addition,the Branchiostoma also has high nutritional value.LiuWuDian,located on northeast of Xiamen Island,as was once the world's only Branchiostoma fishery is famous all over the world.However,its natural resource has decreased sharply due to the effect of habitat destruction and overfishing in recent decedes.It is not only one of the worldwide endangered specids,but also is under state second-class protection in China.In this study,in order to provide relevant conservation genetic background for develop reasonable conservation strategies of endangered species Branchiostoma,we analysised genetic diversity and genetic differentiation of Branchiostoma by microsatellite markers and AFLP and mitochondrial DNA COI gene markers.(1)The microsatellite loci of Branchiostoma were developed following fast isolation by amplified fragment length polymorphism of sequences containing repeats(FIASCO)protocol.Twelve polymorphic loci were successfully amplified.The number of alleles ranged from 3 to 7.The polymorphism information content ranged from 0.309 to 0.778.The levels of observed and expected heterozysities varied from 0.1786 to 0.8571 and from 0.2449 to 0.7825,respectively.The results showed that the twelve loci exhibited highly polymorphic,and it can be used in the population genetic analysis.(2)Ten primer pairs from previous step were used to analyze the genetic diversity and differentiation of three wild Branchiostoma populations which collected from Qingdao Shangdong,Xiamen Fujian,Beihai Guangxi.The mean number of alleles per locus ranged from 3.3000 to 4.5000,with the average of 3.8667;the mean effective number of alleles per locus ranged from 2.6731 to 3.3584,with the average of 3.0249;the average of observed heterozysities was 0.4243,ranging from 0.3087 to 0.5052,and the average of expected heterozysities was 0.4394,ranging from 0.3528 to 0.5052.The genetic distance among the three populations ranged from 0.0388 to 0.0709,and the coefficient of genetic differentiation ranged from 0.0352 to 0.1100.The gene flow among the three populations ranged from 2.0222 to 6.8522.The UPGMA clustering analysis shows that Xiamen and Beihai population belonged to one group,and Qingdao population belonged to the other group.(3)According to the results of genetic diversity analysis by AFLP,the mean number of alleles per locus ranged from 1.8571 to 1.9523,with the average of 1.9106;the mean effective number of alleles per locus ranged from 1.4760 to 1.5620,with the average of 1.5512;the percentage of polymorphic loci was from 85.71% to 95.23%,with the average of 91.06%;the mean of Nei's gene diversity was 0.3033,ranging from 0.2802 to 0.3361;the mean of Shannon's information index was 0.4589,ranging from 0.4229 to 0.5082.The results of genetic differentiation analysis show that,the genetic distance among the three populations ranged from 0.0154 to 0.3435;the gengtic identity varied from 0.7073 to 0.98956;the coefficient of gene differentiation ranged from 0.0240 to 0.2686.The gene flow among the three populations varied from 1.3615 to 10.3764.The UPGMA clustering analysis shows that Xiamen and Beihai population belonged to one group,and Qingdao population belonged to the other group.(4)The study of mtDNA COI gene showed that,96 variable sites were founded in 23 individuals in three population of Branchiostoma,included 3 sites with gaps or missing and 93 polymorphic sites.The 93 polymorphic sites contain 8 single variable sites and 85 parsimony informative sites.21 haplotypes were defined from these individuals.In the 23 individuals of Branchiostoma,the average number of differences was 44.087,the nucleotide diversity was 0.08611.Within population,the average number of differences ranged from 1.929 to 42.429,the nucleotide diversity ranged from 0.00520 to 0.08287.The gengtic distance among three populations ranged from 0.0172 to 0.1975.Tajima's D test results showed that the three populations of Branchiostoma did not departured from neutrality model,but Fu's Fs test results showed that the population of Qingdao and Beihai departured from neutrality model significantly.(5)The microsatellite loci of three species of sea cucumber were developed following FIASCO protocol.Eight polymorphic loci were successfully amplified in Holothuria leucospilta;the number of alleles ranged from 2 to 6;the polymorphism information content ranged from 0.371 to 0.694;the levels of observed and expected heterozysities varied from 0.1154 to 0.7000 and from 0.3913 to 0.6701,respectively.Nine polymorphic loci were successfully amplified in Holothuria scabra;the number of alleles ranged from 2 to 5;the polymorphism information content ranged from 0.402 to 0.670;the levels of observed and expected heterozysities varied from 0.1200 to 0.7391 and from 0.2408 to 0.5983,respectively.Nine polymorphic loci were successfully amplified in Stichopus horrens;the number of alleles ranged from 2 to 5;the polymorphism information content ranged from 0.348 to 0.584;the levels of observed and expected heterozysities varied from 0.0554 to 0.8000 and from 0.2014 to 0.5000,respectively.The identified 26 microsatellite loci would be useful for population gengetic structure analysis and genetic diversity assessment of the three species of sea cucumber. |
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