The Development And Characteristics Of Microsatellite Markers From Four Species Of Sea Cucumbers

In recent years,because microsatellite marker has many advantages including codominant inheritance,high polymorphism,good stability and high developing efficiency and so on,the technology is more and more widely used in research of genetics,such as biological group analysis of genetic diversity,gene mapping,genetic linkage map construction,identification of variety and other aspects.We all know,sea cucumber is not only precious food but also rare medicinal ingredient,so the demand for sea cucumber is continuously growing at home and abroad,however,the quantity resouce of wild population in China becomes continuous declination because of the natural environment destruction and other factors.Additionally,the study of genetic diversity about tropical sea cucumbers is rather limited except temperate apostichopus japonicus in our country,let alone about the development of microsatellite marker,which is only on the blank or insufficient stage.Anyway,in order to make the molecular-assistant breeding research and the population genetic structure analysis much better,and then effectively protect wild groups and increase artificial breeding production,so it's an urgent need to further expand microsatellite marker library from sea cucumbers.In this research,four species of representative sea cucumbers in breeding industry in China were selected to serve as experimental samples,namely Apostichopus japonicus?Stichopus horrens?Holothuria scabra and Holothuria leucospilota.The method of FIASCO?Fast Isolation by AFLP Sequences Containing Repeats?was applied to develop microsatellite loci.In the experimental process,restriction enzyme MseI was used to digest the genome DNA,and the mixture oligonucleotides probes Bio-?CT?₁₅ and Bio-?GT?₁₅ were used to hybridize microsatellite sequences.After magnetic beads enrichment,microsatellite library enriching?GA?n and?CA?n from the four species of sea cucumbers was formed.Finally,novel polymorphic microsatellite loci were isolated from the above libraries,and we also made futher analysis about the relative genetic characteristics.From the microsatellite enrichment library of Apostichopus japonicus,we randomly select142 positive clones?400-1000bp?to sequence,and got 80 microsaellite sequences,so the positive rate of cloning was 56.33%.Then,we got 15 microsatellite loci?14 were polymorphic loci and 1 was monomorphic loci?from the designed 36 pairs of specific primers.Among the 14polymorphic loci,the number of allele?Na?ranges 2-6.The heterozygosity observed?Ho?and expected heterozygosity?He?is 0.0345-0.4667 and 0.0339-0.6635,repectively.The polymorphism information content?PIC?is 0.094-0.679,11 polymorphic loci were middle and high polymorphism.Finally,there were 10 polymorphic loci conforming to Hardy-Weinberg equilibrium after test.From the microsatellite enrichment library of Stichopus horrens,161 positive cloneswere randomly select to sequence,and got 129 microsaellite sequences,then the positive rate of cloning was 80.12%.13 microsatellite loci including 11 polymorphic loci and 1 monomorphic loci were isolated from the designed 45 pairs of specific primers.Among the 11 polymorphic loci,the Na was 3-8,the Ho and He was 0.1364-0.8966 and 0.1653-0.7551,repectively.And,all the 11 polymorphic loci showed high polymorphism with the PIC 0.271-0.7311.Finally,9polymorphic loci were for Hardy-Weinberg equilibrium after test except for 2 loci?P<0.004545?.From the microsatellite enrichment library of Holothuria scabra,300 positive cloneswere randomly select to sequence,and got 240 microsaellite sequences.The result showed that positive rate of cloning was 80.00%.20 microsatellite loci including 16 polymorphic loci and 4monomorphic loci were isolated from the designed 70 pairs of specific primers.Among the 16polymorphic loci,the Na ranged from 3 to 8.The Ho and He was 0.0333-0.5172 and 0.0328-0.6901,repectively.And,the all PIC showed 0.032-0.712,11 polymorphic loci were middle and high polymorphism.Finally,14 polymorphic loci were for Hardy-Weinberg equilibrium after test except for 2 loci?P<0.003125?.From the microsatellite enrichment library of Holothuria leucospilota,we randomly got172 positive clones sequenced,and 111 microsaellite sequences were screened,then the positive rate of cloning was 64.53%.In all of 16 polymorphic microsatellite loci,the Na was 3-5;the Ho and He was 0.0370-0.5000 and 0.0776-0.6250,repectively;the PIC was 0.183-0.668 including14 middle and high polymorphic loci.Finally,13 polymorphic loci were for Hardy-Weinberg equilibrium after test except for 3 loci?P<0.003125?.All the results showed that the method of FIASCO is a kind of high effective development technology of microsatellite marker.In this study,on the one hand,I made a brief analysis about population genetic diversity in the four species of sea cucumbers on based of the above information;on the other hand,I emphatically explored and discussed to improve the building efficiency of microsatellite enrichment library from technical degree,so that optimize the related steps in the development process.All the 47 middle and high polymorphic microsatellite loci can be tentatively used to research other different species of sea cucumbers about genetic diversity,and they will further provide candidate microsatellite loci for phylogenetic analysis and molecular assistant breeding,and et al.