Construction On Enriched Microsatellite Library And Genetic Diversity Research In Swimming Crab (portunus Trituberculatus)

In this paper, partial genomic microsatellite library is constructed on swimming crab(Portunus trituberculatus) using the method of screening SSR-enriched library. 30 microsatellite markers are isolated and all of these are polymorphic loci. Eight microsatellite loci that separated out from the swimming crab are selected for analyzing five populations(ZS,HZ,HC,LZ,YL).The main results as follows:1. Genomic DNA is digested by HaeⅢ, then DNA fragments containing microsatellites are captured by a piece of nylon membrane(Hybond N+) bound with the probe combinations of (AC) 15 and (AG)15. After transforming to the E.coli strain DH5a, the clones are regularly re-arrayed in a new agar plate.150 clones are randomly selected for sequencing, of which 103 are independent containing microsatellites. The microsatellite sequences are divided into three categories:perfect repeat(41.75%),imperfect repeat(7.767%),compound repeat(50.48%).105 primer pairs are designed, of which 56 pairs can be amplified scorable PCR products. The polymorphisms of these scorable loci are assessed using 30 swimming crab individuals, and the results show that 30 loci are polymorphic. The values of Ho, He and PIC varied from 0.2222 to 1.0000,0.4367 to 0.9099 and 0.350 to 0.892, respectively.2. Eight microsatellite loci are used to analyze the genetic diversity and genetic differentiation in five wild populations of swimming crab. A total of 72 alleles are obtained from 8 loci among five populations. Alleles number range from 6 to 12; average alleles are 9.0 in every locus. While effective alleles number range from 2.9058 to 7.7901; average effective alleles are 5.4677 in every locus.3. The 8 loci are high polymorphic locus for PIC because the range of eight loci's PIC value is 0.5394 to 0.8003 and the average PIC is 0.6967 which are more than the standard 0.5.4. The average observed heterozygosity(Ho) value of five populations range from 0.6481 to 0.7604. The test of P value shows that only a little of loci deviated significantly or very significantly from the Hardy-Weinberg equilibrium.5. Pairwise D genetic distance and genetic similarity indices are calculated between populations. The genetic distance ranges from 0.2451 to 0.5179 and the genetic similarity index ranges from 0.5958 to 0.7826. the D value between HC and HZ is the shortest genetic distance, while the D value between LZ and ZS is the longest. UPGMA cluster analysis of the five wild populations based on D data shows that HC and HZ are first grouped and the last is LZ.6. Fst analysis is performed by FASTA software. The Fst values range from 0.0548 to 0.1083 among populations, which shows that the differentiation degree among five wild populations is moderate(0.05<Fst<0.15).