Isolation Of Microsatellite Markers From Charybdis Japonica And Its Application In Genetic Diversity Analysis

Charybdis japonica is one of the important fisheries resources in China, but due to overfishing, habitat damage, pollution and so on, the natural stocks have been declining dramatically of late years. To evaluate the genetic diversity and structure of C. Japonica population in the East China Sea and Yellow Sea, and to investigate the resource management, protection and rational development plan. First of all, we constructed an enriched microsatellite library by a magnetic-bead enrichment method used biotinylated (CA)12 probe, 9 polymorphic microsatellite markers were designed and screened. Polymorphism of these loci was assessed by genome scan with labeled microsatellite. The number of alleles per locus ranged from 12 to 25; the observed and expected heterozygosity varied from 0.5806 to 0.9032 and from 0.8383 to 0.9449, respectively; polymorphism information content (PIC) ranged form 0.8092 to 0.9259; After Bonferroni correction (adjusted P value=0.0056), all of the 9 loci accorded with Hardy-Weinberg equilibrium. These results showed that these microsatellite loci were the ideal molecular markers for the genetic diversity and structure studies of C. Japonica.Genetic diversity and structure of six C. Japonica stocks in the East China Sea and Yellow Sea, were investigated using nine microsatellite loci. The results showed that the average alleles of 9 loci were 23.11; the average observed and expected heterozygosity was 0.8144 and 0.9099, respectively; polymorphism information content of six stocks ranged form 0.8092 to 0.9259, with the average of 0.8526. They all indicated that the genetic diversity of C. Japonica was rich. But theχ2 test of Hardy-Weinberg equilibrium, Hardy-Weinberg departure values (d) and Fis values reflected the stocks of C. Japonica with heterozygosity deficiency, prompted that the genetic diversity of C. Japonica in the East China Sea and Yellow Sea was gradually losing, protection is urgently needed. Fst, gene flow and AMOVA analysis across all stocks showed that there was low level of divergence among 6 stocks (Fst<0.05, the average gene flow was 7.4204), the main source of genetic variation was within populations (98.15%), but population differentiation was still significant (P <0.01). The UPGMA clustering tree demonstrated that the stocks of XM, WZ, ZS and QD clustered together first, then clustered with ND and NT stock, in combination with the East China Sea and Yellow Sea flow direction in breeding stage of C. Japonica, the findings indicated that the larval C. Japonica dispersed with ocean current was the main source of genetic exchange between populations in the East China Sea and Yellow Sea, but the Yangtze River estuary separated them; Meanwhile, the relative enclosed bay (eg, Ningde Bay), due to external currents could not flow into, was also the important factor affected genetic exchange among C. Japonica stocks.