Microsatellite DNA Isolation And Genetic Diversity Of The Pearl Oyster Pinctada Fucata

The pearl oyster Pinctada fucata is widely distributed in tropic and subtropic oceans and seas of China, Japan, Indian and Australia. It is an economically important species that is cultured for pearl production in these areas. In the present study, microsatellite-enhanced genomic library of the pearl oyster was constructed. 219 pairs of primers were designed using Primer premier 3.0. After screening, nine polymorphic loci were obtained. Genetic diversity and differentiation in the wild and cultured populations in China as well as within and among five populations from China (Beibu Bay, Daya Bay, Sanya Bay), Japan (Mie Prefecture) and Australia (Port Stephens) were studied using the nine polymorphic loci. The purpose is to provide some information for the protection ofgermplasm resources and genetic improvement of the animal.There are almost 2000 colonies in the enriched library. The second PCR screening was performed using the primers of (CA)₁₅ probe and T vector, and 357 positive clones were obtained. Sequencing results showed that 297 clones contained microsatellite repeat unit. By alignment, 280 microsatellite sequences were obtained after excluding redundancy, including 479 microsatellite doman. The sequence length ranged from 119 to 787bp with an average of 408bp. The (CA/GT)_n repeat unit was the most abundant motifs with a percentage of 76%. Several other repeat types were also detected, such as (AG)_n, (AT)n, (GC)n, (CAA)n, (AAG)n, (ATT)n, (CCT)n, (ATTT)n, (GTTT)n, (CAGA)n, (GAGT)n, (CCGT)n, (GGGT)n, (CAAAA)n, accounting for 24%, 1.7% for each type. Among all the 479 microsatellites, 370 (77.3%) sequences were perfect type, 95 (19.8%) were imperfect type and 14 (2.9%) were compound type. 219 pairs of primers were designed using Primer premier 3.0 and 49 pairs were synthized. As a result, 31 pairs of primers were screened effective for PCR amplification and nine loci showed polymorphism as tested by a population, and the percentage of polymorphic loci accounted for 29.03%. Genetic diversity in the wild and cultured populations from Beibu Bay, Daya Bay, Sanya Bay was studied using the nine polymorphic markers. A total number of 44 alleles were detected and allele numbers varied from 2 to 9 among loci. The average expected heterozygosity ranged from 0.590 to 0.649, while the average observed heterozygosity ranged from 0.393 to 0.516 among populations. PIC value ranged from 0.530 to 0.586. The negative mean value of D indicated an overall deficit of heterozygotes for the six populations. Forty out of 54 tests were significantly deviated from Hardy-Weinberg equilibrium. AMOVA indicated that 94.59% variation resided within population, while genetic vatiation among populations was only 5.41%. The results showed that genetic diversity was high in all of the six populations, and these populations were competent for selective breeding.Genetic diversity within and among five populations of the pearl oyster from China (Beibu Bay, Daya Bay, Sanya Bay), Japan (Mie Prefecture) and Australia (Port Stephens) was studied using nine microsatellite DNA markers. The average numbers of alleles for the five populations were 4.889, 4.889, 4.889, 4.000 and 4.111. The average expected heterozygosities were 0.592, 0.605, 0.649, 0.575, 0.564 respectively. And the mean levels of obsevered heterozygosity were 0.400, 0.516, 0.485, 0.390 and 0.433, respectively. Shannon's information indexes were 1.131, 1.177, 1.251, 1.028, 1.025. The average polymorphism information contents were 0.530, 0.545, 0.586, 0.498 and 0.483, respectively. These results indicated that genetic diversity of Japanese and Australian populations was lower than the three populations of China. Chi-square tests showed that most of the cases (34) in five populations deviated from Hardy-Weinberg equilibrium. Pairwise F_ST ranged from 0.010 to 0.075. Genetic differentiation among the five populations was significant except the values between Beihai and Daya Bay, and between Beihai and Sanya. AMOVA showed that 96.22% variation resided within populations, genetic vatiation among populations was only 3.78%,but significant (F_SC=0.038, P<0.001). After dividing the five populations into three groups, the results showed that genetic differentiation among the groups was 3.04% but not significant (F_CT=0.030, P=0.104). Pairwise genetic distances among populations ranged from 0.062 to 0.269. An UPGMA tree demonstrated that the population of Beihai was clustered into one group, and the other four populations were clustered into another group. All the results showed that genetic differentiation among the five populations was low, and the genetic distance was not correlated with the geographic distance.