Genetic Diversity Of Hatchery-produced Stocks Of Three Economically Important Abalone Species In Southern China

The abalone Halitosis diversicolor supertexta Lischke, 1870, H. discus hannai Ino, 1952, and H. discus discus Reeve, 1846 are main cultured abalone species in costal waters of southern China. H. diversicolor supertexta has been cultured commercially for more than ten years and the cultivating industry are rely exclusively on hatchery-produced seeds drived from cultured broodstocks which may chang genetic diversity of stocks. H. discus hannai and H. discus discus cultured in southern China were introduced from northern China and Japan, and the genetic diversity of hatchery-produced stocks was unknown. To assessing genetic variation of the three economically important abalone species, we analyzed genetic diversity and genetic structure of five hatchery-produced stocks of H. diversicolor supertext using RAPD markers. The five populations were sampled from Dongshan county in Fujian province, Huilai, Shenzhen and Zhanjiang in Guangdong province and Sanya in Hainan province. We also analyzed two hatchery-produced stocks of each of H. discus hannai and H. discus discus sampled from Dongshan county and Huilai, respectively, using microsatellite DNA markers.RAPD analysis on H. diversicolor supertexta showed that 22 of the 54 candidate random primers can produce reproducible bands and were employed for PCR amplification of all samples. For 172 individuals from the five stocks, 22 random primers generated 180 clear identifiable bands with size ranging from 200 bp to 2400 bp, and the average proportion of polymorphic bands was 91.4%. Gene diversity among stocks ranged from 0.259 in Huilai stock to 0.344 in Zhanjiang stock with a mean value of 0.313. Student's t-test indicated that differences of gene diversity among Dongshan, Shenzhen and Sanya stocks are not significant (P＞0.05).The total gene diversity of five hatchery-produced stocks (H_T), the average diversity overstocks (H_S), the average genetic difference (D_ST) and the proportion of genetic differentiation (G_ST) were 0.381, 0.313, 0.068 and 0.179, respectively. Pairwise G_ST ranged from 0.076 to 0.246. Hierarchical AMOVA suggested that 76.7% of the variation was attributed to sampling of individuals within stocks while genetic variation among stocks accounted for 23.3% of the total variation. Pairwise F_ST ranged from 0.102 to 0.297 revealing a similar genetic differentiation pattern to that of G_ST. Cluster analysis of UPGMA demonstrated that the five stocks clustered into two groups, Shenzhen and Zhanjiang stocks were more closely related clustering into one group with Sanya stock while Dongshan and Huilai stocks clustered into another group. But genetic differentiation between the two groups was not significant as revealed by AMOVA (F_CT =0.103, P=0.099). Random permutation test indicated that genetic differentiation among stocks was significant, so each of the five stocks can be treated as independent "management unit" in genetic resource management. Thus transplant of cultured stocks should be closely monitored.Screened from nine pairs of microsatellite primers developed from H. discus hannai and H. discus discus, four pairs developed from H. discus discus can produce PCR bands in both H. discus hannai and H. discus discus stably. Twenty-four alleles were scored at four microsatellite loci. Effective allele number ranged from 2.214 to 6.001 with a mean value of 4.110. PIC values were all higher than 0.5 ranging from 0.512 to 0.812. The observed heterozygosity ranged from 0.382 to 0.677 with a value of 0.521 on average and the expected heterozygosity ranged from 0.550 to 0.836 with a value of 0.705 on average. Expected heterozygosity was higher than observed heterozygisty and D values were negative at each locus. Allele number and the effective allele number in the two H. discus hannai stocks were all higher than those in H. discus discus stocks. According to the PIC and expected heterozygosity, the order of the four stocks was H. discus discus in Huilai＞H. discus hannai in Huilai＞H. discus hannai in Dongshan＞H. discus discus in Dongshan. X² test of Hardy-Weinberg equilibrium showed that all the four loci were in Hardy-Weinberg disequilibrium except H. discus hannai in Dongshan at locus Hdd108C and the two stocks of H. discus discus at locus Hdd115B.Mean value of inbreeding coefficient was 0.228. AMOVA revealed that 95.22% of the variation was attributed to sampling of individuals within stocks and genetic variation among stocks accounted for only 4.78% of the total variation of H. discus hannai and H. discus discus (F_SC=0.048, P＜0.001). But the genetic differentiation among all the stocks was significant based on random permutation test. Pairwise Nei's (1972) genetic distances among stocks ranged from 0.084 to 0.186. Huilai and Dongshn stocks of H. discus discus had the closest genetic distance while Dongshan stocks of H. discus hannai and H. discus discus had the longest genetic distance. Cluster analysis demonstrated that the two stocks of H. discus discus had the closest genetic relationship and clustered into one group with Huilai stock of H. discus hannai while Dongshan stock of H. discus hannai had farther genetic relationship with others.