The Impacts Of Hybrids Farming On Genetic Structure Of Native Populations Of Pacific Abalone And Genetic Analysis Of Interspecific Introgressive Hybridization

Pacific abalone (Haliotis discus hannai Ino) is one of the most valuable commercial mollusks cultured in China. The aquaculture of Pacific abalone ran at a loss due to mass mortality of unknown reason in the mid of 1990s. Since then, the hybridization between Chinese abalone populations and introduced Japanese abalones (e.g. Pacific abalone and Xishi abalone, H. sieboldii Reeve) had been employed extensively. Nowadays, the most extensively cultured hybrids were generated by Chinese and Japanese populations of H. discus hannai. Genetic materials of Japanese gene pools could introgress into Chinese ones through cultured hybrids in the open sea, and then exert negative influences to local gene pools. In this study, populations of Chinese and Japanese Pacific abalone, Xishi abalone and their hybrids were analyzed to assess the genetic impacts of hybrids farming on genetic structure of native Pacific abalone populations, and to discuss genetic diversity and genetic structure of hybrids.1. Isolation and characterization of microsatellite markers for H. discus and H. sieboldiiTwo methods, EST database mining and microsatellite enrichment libraries construction-PCR screening, were used to develop 12 and 18 microsatellite markers for H. discus hannai and H. sieboldii, respectively. The polymorphisms of 30 markers were assessed with at least 36 individuals respectively. The results showed that the number of alleles ranged from two to 13 and four to 14. The values of HE and HO ranged from 0.2449-0.9311, 0.1222-0.8611 and 0.7364-0.9117, 0.3125-0.8571. Compared these two methods, EST database mining was proven to be an efficient and low-costly approach to obtain new microsatellite markers. However, the small amount of ESTs (1476 sequences) and low ratio (3.4%) of microsatellite-containing ESTs hampered the isolation of a large amount of markers. While PCR-based screening of microsatellite enrichment construction method modified in this study was an efficient and low-cost choice for the isolation of microsatellite markers.2. Genetic impacts of introduced Japanese abalone populations (H. discus hannai) on native population structureHybridization between the introduced Japanese and the Chinese native Pacific abalone (Haliotis discus hannai) has been extensively employed in China since the late 1990s. These aquaculture activities including frequent introductions of nonnative species/populations and artificial hybridization threaten the genetic integrity and persistence of native species/populations. Here, we estimated the influence of these activities on local population genetic structure based on seven polymorphic microsatellite markers and 411 amplified fragment length polymorphisms (AFLP) loci. We detected strong evidence of genetic introgression from the Japanese gene pool into the Chinese native one. The Bayesian clustering analyses suggested that a total of 84.1% of the analyzed 163 individuals be hybrids. Surprisingly, all individuals collected from the coasts of Dalian, Yantai and Qingdao were detected to be hybrids. Only one region, Rongcheng, showed a low hybrid ratio (38.0%). The difference in hybrid ratio is consistent with the records of major abalone grow-out methods employed in these areas (i.e. net-bag versus bottom-sowing culture). The results showed clearly that genetic pollution caused by frequent transplants of non-local populations and subsequent hybridization has been exerting negative influences to local gene pools. Given high values of genetic resources, greater attention must be paid to identifying, protecting and utilizing these genetic resources.3. Crossbreeding of Pacific and Xishi abaloneIntrogressive hybridization method between the native Pacific abalone and the introduced Xishi abalone has been employed to generate hybrids (SD). The fertility rate, hatching rate and metamorphosis rate of hybrid progeny have been elevated significantly along with the processing of introgressive hybridization and selective breeding. The growth and survival rates of SD are statistically significant higher than that of the hybrids of Pacific abalones (DD) during the stage of over summering. The growth rate of shell length of 30-180 days old juveniles in SDP1 and SDP2 is 10.03%-14.31% and 10.96%-15.68% higher than DD. Meanwhile the survival rate of SPD1 and SDP2 is 20.0%-39.2% and 25.8%-39.1% higher than DD during the stage of over summering, respectively.4. Genetic analyses of H. discus hannai, H. sieboldii and their hybrids1)Karyotype and GISH analyses of H. discus hannai, H. sieboldii and their hybrids were performed to detect different chromosome components and identify donor chromatin in hybrids. The results showed that the karyotype of H. discus hannai, H. sieboldii and their hybrids were all the same, 2n=20m+16sm, NF=72. GISH analyses of H. discus hannai, H. sieboldii and their hybrids showed that more than 79% of the Metaphase cells contained 36 chromosomes. All the chromosomes were detected with red signals by either H. discus hannai or H. sieboldii genome DNA as probes. In this study, no abnormal behaviors of chromosomes such as deletion, breakage and recombination were detected. There was also no haploid metaphases (n=18) detected in this study.2)Population differentiation and genetic diversity of H. discus hannai, H sieboldii and their hybrids (SDF1, SDBC1 and SDBC2) were assessed using 14 polymorphic microsatellite loci. The results showed very high genetic diversity in HD. A total of 195 alleles were detected over all loci, while 120 alleles were detected in HS. The average values of HO and HE in HD and HS were 0.5892, 0.7389 and 0.3588, 0.7806, respectively. Genetic analyses indicated that there was statistically significant differentiation between HD and HS (FST=0.1935). The results also showed that genetic diversity index of hybrids was between HD and HS. Genetic diversity of SDF1, SDBC1 and SDBC2 became higher and higher and showed statistically significance with HS (P<0.05). After hybridization with H. discus hannai, SDBC1 showed a significant higher genetic diversity than SDF1, while the increasing of genetic diversity of later generation (SDBC2) was not clear any more.