Molecular Population Genetics Of The Pacific Abalone, Haliotis Discus Hannai

The Pacific abalone, Haliotis discus hannai, naturally occurs in China, Korea and Japan, and is one of the commercially important species of shellfish aquaculture in East Asia. In the study isolation of microsatellite DNA markers, the pattern of inheritance mitochondrial, the genetic variability and population differentiation within and between natural and cultured populations, the effective population size of cultured population and the monthly dynamic change of genetic variability in hatchery offspring during early breeding procedures were investigated. The results obtained here are as follows:1. A total of 1560 ESTs of H. discus hannai and H. discus discus were screened based on expressed sequence tags deposited in public sequence database. Eighteen polymorphic microsatellite DNA markers were developed from H. discus hannai. Subsequent screening on 30 individuals of pacific abalone wild population from Japan revealed the numbers of alleles ranged from two to 17, and the expected and observed heterozygosities ranged from 0.159 to 0.928 and 0.132 to 0.922, respectively. Due to heterozygote deficiency and null alleles existing, seven loci were found to deviate significantly from Hardy–Weinberg equilibrium. The results indicated that these EST-SSR markers are potentially valuable tools used in population genetic study, pedigree analysis, construction of genetic map and location of function gene in H. discus hannai.2. The denatured gradient gel electrophoresis (DGGE) technology was applied to analysis the inheritance pattern of mitochondrial of H. discus hannai. Mitochondrial COI gene was amplified in twenty crosses. According to the electrophoresis, different haplotypes between male and female parent were detected in five crosses. In all five informative mating, the mitochondrial haplotype of 100 offspring individuals were consistent with their female parents. No heteroplasmic individual was observed. This result first confirmed that the mitochondrial of H. discus hannai was strict maternal inheritance.3. Genetic diversity of 5 natural populations of the H. discus hannai from Dalian (Liaoning Province, China), Changdao, Rongcheng (Shandong Province, Chian), Iwata (Japan) and Inchon (South Korea) was analyzed using microsatellite markers and mitochondrial COI gene. The microsatellite loci were polymorphic for all the populations. Number of allele per locus ranged from 3.2 to 14.7, while the observed and expected heterozygosities ranged from 0.599 to 0.878, and from 0.751 to 0.854, respectively. The comparison of genetic variability among the five populations were Japan > South Korea > Changdao > Rongcheng > Dalian. The mitochondrial COI gene fragments from H. discus hannai were amplified via PCR. 580 bp sequences data were retrieved with 27 polymorphic sites and 21 haplotypes. The nucleotide diversity and average number of nucleotide difference among the 5 populations ranged from 0.0031 to 0.0142 and from 1.056 to 4.110 respectively. Similar to the data obtained from microsatellite markers, the genetic variability of Japanese and Korean populations was obviously higher than the 3 Chinese populations. The lower genetic diversity in the 3 Chinese populations is possibly related to the large-scale cultivation of H. discus hannai in China which might affect the genetic structure of natural populations. The Fst value from microsatellite markers and mitochondrial COI gene showed significant genetic structuring between the Japanese population and other 4 populations. The significant correlation between genetic distance and geographic distance were revealed by Mantel test. This could be the result of short planktonic phase and limited diffusivity of H. discus hannai.4. Genetic diversity of cultured populations of the H. discus hannai from northern China was analysed using seven microsatellite markers. The microsatellite loci with alleles range 8.0-9.4. The mean observed and expected heterozygosities were 0.547 (range 0.500-0.596) and 0.774 (range 0.754-0.787), respectively. The allelic diversity in terms of number of alleles per locus was considerably lower than that previously found in wild populations, the small number and the biased sex ratio of abalone for hatchery broodstock might be responsible for the remarkable reduction of genetic variation in the studied populations. Significant genetic differentiation among the five cultured populations was showed using Fst and Rst values, and pairwise comparison based on allelic distribution. The results did not show the consistent relationship between the geographic and the genetic distances, suggesting the existence of exchanges of breeds and eggs between the hatcheries.In order to study the influence of effective parent number on the genetic diversity of cultured population, 185 individuals of 4 controlled crosses were genotyped by 7 polymorphic microsatellite loci. As few as 5 loci could determine the parents of individual larvae in 4 crosses. The effective parents occupied 50% in total 16 broodstock. The contribution of effective broodstock to offspring differed among various parents. The numbers of alleles and low-frequency alleles per locus in the offspring were lower than those of their parents. Effective parent number was positively correlated with heterozygosity and polymorphism information content of progeny. These results reveal that small effective parent size could result in the loss of low-frequency allele and cause drift of allele frequency, and the difference of effective parental contribution could lead to loss of genetic variability in cultural populations. Increasing the number of broodstock and unbiased sex ratio during breeding procedure could effectively maintain the genetic variability in cultured populations.In order to assess the dynamic change of genetic variability in hatchery offspring during breeding procedure, offspring of a hatchery H. discus hannai population were sampled monthly and analyzed using 6 polymorphic microsatellites. With the growth, genetic variability of the progeny decreased. The significant genetic structure was found between the populations sampled on day 3, day 30 and day 60 after spawning; no evidence showed the genetic differentiation between the populations of day 30 and 60. The reduction in effective parent number and unequal family size could result in genetic change of cultural population in breeding procedure.