【作者】 束靖；

【导师】 李琪；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2007， 博士

【摘要】 本论文以中国重要海洋经济贝类皱纹盘鲍(Haliotis discus hannai)为实验对象,研究了皱纹盘鲍微卫星标记的分离与线粒体DNA的遗传模式,分析了皱纹盘鲍野生群体与养殖群体的遗传多样性与遗传分化,探讨了人工养殖群体中的有效亲本数量,研究了育苗生产中养殖群体遗传多样性的动态变化。主要研究结果如下:1.利用生物信息学方法,在1560个皱纹盘鲍(H. discus hannai)和日本盘鲍(H. discus discus)的表达序列标签(ESTs)数据库中进行了微卫星序列的筛选,并根据微卫星序列设计了30对引物。通过在30个野生皱纹盘鲍个体上的扩增,筛选出18个有多态性的皱纹盘鲍微卫星位点,其等位基因数为2-17,期待杂合度与观察杂合度范围分别为0.159-0.928、0.132-0.922,其中有7个位点因杂合子缺失呈现显著偏离哈迪·温博格平衡。研究结果表明这18个位点可以在皱纹盘鲍群体遗传、家系分析、遗传图谱构建、功能基因定位等研究中应用。2.利用皱纹盘鲍单对交配家系,通过可检测单碱基突变的变性梯度凝胶电泳(DGGE)技术对皱纹盘鲍鲍线粒体COI基因序列进行线粒体遗传模式分析,供试的20个家系中有5对亲本组合存在不同的单倍型,在来自这5组家系的100个后代个体中仅检测到母本的单倍型,首次证实皱纹盘鲍线粒体的遗传模式为严格的母性遗传。3.利用微卫星标记与线粒体COI基因,首次对皱纹盘鲍主要分布区的辽宁大连、山东长岛、荣城、日本岩手、和韩国仁川5个野生群体进行了遗传多样性分析与遗传分化的研究。微卫星分析结果表明,5个野生群体有效等位基因数范围为3.2-14.7,期待杂合度与观察杂合度范围分别为0.751-0.854和0.599-0.878,遗传多样性比较为日本群体>韩国群体>长岛群体>荣城群体>大连群体。对线粒体COI基因进行测序,共检出27个多态性核苷酸位点,21种单倍型,5个群体核苷酸多样性指数范围为0.0031-0.0142,平均核苷酸差异数为1.056-4.110,日本群体、韩国群体遗传多样性明显高于中国3个群体;中国3个群体遗传多样性较低可能与中国皱纹盘鲍大规模养殖对野生群体产生影响有关。微卫星标记与线粒体COI基因序列的Fst值分析结果均表明日本群体与其他4个群体间存在显著遗传分化。Mantel检验显示遗传距离与地理距离显著相关。皱纹盘鲍短暂的浮游期与有限的扩散能力可能是导致群体间出现显著分化的主要原因。4.采用微卫星标记对辽宁大连、山东烟台、荣城、崂山和胶南5个皱纹盘鲍养殖群体进行了群体遗传多样性与分化的研究。结果表明:等位基因数为8-9.4,期待杂合度0.754-0.787,观察杂合度0.500-0.596,与野生群体相比整体遗传多样性较低,亲鲍选用数量少与性别比例不均衡可能是产生养殖群体中遗传多样性显著减少的原因。通过对等位基因频率比较分析,在5个群体间Fst与Rst值显著差异,表明养殖群体中由于亲鲍数目的降低加剧了遗传漂变的影响;养殖群体间遗传距离与地理距离不相关,可能是皱纹盘鲍人工养殖过程中各育苗厂之间亲鲍和苗种频繁交流交换所造成的结果。为研究人工养殖群体的有效亲本数量,建立了4个皱纹盘鲍混交家系,利用7个微卫星标记分析了这4个家系185个后代个体。使用5个多态性微卫星标记可以对所有子代进行亲子鉴定,有效亲本数量仅占亲贝的50%,各家系中亲鲍对后代贡献率存在差异;子代等位基因数与亲鲍相比数量较少,亲鲍低频基因在子代中有所丢失;有效亲本数量与子代杂合度以及多态性信息含量呈显著正相关。结果表明,养殖群体中有效亲本数量过少会导致群体中一些低频基因缺失,使群体产生遗传漂变;亲鲍对后代群体贡献率的差异也是导致遗传多样性下降的原因之一;在皱纹盘鲍育苗过程中,增加亲鲍使用数量与平衡雌雄比例可以防止后代遗传多样性下降。为研究苗种生产过程中养殖群体遗传多样性的动态变化,采用6个微卫星位点对1混合家系后代3个不同发育时期的遗传多样性进行了分析。遗传多样性随发育时期呈下降趋势,孵化后第3天与第30天、第60天间群体存在显著遗传分化;第30天与第60天群体之间没有明显遗传分化。研究结果表明,在皱纹盘鲍苗种培育过程中,有效亲本数量随着发育时间的延长而减少是导致不同时期养殖群体遗传多样性变化的原因之一。更多还原

【Abstract】 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.更多还原