【作者】 李荣华；

【导师】 李琪；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2011， 博士

【摘要】 我国是海水养殖大国,海洋贝类的养殖是其中重要的组成部分。对于养殖群体来说,较低的有效群体大小(或有效繁殖群体大小)会使得群体的遗传多样性降低,增加近交和遗传漂变的比率,特别在贝类等高产卵能力的生物中,使用较少的亲本即可以获得足够的苗种,育苗所用的有效繁殖群体数量对养殖群体的遗传结构具有重要影响。本研究以分子遗传学理论为基础,在贝类中进行遗传标记的开发并检测了他们在家系鉴定中的效率。此外,应用分子家系鉴定的方法,调查了北方沿海四种具有不同生活方式的重要经济贝类:太平洋牡蛎(固着型),海湾扇贝(雌雄同体底栖型),虾夷扇贝(雌雄异体底栖型),魁蚶(埋栖型)等在常规苗种繁育过程中有效繁殖群体的大小、遗传多样性的变化以及近交程度,旨在为我国海水养殖贝类遗传多样性保护和健康苗种培育提供指导依据。主要研究结果如下:1.海湾扇贝EST-SSR标记分离及特性分析利用公共数据库资源,在海湾扇贝EST数据库中查找包含微卫星的序列,开发了15个EST-SSR分子标记,采用群体多态性分析结果显示,位点的等位基因数平均值为5.5,观测和期望杂合度范围分别为0.000-0.781和0.155-0.885。平均多态信息含量为0.548。进行Bonferroni校正后,发现15个位点中有8个位点显著偏离哈迪-温伯格平衡。在各位点之间未发现有连锁现象。5个多态性最高的微卫星位点在已知单亲时的累积排除概率超过99%。利用筛选出的15对引物在栉孔扇贝、虾夷扇贝和长肋日月贝中进行PCR扩增,其中9对引物在栉孔扇贝和虾夷扇贝中能扩增出产物,有5对引物扩出的微卫星标记呈多态。在长肋日月贝中有3对引物有扩增产物,有1对引物扩出的微卫星标记呈多态。2.海湾扇贝EST-SNP标记分离及特性分析通过对海湾扇贝EST数据库中4968条ESTs进行聚类及拼接后,共发现3905个候选SNP位点,平均密度为1/118.2bp。从中选择30个候选SNPs进行tetra-primer ARMS-PCR引物设计和优化,17组引物扩增产生与预期片段长度相符的多态性位点,多态性位点最小等位基因频率从0.016到0.484。对含有17个多态SNPs的contigs序列在GenBank中进行BLASTX比对,除一个位点所在序列外均有匹配的功能序列,其中11个已验证SNPs定位于编码区(外显子区),导致同义氨基酸替换。家系模拟鉴定结果表明,SNP标记对鉴定所用亲本的数量比微卫星标记更为敏感,因此需要更多的SNPs来抵消多态性低带来的负面效果以达到较高并且稳定性较好的鉴定成功率。通过对海湾扇贝EST数据库的分析构建了最优密码子使用表,以解释某一特定SNP不同等位基因频率上的差异,分析发现对密码子使用的选择性可能会影响编码区SNP不同等位基因的频率。这些对于海湾扇贝SNP的初步研究是对现有遗传标记的有用补充。3.基于微卫星家系分析的海湾扇贝交配系统和繁殖成功研究运用微卫星家系鉴定的方法对群体产卵海湾扇贝的交配系统进行了研究。对14个候选亲本和237个子代的鉴定结果表明,采用6个多态微卫星位点能够将95%的子代鉴定到亲本。根据家系鉴定结果,发现既有通过自体受精产生的子代也有通过非自体受精产生的子代,但是自体受精率在亲本个体间巨大的变异表明海湾扇贝具有混合交配模式。繁殖成功的变异造成了有效繁殖亲本数量的降低(Nb/N = 0.71)。群体的自体受精率为32.89%,近交系数为16.5%。在不考虑自交子代的情况下,半同胞和非同胞亲本在亲缘关系和繁殖成功之间没有显著的相关关系,表明可能是选择的压力而不是对近交的避免机制支持着海湾扇贝自交兼容性交配系统的进化和维持。本研究结果使我们对目前我国海湾扇贝的养殖状况有了深入了解,并且为以后对其进行繁殖技术改良提供了知识基础。4.太平洋牡蛎EST-SSR多重PCR的开发及其在家系鉴定中的检验我们从以往报道的太平洋牡蛎EST-SSRs中开发出了4组multiplex PCRs,并在12组单对交配家系中检验了其在家系鉴定中的效率。位点的基因丰富度和多态信息含量平均值分别为11.1和0.811,4组multiplex PCRs在家系鉴定中的累积排除能力大于99.99%。对143组分离模式的分析发现,有11个基因型分离比偏离孟德尔分离定律。通过分析位点在家系内的Mendelian分离模式,发现无效等位基因频率为4.9%。对12组单对交配家系的实际鉴定结果表明,使用2组多态信息含量最高的Multiplex PCRs,可以将97%的子代鉴定到亲本,其结果与真实的家系信息对比,错误率为4%;如果使用3组或者4组Multiplex PCRs,则可以将100%的子代鉴定到亲本,与真实的家系信息对比错误率为0。5.太平洋牡蛎养殖群体的微卫星家系分析和有效群体大小估算运用7个高度多态的微卫星位点对群体产卵的太平洋牡蛎进行家系分析,并比较亲本和子代的遗传多样性变化。对8个候选母本、6个候选父本和155个子代的鉴定结果与模拟鉴定一致,运用6个微卫星位点即可获得超过99%的鉴定成功率。等位基因数和观察杂合度(Ho)在亲本和子代中没有明显差异,但是,子代的期望杂合度(He)和亲本相比有显著降低。虽然所有亲本均参与子代繁殖过程,但是亲本繁殖成功的变异和不平衡的性别比例导致了Ne的降低(Ne = 11.42)。群体近交率的估算值为16.5%。这些结果表明在未来制定太平洋牡蛎群产育种方案时,需要考虑到较高的近交率和较低有效群体的因素。6.基于微卫星家系分析的虾夷扇贝有效繁殖群体研究运用微卫星家系鉴定的方法对群体产卵虾夷扇贝幼虫发育两个不同时期的有效繁殖群体的变化及其与亲本性状特征的相关性进行了研究。对14个候选母本、14个候选父本和1080个子代的鉴定结果表明,采用5个多态微卫星位点能够获得97%以上的鉴定成功率。亲本繁殖成功的变异是D形幼虫时期有效群体大小降低的主要原因(Ne/N = 0.87),近交率的估计为0.081。稚贝时期的有效群体大小进一步降低(Ne/N = 0. 78),近交率上升至0.090。这一变化可能与育苗过程中的幼虫分级选择有关,人为的改变了群体的遗传结构,加剧了亲本繁殖成功的变异。也可能与不同亲本组子代的存活力差异有关。回归分析表明,D形幼虫阶段母本的繁殖成功与母本壳长和壳宽存在显著正相关关系,父本的繁殖成功与父本壳高存在显著正相关关系,由此推断在群体产卵时,亲本个体大小在繁殖成功上有显著优势。然而在稚贝阶段,未检测到亲本的繁殖成功与性状特点的相关性。表明随着苗种的养成,亲本个体大小在产卵阶段的繁殖成功优势可能会逐渐消失。7.基于微卫星同胞重建的魁蚶有效繁殖群体研究运用5个微卫星标记,在没有亲本信息的情况下,采取同胞重建的方法研究了群体产卵魁蚶幼虫发育两个不同时期遗传多样性以及有效繁殖群体大小的变化,并对亲本的繁殖成功与交配对象数目的关系进行了相关性研究。等位基因数和观察杂合度在两个采样阶段均没有显著差异。但是对D形幼虫时期以及稚贝时期各180个个体的同胞重建结果表明,D形幼虫时期有效繁殖群体的估算值为20,稚贝时期有效繁殖群体的估算值为16,出现了明显的降低,而且稚贝时期可能的亲本数量也比D形幼虫时期降低了16.13%,?F增加了0.6%。多配繁殖在父/母本中均存在,而且在两个采样阶段,魁蚶父/母本繁殖成功与交配对象数量均存在显著正相关关系。综上,本研究证明基于微卫星的家系分析方法具有较高的鉴定效力。运用5-6个微卫星位点即能够成功地对混合种群进行家系鉴定。在本研究的所有养殖贝类中均检测到有效繁殖群体数量(Nb,或有效种群大小Ne)的降低。交配系统,不平衡的性别比例,亲本贡献的高度变异以及家系规模的变异是导致Nb降低的主要原因。研究结果对我国海水养殖贝类遗传多样性保护和健康苗种培育具有指导作用。更多还原

【Abstract】 The marine shellfish has served as an important component of aquaculture in China. The restricted number of effective population size (or effective number of breeders) used in culture operations can lead to a loss of genetic variability, increase the random drift in gene frequencies and inbreeding in a population. Especially, the high fecundity of some organisms in a culture environment (such as shellfish) ensure that a small number of stocks are used for production to generate sufficient offspring without concerns that genetic variability would decrease in farmed populations. In the present study, we developed different kinds of molecular markers to evaluate their utility in the parentage analysis. Furthermore, the effective population size, the difference of genetic variability and inbreeding rate were assed in four important hatchery mollusks of the northern coasts who live with different styles of life, including Crassostrea gigas (sessile), Argopecten irradians irradians (bentonic, hermaphroditic), Mizuhopecten yessoensis (bentonic, dioecious) and Scapharca broughtonii (infauna) based on microsatellite-based parentage analysis to better facilitate the protection of the genetic diversity and healthy aquaculture of seed production in these species. The main results of these studies are listed as follows:1. A set of polymorphic expressed sequence tag–derived microsatellites from the bay scallop, Argopecten irradians irradians, and their transportability in three other scallop speciesA set of 15 new EST-SSRs for Argopecten irradians irradians was developed from ESTs database. The average number of alleles was 5.5 per locus, observed heterozygosities ranged from 0.000 to 0.781, while expected heterozygosities varied from 0.155 to 0.885. The average PIC was 0.548. Significant departures from HWE were observed in 8 of the 15 single-locus (exact tests after sequential Bonferroni correction). No significant linkage disequilibrium was detected between loci. The simulated combined assignment success rate in parentage analysis for the five highest polymorphic loci was over 99% when one parent known. Cross-species amplification was examined in three other species, Chlamys farreri, Patinopecten yesoensis and Amussium pleuronectes. In both C. farreri and P. yesoensis, nine loci gave successful amplifications, although only five loci were polymorphic. In A. pleuronectes, three loci produced unambiguous PCR products, but only one was variable.2. Characterization of expressed sequence tag-derived single nucleotide polymorphisms in the bay scallop, Argopecten irradians irradians (Lamarck 1819)We described here the discovery of 3905 putative single-nucleotide polymorphisms (SNPs) from the alignment of 4968 sequences in a bay scallop Argopecten irradians irradians expressed sequenced tag (EST) database. The observed frequency of SNPs was estimated at one every 118.2 bp of contig sequences. Thirty of the SNPs were chosen for validation by tetra-primer amplification refractory mutation system-polymerase chain reaction procedure, and 17 of them were polymorphic with the minor allele frequency ranged from 0.016 to 0.484. BLASTX gave significant hits for all but one of the 17 genotyped SNP-containing contigs, 11 of which located in coding regions and all resulted in a synonymous substitution. Parentage simulations demonstrated that SNP markers were more sensitive to the number of parents compared with microsatellites, thus more SNPs were needed to counteract low polymorphism. A table of optimal codons was deduced from the analysis of A. i. irradians EST dataset to explain the frequency difference for a specific SNP. It seems that the selection of codon usage may be partly responsible for the frequency difference between the two alleles of the coding SNPs. These are the first SNPs developed for the bay scallop and will provide a useful complement to currently available genetic markers. 3. Mating systems and reproductive success in hermaphroditic bay scallop, Argopecten irradians irradians (Lamarck 1819) inferred by microsatellite-based parentage analysisIn this study we used microsatellite-based parentage analysis to assess the mating system of a mass spawning bay scallop, Argopecten irradians irradians. Analyses were performed on 237 offspring and 14 candidate parents. Overall, 95% of all offspring were unambiguously allocated to their putative parents based on information from six highly polymorphic loci. Offspring from both the self-fertilization and cross-fertilization style were observed, the large variation of self-fertilization rate among individuals displays a mixed mode of mating. The high variances in reproductive success resulted in a decline in effective breeding numbers (Nb/N = 0.71). The self-fertilization rate was 32.89% for this mass spawning and the coefficient of inbreeding was estimated at around 16.5% per generation. When larvae resulting from selfing were removed, the relationship between relatedness and individual reproductive success was proved insignificant between half-sibings and unrelated, which suggest that selection pressures other than avoidance of inbreeding may be responsible for the evolution and maintenance of the self-compatible mating system of A. i. irradians. These results lend insight to A. i. irradians cultivation in present China and provide a genetic basis for the development of more effective spawning techniques.4. Development of four EST-SSR multiplex PCRs in the Pacific oyster, Crassostrea gigas and their validation in parentage assignmentWe report four highly informative multiplex PCRs developed from twelve previously described EST-SSRs in Crassostrea gigas. We evaluated and validated these multiplex PCRs in twelve full-sib families. The average allelic richness and the polymorphism information content (PIC) were 11.1 and 0.811 respectively. The combined power of exclusion was greater than 99.99% using all four multiplex assays. A hundred and forty three tests of segregation ratios revealed 11 significant departures from expected Mendelian ratios. The frequency of null alleles was estimated as 4.9% of all the alleles segregating based on a within-family analysis of Mendelian segregation patterns. Parentage analysis of real offspring demonstrated that 97% of all offspring were unambiguously allocated to a pair of parents based on two multiplex PCRs with only a 4% error rate, and 100% of the offspring were correctly allocated to their parents when three multiplex PCRs were used.5. Parentage determination and effective population size estimation in mass spawning Pacific oyster, Crassostrea gigas based on microsatellite analysisSeven high polymorphic microsatellite loci were utilized to determine the pedigrees in a mass spawning of Pacific oyster, Crassostrea gigas and to estimate the genetic variability between broodstock and offspring. Analyses were performed on a total of 155 individuals, including 141 offspring, 8 candidate mothers and 6 candidate fathers. The assignment results of real offspring were generally in agreement with simulation with a success rate over 99% using only six of these loci. The allelic diversity and observed heterozygosity (Ho) exhibited similarity between parents and offspring populations, but the expected heterozygosity (He) had a significant decrease in offspring. Although all the males and females contributed to the next generation, the variances of reproductive success and unequal sex ratio resulted in a decline in effective population size (Ne = 11.42). The inbreeding rate of this small-scale, mass spawning population was estimated at around 16.5% per generation. This gave us an insight that when designing breeding programmes based on mass spawning for future oyster cultivation generations, the higher inbreeding and lower effective population size should be considered.6. Effective population size in hatchery mass-spawning Japanese scallop, Mizuhopecten yessoensis based on microsatellite parentage analysisThe microsatellite-based parentage analysis was used to assess the effective population size and its relationship with parental phenotypes in two different developing times of larvae from hatchery mass-spawning Japanese scallop, Mizuhopecten yessoensis. Analyses were performed on 1080 offspring, 14 candidate mothers and 14 candidate fathers. Overall, more than 97% of all offspring were unambiguously allocated to their putative parents based on information from five highly polymorphic loci. For D-shaped larvae phase, the variances of parental reproductive success resulted in a decline in effective population size (Ne/N = 0.87), and the inbreeding rate was estimated at 0.081. The Ne decreased further for post-larval phase (Ne/N = 0. 78), and the inbreeding rate increased to 0.090, this may be a result of culling during the rearing process, which could have an impact on the population genetics, and bias the variances of reproductive success. Also, the different survival rates between families could be responsible for it. For D-shaped larvae phase, maternal shell length and shell width had a significant positive effect on reproductive success, the same was observed for paternal shell, which indicated that parental reproductive success was dependent on the magnitude of the size difference. While there was no such relationship discovered for post-larval phase, which might suggest that the effect of advantage in parental size difference would decrease along with culture process.7. Sibship reconstruction and effective population size estimation in mass spawning ark shell, Scapharca broughtonii based on microsatellite analysisFive high polymorphic microsatellite loci were utilized to reconstruct the sibship in a mass spawning of ark shell,Scapharca broughtonii and to estimate the genetic variability between D-shaped larvae phase and postlarvae phase. The allelic diversity, observed (Ho) and expected (He) heterozygosity exhibited similarity between the two larvae phase. But the sibship reconstruction results of 180 offspring in each larvae phase demonstrated that the effective population size (Ne) was 20 in D-shaped larvae phase and decreased to 16 in postlarvae phase, besides, the inferred number of parents decreased 16.13% and the inbreeding rate increased 0.6% in the latter. Multiple matings were detected both in males and females, and the reproductive success of both sexes in the two larvae phase was positively correlated to the number of individuals with whom it had mated.In general, the microsatellite-based pedigree analysis has been approved to be very efficient that the pedigree of mixed populations could be determined through the use of five to six microsatellite markers. The decline of the effective number of breeders (Nb) or the effective population size (Ne) was detected in all the aquaculture mollusks in this study. The mating system, unequal sex ratios, high variance in parental contributions and family size variations were proved to be the main reasons for the decline of Nb. These results will be useful guides for the protection of the genetic diversity and healthy aquaculture of seed production in the marine shellfish.更多还原