【作者】 张振；

【导师】 刘晓；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2010， 博士

【摘要】 本研究通过构建皱纹盘鲍微卫星富集文库、筛查EST文库获得301个微卫星(SSR)标记,利用含有37个个体的皱纹盘鲍野生群体对其多态性进行评价,其中245个位点多态,扩增得到的等位基因数目从2到18个不等,期望杂合度和观测杂合度的范围分别为0.053～0.873和0.054～1.000。实验验证了EST文库筛查法是简便、低耗的方法,但不适合大规模筛选目标物种的微卫星标记,而富集文库法则是规模化开发微卫星标记的首选方案。通过筛查皱纹盘鲍的EST序列、PCR直接测序并借助多特异性等位基因PCR(PMASA)方法开发了15个单核苷酸多态(SNP)标记,其中核基因组SNP14个。利用上述野生群体对其多态性的评价,结果表明:最小等位基因频率范围为0.1216～0.4865。14个核基因组SNP的观测杂合度和期望杂合度的范围分别为0.189～0.865和0.217～0.507。经Bonferroni校正后,只有位点HdS82偏离哈迪-温博格平衡(Hardy-Weinberg equilibrium,HWE)。本研究开发的这些多态的分子标记为皱纹盘鲍的种群遗传学、系谱分析、遗传图谱构建和分子标记辅助育种提供有力工具。利用342个SSR标记、11个SNP标记和一个壳色标记,以皱纹盘鲍2个作图家系成功构建了皱纹盘鲍遗传连锁图谱。经过Bonferroni校正之后,整合的皱纹盘鲍连锁图谱含有324个标记,覆盖了18个连锁群,每个连锁群含有的标记数目从6到29个不等,平均每个连锁群上有18.0个标记,图谱总长度为810.32 cM,标记间的平均间隔为2.65 cM,图谱的覆盖率为88.8%。壳色标记被定位到LG9。利用该图谱进行了生长相关性状的QTL定位。在整合图谱上共定位了11个关于壳长、壳宽、全湿重和软体部重相关的QTLs。其中检测得到的壳长、壳宽、全湿重和软体部重相关的QTLs的数目分别为3、2、2和4个。这些QTLs成簇的分布于2个连锁群上,单个QTL可以解释表型方差范围为13.4%～29.1%。对该图谱上所有标记的原始序列进行分析,皱纹盘鲍14个的功能基因被定位在图谱上。这些生长相关性状的QTL及功能基因的定位为我们从事分子标记辅助育种和功能基因的图位克隆提供了参考区间。更多还原

【Abstract】 A total of 301 novel microsatellite markers (SSR) of the Pacific abalone were developed by microsatellite-enriched libraries and Expressed Sequence Tag (EST) database mining. The polymorphisms of 245 markers were characterized by genotyping a wild population comprising 37 unrelated individuals. The result showed the number of allele ranged from two to 18, and the values of expected and observed heterozygosities ranged from 0.053 to 0.873 and from 0.054 to 1.000, respectively. EST database mining was proven to be efficient and low-costly approach to obtaining new microsatellite markers, and microsatellite enrichment libraries construction was efficient and suitable to develop a large number of microsatellite markers.Fifteen single nucleotide polymorphism (SNP) markers were developed by EST database mining and PCR direct sequencing. The PCR Primers were designed by the method of polymerase chain reaction amplification of multiple specific alleles (PMASA). The polymorphisms of 15 markers were assessed with 37 unrelated individuals and the result showed that the minor allele frequency ranged from 0.1216 to 0.4865. For the fourteen nuclear SNPs, the value of observed heterozygosity ranged from 0.189 to 0.865, while the expected heterozygosity ranged from 0.217 to 0.507. Only one locus (HdS82) deviated significantly from Hardy-Weinberg equilibrium after Bonferroni correction.These polymorphic markers presented in this study provide a useful tool for population genetics, pedigree analysis, linkage map construction and marker-assisted selection (MAS) of H. discus hannai.The genetic linkage map of H. discus hannai was constructed using 342 microsatellite markers, 11 SNP markers and one shell color marker. Linkage mapping was performed using two F1 outbred families, and the integrated linkage map was generated by incorporating map information from the two families. The integrated linkage map contained 324 markers covering 810.32 cM with an average spacing of 2.65 cM and 88.8% of genome coverage. The number of linkage groups in the integrated linkage map was 18, which was consistent with the haploid chromosome number of H. discus hannai. The shell color marker was located in LG 9. The detection and location of QTLs were performed based on the composite linkage map. Ten putative QTLs (LOD≥3.8) associated with the growth of Pacific abalone were detected and located in the composite linkage map, among which three QTLs for the shell length, two for the shell width, two for the gross weight and four for the flesh weight, respectively. They were scattered on LG5 and LG6, explaining 13.4% to 29.1% of the trait variation. A total of 14 functional genes were located in the composite linkage map through the molecular markers. The location of functional genes and QTLs associated with the growth would be very useful for molecular marker-assisted selection and map-based cloning of functional genes.更多还原