【作者】 鄢婧婧；

【导师】 包振民； 胡景杰；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2013， 博士

【摘要】 仿刺参主要分布于我国北方，日本，朝鲜半岛及俄罗斯远东地区，由于其具有较高的食用和保健价值，成为我国海水养殖的重要物种。为了实现仿刺参养殖业稳定发展，选择快速生长及抗病性强的刺参新品种是迫切需要的。分子标记辅助选择（Marker-assisted selection，MAS）能加快育种进程，促进刺参遗传改良和新品种的培育。本研究探讨了仿刺参分子标记的有效筛选方法及其在仿刺参分子育种中的应用。1仿刺参分子标记的筛查及标记开发本文利用仿刺参EST文库的序列共得到含有微卫星的序列730条（2.1%）。将所得序列独立的212条微卫星序列设计引物，成功扩增率为78.8%。成功扩增的位点在一个群体的48个个体中检测多态性并利用GENE-POP4.0统计主要遗传学参数，结果显示137对具有不同程度的多态性，多态比例为82%。等位基因数为2-7个，平均等位基因3.3个。群体观测杂合度和期望杂合度分别为0.0667-0.6670，0.1769-0.7200。经过Bonferroni校正之后26个标记偏离哈迪温伯格平衡。经过基因注释(BLASTX比对)显示，共有12个标记有注释信息，分别位于不同的12个已知基因。另外通过转录组测序共获得54,185个候选SNP位点。利用高分辨率溶解曲线技术（High Resolution Melting，HRM）技术对其中678个候选SNP进行标记的筛查并在群体的48个个体中进行主要遗传学参数的估计。结果显示249个为多态位点。群体观测杂合度Ho和预期杂合度He的分布范围分别为0.050-0.833和0.073-0.907。33个位点偏离哈代-温伯格平衡。经过基因注释显示，共有70个标记有注释信息。这些来源于EST的分子标记为将来仿刺参遗传连锁图谱的构建提供基础。2基于微卫星标记的仿刺参家系鉴定本研究利用软件模拟及实际混养家系分析，建立了一套可以用于仿刺参家系鉴定的微卫星分子标记系统。我们利用12个微卫星标记来模拟排除率，结果显示9个标记的排除率是和12个标记相当的，均达到99%以上。并用9个微卫星标记在九个已区分家系的180个幼虫中进行评价。最终所有幼虫均准确地分到各自的家系。说明这个标记组合足以达到区分个体的目的。我们又利用9个微卫星标记在混合家系的1000头仿刺参进行家系鉴定，最终共有810头仿刺参确定了各自的亲本，混养家系的鉴定率81.0%。3仿刺参遗传图谱的构建及QTL定位本文采用两个F1作图群体均是根据拟测交策略构建遗传连锁图谱。利用的微卫星标记313个，SNP标记249个。我们分别构建雌雄连锁图谱，连锁群数目均为22，与最近报道的仿刺参单倍染色体数目一致。雌性图谱重组率显著比雄性图谱的高(G检验P<0.01)。家系间重组率的比较通过连锁群之间共享标记间隔的差异来显示，结果表明，大部分标记是相似的，但是也有差异显著的连锁群如（LG2-M， LG11-M，LG9-F， L13-F，L18-F）。性别平均图谱总共含有215个标记（121个微卫星和94个SNP），图谱总长为1498.9cM，平均密度为7.0cM。连锁群长度为44.4cM到121.4cM不等，连锁群标记数目为5-18个不等。雌雄性遗传连锁图谱的基因组覆盖率分别为74.8和72.8%，整合连锁图谱的图谱覆盖率为79.2%。所有EST来源的分子标记进行基因注释，结果显示16个包含标记的EST序列与已知功能的基因同源，而其他的没有注释信息。并对其进行Tblastx比对，结果显示在134条比对序列中，24条序列（17.9%）在海胆海参中同源(e <10-7)。采用两个家系作为材料，以构建的遗传连锁图谱为基础，对仿刺参体重性状进行QTL定位，但无明显结果，可能是表型测量误差较大引起的。更多还原

【Abstract】 The sea cucumber, Apostichopus japonicus, is a common species distributed inshallow waters along northern Asian coasts in China, Japan, Russia and Korea, and isregarded as one of the most valuable aquaculture species due to its potential as adelicacy and traditional medicine. In order to achieve sustainable development of A.japonicus culture industry, selection of bloodstocks with the desired traits, such asrapid growth and disease resistance are urgently needed. It is generally accepted thatmarker-assisted selection (MAS) could provide valuable information to efficientlyimprove desirable traits and accelerate progress in selective breeding. In the presentstudy, we developed and characterized a panel of SSR (Simple Sequence Repeat) andSNP (Single Nucleotide Polymorphism) markers, and also studied their application inparentage assignment and genetic mapping of A.japonicus.1. SSR and SNP mining and marker development for A.japonicusEST (Expressed Sequence Tags) database mining was used to develop novelmicrosatellite markers for A. japonicus. A total of730ESTs containing SSRs wereidentified. Primers were designed for212EST-SSRs according to the flankingsequences using Primer Premier5program. Polymorphism of these loci wereevaluated in48individuals collected from the coast of Shandong province, China.The results showed that expected products were amplified with167of the212primerpairs, of which137loci were polymorphic. The number of alleles ranged from2to7,and the observed and expected heterozygosities varied from0.0667to0.6670andfrom0.1769to0.7200, respectively. Twenty-six of the137loci significantly deviatedfrom the Hardy-Weinberg equilibrium after Bonferroni correction. Gene annotation(BLASTX comparison, e-value threshold of1e-6) indicated that twelve of137polymorphic SSR-bearing ESTs matched to genes with known functions, while otherseither have no significant matches or matched to uncharacterized genes. Throughanalyzing the A. japonicus transcriptome, a large number of putative SNPs were previously identified, among which678were selected for marker development usinghigh-resolution melting (HRM) method. In the analyzed population,249SNP lociwere polymorphic. The observed and expected heterozygosities varied from0.050to0.833and from0.073to0.907, respectively. Thirty-three loci significantly deviatedfrom the Hardy-Weinberg equilibrium. Seventy of249polymorphic SNP-bearingESTs matched to genes with known functions.2Application of SSR markers in parentage assignment of the sea cucumberA.japonicusWe first conducted computer simulation analyses using the developed EST-SSRmarkers to establish guidelines for effective parentage analysis. Real mixed familieswere also utilized for evaluation of the performance of SSR-based parentage analysisin practice. Simulation results demonstrated that the exclusion probability of theselected nine microsatellite markers was0.99which was still satisfactory comparedwith12microsatellites. To evaluate the performance of parentage analysis in a realcase, a total of180larvae from distinguished nine families were genotyped for thepreviously-selected9microsatellite loci. Eventually all the larvae were accuratelyassigned to each family. Then1000offsprings from mixed families were assigned forparentage assignment for9microsatellite loci. The exclusion probability of progenyto their parental couple was81.0%.3Construction of genetic linkage map and identification of quantitative trait loci forthe sea cucumber, A.japonicusWe constructed the genetic linkage map in two F1outbred families of A.japonicususing pseudo-testcross strategy. The sex-specific linkage maps for two mappingfamilies were constructed separately using313microsatellites and249SNPs.22linkage groups (LGs) were identified, which were consistent with the haploidchromosome number of A. japonicus. A significantly higher recombination rate wasobserved in the female map. Significant family-specific differences in recombinationratios were also observed for some LGs between two female maps and two male mapssuch as（LG2-M， LG11-M，LG9-F， L13-F，L18-F）. The sex-averaged mapconsisted of215markers (121microsatellite markers and94SNPs) covering1498.9 cM with an average inter-marker spacing of7.0cM. The length of the linkage groupsranged from44.4cM to121.4cM and the number of markers varied from5to18perlinkage group. On the basis of the expected genome lengths, genome coverage for theconsensus, female, male maps including the triplets was79.2,74.8and72.8%. Geneannotation (BLASTX comparison, e-value threshold of1e-7) indicated that16ESTscontaining markers in maps matched to genes with known functions, while otherseither have no significant matches or matched to uncharacterized genes. Tblastxcomparisons revealed that24hits of the134blasted sequences were significantlyconserved between A. japonicus and sea urchin. Preliminary QTL mapping wereconducted for the growth traits of A.japonicus but no significant results have beendetected. It is possibly due to phenotypic errors caused by measurement.更多还原