【作者】 杜慧霞；

【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】 1仿刺参的基因组大小测定本实验以栉孔扇贝Chlamys farreri为内标，用流式细胞仪测定了仿刺参Apostichopus japonicus (Selenka,1867)的不同组织（体壁、肌肉、肠道、呼吸树）的基因组大小（C值），结果发现用不同组织测出的仿刺参基因组大小均比较接近，最终得出仿刺参的基因组大小为0.878±0.02pg，即859Mb（以1pg DNA=978Mb计算）。仿刺参基因组大小的测定将有助于该物种基因组方面的研究。2仿刺参的转录组测序和分析为系统了解仿刺参转录组的特征，我们选取了不同发育时期的仿刺参，以及成体仿刺参的不同组织为材料，共构建了8个cDNA测序文库，进行了454GS FLX测序及生物信息学分析。测序共得到1,061,078条平均长度为344bp的序列。经过小片段去除和质量筛查后，共获得92%的高质量序列。这些高质量的序列经过拼接得到33,835条Contigs，以及199,011条Singletons。Contigs进一步拼接成29,666条Isotigs，这些Isotigs聚类成21,071个Isogroups。通过与公共蛋白数据库（Swiss-Prot和nr）及核酸数据库（nt）比对，47%的Isogroups获得了注释信息。对于被注释的基因序列，我们进一步根据基因功能作了分类。Gene Ontology（GO）注释和Kyoto Encyclopedia of Genes and Genomes（KEGG）代谢通路分析结果表明，这些基因参与了多种生物学过程。此外，通过比较分别以夏眠前和夏眠中仿刺参的多个组织构建的两个cDNA文库，还获得了一些与夏眠相关的候选基因。随机选取了12个表达有明显差异的基因进行Q-PCR验证，结果发现在测序中有明显表达差异的基因经Q-PCR验证也有明显差异，说明测序结果比较真实地反应了文库中基因的表达信息。通过比较仿刺参与紫海胆Strongylocentrotus purpuratus的转录组发现，两个物种的GO分布非常相似，同时获得4,882个同源性较高的基因，其中，有202个基因在非棘皮动物未被发现。此外，通过生物信息学预测，共获得了730个SSR和54,185个SNP。仿刺参转录组的获得，将有助于仿刺参遗传学和基因组学研究工作的开展。3仿刺参的遗传图谱构建应用2b-RAD技术，以仿刺参90个F1子代为作图群体，以拟测交理论为作图策略，构建了仿刺参的遗传连锁图谱。研究中，共得到2,010个可以被分型的SNPs，其中符合孟德尔分离比(P≥0.05)的标记有1,484个。构建的图谱包含917个标记，分布于22个连锁群上。每个连锁群标记数目为16-98不等，平均标记数为42，连锁群长度在13.5cM-113.8cM之间，相邻标记的平均间隔为1.64cM。图谱的总长度为1,467cM，覆盖率为94.89%。该图谱为首张基于SNP标记的仿刺参遗传连锁图谱，将为今后仿刺参的遗传学研究如功能基因定位、分子标记辅助育种等提供方便。更多还原

【Abstract】 1. Determination of genome size of Apostichopus japonicusThe C-values of four different tissues (body wall, muscle, intestines and respiratorytrees) of A. japonicus were measured in a flow cytometer using Chlamys farreri as aninternal standard. It was noted that the C-values of different tissues were closebetween one another. The results indicate that the C-value of A. japonicus is0.878±0.02pg with the genome size of859Mb. The results of this study will be helpful infuture genomic study of the sea cucumber.2. Transcriptome sequencing and characterization for the sea cucumber A.japonicusWe performed the large-scale transcriptome profiling and characterization bypyrosequencing diverse cDNA libraries from A. japonicus. In total,1,061,078readswith an average length of344bp were obtained by454sequencing of eight cDNAlibraries representing different developmental stages and adult tissues of A. japonicus.Of all these reads,92%(974,004reads) passed through our quality filters andrepresented high-quality (HQ) reads. The HQ reads were assembled into33,835contigs and199,011singletons. Contigs were then assembled into29,666isotigs,which were further clustered into21,071isogroups. About47%of the isogroupsshowed significant matches to known proteins or nucleotide based on sequencesimilarity. As determined by Gene ontology (GO) annotation and Kyoto Encyclopediaof Genes and Genomes (KEGG) pathway mapping, functional annotation of theunigenes recovered diverse biological functions and processes. Candidate genes that were potentially involved in aestivation were identified. Twelve candidate DGEs wererandomly selected for Q-PCR validation. Q-PCR results verified the differentialexpression of these genes between the active and aestivating sea cucumbers.Transcriptome comparison with the sea urchin Strongylocentrotus purpuratusrevealed similar patterns of GO term representation. In addition,4,882putativeorthologous genes were identified, of which202were not present in thenon-echinoderm organisms. More than700simple sequence repeats (SSRs) and54,000single nucleotide polymorphisms (SNPs) were detected in the A. japonicustranscriptome.This transcriptome resource would lay an important foundation forfuture genetic or genomic studies on this species.3. Construction of SNP-based linkage map for A. japonicusLinkage mapping was performed using an F1family of90A. japonicus individuals,and a genetic linkage map was constructed using a2b-RAD method. Two thousandsand ten SNP markers were genotyped and1,484markers that conformed to theexpected Mendelian ratios (P≥0.05) were included in the linkage analysis. The geneticlinkage map contained917markers ordered on22linkage groups and spanned1,467cM, with an average intermarker spacing of1.64cM and94.89%of genome coverage.The size of the linkage groups ranged from13.5cM to113.8cM, and the number ofmarkers per linkage group varied from16to98, with an average number of42. Thisis the first SNP-based linkage map of A. japonicus and will be useful for furthergenetic analyses such as gene mapping and molecular marker-assisted selection(MAS).更多还原