文蛤弧菌抗性相关标记/基因的确定及遗传互作效应分析

IOCAS-IR > 实验海洋生物学重点实验室

	文蛤弧菌抗性相关标记/基因的确定及遗传互作效应分析
	聂庆
学位类型	博士
导师	刘保忠
	2015-05-15
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物
关键词	文蛤弧菌抗性微卫星单核苷酸多态性标记-性状关联分析多毛蛋白 Dna分化抑制因子2
其他摘要	文蛤隶属于软体动物门瓣鳃纲，是我国重要的海产经济贝类。然而，频繁发生的病害问题，尤其是弧菌的感染，限制了文蛤养殖业的发展。文蛤生活在开放海域，难以直接通过改善环境条件、隔离病原菌等方式来减少病害的暴发。因此，培育抗性品系是文蛤育种工作中的重要课题。选择育种和杂交育种是文蛤育种中常用的方法，但这些方法存在育种周期长、效率低和易受环境影响等问题。近些年来，分子标记辅助育种技术（MAS）的提出，为文蛤的抗性育种提供了新的思路。由于MAS具有不受基因表达和环境的影响、可在生物发育早期进行选择等优势，将其与传统的育种方法相结合，必将提高文蛤的抗性育种效率。开发可靠的抗性相关分子标记是MAS能够成功应用于文蛤抗性育种工作的前提。本研究开展了文蛤弧菌抗性相关分子标记的开发和验证工作。首先通过弧菌感染实验构建了副溶血弧菌（Vibrio parahaemolyticus）抗性差异群组11-R、11-S及14-R、14-S，和哈氏弧菌（V. harveyi）抗性差异群体09-R、09-C，作为弧菌抗性相关分子标记开发的遗传材料。利用生物信息学方法，分别从文蛤幼虫转录组数据库中挖掘出了有免疫功能注释的contigs，其中包含微卫星标记48个和SNP标记323个，这些标记将作为候选的分子标记应用于后续的关联分析。在弧菌抗性相关微卫星标记的开发中，对48个候选的微卫星标记进行了分型，确认其中有27个标记在2011山东文蛤群体中具有多态性。采用卡方检验进行标记-性状关联分析，在11-R和11-S群组间获得了3个与文蛤弧菌抗性存在极显著相关性的微卫星标记（P < 0.001），即MM959、MM4765和MM8364。而且，该结果也在09-R和09-C群体中得到了证实。多维尺度分析发现，这3个微卫星标记能够区分易感群组11-S中按照存活时间分成的三个亚组。序列功能注释提示，包含这3个微卫星标记的基因可能在文蛤的免疫方面具有重要作用。在弧菌抗性相关SNP标记的开发中，首先利用混合模板对已获得的候选SNPs进行了扩增和测序，获得了66个多态性的SNPs，其中有40个SNPs可成功应用于多重SNaPshot分型。采用卡方检验进行标记-性状关联分析，在11-R和11-S群组中鉴定了8个与弧菌抗性显著相关的SNPs（P < 0.05）。其中，有4个SNPs在09-R和09-C群体中得到了验证，这些标记分别位于contig16077、contig24215和contig34222上。序列功能注释提示，包含这4个SNPs的基因可能在文蛤的免疫方面具有重要作用。针对在11-R和11-S文蛤群组中获得的微卫星和SNP标记的多态信息，采用多因子降维法（MDR）分析了这些标记间的交互作用。结果显示，标记间存在不同程度的协同作用。采用一般线性模型分析了主要的协同作用对文蛤弧菌抗性性状的影响，确认其中有4对微卫星和3对SNPs间的协同作用对文蛤弧菌抗性性状有显著的影响。利用二元逻辑回归模型建立标记与文蛤弧菌抗性性状的回归方程，该回归方程对11-R和11-S群组的个体检出率分别达到了88.6%和88.3%。在获得弧菌抗性相关SNPs的基础上，分析了位于文蛤Hairy基因（MmeHairy）上的2个弧菌抗性相关SNPs（SNP1066和1067）在基因表达中的调控作用。首先利用末端序列扩增技术获得了长为1403bp的Hairy cDNA序列。其ORF长1029bp，可编码342个氨基酸残基。进化树（NJ-tree）显示，MmeHairy属于Hairy蛋白家族。定量分析显示MmeHairy在文蛤的肝胰腺中表达量最高，而且副溶血弧菌感染会导致肝胰腺中MmeHairy的表达量升高。采用SNaPshot分型法对122个文蛤样品进行分型，并按照分型结果将文蛤个体分成不同的组。实时荧光定量检测结果显示，不同基因型所对应的个体中的MmeHairy的表达量存在显著差异（P < 0.05），提示这2个位点可能参与MmeHairy的表达调控。实验克隆了长为1936bp的文蛤DNA分化抑制因子（MmeId2）基因序列，其中包含一个227bp的内含子。通过直接测序的方法，在MmeId2基因上鉴定出了32个SNPs。采用标记-性状关联分析的方法，在09-R和09-C群体中获得5个与哈氏弧菌抗性相关的SNPs，在14-R和14-S群组中获得3个与副溶血弧菌抗性相关的SNPs，两者之间重合的标记是SNP6和SNP15，提示这两个标记与文蛤的弧菌抗性相关。此外，通过连锁不平衡和单体型分析，鉴定了1个与弧菌抗性相关的单体型，即SNP6A/SNP8G/SNP9A。弧菌感染后，肝胰腺中的MmeId2的mRNA和蛋白均呈现上调表达趋势，提示该基因参与了文蛤的弧菌免疫。; The clam Meretrix meretrix belongs to Lamellibranchia, Mollusca, and has broad market prospects. However, the development of M. meretrix aquaculture is limited by disease problems, especially Vibrio infection. Given that M. meretrix live in the open sea, it is difficult to reduce disease outbreaks by isolating the pathogens. Therefore, it is important to cultivate resistant strains in genetic breeding of M. meretrix. Recently, the development of molecular marker-assisted breeding (MAS) provides a new approach for resistance breeding of M. meretrix. Molecular markers are not affected by gene expression and environment, and can be detected in the early biological development. So the combination of MAS and traditional breeding methods will improve the efficiency of resistance breeding of M. meretrix. Development of reliable molecular markers associated with resistance is premise for MAS in resistance breeding. The vibrio-resistance related molecular markers in M. meretrix were developed and validated in the present study. Firstly, the stock materials of M. meretrix with different vibrio-resistance were constructed through vibrio infection experiments (i.e. 09-R, 09-C to Vibrio harveyi; 11-R, 11-S, 14-R and 14-S to V. parahaemolyticus). Secondly, the contigs involved in immune were excavated from M. meretrix transcriptome by bioinformatics methods, which contained 48 SSRs and 323 SNPs. These molecular markers would be applied as candidate markers for subsequent marker-trait association analysis. For the development of SSRs associated with vibrio-resistance, 8% nondenaturing polyacrylamide gels electrophoresis was used for genotyping, and 27 SSRs were verified to be polymorphic in 2011 Shandong M. meretrix population. Chi-square test was used for marker-trait association analysis, and MM959, MM4765 MM8364 were identified to be associated with vibrio-resistance. Multi-dimensional scaling (MDS) analysis showed that clusters generated by genetic similarity revealed by these 3 SSRs were consistent with the subgroups distinguished by survival time in 11-S. The putative functions also suggested that the three SSR-involved genes might play important roles in immunity of M. meretrix. For the development of SNPs associated with vibrio-resistance, Multiplex SNaPshot genotyping method was used and the genotypes of 40 SNPs were obtained in 2011 Shandong M. meretrix population. Chi-square test was applied for marker-trait association analysis, and 8 SNPs were found to be associated with vibrio-resistance trait, among which 4 SNPs were further validated in 09-R and 09-C population. Sequence alignments and annotations indicated that the contigs containing the associated SNPs had high similarity to the immune related genes. The interactions between molecular markers were also analyzed by multifactor dimensionality reduction (MDR), and the results showed that there were different levels of interactions between markers. The general linear model (GLM) indicated the interactions between 4 pairs of SSRs and 3 pairs of SNPs had significant influence on vibrio-resistance trait in M. meretrix. The binary logistic regression model contained both SSRs and SNPs could distinguish 11-R and 11-S groups. The full-length cDNA of MmeHairy was obtained and phylogenetic tree revealed the MmeHairy belongs to the Hairy protein subfamily. Real-time quantitative PCR showed that MmeHairy had the highest expression in hepatopancreas, and the expression of MmeHairy up-regulated in hepatopancreas after V. parahaemolyticus challenge. Quantitative analysis also showed that the MmeHairy mRNA expression levels had significant differences among clams with different genotypes at SNP1066 and 1067 (P < 0.05), which indicated that these two SNPs may affect the expression of MmeHairy. The 1936bp of MmeId2 gene sequence was cloned which contained one 227bp of intron, and 32 SNPs of MmeId2 were detected by direct sequencing. Marker-trait association analysis was applied and 5 V. harveyi resistance related SNPs and 3 V. parahaemolyticus resistance related SNPs were indentified, among which SNP6 and SNP15 were coincident. In addition, linkage disequilibrium and haplotype analysis identified a haplotype associated with vibrio-resistance. Both mRNA and protein of MmeId2 were up-regulated after V. parahaemolyticus infection also indicated MmeId2 was involved in vibiro immune of M. meretrix.
学科领域	主要研究方向:海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/22742
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	聂庆. 文蛤弧菌抗性相关标记/基因的确定及遗传互作效应分析[D]. 北京. 中国科学院大学,2015.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
博士毕业论文-聂庆.docx（4228KB）	学位论文		限制开放	ODC PDDL	浏览