长牡蛎品质性状和盐度适应的遗传基础研究

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	长牡蛎品质性状和盐度适应的遗传基础研究
	佘智彩
学位类型	博士
导师	张国范
	2015-05-23
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	水产养殖
关键词	长牡蛎 Snp 品质性状盐度适应关联分析
摘要	长牡蛎（Crassostrea gigas），俗称太平洋牡蛎，做为一种重要的经济贝类，在我国及世界范围内被广泛养殖。牡蛎肉中含有丰富的糖原、牛磺酸及其他游离氨基酸（Free amino acids, FAAs），营养丰富，肉味鲜美。但是我国牡蛎人工养殖过程中的遗传管理不善，对种质结构造成一定的负面影响，继而也影响了生产性性状，如大规模死亡频繁发生，产品品质不断下降。这是导致牡蛎养殖产业整体经济效益长期在低位徘徊的重要原因之一。另外，牡蛎生活在潮间带，潮间带环境潮水涨落交替，受降雨和陆地环境影响大，是海洋温盐变化最大的环境类型。虽然牡蛎已进化出了一套自己的环境适应机制，可以适应潮间带复杂多变的环境，具有广温、广盐性等生物学特征，但在夏季若逢久旱无雨，海水温度和盐度都会处于较高的状态，处于此环境中的牡蛎就会因抵抗力下降而爆发多种疾病，从而造成大规模死亡，对经济带来损失。因此，对牡蛎的品质性状及抗性性状育种研究成为迫切需求。然而传统的育种方法周期长、效率低，与之相比，后来发展起来的分子标记辅助选择方法可以大大的缩短育种年限，提高育种效率，并且可以定向的进行选择，目标明确。本研究选定牡蛎糖原和游离氨基酸含量及盐度适应为目标性状，分别利用长牡蛎自然群体和人工群体进行关联分析，鉴定出与牡蛎糖原和游离氨基酸含量以及盐度适应相关的分子标记和基因，以期解析长牡蛎高糖原含量、高游离氨基酸含量和盐度适应的分子机制，从而进一步推动牡蛎品质性状和抗性性状育种研究。本研究的主要内容及结果和结论如下： 1.长牡蛎糖原含量的遗传基础利用一个野生长牡蛎群体（JN）进行候选基因关联分析，并在另外一个独立群体（STG）中利用目标基因重测序和mRNA表达分析的方法对分析结果进行了进一步的验证。通过HRM分析，在90个糖原含量相关候选基因中验证了295个SNPs，其中86个在JN群体144个个体中成功分型。另外，通过目标基因重测序，在STG群体64个个体中获得732个高质量的SNPs并成功分型。通过关联分析鉴定出位于糖原脱支酶（Cg_GD1）上的两个SNPs（Cg_SNP_TY202 和Cg_SNP_3021）和位于糖原磷酸化酶（Cg_GP1）上的一个SNP（Cg_SNP_4）与糖原含量相关联。在Cg_SNP_TY202基因型为TT或TC的个体糖原含量显著高于基因型为CC的个体，且两个糖原含量相关基因在高糖原含量组和低糖原含量组中表达产生分化。 2.长牡蛎游离氨基酸含量的遗传基础利用一个野生长牡蛎群体（JN）进行候选基因关联分析，并在另外一个独立群体（STG）中对关联分析结果进行了进一步的验证。通过对氨基酸代谢通路的总结整理，筛选出代谢通路上的30个关键基因做为关联分析的候选基因。利用HRM分析的方法，对候选基因进行多态性的研究。结果在30个游离氨基酸含量相关候选基因中验证了30个SNPs，其中12个在JN群体72个个体中成功分型。通过关联分析鉴定出位于谷氨酸脱氢酶Cg_GluDe（CGI_10017227）上的SNP位点Cg_SNP_AA41（R(A/G)）与甘氨酸含量显著关联，且该结论在独立群体STG中得到了验证。 3.长牡蛎盐度适应的遗传基础利用性状分化的（适应不同盐度）实验群体进行全基因组关联分析，寻找与长牡蛎盐度适应相关的基因和SNPs。对实验群体进行全基因组重测序及表达谱测序分析，在全基因组范围内检测SNPs和组间差异表达基因，结合SNP等位基因频率分析、群体分化指数（F_ST）分析和表达谱分析三种分析方法鉴定目标性状相关位点。通过对低盐组和对照组的分析，得出可能与长牡蛎低盐适相关的基因集。共24个基因，分为六大类：离子通道/水通道蛋白、转运蛋白、FAA代谢相关基因、抗氧化相关基因、免疫相关基因和化学防御相关基因。其中有氯离子通道蛋白(Chloride channel protein 7)、钾离子电压门控通道蛋白 (Potassium voltage-gated channel protein)、牛磺酸转运蛋白 (Taurine transporter)、凋亡抑制因子 (Apoptosis inhibitor) 等基因。这些基因在组间分化较大，可能在长牡蛎低盐适应过程中起重要作用。通过对高盐组和对照组的分析，得出可能与长牡蛎高盐适相关的基因集，并且富集到了与长牡蛎生长发育调控相关的生物过程 (positive regulation of developmental process, P＜0.05)。富集到的基因集中，基因功能主要集中在转录调控和信号转导两个方面，其次是免疫反应和应激反应，最后是细胞间的相互作用以及细胞生长和分化。其中钙调蛋白 (Calmodulin) 和血凝素 (Ficolin-2) 在群体间分化程度较大，可能在长牡蛎高盐适应过程中起重要作用。人工构建性状分化的验证群体，对其进行不同盐度应激处理后取样，利用其对上述关联分析结果中某些重要基因进行验证，主要从两个水平上进行：1）从DNA水平上利用HRM的方法对基因型进行验证；2）从mRNA水平上利用Q-PCR的方法对基因表达进行验证。结果表明位于凋亡抑制因子 (Apoptosis 1 inhibitor) 上的SNP位点Cg_SNP_SV5、位于氯离子通道蛋白 (Chloride channel protein 7) CDS区的SNP位点Cg_SNP_S1和位于氯离子通道蛋白下游的Cg_SNP_SV34、位于钾离子电压门控通道 (Potassium voltage-gated channel protein) 上游的SNP位点Cg_SNP_SV66和Cg_SNP_S2等位基因频率在低盐组和对照组间均产生很大差异，可能是与长牡蛎低盐适应相关联的重要多态性位点，上述三个基因可能在长牡蛎低盐适应过程中起重要作用。Q-PCR研究表明氯离子通道蛋白在低盐组和对照组间的表达产生分化，且差异达到显著水平（P < 0.01），进一步支持氯离子通道蛋白在长牡蛎低盐适应中起着重要作用。
其他摘要	The Pacific oyster Crassostrea gigas is an important cultivated shellfish thatis rich in nutrients. It contains high levels of glycogen and FAAs, which are of high nutritional value. Besides, oyster is rich in unsaturated fatty acids, vitamins and trace elements and other nutrients too. But the poor genetic management during the long time industrial seedling rearing has a negative impact on the genetic structure and production traits, such asthe frequent occurrence of mortality, the continous decline of quality and so on, which is one of the main causesof the relatively loweconomic benefit of oyster farming industry. In addition, oyster lives in the intertidal zone, in which the environment is complex and changeable, it can be easily affected by rainful and land environment, and has the largest variation of temprature and salinity. Although oyster is eurythermic and euryhaline, and has evolved its own mechanism to adapt the complex and changeable intertidal environment, in summer, the temperature and salinity is easily to rise because of drought, and thus lead to the decrease of resistance and mortality of oyster, bring losses to the economy. Therefore, oyster quality traits breeding and resistance breeding research has become an urgent demand. However, the traditional breeding method is time consumingand inefficient.In contrast, the molecular marker assisted selection method can greatly shorten the breeding period, improve breeding efficiency, and can be targeted to choose. To investigate the genetic basis of this high glycogen and FAAs content, as well as the mechanism of the salinity adaptation, we conducted a candidate gene association study and a genome-wide association study respectively, identifying the polymorphsms associated with glycogen content, FAAs content and salinity adaptation, expecting tointerpretthe mechanism of the high glycogen and FAAs contentand the salinity adaptation, and to promote the process of oyster quality traits breeding and resistance breeding. The main results and conclusions of this research were as follows: 1. Genetic basis of glycogen content of the Pacific oyster We conducted a candidate gene association study in a wild oyster population (JN), and confirmed our results using an independent population (STG),via targeted gene resequencing and mRNA expression analysis. We validated 295 SNPs in the 90 candidate genes surveyed for association with glycogen content, 86 of were ultimately genotyped in all 144 experimental individuals from Jiaonan (JN). In addition, 732 SNPs were genotyped via targeted gene resequencing. Two SNPs (Cg_SNP_TY202 and Cg_SNP_3021) inCg_GD1 (glycogen debranching enzyme) and one SNP (Cg_SNP_4) in Cg_GP1 (glycogen phosphorylase) were identified as being associated with glycogen content. The glycogen content ofindividuals withgenotypes TT and TC in Cg_SNP_TY202 was higer than that of individualswith genotype CC.The transcript abundance of both glycogen-associated genes was differentially expressed in high glycogen content and low glycogen content individuals, which suggests that it is possible that transcript regulation is mediatedby variations of Cg_SNP_TY202, Cg_SNP_3021, and Cg_SNP_4. 2. Genetic basis of FAAs content of the Pacific oyster We conducted a candidate gene association study in a wild oyster population (JN), and confirmed our results using an independent population (STG). We screened 30 genes from the summarized FAAs metabolism pathway as the candidate genes for the association study. We validated 30 SNPs using HRM analysis in the 30 candidate genes surveyed for association with FAAs content, 12 of were ultimately genotyped in all 72 experimental individuals from Jiaonan (JN). Based on the association analysis, one SNP (Cg_SNP_AA41, (R(A/G)) located inCg_GluDe (Glutamate dehydrogenase) was identified as being associated with glycine content, and this was confirmed in STG population. 3. Genetic basis of salinity adaptation of the Pacific oyster We conducted a genome-wide association study using the phenotypic differentiation experimental groups (hypo-salinityadaptationgroup, hyper-salinityadaptationgroup and control group) to search for salinity adaptationassociated genes and SNPs of the Pacific oyster.We performed the whole genome sequencing and the expression profile sequencing analysis, detecting SNPs and the differentially expressed genes between groups, and identified the target trait loci through SNP allele frequency analysis, F_ST analysis and expression analysis. Based on the analysis between the hypo-salinity group and the control group, we got the gene set that might be associated with hypo-salinity adaptation. There were 24 genes in total , and the functions related could be divided into the following six categories: ion/water channel, transporter, FAAs metabolism, ROS related, immune response and chemical defense. Genes such as chloride channel protein 7, potassium voltage-gated channel proteintaurine, transporter and apoptosis inhibitor were greatly differentiated between groups, thus they might play an important role in the hypo-salinity adaptation of oyster. Based on the analysis between the hyper-salinity group and the control group, we got the gene set that might be associated with hyper-salinity adaptation, and the biologycol process positive regulation of developmental process was enriched. Functions of genes enriched in the process were focused on transcriptional regulation and signal transduction, followed by immune response and stress stimulus, andthen were cellinteraction and cell growth and differentiation. Among these genes, calmodulin and ficolin-2 were greatly differentiated between groups, thus they migh play an important role in the hyper-salinity adaptation of oyster. We sampled another three phenotypic differentiation (adapted to different salinity) experimental groups after the different salinity treatment for the confirmation of the results of the association analysis. We conducted the confirmation from two levels: the genotype confirmation in DNA level by HRM analysis and the expression confirmation in mRNA level by Q-PCR. The allele frequncy between groups of Cg_SNP_SV5, located in apoptosis 1 inhibitor, Cg_SNP_S1 and Cg_SNP_SV34, located inand downstream of chloride channel protein 7, Cg_SNP_SV66 and Cg_SNP_S2, located upstream of potassium voltage-gated channel proteintaurine, was significantly different, thus these SNPs may be the important polymorphisms associated with the hypo-salinity adaptation, and the genesrelated may have an important influence on the hypo-salinity adaptation of oyster. In addition, chloride channel protein 7 was differentially expressed between groups, and the difference reached to the significant level (P < 0.01), which further confirmed that chloride channel protein 7may play an important part in the hypo-salinity adaptation of oyster.
学科领域	水产养殖
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/23247
专题	海洋生物技术研发中心
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	佘智彩. 长牡蛎品质性状和盐度适应的遗传基础研究[D]. 北京. 中国科学院大学,2015.

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