【作者】 代平；

【导师】 刘保忠；

【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， 2014， 博士

【摘要】 文蛤作为我国重要经济贝类之一，其养殖产业的快速发展对优质苗种资源的需求日益迫切。提高养殖产量和效益一直是文蛤养殖业的焦点，这要求我们选育高产、抗逆并且稳定遗传的文蛤良种。本研究利用开发的微卫星标记，结合传统的选择和杂交手段，对文蛤不同选育群体进行了遗传测定，另外开发了与生长性状相关的SNP标记，以期为文蛤新品系培育提供基础数据和理论依据，推动文蛤养殖业健康持续的发展。利用文蛤野生群体构建了一批家系，从中选取3个家系（P1、P2、P3）按3×3完全双列杂交设计得到9个组合。利用8个微卫星标记对9个组合进行亲权鉴定，统计各个组合中亲本对子代的贡献率。结果显示，同一个家系来源的不同亲本间的子代贡献率差异很大，同一个亲本在不同组合中的贡献率也明显不同。根据亲本贡献率估算了有效亲本数量（Ne），发现所有组合的有效亲本数量都有所偏小，Ne/N值从0.58到0.86不等。另外，大部分组合的子代遗传多样性较其亲本都有所下降。从9个组合中选取3个高存活率的杂交组合和3个近交组合分别构成对照组和近交组，利用8个微卫星标记评估它们的遗传多样性。结果显示，与对照组相比，近交组的平均等位基因数（Na）下降19.4%，平均观测杂合度（Ho）和期望杂合度（He）分别下降20.8%和9.3%。近交组的平均近交系数（FIS）显著高于对照组以及野生群体。在12月龄时，近交组在壳长、壳高、壳宽和湿重4个性状上的近交衰退系数分别为0.803、0.747、0.826和2.073。另外，通过研究家系P1和其子代组合P1×P1发现，在繁育过程中出现了严重的遗传漂变现象。模拟分析显示，较大的亲本数量可以控制闭锁群体杂合度的降低。将9个组合的子代分别养殖在室内池和室外池两种环境中，测量其12月龄时的生长数据并利用一般线性模型进行方差分析，结果显示存在显著的组合效应、环境效应以及组合环境互作效应。配合力分析显示，3个家系中P2在生长性状上的一般配合力（GCA）最高，P1和P3组合的特殊配合力（SCA）最高，而且组合间的反交效应显著。另外，计算了在单个环境内和综合环境间6个杂交组合的杂种优势（MPH）。结果显示，有半数的杂交组合存在有利的杂种优势（>1%）。基于文蛤EST数据库和生物信息学的方法预测了一批EST-SNP标记，经混合测序验证后，将确认的16个SNPs通过SNaPshot的方法在一个生长优势群体和一个对照群体中分型，利用标记-性状关联分析鉴定出7个与生长性状显著相关的SNPs（SNPg3、SNPg4、SNPg5、SNPg6、SNPg8、SNPg9和SNPg16）（P<0.05），又利用一个独立群体进行双向选择性分型验证了其中3个标记（SNPg5、SNPg6和SNPg9）与生长性状的相关性。这是首次在文蛤中进行生长相关EST-SNP标记的批量开发，为文蛤标记辅助选育提供了有价值的信息。利用生长差异的群体材料，开展了重要功能基因多态性与文蛤生长的关联分析。从文蛤中克隆到一个长链酯酰辅酶A合成酶基因（MmeACSL1），全长1867bp，编码475个氨基酸。利用Real-time PCR检测了在不同营养条件下文蛤该基因的表达，发现MmeACSL1在禁食条件下的表达水平显著高于正常进食下的表达水平（P <0.05），提示其参与了文蛤的能量代谢。通过生物信息学预测和直接测序，在此基因中发现2个外显子SNPs和6个内含子SNPs。基于标记-性状关联分析，从这些SNPs中鉴定出5个显著生长相关的SNPs（mmACSLe-2、mmACSLi-2、mmACSLi-3、mmACSLi-5和mmACSLi-4）（P <0.05），而且它们所构建的单倍型也与生长性状显著相关（P <0.05），表明MmeACSL1在文蛤能量代谢中具有重要作用，并与文蛤生长存在密切联系。克隆获得了文蛤细胞周期蛋白依赖性激酶基因（CDK10），cDNA全长1854bp，编码390个氨基酸。此基因与哺乳动物中CDK10基因的同源性最高，将其命名为MmeCDK10。通过对该基因测序，发现5个外显子SNPs和5个内含子SNPs。利用两个生长差异显著的群体对这些SNPs进行标记-性状关联分析，发现4个外显子SNPs（mmCDKe-1、mmCDKe-3、mmCDKe-4和mmCDKe-5）和1个内含子SNP（mmCDKi-1）与生长性状显著相关（P <0.05），提示MmeCDK10参与了文蛤的生长。更多还原

【Abstract】 The clam Meretrix meretrix is one of the most important commercial mollusks inChina. With the development of its artificial culture, the demand for quality seedresources increasingly urgent. High yield and benefit have been the focus of itsaquculture, which requires us to raise improved seeds with high-yield, resistance andgenetic stability. In the present study, with the microsatellite markers and thetraditional methods of selection and hybridization, we conducted genetic test fordifferent populations of M. meretrix. And we further developed SNP markersassociated with growth traits. These are expected to provide basic data and theory forbreeding new strains and promote the sustainable and healthy development of clamaquaculture.A natural population of M. meretrix was used to found a number of families, fromwhich three (P1, P2, P3) were chosen to set a complete3×3diallel cross comprisingnine crosses. Eight microsatellite markers were used for parentage analysis in the ninecrosses. Estimation of parental contributions to offspring showed that thecontributions of different sires or dams from one family to offspring had greatdifference and the contributions of the same parent to offspring in different crosseswere markedly unequal. Effective parent number (Ne) was calculated based onparental contribution. It was found that there was a significant reduction of Nein allthe crosses. The Ne/N ratio for the nine crosses ranged from0.58to0.86. In addition,the genetic diversity of all the parents was some degree higher than that of theirrespective offspring in most crosses.Three outbred crosses with high survival rate and three inbred crosses wererespectively chosen from the nine crosses to establish a control group and an inbredgroup. After evaluated by eight microsatellite markers, genetic variability of the inbred group diminished relative to the control group, as evidenced by drop of19.4%,20.8%and9.3%on average number of alleles, observed and expected heterozygosity,respectively. The inbred line had a higher average FISvalue than the control group andnatural population. The estimated inbreeding depression coefficients for shell length,shell height, shell width and whole body weight at12months post-fertilization were0.803,0.747,0.826and2.073per10%increase in FIS. Investigation of familyP1and its progeny cross P1×P1indicated that a serious genetic drift occurred in thebreeding. Simulation results showed that a large Necould control reduction ofheterozygosity in a closed population.Progeny of the nine crosses were reared in two environments, indoor ponds andopen-air ponds. After cultured for12months, the growth data were measured and ageneral linear model was used for analysis of variance. The results indicated highlysignificant cross effects, environment effects and the interaction of cross byenvironment. Analysis of combining ability showed that P2was the top combineramong the three parental families for growth traits, the cross of P1and P3had thehighest specific combing ability (SCA) and there were significant reciprocal effects.In addition, Mid-parent heterosis (MPH) was estimated for the six crossbredcombinations in individual environment and over environments. The results showedthat about half of the crossbred combinations had favorable MPH (>1%).Based on EST database of M. meretrix and bioinformatic methods, a batch ofEST-SNPs were predicted, and then validated by pooled sequencing. A total of16confirmed SNPs were genotyped with SNaPshot assay between a fast-growingpopulation and a control population. A marker-trait association analysis identifiedseven SNPs (SNPg3, SNPg4, SNPg5, SNPg6, SNPg8, SNPg9and SNPg16)significantly associated with growth (P <0.05). After bidirectional selectivegenotyping in an independent population, three SNPs (SNPg5, SNPg6and SNPg9) ofthe seven markers were further confirmed to be associated (P <0.05). This is the firstreport on identification of a batch of growth-associated SNPs in M. meretrix, whichprovides valuable information for marker-assisted selection for this species. Based on two populations with significant difference in growth, association studyof SNPs in important functional genes with the growth traits of M. meretrix was made.A long-chain fatty acyl-CoA ligase gene (MmeACSL1) was cloned in M. meretrix,with a full-length cDNA of1876bp encoding475amino acids. Examination ofMmeACSL1gene expression under different nutrient conditions by Real-time PCRshowed that the expression of MmeACSL1gene under fasting condition had asignificant increase compared with that under feeding condition (P <0.05), whichsuggested that MmeACSL1was involved in energy metabolism of M. meretrix. Bymeans of bioinformatic prediction and direct sequencing, two exon SNPs and sixintron SNPs were detected in this gene. A marker-trait association analysis identifiedfive SNPs (mmACSLe-2, mmACSLi-2, mmACSLi-3, mmACSLi-5and mmACSLi-4)significantly associated with growth (P <0.05), and haplotypes comprising these fiveSNPs were also significantly growth-associated (P <0.05), which implied theimportant role of MmeACSL1in energy metabolism and its association with thegrowth of M. meretrix.A cyclin-dependent kinase (CDK) gene was cloned in M. meretrix, with afull-length cDNA of1854bp, which encoded390amino acids. This gene showed thehighest homology with CDK10in mammals, so it was named MmeCDK10. Directsequencing of this gene detected five exon SNPs and five intron SNPs, from whichfour exon SNPs (mmCDKe-1, mmCDKe-3, mmCDKe-4and mmCDKe-5) and oneintron SNP (mmCDKi-1) were found to be significantly associated with growth traits(P <0.05), by a marker-trait association analysis between two populations withsignificant difference in growt, which suggested that MmeCDK10was involved in thegrowth of this clam.更多还原