IOCAS-IR  > 海洋生物技术研发中心
海湾扇贝主要经济性状的遗传解析
杜雪地1,2
学位类型博士
导师张国范 ; 李莉
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
关键词海湾扇贝 生长 高温耐受性 营养品质 关联分析 Qtl定位
摘要
海湾扇贝原产于美国东海岸和墨西哥湾,有四个地理亚种。20世纪80年代和90年代,北方亚种和南方亚种先后引种到中国。海湾扇贝引种和人工育苗成功曾引领了我国海水养殖的一次浪潮。利用选择育种的方法,育种学家成功培育出“中科红”和“中科2号”两个海湾扇贝养殖新品种,有效缓解了养殖户对良种的需求。然而,传统选择育种周期长,而且难以对不能活体测量的性状开展选择。深入研究重要经济性状的遗传基础,发展标记辅助育种和基因组设计育种是实现海湾扇贝养殖业可持续发展的必经之路。在本研究中,我们对海湾扇贝北方亚种和南方亚种分别进行了转录组测序,筛选了大量的基因区的SNP标记;利用候选基因关联分析法对海湾扇贝高温耐受性进行了关联分析,鉴定到一个与高温耐受显著相关的SNP位点;以中科红自交家系的子代为材料开展了海湾扇贝基因组调查;利用精简基因组测序技术构建了海湾扇贝遗传图,并对成贝规格和闭壳肌中EPA与DHA含量性状进行了QTL定位;以自交分离家系的表型极端个体为材料研究了闭壳肌中类胡萝卜素性状的遗传基础。主要结论如下:
1. 海湾扇贝转录组测序的总体分析:
组装获得了海湾扇贝82,267条unigene序列,通过与Nr、SWISSpro数据库比对共到32,595条unigene的注释信息。对海湾扇贝北方亚种和南方亚种进行SNP筛选,分别得到32,205和23,312个SNP。对两个亚种的SNP进行比较,进一步将SNP分为4类:南北亚种共有、北方亚种特有、南方亚种特有和亚种间特有。随机挑选了36个SNP进行了HRM验证,其中31个具有多态性,表明预测的SNP具有很高的准确性。对亚种特有的SNP所在的基因进行KEGG通路富集分析,发现在与能量代谢和与环境因子互作的两个通路上显著富集,这表明两个亚种在这两个通路上分化程度更高。GO富集分析的结果与KEGG富集分析结果一致。
2. 候选基因高温耐受性关联分析
在海湾扇贝南北亚种杂交的基础上,进行杂交贝的小群繁育得到两个群体(杂交贝与杂交贝杂交得到ZZ,杂交贝与北方亚种杂交得到ZN)。与北方亚种小群繁育的群体相比,ZZ和ZN具有更高的高温耐受性,但同时群体内也表现出极大的个体间差异。在ZZ和ZN群体中分别挑选热敏感的48只和高温耐受的48只组成两个关联群体,ZZ96 和ZN96。在399个候选基因中开发SNP并在关联群体中分型,关联分析发现SNP all-53308-760 T/C与海湾扇贝高温耐受性极显著相关,且等位基因C不利于海湾扇贝抗热。基因表达定量研究显示,该基因的不同基因型间表达差异显著,且等位基因C的表达强度达到等位基因T的四倍,推测该基因的表达量与海湾扇贝抗热性呈负相关。
3. 海湾扇贝基因组调查
构建了180 bp和500 bp两种插入片段的文库,使用Illuminar Hiseq2000平台进行测序,共获得94 GB双端测序reads。K-mer分析估计海湾扇贝基因组约1.2G,比已全基因组测序的栉孔扇贝和虾夷扇贝稍小;估计的基因组杂合度较低,仅有0.3%。经从头组装得到scaffold序列217,310条,N50达6.8k,总长700M,覆盖基因组的58.3%。
4.遗传图谱构建及主要经济性状的QTL定位
采用EcoRI-MspI双酶切双barcode策略构建了海湾扇贝作图家系两个亲本及180个子代的精简基因组文库,利用Hiseq2000平台进行2×100 bp测序。分析测序数据共获得7,047个可用于作图的SNP标记,但卡发检验发现大量的SNP标记发生偏分离。筛选符合孟德尔分离比的SNP标记,利用拟测交策略构建了海湾扇贝雌性和雄性图谱,其中雄性图谱总长1426.947 cM,包含654个标记,平均标记间距2.237cM,雌性图谱总长877.455 cM,包含115个标记,平均标记间距8.954cM。
对全湿重、闭壳肌重和出柱率三个生长相关性状及EPA和DHA含量两个营养品质性状进行QTL定位,得到了18个QTL,其中出柱率的两个QTL与闭壳肌重完全重叠而全湿重与闭壳肌重和EPA与DHA分别有一个QTL重叠。全湿重2个QTL解释19.9%的表型变异,闭壳肌重2个QTL可解释25.6%的表型变异,出柱率两个QTL共解释20.4%的表型变异,EPA6个QTL共可解释77.4%的表型变异,DHA6个QTL共可解释61.6%的表型变异。
5. 自交系内闭壳肌中类胡萝卜素含量性状的分析
在海湾扇贝自交系发现闭壳肌颜色性状呈“白色:黄色=3:1”孟德尔分离比,推测该性状为受单基因控制的质量性状,且黄色闭壳肌为隐性。利用nanodrop对色素抽提液进行连续扫描,发现在455 nm为最高吸收峰,与虾夷扇贝和华贵栉孔扇贝中报道的类胡萝卜素吸收峰接近。通过对自交系亲本及白色闭壳肌和黄色闭壳肌个体进行精简基因组测序,在21,300个SNP中筛选到23个SNP与该性状相关。
其他摘要The bay scallop, Argopecten irradians, is naturally distributed along the eastern coast of the United States and the Gulf of Mexico, and it includes four subspecies: A. i. irradians (Lamarck), A. i. concentricus (Say), A. i. amplicostatus (Dall) and A. i. taylorae (Petuch). It was introduced into China in the 1980s and 1990s, and are now major aqualculture molluscs in China. Two varieties have been cultiviated by selective breeding, and played important roles in the bay scallop industry. However, traditional selective breeding suffers from long term of investment and is not suitable for traits that can’t be measured easily. Marker assisted breeding, which relies on the linkage disequilibrium of genes controling trait and molecullar markers is of great application value in the fulture. Thus, in this study, we mainly focused on the molecullar basis of key economic traits including heat tolerance, growth and nutritional content, aimming to identify functional markers for breeding.
1.      Transcriptome sequecing for genic SNP identification.
RNA from six tissues of 67 and 42 individuals of northern and southern bay scallops, respectively, were used and 55.5 and 34.9 million raw reads were generated, respectively. There were 82,267 unigenes produced in total, of which 32,595 were annotated. Altogether, 32,206 and 23,312 high-quality SNPs were identified for northern and southern bay scallops, respectively. SNPs of the two subspecies were compared and classified into four classes: shared SNPs, northern specific, southern specific and inter-specific. A subset of the shared SNPs were selected randomly for pridiction virification, and over 86% were confirmed polymophic. KEGG enrichment analysis was carried out for the subspecies specific SNPs, and significant inrichment was detected in the pathways related to energy metabolism and enviroment interaction.
2.      Candidate gene based association on heat tolerance.
Based on the inter-cross of the northern and southern bay scallops, two populations, ZZ and ZN, were produced. To compare the heat tolerance capacity of ZZ, ZN and northern bay scallops NN, chronic heat stress exprement was carried out. The experiment showed that the ZZ and ZN which containing the genetic background of southern subspecies were significantly more heat resistant than NN. Individuals showed exterme heat sensitive and heat resistant from the ZZ an ZN populations were collected and made up two association populations for heat tolerance. According to literature and allelic imbalance analysis of the shared SNPs, 399 SNPs were developed and genotyped in the two association populations followed by case-control association analysis. One SNP, all-53308-760 T/C very significantly associated with heat tolerance was identified, with allele C being disadvantage for heat tolerance. Quantitative expression analysis showed that the expression level of this gene was negatively correlated with heat tolerance capacity, and that allele C and allele T in the heterozygous individuals was differently transcribed
3.      Genome survey of the bay scallop
Genome survey is of great importance to get the fundamantal imformation of the genome of a species. Zhongkehong was the et al. 1 cultivated veriaty of bay scallop, and have passed over ten years. It was thought that the genome of zhongkehong is of low heterozygosity, and thus is suitable material for genome sequencing in the fulture. To confirm our hypothesis, a self family was established and the DNA of a progeny was used for library construction and next generation sequencing on Hiseq 2000 platform. Totally, 94 GB paired end reads were obtained. K-mer analysis showed the genome is about 1.2 G in size and the heterozygosity is about 0.3%, which is quite low for marine animals. Beside that, denovo assembly produced 217,310 scaffold sequences with an N50 size of 6.8 kb.
4.      Linkage map construction and QTL mapping for growth and nutritional traits
Double-digest double-barcode genotyping by sequencing method was used for SNP development and genotyping in the mapping family, and 7,047 SNPs genptyped in more than 80% of the peogenies were obtained. Chi-squere test was conducted and most SNPs were found to be distorted in segregation. Male- and female-genetic map were constrcted respectively, using the so called pseudo-testcross strategy. The male-map was 1426.947 cM in length and contained 654 SNPs with an average marker distance of 2.237 cM while the female-map was 877.455 cM in length and contained 115 SNPs with an average marker distance of 8.954 cM. QTL mapping was carried out for total weight, muscle weight, cutability, EPA and DHA content, and 18 QTLs were identified in total with each explaining about 10% of the phenotypic variation.
5.      Bulked segregation analysis of carotenoid content in self family
Mendelian segregation of the carotenoid content was observed in one self family, with the high content being a ressesive trait. To illustrate which gene control the mendelian trait, reduced representation sequencing using barcode strategy was carried out for the parent, the low content progenies and the high content progenies. Totally, 21,300 SNPs were identified, among which 23 SNPs were homozygous in the high content progenies and the allele frequency difference between low- and high-content progenies was greater than 0.5. These 23 SNPs were thought in linkage disequilibrium with the gene controlling the trait.
学科领域海洋生物学 ; 海洋生物技术
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/22786
专题海洋生物技术研发中心
作者单位1.中国科学院海洋研究所
2.中国科学院大学
第一作者单位中国科学院海洋研究所
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杜雪地. 海湾扇贝主要经济性状的遗传解析[D]. 北京. 中国科学院大学,2015.
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