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海湾扇贝壳色性状遗传分析
其他题名Genetic analysis of shell color trait in the bay scallop Argopecten irradians
腾文
学位类型硕士
导师阙华勇
2017-05-20
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物工程
关键词海湾扇贝 壳色 Snp分子标记 简化基因组 转录组
摘要海湾扇贝源起于美国东海岸沿线,在二十世纪八十年代到九十年代,其南北方亚种被引进我国,一经推广,迅速成为我国重要的海水养殖贝类,成功掀起了一次海水养殖浪潮。为了更进一步提高提养殖产量,提升产品质量,中国育种学家们采用“壳色——生长性状协同选择”的创新育种方法,成功培育出新品种“中科红”(登录号:GS-01-004-2006),并于2007年通过了全国水产原种和良种审定委员会的新品种审定(张国范和郑怀平, 2009)。该新品种不仅存活率提高,生长速度加快,出柱率增加,且拥有醒目的壳色标记,左右壳均为纯正的橙色。为了揭示其橙色遗传机制,为海湾扇贝的育种工作提供理论指导,同时推进海洋贝类壳色研究,在本研究中,采用橙色壳海湾扇贝三个自交家系(来自“中科红”)和白壳色三个自交家系,利用简化基因组技术(2b-RAD)开发SNP分子标签;另外,使用“中科红”一个自交家系,通过转录组测序技术分析成色前后两个阶段的样品差异表达情况,筛查参与海湾扇贝贝壳成色(橙色)过程候选基因。主要结论如下:
1.海湾扇贝简化基因组测序分析开发SNP分子标记
样品来自于橙色壳(“中科红”)三个自交家系子二代和白色壳三个自交家系子二代,每个家系10个个体,构建文库60个,使用Illumina Hiseq2500平台测序,每个文库获得平均unique标签数目101,413,个体平均测序深度为52X。个体的测序深度能够达到准确分型的标准,SNP标记分型分析得到位点数44,286,通过关联分析发现14个SNP位点与海湾扇贝橙色壳紧密关联。最后,在养殖大群中随机挑取橙色壳和白色壳海湾扇贝各50个个体,使用SNapShot技术进行后期验证,发现有5个位点在80%以上个体中,其验证结果同实验结果一致,它们是ref-2988-22、ref-125669-21、ref-40155-24、ref-40155-26和ref-82377-26。据此认为以上5个SNP位点与海湾扇贝壳色(橙色和白色)这一表型紧密关联。
2.海湾扇贝转录组测序分析筛查成色相关候选基因
以一个自交了11代的 “中科红”家系作为实验材料,在其贝壳成色前后两阶段分别取三个重复样,构建了六个(2×3重复)文库,利用Illumina HiSeq2500平台进行双端测序,得到了289,839,646 条reads和70,929个转录本。在SWISSPROT, NR和KOG数据库中比对,最终成功注释到30,896个基因。KEGG和GO 注释,富集到了许多与生物矿化和成色相关的基因。在差异表达分析中,我们发现了黑色素和微量金属元素等相关基因在海湾扇贝贝壳成色后显著上调表达。利用电感耦合等离子体原子发射光谱法(ICP-AES)对海湾扇贝橙色壳和白色壳棱柱层微量金属(Cu, Fe, Mn, Zn)含量进行测定,发现两种壳色的贝壳之间没有显著差异,据此猜测卟啉代谢等相关基因没有参与贝壳的成色过程,而黑色素的作用需进一步实验加以阐释。
 
关键词:海湾扇贝,壳色,SNP分子标记,简化基因组,转录组
其他摘要Historically, bay scallop (Argopecten irradians) was widely distributed in the east coast of the United States and the Gulf of Mexico. In 1980s and 1990s, Argopecten irradians irradians and Argopecten irradians concentricus, both are subspecies of Argopecten irradians, were introduced to China. With the efforts of Chinese scientists from the Institute of Oceanology Chinese Academy of Sciences, bay scallop was cultivated as one of the most important sea products in China and have provoked a boom in mariculture. In order to ramp up production and improve the quality, Chinese researchers cultivated a new bay scallop strain named “Zhongkehong” with wonderful performance. “Zhongkehong” not only has improved survival, growth rate and meat yield, but also has pure orange shell color. In order to understand the genetic control and molecular mechanism of this orange shell color of A. irradians, this study was performed. We employed two main explore methods and one is streamlined restriction site-associated DNA genotyping for genic SNP identification and the other one was transcriptome sequencing for candidate genes associated with orange shell color. The main results were as follows:
1.      Streamlined restriction site-associated DNA genotyping for genic SNP identification
Three self-bred “zhongkehong” family, and three self-bred A. irradians with white shell color (both the lef and right shell) were set up. Genomic DNA from ten individuals of the F2 generation from each family was equally pooled and the restriction-site associated DNA sequencing approach, 2b-RAD, was used to generate genome-wide single nucleotide polymorphism (SNP) genotypes for all samples. We identified 14 SNPs associated to orange shell color of A. irradians. To obtain more gene sequences, these 2b-RAD tags were mapped to a genome survey of A. irradians and 9 unique 2b-RAD tags were left for a further verification test. In the verifying experiment, 50 individuals with orange shell color and 50 individuals with white shell color of A. irradians were randomly selected from a workshop’s population and SNP detection of every individual was performed using SNaPshot. The results indicated that five of the nine SNPs were highly associated to orange shell color of A. irradians.
2.      Transcriptome sequencing for candidate genes
A selective variety, named as “zhongkehong”, characteristic of consistently orange-color shells were successfully obtained in 2006 and has been propagated for 11 generations in the bay scallop Argopecten irradians. In this study, samples were taken from this variety right before and after the appearance of orange shell color with 3 replications. A total of 6 Transcriptome libraries were used for pair-end sequencing. 289,839,646 paired-end reads were assembled into 70929 transcripts. Annotated against SWISSPROT, NR and KOG database using BLASTX and BLASTN, 30896 Unigenes were successfully annotated. Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes classification identified numbers of unigenes involved in biomineralization and pigmentation. The digital gene expression analysis revealed that melanins and trace metal elements may be potentially involved in shell coloration of A. irradians. But the detection of trace metal of prismatic layer revealed no significant difference between orange shell and white shell. This result may eliminate the function of the trace metal in bay scallop shell pigmentation.
 
Key Words: Argopecten irradians, shell color, SNPs Marker, 2b-RAD, RNA-seq
学科领域水产学 ; 水产学基础学科 ; 水产生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136527
专题实验海洋生物学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
推荐引用方式
GB/T 7714
腾文. 海湾扇贝壳色性状遗传分析[D]. 北京. 中国科学院大学,2017.
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