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中华绒螯蟹(Eriocheir sinensis)高密度遗传图谱的构建和基因组框架图的绘制
宋呈文
学位类型博士
导师崔朝霞
2016-04-23
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词中华绒螯蟹 进化 遗传图谱 基因组测序 Qtl
其他摘要        中华绒螯蟹是一种广盐性物种,具有重要的经济价值和生态价值。它的一些生理特性已经成为人们研究的热点,例如,早期发育过程从浮游到底栖的生活方式转变和短尾化,生长过程中的溯河洄游,繁殖期的降河洄游等。然而,人们对于调节这些生理过程的分子机制的理解是非常有限的。
        运用二代测序技术,我们对中华绒螯蟹大眼幼体和仔蟹I期进行了比较转录组分析,鉴定出22,622个注释unigenes和1,016个差异表达基因,尤其是参与形态发生、感觉认知和免疫系统的基因。在这些unigenes中,169个基因被注释到形态发生信号通路中(Wnt、Hedgehog和hormone biosynthesis)。对于变态过程,38个与细胞骨架构建相关的基因在大眼幼体中表达上调,而52个与表皮发育和蛋白降解相关的基因在仔蟹中表达上调。几个参与光转导通路的关键基因和两个嗅觉相关基因在大眼幼体中显著上调。此外,44个免疫相关基因在大眼幼体中高度表达。本研究对中华绒螯蟹短尾化和底栖适应性提供了一个全面的了解。
        在本研究中,我们构建了首张中华绒螯蟹高密度遗传图谱,由73条连锁群构成,平均标记间隔0.49 cM,基因组覆盖度~98.5%。对10个生长性状进行全基因组范围的QTL,结果定位了61个显著的QTLs区域。大多数的QTLs位于一个较窄的区域内(< 1 cM),并且获得了全基因组关联分析(GWAS)的验证。QTL分析发现所有与性别连锁的标记均位于连锁群LG60上,GWAS分析肯定了这一结果。
        我们采用Illumina和PacBio数据混合组装的策略绘制了中华绒螯蟹基因组框架图。组装基因组大小为1.55 Gb,contig和scaffold N50分别为44.90 kb和490.42 kb。利用已经构建的高密度遗传图谱,将1,584个scaffolds锚定到了73条连锁群上,覆盖809 Mb(52.23%)基因组序列。对两个普通雌蟹进行重测序,与组装的杂交系雄蟹基因组比较,发现杂交导致了SNP杂合度的不均匀分布。运用不同的方法对组装的基因组进行了评估。96%的转录组unigenes被scaffolds覆盖;211条CDS序列中有202条被基因组覆盖;真核生物中高度保守的458条基因中,87.78%的基因被基因组覆盖。通过多种预测方法共鉴定出35,089个蛋白编码基因,其中78.07%的基因获得了注释信息。超过40%的插入缺失与检测的转座子元件重叠,说明转座子元件可能在改变中华绒螯蟹基因组大小方面具有积极的作用。进化分析证明了甲壳动物并系发生的进化关系。发现了Hox基因家族的10个成员,它们位于不同的scaffold上。作为甲壳动物特有的insulin-like蛋白编码基因,IAG通过标记m4821被定位在连锁群LG61上。显著扩张的transporters可能与蟹的渗透压调节相关。Fem-1基因家族成员序列结构和表达模式分析表明,它们可能参与蟹的早期性别决定和性腺的发育。
        作为一个新的短尾类动物基因组,中华绒螯蟹基因组框架图为蟹类生物学研究提供了新思路。借助于全基因组序列和转录组数据,越来越多的基因将会被人们所熟知,从而更全面的理解中华绒螯蟹乃至十足目甲壳动物的生物特性及其分子机制。
;         The Chinese mitten crab (Eriocheir sinensis) is a euryhaline species with important economical and ecological value. Its transition from pelagic to benthic lifestyle and brachyurization during early development, anadromous migration during growth, and catadromous migration during breeding have been attractive features for research. However, knowledge of the underlying molecular mechanisms that regulate these processes is still very limited.
        Using next-generation RNA sequencing, we performed the comparative transcriptomic analysis between the megalopa and the first juvenile crab. 22,622 annotated unigenes and 1016 differentially expressed genes were identified, especially involved in metamorphosis, sensory perception and immunity in the two stages. Among 22,622 unigenes, 169 unigenes were assigned tomorphogenic pathways (Wnt, Hedgehog and hormone biosynthesis). For metamorphosis, 38 up-regulated genes in megalopae were associated with cytoskeleton construction, while, 52 up-regulated genes in juvenile were related to cuticle development and protein degradation. Several crucial genes involved in phototransduction and two olfactory-related genes were significantly up-regulated in megalopae. Moreover, 44 immunity-related genes were highly expressed in megalopae. This study provides a comprehensive view of brachyurization and benthic adaptation mechanisms in E. sinensis.
        Here, we reported a high-density (0.49 cM for average marker interval) genetic map covering ~98.5% of the genome, with 73 linkage groups corresponding to the haploid chromosome number. A genome-wide quantitative trait loci (QTLs) scan for 10 growth traits was performed, resulting in 61 significant QTLs with phenotypic variance explained by a QTL from 11.2% to 42.2%. Most of the QTLs were restricted to narrow regions (< 1 cM) and confirmed by genome-wide association study (GWAS). All sex-linked markers in the family we used were located on a single linkage group LG60, and sex linkage was confirmed by GWAS.
        We reported a draft genome of male E. sinensis using a hybrid assembly strategy incorporating Illumina short reads and PacBio long reads. The final assembly spanned 1.55 Gb with a contig N50 size of 44.90 kb and a scaffold N50 size of 490.42 kb. Using our constructed high-density genetic map, we anchored 1,584 scaffolds on 73 linkage groups, covering 809 Mb (52.23%) of the genome assembly. Genomes from two common females were re-sequenced and comparison with the assembled inbred male genome showed that inbreeding could cause a heterogeneous distribution of the heterozygosity rates. The assembly was evaluated by various methods. 96% transcriptomic unigenes were covered by scaffolds; 202 of 211 CDS were covered; 87.78% of 458 highly conserved Core Eukaryotic Genes were covered by genome. From the assembled genome, we annotated a total of 35,089 protein-coding genes by combining de novo prediction, homology and transcript-based evidence, of which 78.07% were annotated in GO, KEGG, InterPro, Swiss-Prot and TrEMBL. Over 40% of indels were overlapped with detected transposable elements, which suggested that transposable elements with relatively low sequence divergence might have an active role in shaping genome variation in E. sinensis. Phylogenetic analysis confirmed the paraphyly evolution relationship in crustaceans. Ten members of Hox family were identidied in this study and they located on different scaffolds. As a crustacean specific insulin-like gene, EsIAG with maker m4821 was located on scaffold haploid_15 of LG61. Significant expansion of transporters might correlate with osmoregulation. The analysis for sequence and expression patterns of Fem-1 genes revaelded that they might involved in early sex determination and gonadal development of crab. 
        As the new genome assembled from the Brachyura, the E. sinensis genome offers new insights into crab biology. With complete genome sequences and transcriptomic data, more genes will be characterized, thus leading towards a comprehensive understanding of the biological characteristics and molecular mechanisms of E. sinensis and decapod crustaceans.
学科领域海洋科学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/112526
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
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宋呈文. 中华绒螯蟹(Eriocheir sinensis)高密度遗传图谱的构建和基因组框架图的绘制[D]. 北京. 中国科学院大学,2016.
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