三疣梭子蟹生长相关 SNP 位点的鉴定
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1. 中国水产科学研究院 黄海水产研究所, 山东 青岛 266071; 2. 上海海洋大学 水产与生命学院, 上海 201306

作者简介:

张德宁(1988–), 男, 硕士研究生, 研究方向为三疣梭子蟹生长和低盐适应相关 SNP 筛选及功能验证研究. E-mail: youzaiyouzaihappy@163.com

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S917

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国家 863 计划项目(2012AA10A409); 农业科技成果转化资金项目(2013GB23260589); 山东省自主创新专项(2013CX80202); 国家自然科学基金项目(41306177); 中国博士后科学基金特别资助项目(2014T70668).


Identifying SNP markers correlated with growth of swimming crab (Portunus trituberculatus) based on a comparative transcriptome
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1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; 2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

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    摘要:

    为了发掘三疣梭子蟹(Portunus trituberculatus)与生长相关的SNP, 本研究基于三疣梭子蟹比较转录组学数据, 筛选出生长相关候选基因片段19条, 利用该基因序列设计引物进行PCR扩增, 得到总长为17 387 bp的DNA片段; 通过直接测序法发现 74 个 SNP 位点, 分布频率为 0.43/100 bp, 其中转换突变的比例为 80%, 颠换突变的比例为 20%, 转换的比例远远大于颠换, 符合“transition bias”原理。C/T(G/A)突变所占比例为 60%, G/T(C/A)突变占 20%, A/T 突变占 9.33%, G/C突变占 10.67%, C/T(G/A)突变所占比例最大。内含子上突变发生的频率为 1.34/100 bp, 外显子 上仅为 0.17/100 bp, 说明外显子上的碱基更加保守。通过飞行质谱法, 在三疣梭子蟹生长性状分离群体中成功分型 了 10 个 SNP 位点; 通过卡方检验和多元方差分析的方法进行性状关联分析, 发现 3 个 SNP 位点与生长性状显著 相关(P<0.05), 并且 comp58070-R31 位点与生长性状极显著相关(P<0.01); 一般线性模型的多元方差分析显示, comp58070-R31位点与体重、全甲宽、甲宽3个性状极显著相关(P<0.01), 与体长、体高2个性状为显著相关(P<0.05); comp46623-F49 位点与甲宽显著相关(P=0.05); comp49193-R333 位点与甲宽显著相关(P<0.05)。通过对 SNP 位点的 多态性分析发现, 三疣梭子蟹 SNP 标记观测杂合度(Ho)范围为 0.162 8~0.833 3, 期望杂合度(He)范围为 0.189 6~ 0.591 2, 并且显示 SNP 标记的信息量低于微卫星标记。该研究的结果将有助于推进三疣梭子蟹分子标记辅助育种 进程。

    Abstract:

    Comparative transcriptomics data have become one of the most important ways to study single nucleotide polymorphisms (SNP). We have identified 129 gene segments associated with growth. SNP is a third-generation mo- lecular marker and is useful for breeding rapidly growing species, such as the swimming crab, Portunus trituberculatus. We developed an efficient method to selectSNPs correlated with P. trituberculatus growth. Time-of-flight-mass spec- trometry was used for SNP typing, and the chi-square test was used for the correlation analysis. Nineteen gene segments were amplified based on comparative transcriptomics, and a total of 17 387 bp DNA fragments were amplified. SNP frequency was estimated to be 0.43/100 bp of DNA sequence, the conversion mutation rate was 80%, and the transver- sion mutation rate was 20%. The conversion ratio was far greater than the transversion ratio to comply with the princi- ple of “transition bias”. The G/T (C/A) rate was 20%, the A/T rate was 9.33%, the G/C rate was 10.67%, and the C/T (G/A), which is the most common gene mutation type, was 60%. The intron mutation frequency was 1.34/100 bp and that of exons was 0.17/100 bp, indicating that the exon mutation frequency was more conserved. Three SNP markers were significantly correlated with growth traits, and P-values of 10 SNPs were < 0.05. The chi-square test result re- vealed that comp58070-R31 was closely related to growth traits (P<0.01). A multivariate analysis of variance showed that comp58070-R31 was closely related to body weight, full carapace width, and carapace width (P<0.01). A signifi- cant correlation was detected between body length and body height(P<0.01). Loci comp46623-F49 and comp49193-R333 were significantly correlated with carapace width (P≤0.05). Observed heterozygosity of the P. trituberculatus SNPs was 0.162 8–0.833 3, and expected heterozygosity was 0.189 6–0.591 2, indicating less information content than that from microsatellite markers. These SNPs will be helpful for pedigree analysis, association studies, and marker-associated selection in this crab species.

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张德宁,吕建建,刘萍,冯艳艳,高保全,李健.三疣梭子蟹生长相关 SNP 位点的鉴定[J].中国水产科学,2015,22(3):393-401
ZHANG Dening, LV Jianjian, LIU Ping, FENG Yanyan, GAO Baoquan, LI Jian. Identifying SNP markers correlated with growth of swimming crab (Portunus trituberculatus) based on a comparative transcriptome[J]. Journal of Fishery Sciences of China,2015,22(3):393-401

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  • 在线发布日期: 2015-05-14
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