基于微流控芯片的鲑科鱼类单核苷酸多态性分型系统构建

CONSTRUCTION OF A SINGLE NUCLEOTIDE POLYMORPHISM GENOTYPING SYSTEM FOR SALMONID SPECIES BASED ON MICROFLUIDIC CHIP

  • 摘要: 为开发针对大规模样本、低通量位点的单核苷酸多态性(Single nucleotide polymorphism, SNP)分型技术, 研究依据虹鳟高通量SNP芯片检测鲑科4个属不同物种群体样本的结果, 筛选获得了96个高质量共享多态性位点, 应用Fluidigm 96.96微流控动态芯片平台, 构建了用于鲑科物种增殖放流个体识别的SNP分型系统。以细鳞鲑为例评估芯片分型结果可靠性, 分型成功率为98.63%, 与Affymetrix高通量芯片分型一致性达到97.92%。基于该芯片分型结果, 使用CERVUS 3.0.7软件对96尾细鳞鲑子代样本及其候选亲本和干扰亲本进行亲权鉴定, 结果能够准确重现复杂家系的真实系谱, 在用于单亲本亲权鉴定时, 第一亲本非排除率(Non-exclusion probability for first parent, NE-1P)为4.362×10–4, 用于双亲本亲权鉴定时, 双亲非排除率(Non-exclusion probability for parent pair, NE-PP)为6.538×10–12, 完全满足增殖放流回捕个体分子鉴定的需求。基于该芯片分型数据进行STRUCTURE遗传结构分析, 可以明确区分不同来源野生群体的遗传组分, 并对待测个体的遗传组分进行初步判别, 能够满足种群遗传结构初步评估的需求。研究构建的包含96个位点的SNP分型系统, 适合应用于鲑科鱼类增殖放流个体识别、种群遗传结构动态评估, 以及在此基础上开展的增殖放流效果评估。

     

    Abstract: To develop reliable single nucleotide polymorphism (SNP) genotyping technologies suitable for large sample number and limited marker number, a microfluidic platform, namely Fluidigm 96.96 dynamic chip, was used to conduct the SNP genotyping system for identification of released individuals from stock enhancement projects. This study screened 96 high-quality polymorphic markers shared among salmonid species based on genotyping results of fish samples from 4 genera of Salmonidae using rainbow trout 57K SNP array. The microfluidic system was proved with high accuracy using Brachymystax lenok as testing populations, with a call rate of 98.63%, and the consistency with the previous Affymetrix 57K array reached 97.92%. Parentage assignments for 96 offspring individuals to their candidate parents and interfering parents were performed using CERVUS 3.0.7 software and the 96 SNP genotyping data. The results matched perfectly with the true pedigrees among complicated families, proving high accuracy in pedigree assignment application for the constructed microfluidic system. The non-exclusion probability for first parent (NE-1P) was 4.362×10–4 during a single parent assignment, while the non-exclusion probability for parent pair (NE-PP) was as low as 6.538×10–12. These results fully met the needs for molecular identification of released individuals, to determine if one captured fish was an offspring of documented broodstocks, which meant it’s hatched and released from certain stock enhancement projects. STRUCTURE analysis was also performed based on the 96 SNP genotyping data. The genetic components of 4 wild populations clearly distinguished, and the genetic composition of testing individuals were well demonstrated, suggesting that the constructed system is suitable for preliminary population genetic analysis for captured salmonid species. Thus, this study has great application potentials in fishery stock enhancement, to monitor the survival rate of released individuals, and to evaluate the supplemental effects on natural fishery resources.

     

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