中华绒螯蟹基因组30 K液相芯片的开发与验证

Development and validation of the 30 K liquid chip for Chinese mitten crab (Eriocheir sinensis)

  • 摘要:
    目的 为提高中华绒螯蟹基因组选育和种质资源评价的效率,本研究旨在开发一款中密度、高精确度的中华绒螯蟹全基因组单核苷酸(SNP)液相芯片。
    方法 基于136只中华绒螯蟹重测序数据,经参数过滤及质控等筛选出高质量SNP位点用于后续探针设计及合成;为初步测试芯片性能,分别从用于芯片开发的重测序样本及待测样本中选取3份和10份样品进行基因分型,其中设置2份技术重复,并将3份芯片分型结果与对应重测序结果比对;使用96份中华绒螯蟹样本并设置2次技术重复开展生产性测试,基于获得的分型数据评估芯片SNP位点的染色体分布及芯片分型性能;采用液相芯片对724份中华绒螯蟹样本进行SNP分型,采用BayesB模型进行色泽性状的全基因组育种值预测,评估准确性及无偏性。
    结果 ①初步验证试验中,13份样本的位点平均检出率为99.33%,两次技术重复检出率分别相差0.04%和0.03%,芯片分型结果与重测序结果平均一致率为93.25%;②生产性测试中,位点检出率为98.61%~99.54%,两次技术重复基因型结果一致率为99.78%和98.76%;③芯片SNP位点均匀分布于72条染色体上,平均密度25.5 SNPs/Mb,各染色体SNP密度20~32 SNPs/Mb且与染色体长度呈正相关;④SNP的次等位基因频率(MAF)和哈温平衡(Hardy-Weinberg)等质控指标显示芯片具有较高的分型质量,其中共归纳出21 680个多态性位点;⑤色泽性状的全基因组选择结果表明,四个色泽参数预测准确性在0.153 5~0.557 6,无偏性为0.691 7~1.002 5,红度(a*)预测准确性最高,表明该芯片在实际育种中具有良好的应用前景。
    结论 本研究开发的中华绒螯蟹30 K液相芯片,具有检出率高、分型准确性和技术稳定性,可满足中华绒螯蟹全基因组选育和种质资源评价等应用需求,可为中华绒螯蟹的分子选育、种质鉴定和种群遗传等提供可靠工具。

     

    Abstract: The Chinese mitten crab (Eriocheir sinensis) is one of the most widely cultured aquatic species in China and has high economic and practical value. However, its development has been severely constrained by germplasm degradation and inbreeding caused by long-term unregulated breeding practices. Therefore, genetic improvement through selective breeding has become an effective approach to address these challenges.To improve the efficiency of genomic selection and germplasm resource evaluation in the E. sinensis, this study aimed to develop a medium-density and high-accuracy whole-genome single nucleotide polymorphism (SNP) liquid-phase genotyping chip. Based on whole-genome resequencing data from 136 E. sinensis individuals, high-quality SNPs were identified through parameter filtering and quality control and subsequently used for probe design and synthesis. In the preliminary validation of the SNP array, three resequenced individuals used in array development and ten individuals randomly selected from 96 genotyped samples were analyzed, including two technical replicates. Genotypes generated by the SNP array were compared with corresponding whole-genome resequencing results to assess concordance. A production-scale evaluation was then conducted using 96 E. sinensis individuals with two technical replicates, and the resulting genotyping data were used to assess chromosomal distribution and genotyping performance of SNP loci. Liquid-based SNP array was used to genotype 724 E. sinensis individuals. Genomic estimated breeding values for color traits were predicted using the BayesB model, and the prediction performance was assessed in terms of accuracy and bias. ① In the preliminary validation experiment, the average call rate of 13 samples was 99.33%, with call rate differences between two technical replicates of 0.04% and 0.03%, respectively, and the average concordance rate between chip genotyping and resequencing results for three samples was 93.25%; ② In the production-scale test, SNP call rates ranged from 98.61% to 99.54%, and genotype concordance rates between two technical replicates were 99.78% and 98.76%, respectively; ③ SNP markers on the chip were evenly distributed across 72 chromosomes, with an average SNP density of 25.5 SNPs/Mb; SNP density across chromosomes ranged from 20 to 32 SNPs/Mb and showed a positive correlation with chromosome length; ④ Quality control indicators, including minor allele frequency (MAF) and Hardy–Weinberg equilibrium (HWE), demonstrated the high quality of SNP markers on the chip, with a total of 21 680 high-quality polymorphic loci identified;⑤The genomic selection results for color traits showed that the prediction accuracy of the four color parameters ranged from 0.1535 to 0.5576, while the prediction bias ranged from 0.6917 to 1.0025. Among these traits, redness (a*) exhibited the highest prediction accuracy, indicating that the SNP array has good potential for practical application in breeding programs. The 30 K liquid-based SNP array developed in this study for the E. sinensis exhibits a high detection rate, high genotyping accuracy, and strong technical stability. It meets the requirements for genome-wide selection and germplasm resource evaluation in E. sinensis, and provides a reliable tool for molecular breeding, germplasm identification, and population genetic studies.

     

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