Validation of SSR-tetranucleotide multiplex panel for kinship evaluation in tilapia breeding programs

ABSTRACT This study aimed to develop PCR assays, reaction combinations, and validation of tetranucleotide SSR loci for tilapia, to minimize the impact of erroneous allele inferences on genotype determination of this marker. Microsatellites containing tetranucleotide repeats were obtained from tilapia genome, version 2.1, avoiding loci in the same linking group. Primers were designed for different fragment sizes, and fluorescence added to each locus. A total of 10 loci were amplified, separately and in combination, and loaded into a single capillary sequencer panel. Alleles were amplified without stutters and easily interpreted. PCR amplifications of DNA repeatedly extracted from samples, and genotyping at different PCR rounds were performed to infer allele signaling errors. The panel obtained in this study is currently used in kinship analyses and pedigree corrections in a tilapia breeding program.摘要 本研究旨在开发针对罗非鱼的四核苷酸SSR（Simple Sequence Repeats）位点的PCR检测方法与反应组合体系，并对该类位点进行验证，以降低错误等位基因推断对该标记基因型判定的不利影响。从罗非鱼2.1版参考基因组中筛选得到含四核苷酸重复的微卫星位点，且规避了位于同一连锁群的位点。针对不同扩增片段长度设计引物，并为每个位点添加荧光标记。共获得10个可单独或组合扩增的位点，将其加载至同一毛细管测序仪检测体系中。扩增所得等位基因无非特异性拖尾峰，且易于判读。通过对样本重复提取的DNA开展PCR扩增，并在多轮PCR反应中进行基因分型，以此推断等位基因信号的错误来源。本研究构建的该检测体系目前已应用于罗非鱼育种项目中的亲缘关系分析与系谱校正工作。