The novel non-targeted PCR-based genotyping system, namely Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di), is characterized by the simplicity in library construction and robustness against DNA degradation and is expected to facilitate advancements in genetics, in both basic and applied sciences. In this study, we tested the utility of GRAS-Di for genetic analysis in a cultured population of the tiger pufferfish Takifugu rubripes. The genetic analyses included family structure analysis, genetic map construction, and quantitative trait locus (QTL) analysis for the male precocious phenotype using a population consisting of four full-sib families derived from a genetically precocious line. An average of 4.7 million raw reads were obtained from 198 fish. Trimmed reads were mapped onto a Fugu reference genome for genotyping, and 21,938 putative single-nucleotide polymorphisms (SNPs) were obtained. These 22 K SNPs accurately resolved the sibship and parent–offspring pairs. A fine-scale linkage map (total size: 1,949 cM; average interval: 1.75 cM) was constructed from 1,423 effective SNPs, for which the allele inheritance patterns were known. QTL analysis detected a significant locus for testes weight on Chr_14 and three suggestive loci on Chr_1, Chr_8, and Chr_19. The significant QTL was shared by body length and body weight. The effect of each QTL was small (phenotypic variation explained, PVE: 3.1–5.9%), suggesting that the precociousness seen in the cultured pufferfish is polygenic. Taken together, these results indicate that GRAS-Di is a practical genotyping tool for aquaculture species and applicable for molecular breeding programs, such as marker-assisted selection and genomic selection.

新的基于非靶向 PCR 的基因分型系统，即通过随机扩增子测序直接 (GRAS-Di) 进行基因分型，其特点是文库构建简单且对 DNA 降解具有稳健性，预计将促进遗传学的进步，包括基本的和应用科学。在这项研究中，我们测试了 GRAS-Di 在虎河豚Takifugu rubripes的养殖种群中进行遗传分析的效用. 遗传分析包括家族结构分析、遗传图谱构建和雄性早熟表型的数量性状基因座 (QTL) 分析，使用由来自遗传性早熟品系的四个全同胞家族组成的种群。从 198 条鱼中平均获得了 470 万条原始读数。修剪后的读数被映射到府谷参考基因组上进行基因分型，并获得了 21,938 个假定的单核苷酸多态性 (SNP)。这 22 K SNP 准确地解析了同胞和亲代-后代对。精细尺度连锁图（总大小：1,949 cM；平均间隔：1.75 cM）由 1,423 个有效 SNP 构建，其等位基因遗传模式是已知的。QTL 分析在 Chr_14 上检测到睾丸重量的显着位点，在 Chr_1、Chr_8 和 Chr_19 上检测到三个提示性位点。显着 QTL 由体长和体重共享。每个 QTL 的影响很小（表型变异解释，PVE：3.1-5.9%），表明在养殖河豚中看到的早熟是多基因的。总之，这些结果表明 GRAS-Di 是一种实用的水产养殖物种基因分型工具，适用于分子育种计划，例如标记辅助选择和基因组选择。