Table6_Genetic diversity analysis and development of molecular markers for the identification of largemouth bass (Micropterus salmoides L.) based on whole-genome re-sequencing.XLSX

Largemouth bass (Micropterus salmoides L.) is generally considered to comprise two subspecies, Florida bass (M. floridanus) and Northern Largemouth bass (M. salmoides), which have biological characteristic differences because of their geographical distribution. In this study, whole-genome re-sequencing was performed among 10 Florida and 10 Northern largemouth bass, respectively. In total, 999,793 SNPs and 227,797 InDels were finally identified, and 507,401 SNPs (50.75%) and 116,213 InDels (51.01%) were successfully mapped to annotated 18,629 genes and 14,060 genes, respectively. KEGG classification indicated that most of these genes were focused on the pathways including signal transduction, transport and catabolism, and endocrine system. Genetic diversity analysis indicated that Florida largemouth bass had higher genetic diversity than Northern largemouth bass, indicating that the germplasm quality of Northern largemouth bass had markedly reduced in China. To examine the accuracies of the identified markers, 23 SNPs and eight InDels (the insertions or deletions of more than 45 bp) were randomly selected and detected among Florida largemouth bass, Northern largemouth bass, and their F1 hybrids. The detection efficiencies of all the markers were higher than 95%; nineteen SNPs and three InDels could accurately distinguish the two subspecies and their F1 hybrids with 100% efficiencies. Moreover, the three InDel markers could clearly distinguish the two subspecies and their F1 hybrids with a PCR-based agarose gel electrophoresis. In conclusion, our study established a simple PCR-based method for the germplasm identification of largemouth bass, which will be useful in the germplasm protection, management, and hybridization breeding of largemouth bass.

大口黑鲈（*Micropterus salmoides* L.）通常被认为包含两个亚种：佛罗里达大口黑鲈（*M. floridanus*）与北方大口黑鲈（*M. salmoides*），二者因地理分布差异呈现生物学特性分化。本研究分别对10尾佛罗里达大口黑鲈和10尾北方大口黑鲈开展全基因组重测序（whole-genome re-sequencing）。最终共鉴定得到999793个单核苷酸多态性（Single Nucleotide Polymorphism，SNP）位点与227797个插入缺失变异（Insertion-Deletion，InDel）；其中分别有507401个SNP位点（占比50.75%）与116213个InDel变异（占比51.01%）成功比对注释至18629个基因与14060个基因。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）分类分析显示，此类基因主要富集于信号转导、转运与分解代谢、内分泌系统等通路。遗传多样性分析结果表明，佛罗里达大口黑鲈的遗传多样性高于北方大口黑鲈，提示中国境内北方大口黑鲈的种质质量已显著下降。为验证所鉴定分子标记的准确性，本研究随机选取23个SNP位点与8个长度大于45 bp的InDel变异，在佛罗里达大口黑鲈、北方大口黑鲈及其F1杂交后代中开展检测。所有标记的检测效率均高于95%；其中19个SNP位点与3个InDel变异可实现对两个亚种及其杂交后代的100%精准区分。此外，依托聚合酶链式反应（Polymerase Chain Reaction，PCR）结合琼脂糖凝胶电泳，即可通过这3个InDel标记清晰区分两个亚种及其杂交后代。综上，本研究建立了一种简便的基于PCR的大口黑鲈种质鉴定方法，该方法可应用于大口黑鲈的种质保护、资源管理及杂交育种工作。