Development and characterization of 37 SNP markers for the largemouth bass (Micropterus salmoides) by using PCR–RFLP method

Abstract

Largemouth bass (Micropterus salmoides) is an economically important species in China. Contrary to its rapidly increasing yield during the last decades, the domestic genetic diversity of largemouth bass has gradually declined. For further rationally excavation and utilization of largemouth bass germplasm resources, 37 single nucleotide polymorphism (SNP) markers were developed based on genotyping-by-sequencing (GBS) data and characterized by genotyping 32 individuals using the PCR-RFLP method. The effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC) of these SNPs ranged from 1.168 to 1.998, 0.156 to 0.844, 0.146 to 0.507, and 0.134 to 0.375, respectively. Totally, ve loci deviated signi cantly from Hardy-Weinberg equilibrium (p < 0.05), while there existed no linkage disequilibrium at all loci. These novel polymorphic markers will lay the foundation for future population and conservation genetics of M. salmoides. Main TextLargemouth bass (Micropterus salmoides) is a wildly distributed and indigenous species in North American freshwaters, and can be considered a typical representative carnivorous sh (Chen et al. 2015; Gaeta et al. 2015). As famous for its superior growth rate and broad adaptability, largemouth bass has been introduced into Chinese mainland in the 1980s. Nowadays, the annual production of largemouth bass exceeds 500,000 tons, and its cultivation areas are almost all over China. Correspondingly, the conservation of its germplasm resources has gradually attracted the attention of researchers. It is worth mentioning that molecular markers about largemouth bass were mainly about simple sequence repeat (SSR) markers (Kubota et al. 2014), while few single nucleotide polymorphism (SNP) markers have been developed and reported, which may be caused by the high SNP-genotyping costs.SNPs represent the most profuse form of genetic variations that can be wildly used in molecular marker-assisted selection (MAS) breeding (Qu et al. 2019). At present, the common SNP genotyping methods include direct sequencing, high resolution melting-curve (HRM) (Guo et al. 2018), denaturing high-performance liquid chromatography (DHPLC) (Wolford et al. 2000), kompetitive allele speci c PCR (KASP) (Semagn et al. 2014), etc. However, due to the high requirements for instruments or high costs, most of the above genotyping methods are di cult to be widely used in aquatic animals. PCR-RFLP is a cheap and e cient SNP genotyping method with PCR technology as the core to improve the resolution of traditional RFLP, and this genotyping method has low requirements for instruments, which can be carried out in most laboratories. In addition, with the publication of largemouth bass genome (Sun et al. 2020), a large number of restriction enzyme sites could provide the basis for SNP mining and application, especially using PCR-RFLP genotyping method (Lozano-Duque et al. 2018).Pectoral ns from 32 cultured largemouth bass (Zhoukou, Henan) were randomly sampled. Gen...

Eight single nucleotide polymorphisms and their association with food habit domestication traits and growth traits in largemouth bass fry (Micropterus salmoides) based on PCR-RFLP method

“…Fortunately, several methods have been developed and improved to decrease SNP genotyping cost, including allele-specific PCR (AS-PCR), SNaPshot, and PCR-RFLP ( Zhao et al, 2017 ). The PCR-RFLP is a cost-effective method and has been successfully applied in SNP genotyping of many species ( Forche, Steinbach & Berman, 2009 ; Jiang et al, 2021 ). In the 1980s, Botstein et al (1980) used DNA RFLP to construct a genetic linkage map of human genes, which pioneered the use of DNA polymorphic genetic markers.…”