Largemouth bass (Micropterus salmoides) is a wildly distributed and indigenous species in North American freshwaters, and can be considered a typical representative carnivorous fish (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 specific 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 difficult to be widely used in aquatic animals. PCR-RFLP is a cheap and efficient 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 fins from 32 cultured largemouth bass (Zhoukou, Henan) were randomly sampled. Genomic DNA was extracted by “Rapid Animal Genomic DNA Isolation Kit” (Sangon Biotech (Shanghai) Co., Ltd.) and diluted to 50 ng/μL. According to our previous GBS data (unpublished), 37 SNP loci, which could be used for PCR-RFLP genotyping method, were randomly selected and primers were designed according to the flanking sequences (Electronic supplementary 1). The amplification reaction volume was 20 μL, which contained 10.0 μL of 2×Taq PCR Mastermix (Tiangen Biotech (Beijing) Co., Ltd.), 1 μL of primer pairs, 2 μL of genomic DNA and 7 μL of ddH2O. The amplification parameters were as follows: an initial denaturation at 95 °C for 5 min; total of 35 cycles of denaturation at 95 °C for 30 s, annealing at 46.6-63 °C (Table 1) for 30 s, extension at 72 °C for 30 s; and finally extension at 72 °C for 5 min. Subsequently, PCR products were digested by specific enzyme at 37 °C for 45 min. The digestion system included 5 μL of PCR product, 1 μL of 10×SpeedyOne Buffer, 0.5 μL of SpeedyCut enzyme and 3.5 μL of ddH2O. Finally, the digestion products were genotyped by 2% agarose gel electrophoresis.
The effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC) of these SNP loci were analyzed by software Cervus 3.0. Results showed that the Ne, Ho, He and PIC of 37 SNP loci ranged from 1.168 to 1.998 (mean 1.794), 0.156 to 0.844 (mean 0.470), 0.146 to 0.507 (mean 0.443) and 0.134 to 0.375 (mean 0.338), respectively (Table 1). Results from software Popgen 32 showed that five SNP loci significantly deviated from Hardy–Weinberg equilibrium (Table 1, superscript with ‘*’), while there was no linkage disequilibrium at all loci. In summary, 37 SNP loci, of which 35 were moderate polymorphic, of largemouth bass were successfully developed and characterized by PCR-RFLP genotyping method. These polymorphic SNP loci could provide reference for the excavation and rational utilization of largemouth bass germplasm resources.