Screening and Characterization of Polymorphic SSR Markers Based on Whole Genome Sequencing of Cobia (Rachycentron canadum)

The cobia, Rachycentron canadum (Linnaeus, 1766), is an important aquaculture species in cage and other intensive systems. This species has many advantages such as fast growth rate, excellent meat quality, and high market value, making cobia an excellent candidate species for commercial aquaculture. However, long-term artificial breeding of cobia has reduced gene exchange and population genetic diversity. To better protect germplasm resources, molecular markers provide a powerful tool in developing the breeding industry of cobia. Microsatellites are widely distributed, large in number with high polymorphism, and have long been considered important molecular markers for genetic diversity and marker-assisted breeding. More polymorphic microsatellite markers need to be developed for cobia because the number of published polymorphic simple sequence repeat (SSR) loci is very limited. In this study, Micro Satellite (MISA) software was used to identify SSR loci based on the genome sequencing data of cobia. We analyzed the distribution, quantity, and composition characteristics of the SSR loci to develop polymorphic microsatellite markers. The identified markers were used to evaluate the genetic diversity in five cultured populations.In this study, a total of 424 827 SSR loci were identified in the genome data of cobia, among which mononucleotides, dinucleotides, and trinucleotides accounted for 50.50%, 30.23%, and 14.02% of the total SSRs, respectively. Among all the repeat units contained in the total SSRs, A/T was the predominant repeat type of the mononucleotide repeats; AT/AT and AC/GT were the dominant repeat types of dinucleotides; AAT/ATT and AGG/CCT were the dominant repeat types of trinucleotides. Repeat numbers of the SSR core sequences in the genome of cobia ranged from 4 to 275 times. The predominant repeat number of the mononucleotide SSR was ten and the predominant number of the dinucleotide and trinucleotide SSR were six and four, respectively. A total of 173 518 SSR loci had a length of ≥20 bp, accounting for 47.98% of the total number of SSRs in the genome. These results indicated that the SSR loci in the genome of cobia were of a high frequency, rich variety, and with high polymorphic potential.Unigenes obtained by the genome sequencing of cobia were used to detect and analyze the SSR loci information using MISA software. The numbers and types of SSR sequences on the single-stranded DNA of the genome were counted. SSR sites in cobia genome mainly contain mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats. The screening criteria for the polymorphic SSR loci was set as "mononucleotide repeat units with repeats at least 10 times; dinucleotides with repeats at least six times; trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides with repeats at least four times". Subsequently, based on the information of SSR loci screening, 100 candidate loci were randomly selected to design and synthesize primers for amplification. A total of 16 DNA samples from different cultured populations were used as templates. Multiple PCR amplifications were performed to screen ideal polymorphic SSR loci and suitable PCR primers. PCR products were detected using capillary fluorescence electrophoresis. The GeneMapper 4.1 software was used to analyze the accurate sites of the amplified sequences. Based on the genotyping data of the 100 SSR loci, SSR loci with high polymorphism were selected to analyze the genetic diversity of five cultured populations of cobia.The screening analysis results revealed a total of 344 820 SSR loci were detected in the genome of cobia. Among these SSR (with 1–6 nucleotide as repeat units), the top three repeat types were mononucleotide, dinucleotide, and trinucleotide, accounting for 50.49%, 30.23% and 14.02% of the total detected SSRs, respectively. Among the repeating units included in the detected SSRs, the mononucleotide repeats were dominated by A/T type, accounting for 46.34% of the total detected SSRs; AC/GT was the dominant repeat unit type of dinucleotide, accounting for 21.81% of the total detected SSRs. The number of SSR core sequence repeats in the total detected SSRs fluctuated in the range of 4 to 275 times. The predominant number of repeats of mononucleotide SSR was ten, and the predominant number of repeats of dinucleotide SSR was six. In this study, the length of ≥12 bp was set as the standard for screening high polymorphic SSR loci. A total of 361 684 SSR loci were obtained. However, the SSR loci with fragment lengths of 12–19 bp accounted the largest number, with a total of 188 166; these loci accounted for 52.02% of the total number of SSRs. Among the selected 100 candidate loci for genotyping, a total of ten polymorphic SSR markers were obtained. These markers were used in the genetic diversity analysis of five cultured populations collected in Beihai (RC-BH), Lingshui (RC-LS), Naozhou (RC-NZ), Xuwen (RC-XW), and Sanya (RC-SY). A total of 69 alleles were detected from 145 individuals, the average observed heterozygosity (Ho) was 0.628, and the average expected heterozygosity (He) was 0.706, and the mean polymorphism information content (PIC) was 0.653. The inbreeding coefficient (Fis) of the ten loci in the five cultured populations ranged from –0.317 to 0.270. The genetic distance between the five cultured populations ranged from 0.141 to 0.464, and the genetic similarity was 0.629–0.868. The results were similar to that of previous research using published markers, indicating that the polymorphic markers screened from the genome of cobia were of high accuracy and reliability. These polymorphic SSR markers provide strong support for population genetic diversity evaluation and molecular marker-assisted breeding of cobia, and provide effective technical support for the development of the cobia aquaculture industry.