军曹鱼全基因组微卫星特征分析与多态性标记的筛选及应用

Screening and characterization of polymorphic SSR markers based on whole genome sequencing of cobia (Rachycentron canadum)

中文关键词: 军曹鱼  基因组  SSR  分子标记  遗传多样性

英文关键词: Rachycentron canadum  Genome  SSR  Molecular marker  Genetic diversity

中文摘要:

      为更好地开发军曹鱼(Rachycentron canadum)高多态性微卫星分子标记，本研究基于军曹鱼全基因组测序结果，利用MISA1.0软件对其简单重复序列(simple sequence repeat, SSR)的位点信息进行检索及分析。结果显示，基因组中共筛选出1~6个核苷酸为重复单元的SSR位点344 820个，其中，以单核苷酸重复序列最多(174 146个)，占SSR总数的50.50%；其次为二核苷酸重复和三核苷酸重复序列，分别占SSR总数的30.23%和14.02%。在SSR包含的重复单元中，单核苷酸重复以A/T类型为主，AC/GT是二核苷酸的优势重复单元类型。军曹鱼基因组SSR核心序列重复次数在4~275次范围内波动，单核苷酸SSR重复数为10的最多，二核苷酸SSR重复次数为6的最多。本研究设置长度≥12 bp为筛选高多态性SSR位点的标准，共获得361 684个位点。在上述位点中随机选取100个候选位点进行基因分型检测，利用从中筛选到多态性较高的10个SSR标记分别对北海、陵水、硇洲、徐闻和三亚5个养殖群体进行遗传多样性分析，145尾个体中共检测到69个等位基因，观测杂合度(Ho)、期望杂合度(He)和多态信息含量(PIC)平均值分别为0.628、0.706和0.653。相关研究表明，军曹鱼基因组中SSR位点类型较为丰富、多态性潜能较高，从中筛选获得的多态性SSR标记可为军曹鱼分子标记辅助育种、群体遗传多样性评价等研究提供有力支持。

英文摘要:

      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.