背景:使用性别特异性分子标记已成为提高鱼类产量和经济价值的一种突出方法,以及为理解鱼类性别决定中涉及的复杂分子机制提供了基础。在过去的几十年里,男性和女性性别鉴定的研究主要采用分子生物学方法,如限制性片段长度多态性,多态性DNA的随机扩增,简单序列重复,和扩增片段长度多态性。高通量测序技术的出现,尤其是Illumina,已导致利用单核苷酸多态性和插入/缺失变体作为研究鱼类性别鉴定的重要分子标记。性别控制育种的发展遇到许多挑战,包括当前方法的低效率,复杂的实验方案,高开发成本,假阳性率升高,标记不稳定,和繁琐的现场测试程序。然而,PacBio高通量测序技术的出现和迅速发展,其特点是长读取输出能力,提供了克服这些障碍的新机会。
结果:利用男性/女性组装的基因组信息,结合短读测序数据调查和长读PacBio测序数据,通过具有双向比较的全基因组变异位点扫描方法,生成了大片段(>100bp)插入/缺失遗传变异的目录.基于插入/缺失位点的长读段深度对序列标记位点进行分级,具有较低的长读段深度的标记被认为对大段缺失变体更有效。随后,开发了雄性/雌性变异基因座的批量引物和模拟PCR目录,结合针对目标区域的引物设计和电子PCR(e-PCR)技术。日本鹦嘴鱼(Oplegnathusfasciatus),属于Centrarchiformes命令中的Oplegnathidae家族,作为东亚本土的岩石礁鱼,具有重要的经济价值。通过琼脂糖凝胶电泳建立了快速鉴定日本鹦嘴鱼雌雄差异的标准,揭示了男性的2条扩增带和女性的1条扩增带。然后利用该方法构建了性别特异性标记的高通量鉴定目录,导致3,639(2,786INS/853DEL,作为参考)和3,672(2,876INS/833DEL,作为参考)标记与1,021和894个高质量的遗传性别鉴定标记相结合,分别。从目录中随机选择16个差异位点进行验证,其中11人符合男女区别标准。实施具有成本效益和效率的技术过程将通过加快各种物种的性别遗传标记的高通量开发来促进遗传育种的快速发展。
结论:我们的研究利用了从PacBio获得的男性和女性个体的组装基因组信息,除了来自短读测序数据调查和长读PacBio测序数据的数据。我们广泛采用全基因组变异位点扫描和鉴定,靶区高通量引物设计,和e-PCR批量扩增,以及变异位点的长阅读深度的统计分析和排名。通过这种综合方法,我们成功地编制了一个大的插入/缺失位点目录(>100bp)在雄性和雌性日本鹦鹉鱼。
The use of sex-specific molecular markers has become a prominent method in enhancing fish production and economic value, as well as providing a foundation for understanding the complex molecular mechanisms involved in fish sex determination. Over the past decades, research on male and female sex identification has predominantly employed molecular biology methodologies such as restriction fragment length polymorphism, random amplification of polymorphic DNA, simple sequence repeat, and amplified fragment length polymorphism. The emergence of high-throughput sequencing technologies, particularly Illumina, has led to the utilization of single nucleotide polymorphism and insertion/deletion variants as significant molecular markers for investigating sex identification in fish. The advancement of sex-controlled breeding encounters numerous challenges, including the inefficiency of current methods, intricate experimental protocols, high costs of development, elevated rates of false positives, marker instability, and cumbersome field-testing procedures. Nevertheless, the emergence and swift progress of PacBio high-throughput sequencing technology, characterized by its long-read output capabilities, offers novel opportunities to overcome these obstacles.
Utilizing male/female assembled genome information in conjunction with short-read sequencing data survey and long-read PacBio sequencing data, a catalog of large-segment (>100 bp) insertion/deletion genetic variants was generated through a genome-wide variant site-scanning approach with bidirectional comparisons. The sequence tagging sites were ranked based on the long-read depth of the insertion/deletion site, with markers exhibiting lower long-read depth being considered more effective for large-segment deletion variants. Subsequently, a catalog of bulk primers and simulated PCR for the male/female variant loci was developed, incorporating primer design for the target region and electronic PCR (e-PCR) technology. The Japanese parrotfish (Oplegnathus fasciatus), belonging to the Oplegnathidae family within the Centrarchiformes order, holds significant economic value as a rocky reef fish indigenous to East Asia. The criteria for rapid identification of male and female differences in Japanese parrotfish were established through agarose gel electrophoresis, which revealed 2 amplified bands for males and 1 amplified band for females. A high-throughput identification catalog of sex-specific markers was then constructed using this method, resulting in the identification of 3,639 (2,786 INS/853 DEL, ♀ as reference) and 3,672 (2,876 INS/833 DEL, ♂ as reference) markers in conjunction with 1,021 and 894 high-quality genetic sex identification markers, respectively. Sixteen differential loci were randomly chosen from the catalog for validation, with 11 of them meeting the criteria for male/female distinctions. The implementation of cost-effective and efficient technological processes would facilitate the rapid advancement of genetic breeding through expediting the high-throughput development of sex genetic markers for various species.
Our study utilized assembled genome information from male and female individuals obtained from PacBio, in addition to data from short-read sequencing data survey and long-read PacBio sequencing data. We extensively employed genome-wide variant site scanning and identification, high-throughput primer design of target regions, and e-PCR batch amplification, along with statistical analysis and ranking of the long-read depth of the variant sites. Through this integrated approach, we successfully compiled a catalog of large insertion/deletion sites (>100 bp) in both male and female Japanese parrotfish.