Innovative approach for high-throughput exploiting sex-specific markers in Japanese parrotfish Oplegnathus fasciatus

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.