Supporting data for "Innovative approach for high-throughput exploiting sex-specific markers in Japanese parrotfish <i>Oplegnathus fasciatus</i>"

The utilization of sex-specific molecular markers has emerged as a significant technical approach to augment fish production and enhance economic value, while also establishing a fundamental basis for unraveling the intricate molecular mechanisms governing fish sex determination. In the past ten years, the field of genetic sex marker mining has primarily relied on first-generation development methods such as RFLP, RAPD, SSR, and AFLP, as well as second-generation techniques utilizing Illumina's SNP/InDel markers. However, the progress of sex-controlled breeding has been hindered by several factors, including the limited efficiency of the aforementioned methods, complex experimental procedures, high development costs, a high incidence of false positives, unstable markers, and inconvenient on-site testing. Nevertheless, the emergence and rapid advancement of third-generation sequencing technology offers new opportunities for overcoming these limitations. <br>Using male-female linear genomic information combined with Illumina survey and PacBio CLR/CCS data, a database of large-segment (&gt;100 bp) insertion-deletion genetic variants was constructed using a genome-wide variant site scanning method with bidirectional comparisons. A database of bulk primers and simulated PCR for the male-female variant loci were then constructed, employing primer design for the target region and e-PCR technology. Finally, the criteria for rapid identification of male and female differences were established based on agarose gel electrophoresis with two amplified bands for males and one amplified band for females. A high-throughput identification database of sex-specific markers for <i>Oplegnathus fasciatus</i> was constructed utilizing this method, yielding 3,645 (2,791 INS/854 DEL, <em> </em>as reference) and 3,872 (3,039 INS/833 DEL, <em> </em>as reference) genetic sex identification markers. Four differential loci were randomly selected from the database for validation, and the results met the criteria for male-female differences. Utilization of this technology will accelerate the identification of genetic sex markers for species, contributing to the rapid development of genetic breeding. <br>By utilizing the linear genomic information of male and female individuals acquired through PacBio sequencing, along with data from Illumina surveys and PacBio CLR/CCS. Our research extensively employed whole-genome variant site scanning and identification, high-throughput primer design for the target regions, and e-PCR batch amplification. By employing this comprehensive approach, which encompassed genome-wide variant site scanning, high-throughput primer design within the desired region, and e-PCR batch amplification and validation methodologies, we successfully developed a database encompassing insertion/deletion loci in large segments (&gt;100 bp) for both male and female <i>O. fasciatus</i>.

性别特异性分子标记的应用已成为提升鱼类养殖产量、增加经济价值的重要技术手段，同时也为解析调控鱼类性别决定的复杂分子机制奠定了基础。近十年来，遗传性别标记挖掘领域主要依赖第一代开发技术，包括限制性片段长度多态性（Restriction Fragment Length Polymorphism, RFLP）、随机扩增多态性DNA（Random Amplified Polymorphic DNA, RAPD）、简单序列重复（Simple Sequence Repeats, SSR）、扩增片段长度多态性（Amplified Fragment Length Polymorphism, AFLP），以及基于Illumina平台的单核苷酸多态性/插入缺失多态性（Single Nucleotide Polymorphism/Insertion-Deletion, SNP/InDel）标记的第二代技术。然而，上述方法存在检测效率有限、实验流程繁琐、开发成本高昂、假阳性率高、标记稳定性差以及现场检测不便等问题，制约了性别控制育种的发展。而第三代测序技术的出现与快速发展，为突破这些局限提供了新的契机。

本研究结合雌雄个体线性基因组信息与Illumina测序调查数据及PacBio连续长读长（Continuous Long Read, CLR）/环形一致序列（Circular Consensus Sequencing, CCS）数据，采用双向比对的全基因组变异位点扫描方法，构建了大片段（>100碱基对，base pair, bp）插入缺失遗传变异数据库。随后针对雌雄变异位点，通过靶区域引物设计与电子PCR（electronic PCR, e-PCR）技术，构建了批量引物与模拟PCR数据库。最终基于琼脂糖凝胶电泳（agarose gel electrophoresis）结果确立了雌雄差异快速鉴定标准：雄性个体呈现两条扩增条带，雌性个体仅呈现一条扩增条带。利用该方法构建了条石鲷（*Oplegnathus fasciatus*）性别特异性标记高通量鉴定数据库，共获得3645个（2791个插入/854个缺失，以参考序列计）与3872个（3039个插入/833个缺失，以参考序列计）遗传性别鉴定标记。随机选取数据库中的4个差异位点进行验证，结果符合雌雄差异鉴定标准。该技术的应用将加速物种遗传性别标记的开发进程，助力遗传育种工作的快速发展。

本研究利用PacBio测序获得的雌雄个体线性基因组信息，结合Illumina测序调查数据与PacBio CLR/CCS数据，全面开展了全基因组变异位点的扫描与鉴定、靶区域高通量引物设计以及电子PCR批量扩增实验。通过这套涵盖全基因组变异位点扫描、靶区域高通量引物设计以及电子PCR批量扩增与验证的完整研究方案，本研究成功为雌雄条石鲷（*O. fasciatus*）构建了包含大片段（>100 bp）插入缺失位点的数据库。