Abstract:While sex determination in animals refers to the mechanism by which individuals develop into females or males, it presents the process by which undifferentiated gonads develop into mature testes or ovaries, with sex determination as a prerequisite. Among vertebrates, fish are at a primitive stage in the process of sex evolution, and their sex-determining mechanisms are primitive, diverse, and variable, and share the sex-determining modalities of all vertebrates. The mechanisms of sex determination in fish are complex and diverse, including genetic sex determination (GSD), environmental sex determination (ESD), or a combination of both. Among them, GSD can be subdivided into two types: chromosomal sex determination and polygenic sex determination. Chromosomal sex determination is the main sex-determining (SD) gene located on the sex chromosomes that determines the sex of the offspring; whereas polygenic sex determination is the combination of multiple genes on the genome or the cumulative effect of multiple alleles on a pair of chromosomes determining the sex of the offspring; virtually all are the result of the direct or indirect action of genes. The cobia (Rachycentron canadum) is an important seawater net-pen cultured fish species in southern China. No heteromorphic sex chromosomes exist in the chromosomal karyotype of cobia, and there are no associated sex markers to distinguish between males and females. Furthermore, the sex determination and differentiation of cobia is limited, and it is difficult to rely on the external morphology of cobia to differentiate the sexes of male and female cobia parents during the breeding process. In this study, based on the whole genome information of cobia obtained in the previous period, some reported fish sex determination and sex differentiation related genes (Amh, Cyp19a1b, Dmrt1, Gdf6a/6b, and Sox9a/9b) were identified. The genes were subjected to chromosomal localization, whole genome and gene annotation files of XX/XY-type Oryzias latipes and Oreochromis niloticus, ZZ/ZW-type Cynoglossus semilaevis and Oreochromis aureus, and polygenic sex-determined zebrafish were then downloaded from the NCBI database for inter-species covariance analysis. Subsequently, phylogenetic analyses were performed, and the amino acid sequences were compared using Protein BLAST on the NCBI database. The amino acid sequences of other Osteichthyes and higher vertebrates were downloaded separately, and the phylogenetic tree was constructed using the neighbour-joining (NJ) method; followed by real-time fluorescence quantitative PCR (qRT-PCR) to detect the distribution of the expression of these genes in the tissues of adult cobia and their expression levels in gonadal tissues of different developmental stages, with the aim of screening for sex-specific genes, which will provide research materials for further exploring the mechanism of sex determination and differentiation of cobia, as well as providing a reference for the screening of sex markers in cobia. Chromosomal localization revealed that the five genes were located on seven different chromosomes of cobia without clustering. The results of covariance analyses showed that three of the five genes in cobia were covariant with XX/XY, ZZ/ZW and polygenic sex-determining fish, of which only the covariant gene for Amh was localized on the Y chromosome of XX/XY fish, all other genes are localized to autosomes. The results of interchromosomal covariance analyses showed homology between Chr7, where Amh is located, and the Y chromosome of XX/XY type fish. Phylogenetic analyses showed that each of the five genes clustered together (Sox9a and Sox9b clustered together, Gdf6a and Gdf6b clustered together), and that the scleractinians formed a separate branch in each of the gene clusters, independent of other higher vertebrates; Amh, Dmrt1, Cyp19a1b and Gdf6a were the closest relatives to Echeneis naucrates, Gdf6b was the closest relative to Lateolabrax japonicus, and Sox9a and Sox9b were the closest relatives to Seriola dumerili. The qRT-PCR results showed that Amh and Dmrt1 were specifically expressed only in the testis of cobia, which represent the male-specific genes of cobia. Gdf6a and Gdf6b were both highly expressed in the testis and skin, which may be male-biased genes. Finally, Cyp19a1b, Sox9a and Sox9b were expressed to varying degrees in tissues such as brain, skin, and heart. In the gonads of cobia at stages Ⅲ to Ⅴ, the expression of all five genes in the testis was extremely significantly higher than that in the ovary at the same period (P<0.01), and all were significant increased followed by a decrease in gonadal maturation, except for a significant decrease in Sox9b. The above results suggest that these genes may play important roles in spermathecae development in cobia, and the results may lay a foundation for revealing the regulatory mechanism of gonadal development in cobia.
