Elucidation of the XX/XY Sex Determination System and Development of a Sex-Linked Molecular Marker in the Freshwater Snail Bellamya purificata.

The freshwater snail Bellamya purificata is both ecologically and economically significant, exhibiting notable sexual dimorphism in growth and nutritional traits that underscore the importance of breeding of monosex stocks. However, the genetic basis of sex determination remains unclear. Herein, genome-wide association studies (GWASs) combined with transcriptomic analysis were conducted to identify sex-linked markers and candidate genes for this species. GWAS generated 571 significantly sex-associated SNPs and 1853 InDels, corresponding to 44 candidate genes. Multiple significant SNP peaks were detected on chromosomes 1 and 2, with mrc2 and mis18bp1 as key candidate genes. A sex-linked InDel marker located within mis18bp1 can distinguish males and females cost-effectively. Genotype analysis of the sex-associated loci revealed that most females were homozygous while males were heterozygous, suggesting that B. purificata has a primarily XX/XY sex determination system. Comparative gonadal transcriptome analyses identified 2996 female-biased and 4281 male-biased genes. Among them, sry, sox8, dmrt1 and dmrt2 may be critical in male sex differentiation, while β-catenin, foxl2, esr1 and nr5a2 may be important in female sex differentiation. Integration of GWAS and transcriptomic data highlighted four pronounced sex-associated candidate genes, including mis18bp1, rnf216, tbx1 and mrc2. These results provide a valuable foundation for elucidating the genetic mechanisms underlying sex determination and for the development of monosex stocks in B. purificata.