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

## TL;DR

This study identifies the XX/XY sex determination system in the freshwater snail Bellamya purificata and develops a cost-effective molecular marker to distinguish males and females.

## Contribution

The study provides the first elucidation of the sex determination system and a sex-linked molecular marker in Bellamya purificata.

## Key findings

- The sex determination system in B. purificata follows an XX/XY pattern with females being homozygous and males heterozygous.
- A sex-linked InDel marker within the mis18bp1 gene was developed for cost-effective sex identification.
- Candidate genes like mrc2, mis18bp1, and dmrt1 were identified as critical for sex differentiation.

## Abstract

Bellamya purificata, a homotypic synonym of Sinotaia purificata, is an ecologically and economically important freshwater snail in China. Elucidation of sex determination mechanism and identification of sex-linked molecular markers are of great importance for breeding of monosex stocks. In this study, we used whole-genome resequencing data and gonadal transcriptome data to develop a sex-linked marker and elucidate the genetic architecture underlying sex determination for this species. Based on the data analysis, we obtained several important sex-associated single-nucleotide polymorphisms (SNPs), insertion–deletions (InDels) and candidate genes. The sex determination system of B. purificata follows a primarily XX/XY pattern, characterized by females being homozygous and males being heterozygous. Moreover, a sex-specific marker was successfully developed, which can identify male and female individuals cost-effectively. Our research findings provide a molecular marker for developing monosex stocks and contribute to understanding the genetic basis of sex determination in B. 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.