Integrated transcriptome and SNP analysis reveals sex-related genetic insights in the black-spotted frog (Pelophylax nigromaculatus).

Pelophylax nigromaculatus is an important aquaculture economic animal in China, with an annual output of more than 80,000 tons in recent years. P. nigromaculatus demonstrates distinct sexual dimorphism, characterized by females being substantially larger than males. Consequently, the cultivation of female populations presents higher economic value. The RNA-Seq technology is harnessed in this study to delve into the sexual differentiation and reproductive mechanisms of these frogs. By analyzing the transcriptomes of mature testes and ovaries, 17.24 Gbp and 23.71 Gbp of clean data were obtained respectively. This analysis yielded 107,897 annotated genes, among which 8099 were differentially expressed; 4370 were upregulated and 3729 were downregulated in ovaries. GO and KEGG analyses highlighted 70 genes exhibiting sexual dimorphism. For validation, 13 differentially expressed genes were subjected to qRT-PCR, corroborating the results of transcriptome sequencing. Furthermore, the transcriptome analysis of P. nigromaculatus' sexual glands revealed 1,062,663 SNPs, with 676,172 (63.63%) and 386,491 (36.37%) being base conversion and base transversion sites, respectively. Utilizing excel for SNP site analysis, 35,406 SNPs were identified as significantly associated with the sex of P. nigromaculatus. In pursuit of sex-related markers, 100 SNP sites, aligning perfectly with male heterozygosity and female homozygosity, were randomly selected for amplification and sequencing. Through this approach, 134 SNPs were identified, 83 (61.94%) of which matched the transcriptome data. The findings of this study provide an invaluable resource for future functional research on sex-related genes of P. nigromaculatus and offer a foundation for the development of sex-related molecular markers. [ABSTRACT FROM AUTHOR]