Multi-tissue RNA-Seq Analysis and Long-read-based Genome Assembly Reveal Complex Sex-specific Gene Regulation and Molecular Evolution in the Manila Clam

Abstract

The molecular factors and gene regulation involved in sex determination and gonad differentiation in bivalve molluscs are unknown. It has been suggested that doubly uniparental inheritance (DUI) of mitochondria may be involved in these processes in species such as the ubiquitous and commercially relevant Manila clam, Ruditapes philippinarum. We present the first long-read-based de novo genome assembly of a Manila clam, and a RNA-Seq multi-tissue analysis of 15 females and 15 males. The highly contiguous genome assembly was used as reference to investigate gene expression, alternative splicing, sequence evolution, tissue-specific co-expression networks, and sexual contrasting SNPs. Differential expression and differential splicing analyses revealed sex-specific transcriptional regulation in gonads, but not in somatic tissues. Co-expression networks revealed complex gene regulation in gonads, and genes in gonad-associated modules showed high tissue specificity. However, male gonad-associated modules showed contrasting patterns of sequence evolution and tissue specificity. One gene set was related to the structural organization of male gametes and presented slow sequence evolution but high pleiotropy, while another gene set was enriched in reproduction-related processes and characterized by fast sequence evolution and tissue specificity. Sexual contrasting SNPs were found in genes overrepresented in mitochondrial related functions, providing new candidates for investigating the relationship between mitochondria and sex in DUI species. Together, these results increase our understanding of the role of differential expression, differential splicing, and sequence evolution of sex-specific genes in an understudied taxon. We also provide resourceful genomic data for studies regarding sex diagnosis and breeding in bivalves.

Sex‐specific gene expression in eastern oyster, Crassostrea virginica, gonad and mantle tissues

“…Advances in our understanding of sex differentiation in bivalves have coincided with the availability of cost‐effective next‐generation sequencing technologies and improved genomic resources. Female and male gonad gene expression patterns examined previously in simultaneous hermaphrodite, sequential hermaphrodite, and gonochoric bivalves highlight the involvement of similar transcripts in gonad development, despite differences in sexual system (Li et al, 2016; Teaniniuraitemoana et al, 2015; Xu et al, 2022; Yang et al, 2016). Furthermore, several female‐ and male‐biased transcripts associated with sex differentiation are consistent across eukaryotic species (N. Zhang, Xu, & Guo, 2014).…”