De Novo Assembly of the Manila Clam Ruditapes philippinarum Transcriptome

By Fabrizio Ghiselli

Despite their biological, ecological and economical importance, very little is known about bivalve genetics/genomics and until recently (see for example Boutet et al. 20081; Craft et al. 20102; Milan et al. 20113), the structure and gene content of bivalve genomes have been poorly understood even in the most important aquacultured organisms. The Manila clam (Ruditapes philippinarum) represents, after oysters, the most important species for Global Aquaculture production.

Exploiting the advantages of Next Generation Sequencing methods we performed a de novo assembly of the R. philippinarum transcriptome, obtaining 22,886 transcripts, of which 17,186 could be annotated to known genes, providing a substantial amount of new genetic data4. However, the most striking feature of R. philippinarum is the Doubly Uniparental Inheritance (DUI), a peculiar system of mitochondrial inheritance, shared with only a few other bivalve molluscs. In DUI species two mitochondrial genomes are present, one transmitted through sperm, the other through eggs. The existence of lineages that show sex-ratio bias associated with specific sperm mitochondrial distribution in embryos suggested a link between DUI and sex-determination mechanism, which is unknown in bivalves. We compared expression patterns between males and females from families with opposite sex-bias and found genes showing sex-bias, family-bias and a sex-family interaction.

A substantial number of biased genes function in the ubiquitination process, which is known to be involved in transcriptional regulation of genes for sex-determination. We propose a preformation mechanism by which transcription factors (such as ubiquitination genes), stored in female oocytes during gametogenesis, would activate sex-gene expression in embryos. The sex-differentiation process would be multifactorial and quantitative: male development would require the crossing of a critical threshold of masculinizing transcripts. Ubiquitination is also associated with sperm mitochondria elimination and a modification of this mechanism has been hypothesized as responsible for the retention of sperm mitochondria in male embryos of DUI species.

This work builds foundations for bivalve genetics. The development of sex-specific markers is a useful tool in selective breeding programs of species with a high economic value. Gene annotation and the analysis of the differential expression between sexes and lineages is fundamental for the characterization of sex-determination and DUI, which represents a valuable model system to understand mitochondrial inheritance.

Cited Literature:

  1. Boutet I, Moraga D, Marinovic L, Obreque J, Chavez-Crooker P. 2008. Characterization of reproduction-specific genes in a marine bivalve mollusc: influence of maturation stage and sex on mRNA expression. Gene 407:130-138. [abstract]
  2. Craft JA, Gilbert JA, Temperton B, Dempsey KE, Ashelford K, Tiwari B, Hutchinson TH, Chipman JK. 2010. Pyrosequencing of Mytilus galloprovincialis cDNAs: tissue-specific expression patterns. PLoS One 5:e8875. [article]
  3. Milan M, Coppe A, Reinhardt R, et al. (11 co-authors). 2011. Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring. BMC Genomics 12:234. [article]
  4. Ghiselli F, Milani L, Chang PL, Hedgecock D, Davis JP, Nuzhdin SV, Passamonti M. De Novo Assembly of the Manila Clam Ruditapes philippinarum Transcriptome Provides New Insights into Expression Bias, Mitochondrial Doubly Uniparental Inheritance and Sex Determination. Mol Biol Evol. 2012 Feb;29(2):771-86. Epub 2011 Oct 5. PubMed PMID: 21976711; PubMed Central PMCID: PMC3258041. [abstract]