De Novo assembly of the Manila clam Ruditapes philippinarum transcriptome provides new insights into expression bias, mitochondrial doubly uniparental inheritance and sex determination

Abstract

Males and females share the same genome, thus, phenotypic divergence requires differential gene expression and sex-specific regulation. Accordingly, the analysis of expression patterns is pivotal to the understanding of sex determination mechanisms. Many bivalves are stable gonochoric species, but the mechanism of gonad sexualization and the genes involved are still unknown. Moreover, during the period of sexual rest, a gonad is not present and sex cannot be determined. A mechanism associated with germ line differentiation in some bivalves, including the Manila clam Ruditapes philippinarum, is the doubly uniparental inheritance (DUI) of mitochondria, a variation of strict maternal inheritance. Two mitochondrial lineages are present, one transmitted through eggs and the other through sperm, as well as a mother-dependent sex bias of the progeny. We produced a de novo annotation of 17,186 transcripts from R. philippinarum and compared the transcriptomes of males and females and identified 1,575 genes with strong sex-specific expression and 166 sex-specific single nucleotide polymorphisms, obtaining preliminary information about genes that could be involved in sex determination. Then we compared the transcriptomes between a family producing predominantly females and a family producing predominantly males to identify candidate genes involved in regulation of sex-specific aspects of DUI system, finding a relationship between sex bias and differential expression of several ubiquitination genes. In mammalian embryos, sperm mitochondria are degraded by ubiquitination. A modification of this mechanism is hypothesized to be responsible for the retention of sperm mitochondria in male embryos of DUI species. Ubiquitination can additionally regulate gene expression, playing a role in sex determination of several animals. These data enable us to develop a model that incorporates both the DUI literature and our new findings.

FIG. 1.

Male proportion in families. The percentage of males per family ranges from 8% to 83%. The chi-square test is highly significant (P < 0.001), supporting the sex ratio heterogeneity across all the families.

FIG. 2.

Radar plots of mean gene expression. (A) Sex-biased genes are more highly expressed in males than in females (P < 2.2 × 10⁻¹⁶; table 4). In males, sex-biased genes are 4.7 times more expressed than unbiased genes, whereas in females, the ratio is 2.9 (see table 3). Male-biased genes in females of Family 2 (which produces more males) show higher transcription in comparison to females of Family 1 (which produces more females) (P = 7.9 × 10⁻³; table 4). Males show higher transcription of female-biased genes than females of male-biased genes (P < 2.2 × 10⁻¹⁶; table 4). (B) Family-biased transcripts are more highly expressed in males and females of Family 2, with males having a higher expression than females. In Family 1, the ratio between family-biased and family-unbiased genes is 2.6, whereas it is 3.6 in Family 2 (see table 3).

FIG. 3.

Proportion of annotated genes. All genes: 8,473 genes, corresponding to the 37% of all the data set (orange color), were annotated with GO. Contig sequences were also aligned using a BLASTX search to the nr protein database available from GenBank: 12,915 nodes resulted in a hit when the length of the alignment was required to be greater than 80% of the length of the query. For 4,176 nodes that did not report any acceptable hits, we were able to find local regions of similarity (40% of the length; light blue color). Overall, 17,091 genes (75%) were annotated with BLASTX, providing 8,713 extra hits in addition to the GO annotation, thus, the nonannotated genes were 5,700, that is, the 25% of the data set (gray color). Dark blue color indicates genes annotated by BLASTX (80%) but not with GO.

FIG. 4.

Distribution of GO terms (Level 2). Biological process domain: The most represented terms are cellular process (23%), metabolic process (17%), and biological regulation (10%), whereas developmental process constitutes 7% and reproduction 1.5%. Molecular function domain: Binding (53%) and catalytic activity (34%) are the principal terms. Cellular component domain: An abundance of the organelle term is present (32%).

FIG. 5.

Scatter plot of reads/numbers of SNPs. Number of reads (FKPM) plotted against number of SNPs (number of SNPs/gene length, per 1,000 bp) for each gene. Black dots: unbiased genes; blue dots: male-biased genes; red dots: female-biased genes. The number of SNPs does not increase at higher coverage.

FIG. 6.

Kernel density plots of Tajima’s D, Fu & Li’s D* and F* values. First two lanes: Kernel density plots for each category showed notably different frequency distribution between biased (red lines) and unbiased genes (black lines). The Wilcoxon rank sum test between biased an unbiased genes is significant in all the cases (P values in table 7), and it shows that sex- and family-biased genes have a higher polymorphism compared with unbiased genes. Third lane: Male-biased genes (blue lines) have a higher value than female-biased genes (red lines) and reproductive genes (green lines) appear to be the most variable among the sex-biased genes. Dashed lines indicate mean value.

FIG. 7.

A simplified model for DUI and sex determination. Transcription factors (e.g., ubiquitination genes) stored in female oocytes would activate sex–gene expression in early embryonic developmental stages, and male development would require the crossing of a critical threshold of masculinizing transcripts. The sperm genotype contributes to F2 sex bias. (A, B) A “female egg” will produce a female regardless the genotype of the spermatozoon. (A) If it is fertilized by a spermatozoon with a “female-biased” genotype (g), the F1 female will produce mostly female eggs. (B) If it is fertilized by a spermatozoon with a “male-biased” genotype (G), the F1 female will produce both egg types (50:50). (C, D) A “male egg” will produce a male regardless the genotype of the spermatozoon. (C) If it is fertilized by a spermatozoon with a “male-biased” genotype (G), the F1 male will produce sperm carrying a male-biased genotype (G). (D) If it is fertilized by a spermatozoon with a “female-biased” genotype (g), the F1 male will produce both sperm types (50:50). Some ubiquination factors could also be involved in mitochondrial inheritance, and their differential expression could be responsible for the different fate of sperm mitochondria in the two families: degradation (A, B) or maintenance (C, D). Note that the genomic sex-determining factors (G and g) probably comprise more than one gene; recombination among these genes and environmental factors could account for the nearly continuous distribution of sex ratios among families (table 1; fig. 1).