Transcriptome Assembly and Molecular Evolutionary Analysis of Sex-Biased Genes in the Guppy (Poecilia reticulata)

Abstract

It is a phenomenon universally seen that males and females of a species show phenotypic differences as they evolve under often diverging sex-specific selection pressures. The evolution and maintenance of their sexual dimorphism is generally associated with gene expression divergence between the sexes. Genes that show enriched expression in one sex, also called sex-biased genes, often show rapid molecular evolution. Furthermore, sex-biased genes have also been found to be over-represented on X or Z chromosomes in several species with differentiated sex chromosomes or neo-sex chromosomes. While research on sex-biased genes in drosophilids, mammals and birds has developed in the last decade, there is relatively little known about sex-biased genes in teleost species with largely undifferentiated sex-chromosomes of recent origin. A case in point is that of the Trinidadian guppy, Poecilia reticulata, which is the focal species of my thesis. In this dissertation, I investigate sex-biased gene expression in guppy, a fresh-water fish with XY sex-determination and Y-linked inheritance of male-advantageous traits. Guppies display sexual dimorphism in size, ornaments, and behavior, traits that are shaped by both natural and sexual selection in the wild. My first task was to assemble a transcriptome reference using deep sequencing of cDNA. I compared several methods of assembly with RNA sequencing (RNA-seq) data and assembled and annotated a reference transcriptome combining a genome-independent and a genome-guided assembly. Subsequently, I analyzed sex-biased gene expression in brain, tail and gonads, tissues with overt sexual dimorphism in adult guppies. I found tissue-specific expression generally related to the phenotypic sexual dimorphism. For example, genes related to signal transduction, pigmentation processes and spermatogenesis were expressed more in males; while female-biased genes related to growth, cell-division, extra-cellular matrix organization, nutrient transport, and folliculogenesis. As male sex-determination and differentiation in guppies is believed to be associated with the male-specific pigment patterns, I analyzed the gene-expression and genomic locations of guppy orthologs of candidate genes functional in these processes in other vertebrates. The list of candidate genes could be specifically aligned to the female genome and no male-limited candidate could be identified. I found tissue-specificity in the magnitude and direction of sex-bias in the expression of several sex-related and pigmentation genes. I then studied the genomic distribution of all sex-biased genes. I observed the accumulation of ovary-biased genes on the putative sex linkage group, LG12. Genome-wide comparison of rates of evolution of sex-biased and unbiased genes, measured by the ratio of non-synonymous substitution rate (dN) to the synonymous substitution rate (dS), indicated faster evolution of testis-biased genes, and female-biased genes in all three studied tissues. Among these, the female-biased genes in brain showed elevated ratios of non-synonymous substitutions irrespective of the breadth and magnitude of expression. In this study, I describe a comprehensive annotated guppy reference transcriptome that is compiled after extensive evaluation of different existing methods for assembly using de novo strategies as well as reference-guided strategies. The reference transcriptome of the guppy provides a resource for investigating the molecular genetics of the guppy’s complex adaptive traits. The methods and pipelines are generally applicable for developing and utilizing transcriptomic resources in organisms with limited molecular resources. Genome-wide differential expression between male and female tissues, allowed us to identify genes with strong characteristic differential expression in the differentiated gonads as well as genes with small but significant expression differences in the somatic tissues. These sets of sex-biased genes may be relevant for the tissue-associated sexual dimorphism. Differential genomic distributions of ovary- and testis-biased genes provide evidence for sex-specific selection pressures acting on the slightly differentiated sex chromosomes of the guppy. Elevated rates of molecular evolution observed in testis-biased and all categories of female-biased genes suggest evolution under distinct selection pressures on the reproductive versus non-reproductive tissues. Overall, these results are useful for guppy researchers and for further understanding the evolution of sex differences in diverse species.