The porcine sex chromosomes: gene content sequence relationships and evolutionary implications for sex chromosome functionality

The X and Y chromosomes in mammals have evolved from a common ancestral pair of identical chromosomes. As they have diverged from one another the Y has acquired a specific function in determining male sex. At the same time, the X and Y chromosomes have accumulated genes that confer advantage to the male, including many genes involved in male fertility and behaviour. Particular variants of these genes are postulated to give advantage to some males over others in the competition for reproductive success. The genes that have evolved on the sex chromosomes that are involved in these functions may differ between different mammalian species. To date, the DNA sequence and gene content of the mouse and human sex chromosomes has been the most intensively studied. This has defined X and Y genes that appear to be shared between most mammals, but has also revealed the presence of lineage specific sex chromosome gene content. These studies have demonstrated a number of what appear to be emerging themes that define sex chromosomes; namely (1) gene amplification on the X and Y, (2) palindromic organisation of amplified sequence, (3) expression of genes specifically in the testis and (4) from studies in the mouse, apparent competition between X-Y homologous amplified gene families that influence the number of males to females in the offspring (sex ratio). As only two well defined mammalian lineages have been sequenced and annotated extensively (with fragmentary data on number of primates and other mammals) it is important to extend studies of the sex chromosomes to encompass all branches of mammalian evolution in order to determine the universality of these themes associated with the X and Y. This project will investigate the porcine sex chromosomes in order (1) to understand their gene content and to determine if the pig harbours testis expressed genes not shared by other mammals, (2) to determine the patterns of X-Y homology, (3) to determine what sequences have become co-amplified on the X and Y that may have a role in influencing sex ratio, (4) to understand how these sequences are organised and (5) to understand the variation between Y chromosomes carried by suids and how this relates to variation in boar fertility.

The resources of the Department of Pathology, University of Cambridge and the Sanger Institute will work to deliver the sequence and annotation of the pig X and Y chromosomes. The initial phase of fingerprinting genomic clones and to complete the sequence of key autosomes carrying important QTL has been funded. The X chromosome remains the most fragmented with respect to contigs. The repeat rich Y chromosome has received fewer resources and poses a greater challenge. This project aims to deliver the finished sequence of the porcine X chromosome and substantial segments of the Y representing transcribed regions. These studies aim to address emerging themes characteristic of mammalian sex chromosomes; (1) Identification of the X-Y homologous genes shared with other mammals and those specific to the pig; (2) Identification of X and Y genes expressed specifically in testis and brain; (3) Identification of amplified sequence families, their organisation on X and Y and their co-evolution in suids; (4) Identification of Y SNP variants to allow future study of boar fertility. The project will isolate the X and Y transcriptomes using cDNA selection with DNA prepared from flow-sorted X and Y chromosomes. These cDNA clones will be used to isolate X and Y genomic clones to complete X contigs and develop Y contigs for sequencing. cDNA clones will be sequenced and mapped on to developing X and Y contig sequence. The expression patterns of X and Y transcripts will be determined using array technology and a range of porcine tissues to identify testis and brain specific transcripts. In situ hybridization of testis-specific transcripts to testis sections will provide more detailed analysis in relation to cell type. This project will generate high quality, well annotated genomic sequence for the X and Y chromosomes, together with preliminary functional assessment of sex chromosome encoded genes in the testis and brain.