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

Lead Research Organisation: University of Cambridge
Department Name: Pathology

Abstract

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.

Technical Summary

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.
 
Description A genome contains the complete DNA sequence of an animal. Just as the letters in a book are separated into chapters, the DNA sequence within a genome is divided into chromosomes. The number of chromosomes varies between species, but there are similarities which reflect their common ancestry. Two of the chromosomes in a mammalian genome are called the sex chromosomes - the X and Y chromosomes. In general, female mammals have two X chromosomes and males have an X and a Y chromosome. Consequently many of the key genes that differentiate males and females are found on the X and Y chromosomes.

We are interested in the sex chromosomes of pigs particularly, for a number of reasons. Firstly, pigs are an important agricultural species. Secondly, they are used as models for a number of human diseases - for example, for research into obesity, and also for puerperal psychosis, similar to a form of postnatal depression that can severely affect human women. Thirdly, pigs represent a potential source of organs for xenotransplantation.

Our project can be broadly divided into three related areas of structure, content, and evolution:

1) Structure: Regions shared between X and Y

X and Y chromosomes look very different now. However, they once looked very similar. The Y has lost much of the material it once shared with the X. Our goal was to find regions between the X and Y that are still shared - so-called regions of XY homology. Identifying the genes in these regions can help with chromosome sequencing, and tell us about evolution amongst different species of mammal.

We found two regions of the X chromosome that we could confirm contained sequences also found on the Y chromosome. First, we were able to find the boundary of a region called the pseudoautosomal region (PAR). PARs are regions shared between the sex chromosomes - that is, they are the part of the chromosomes that are still identical. When comparing the PAR in pig with that of other species, we found broad similarities. However, there are some genes that appear to be unique to pigs, and other genes lacking in pigs that have been found in other species.

The second region contained some sequence that, while only found in a single location on the X chromosome, was highly repeated across half of the Y chromosome. This is the first time any sequence has been found linking the repetitive part of the Y with the X.

2) Content: Functional genes on the Y

We captured from the repetitive part of the Y chromosome genes that are active in testes (Y genes are often fertility related). After capturing these sequences, we studied them to identify which other tissues they are expressed in.

What we found is that there are a number of genes in the repetitive part of the pig Y chromosome that are active in testes, and few other tissues. There seem to be multiple different variants of these genes. This is probably because as they were copied, errors crept in, or pieces of the chromosome were mixed up and reordered. We were also able from extensive sequencing of the short arm of the pig Y chromosome to build the first sequence map of the pig Y and determine the arrangement of gene and non coding sequences. This has demonstrated that most of the genes on the pig Y are found on the short arm.

3) Evolution: The history of a gene, HSFY

The gene HSFY has amplified on the Y in another species - cow. We suspected HSFY might be on the repetitive part of the pig Y also, and wondered if the amplification occurred in pig alone, in an ancestor of modern pig species, or perhaps was shared from the ancestor of modern pigs and cattle.

We studied the copies in pigs, and were able to identify at least two distinct types of HSFY. Each of these types seems to be repeated many times on the pig Y chromosome. When we looked at other pig species (such as warthogs and peccaries), we were able to find the two different types of HSFY in these species as well. By comparing the copies with those seen in cow, we found that HSFY amplified in cow after the ancestors of pigs and cows diverged.



Overall, this study has given us a better insight into the types and organisation of repetitive and gene sequences on the pig Y chromosome, and may prove important for future work by breeding companies.
Exploitation Route A full draft of the pig X and Y chromosome sequence provides the basis for understanding sex chromosome gene content and function in a range of different contexts - this is primarily of interest to animal breeders, but is also of interest to groups using porcine models of human disease.
Sectors Agriculture, Food and Drink
 
Title Supporting data for "An improved pig reference genome sequence to enable pig genetics and genomics research" 
Description The domestic pig ( Sus scrofa) is important both as a food source and as a biomedical model given its similarity in size, anatomy, physiology, metabolism, pathology and pharmacology to humans. The draft reference genome (Sscrofa10.2) of a purebred Duroc female pig established using older clone-based sequencing methods was incomplete and unresolved redundancies, short range order and orientation errors and associated misassembled genes limited its utility. We present two annotated highly contiguous chromosome-level genome assemblies created with more recent long read technologies and a whole genome shotgun strategy, one for the same Duroc female (Sscrofa11.1) and one for an outbred, composite breed male (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy than Sscrofa10.2. These highly contiguous assemblies plus annotation of a further 11 short read assemblies provide an unprecedented view of the genetic make-up of this important agricultural and biomedical model species. We propose that the improved Duroc assembly (Sscrofa11.1) become the reference genome for genomic research in pigs. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
URL http://gigadb.org/dataset/100732
 
Description Collaboration with the Sanger Institute and Genus plc. This collaboration provided key tissues and DNA samples to permit expression studies and the sequencing of Y chromosome material. 
Organisation Genus plc
Country United Kingdom 
Sector Private 
PI Contribution This project was a joint application with the Sanger Institute in which we collaborated with Genus plc. The sanger Institute managed the sex chromosome library construction, sequencing assembly and annotation of the porcine sex chromosomes. We worked with the Sanger to identify regions of biological interest. Genus plc supplied DNA samples and the boar used to provide various tissues and the Y chromosome library. While we have interacted with the Sanger Institute previously, this collaboration has provided extended contact and gave the opportunity of regular meetings and presentations by members of both groups. For example, it facilitated the provision of lectures to our undergraduates by a Sanger Faculty member. This project has extended our longstanding collaboration with Genus plc and has been useful in fostering further collaboration on subsequent (and current) BBSRC funded Industrial Link grant
Start Year 2009