Sequence analysis in Bos taurus reveals pervasiveness of X-Y arms races in mammalian lineages.

Studies of Y Chromosome evolution have focused primarily on gene decay, a consequence of suppression of crossing-over with the X Chromosome. Here, we provide evidence that suppression of X-Y crossing-over unleashed a second dynamic: selfish X-Y arms races that reshaped the sex chromosomes in mammals as different as cattle, mice, and men. Using super-resolution sequencing, we explore the Y Chromosome of Bos taurus (bull) and find it to be dominated by massive, lineage-specific amplification of testis-expressed gene families, making it the most gene-dense Y Chromosome sequenced to date. As in mice, an X-linked homolog of a bull Y-amplified gene has become testis-specific and amplified. This evolutionary convergence implies that lineage-specific X-Y coevolution through gene amplification, and the selfish forces underlying this phenomenon, were dominatingly powerful among diverse mammalian lineages. Together with Y gene decay, X-Y arms races molded mammalian sex chromosomes and influenced the course of mammalian evolution.

Copy number variation in the horse genome.

We constructed a 400K WG tiling oligoarray for the horse and applied it for the discovery of copy number variations (CNVs) in 38 normal horses of 16 diverse breeds, and the Przewalski horse. Probes on the array represented 18,763 autosomal and X-linked genes, and intergenic, sub-telomeric and chrY sequences. We identified 258 CNV regions (CNVRs) across all autosomes, chrX and chrUn, but not in chrY. CNVs comprised 1.3% of the horse genome with chr12 being most enriched. American Miniature horses had the highest and American Quarter Horses the lowest number of CNVs in relation to Thoroughbred reference. The Przewalski horse was similar to native ponies and draft breeds. The majority of CNVRs involved genes, while 20% were located in intergenic regions. Similar to previous studies in horses and other mammals, molecular functions of CNV-associated genes were predominantly in sensory perception, immunity and reproduction. The findings were integrated with previous studies to generate a composite genome-wide dataset of 1476 CNVRs. Of these, 301 CNVRs were shared between studies, while 1174 were novel and require further validation. Integrated data revealed that to date, 41 out of over 400 breeds of the domestic horse have been analyzed for CNVs, of which 11 new breeds were added in this study. Finally, the composite CNV dataset was applied in a pilot study for the discovery of CNVs in 6 horses with XY disorders of sexual development. A homozygous deletion involving AKR1C gene cluster in chr29 in two affected horses was considered possibly causative because of the known role of AKR1C genes in testicular androgen synthesis and sexual development. While the findings improve and integrate the knowledge of CNVs in horses, they also show that for effective discovery of variants of biomedical importance, more breeds and individuals need to be analyzed using comparable methodological approaches.

The Eutherian Pseudoautosomal Region.

The pseudoautosomal region (PAR) is a unique segment of sequence homology between differentiated sex chromosomes where recombination occurs during meiosis. Molecular and functional properties of the PAR are distinctive from the autosomes and the remaining regions of the sex chromosomes. These include a higher rate of recombination than genome average, bias towards GC-substitutions and increased interindividual nucleotide divergence and mutations. As yet, the PAR has been physically demarcated in only 28 eutherian species representing 6 mammalian orders. Murid rodents have the smallest, gene-poorest and most diverged PARs. Other eutherian PARs are largely homologous but differ in size and gene content, being the smallest in equids and human/simian primates and much larger in other eutherians. Because pseudoautosomal genes escape X inactivation, their dosage changes with sex chromosome aneuploidies, whereas phenotypic effects of the latter depend on the size and gene content of the PAR. Thus, X monosomy is more viable in mice, humans and horses than in species with larger PARs. Presently, little is known about the functions of PAR genes in individual species, though human studies suggest their involvement in early embryonic development. The PAR is, thus, of evolutionary, genetic and biomedical significance and a 'research hotspot' in eutherian genomes.

Genome-wide association study implicates testis-sperm specific FKBP6 as a susceptibility locus for impaired acrosome reaction in stallions.

Impaired acrosomal reaction (IAR) of sperm causes male subfertility in humans and animals. Despite compelling evidence about the genetic control over acrosome biogenesis and function, the genomics of IAR is as yet poorly understood, providing no molecular tools for diagnostics. Here we conducted Equine SNP50 Beadchip genotyping and GWAS using 7 IAR-affected and 37 control Thoroughbred stallions. A significant (P<6.75E-08) genotype-phenotype association was found in horse chromosome 13 in FK506 binding protein 6 (FKBP6). The gene belongs to the immunophilins FKBP family known to be involved in meiosis, calcium homeostasis, clathrin-coated vesicles, and membrane fusions. Direct sequencing of FKBP6 exons in cases and controls identified SNPs g.11040315G>A and g.11040379C>A (p.166H>N) in exon 4 that were significantly associated with the IAR phenotype both in the GWAS cohort (n = 44) and in a large multi-breed cohort of 265 horses. All IAR stallions were homozygous for the A-alleles, while this genotype was found only in 2% of controls. The equine FKBP6 was exclusively expressed in testis and sperm and had 5 different transcripts, of which 4 were novel. The expression of this gene in AC/AG heterozygous controls was monoallelic, and we observed a tendency for FKBP6 up-regulation in IAR stallions compared to controls. Because exon 4 SNPs had no effect on the protein structure, it is likely that FKBP6 relates to the IAR phenotype via regulatory or modifying functions. In conclusion, FKBP6 was considered a susceptibility gene of incomplete penetrance for IAR in stallions and a candidate gene for male subfertility in mammals. FKBP6 genotyping is recommended for the detection of IAR-susceptible individuals among potential breeding stallions. Successful use of sperm as a source of DNA and RNA propagates non-invasive sample procurement for fertility genomics in animals and humans.

Serum metabolomics identifies citrulline as a predictor of adverse outcomes in an equine model of gut-derived sepsis.

Acute laminitis is an inflammatory disease of the equine foot that often occurs secondarily to sepsis or systemic inflammation associated with gastrointestinal disease. It has been suggested that laminitis is similar to multiple organ dysfunction syndrome in humans, although in horses the weight-bearing laminar epithelium of the foot appears to be the tissue most sensitive to insult and the first "organ" to fail. Metabolomics performed on serum samples collected before (Con) and after (Lmn) experimental induction of gastrointestinal-associated sepsis in six horses detected 1,177 metabolites of both mammalian and bacterial origin in equine serum. Network and correlation analyses suggested a dysregulation of fatty acid metabolism in the Lmn group, as well as an accumulation of organic acids such as lactate. Furthermore, concentrations of the amino acid citrulline were decreased in Lmn samples from all study animals, suggesting that citrulline might be useful as a biomarker to identify critically ill animals that are at risk of developing laminitis. We therefore established normal ranges of plasma citrulline concentrations in a separate group of horses (n = 36) and tested the ability of citrulline to predict adverse outcomes (laminitis or death) in critically ill horses (n = 23). Plasma citrulline was significantly lower in critically ill horses that went on to experience adverse outcomes (n = 6). Further study is required to accurately determine a diagnostic cutoff, but the present data are suggestive of the predictive value of citrulline as a biomarker for laminar failure in equine sepsis.

Plasma proteomics shows an elevation of the anti-inflammatory protein APOA-IV in chronic equine laminitis.

BACKGROUND: Equine laminitis is a devastating disease that causes severe pain in afflicted horses and places a major economic burden on the horse industry. In acute laminitis, the disintegration of the dermal-epidermal junction can cause the third phalanx to detach from the hoof wall, leaving the horse unable to bear weight on the affected limbs. Horses that survive the acute phase transition into a chronic form of laminitis, which is often termed "founder". Some evidence suggests that chronic laminar inflammation might be associated with alterations in the endocrine and immune systems. We investigated this broad hypothesis by using DIGE to assess global differences in the plasma proteome between horses with chronic laminitis and controls. RESULTS: We identified 16 differentially expressed proteins; the majority of these were involved in the interrelated coagulation, clotting, and kininogen cascades. Clinical testing of functional coagulation parameters in foundered horses revealed a slight delay in prothrombin (PT) clotting time, although most other indices were within normal ranges. Upregulation of the intestinal apolipoprotein APOA-IV in horses with chronic laminitis was confirmed by western blot. CONCLUSIONS: Our results support the hypothesis that localized laminar inflammation may be linked to systemic alterations in immune regulation, particularly in the gastrointestinal system. Gastrointestinal inflammation has been implicated in the development of acute laminitis but has not previously been associated with chronic laminitis.

Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis.

BACKGROUND: The nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis. RESULTS: Fecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks. CONCLUSIONS: Our exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis.

Classification of diet-modulated gene signatures at the colon cancer initiation and progression stages.

BACKGROUND: The effects of dietary polyunsaturated (PUFAs) and monounsaturated fatty acids (MUFAs) on intestinal cytokinetics within the context of colon cancer initiation and progression have been extensively studied. n-3 PUFAs have received the most attention due to their potential protective role. However, further investigation of the epigenetic perturbations caused by fatty acids in the context of colon cancer development is needed. METHODS: We used DNA microarrays to identify discriminative gene signatures (gene combinations) for the purpose of classifying n-3 PUFA-fed, carcinogen-injected, Sprague-Dawley rats at the initiation and progression stages. Animals were assigned to three dietary treatments differing only in the type of fat (corn oil/n-6 PUFA, fish oil/n-3 PUFA, or olive oil/n-9 monounsaturated fatty acid). RESULTS: The effects of diet on colonic mucosal gene expression signatures during tumor initiation and progression were subsequently compared (12 h and 10 weeks after azoxymethane injection). Microarray analysis revealed that the number of differentially expressed (DE) genes in each of the three diet comparisons increased with the progression of colon cancer. Each dietary lipid source exhibited its own unique transcriptional profile, as assessed by linear discriminant analysis. Applying this novel approach, we identified the single genes and the two- to three-gene combinations that best distinguished the dietary treatment groups. For the chemoprotective (fish oil) diet, mediators of stem cell homeostasis, e.g., ephrin B1 and bone morphogenic protein 4, were the top-performing gene classifiers. CONCLUSIONS: These results suggest that dietary chemoprotective n-3 PUFA impact genes that regulate the colon stem cell niche and tumor evolution.

A high-resolution cat radiation hybrid and integrated FISH mapping resource for phylogenomic studies across Felidae.

We describe the construction of a high-resolution radiation hybrid (RH) map of the domestic cat genome, which includes 2662 markers, translating to an estimated average intermarker distance of 939 kilobases (kb). Targeted marker selection utilized the recent feline 1.9x genome assembly, concentrating on regions of low marker density on feline autosomes and the X chromosome, in addition to regions flanking interspecies chromosomal breakpoints. Average gap (breakpoint) size between cat-human ordered conserved segments is less than 900 kb. The map was used for a fine-scale comparison of conserved syntenic blocks with the human and canine genomes. Corroborative fluorescence in situ hybridization (FISH) data were generated using 129 domestic cat BAC clones as probes, providing independent confirmation of the long-range correctness of the map. Cross-species hybridization of BAC probes on divergent felids from the genera Profelis (serval) and Panthera (snow leopard) provides further evidence for karyotypic conservation within felids, and demonstrates the utility of such probes for future studies of chromosome evolution within the cat family and in related carnivores. The integrated map constitutes a comprehensive framework for identifying genes controlling feline phenotypes of interest, and to aid in assembly of a higher coverage feline genome sequence.

Novel gene acquisition on carnivore Y chromosomes.

Despite its importance in harboring genes critical for spermatogenesis and male-specific functions, the Y chromosome has been largely excluded as a priority in recent mammalian genome sequencing projects. Only the human and chimpanzee Y chromosomes have been well characterized at the sequence level. This is primarily due to the presumed low overall gene content and highly repetitive nature of the Y chromosome and the ensuing difficulties using a shotgun sequence approach for assembly. Here we used direct cDNA selection to isolate and evaluate the extent of novel Y chromosome gene acquisition in the genome of the domestic cat, a species from a different mammalian superorder than human, chimpanzee, and mouse (currently being sequenced). We discovered four novel Y chromosome genes that do not have functional copies in the finished human male-specific region of the Y or on other mammalian Y chromosomes explored thus far. Two genes are derived from putative autosomal progenitors, and the other two have X chromosome homologs from different evolutionary strata. All four genes were shown to be multicopy and expressed predominantly or exclusively in testes, suggesting that their duplication and specialization for testis function were selected for because they enhance spermatogenesis. Two of these genes have testis-expressed, Y-borne copies in the dog genome as well. The absence of the four newly described genes on other characterized mammalian Y chromosomes demonstrates the gene novelty on this chromosome between mammalian orders, suggesting it harbors many lineage-specific genes that may go undetected by traditional comparative genomic approaches. Specific plans to identify the male-specific genes encoded in the Y chromosome of mammals should be a priority.

Gene discovery and comparative analysis of X-degenerate genes from the domestic cat Y chromosome.

Mammalian sex chromosomes are the remnants of an ancient autosomal pair present in the ancestral mammalian karyotype. As a consequence of random decay and chromosome rearrangements over evolutionary time, Y chromosome gene repertoires differ between eutherian lineages. To investigate the gene repertoire and transcriptional analysis of the domestic cat Y chromosome, and their potential roles in spermatogenesis, we obtained full-length cDNA sequences for all known Y genes and their X chromosome gametologues and used those sequences to create a BAC-based physical map of the X-degenerate region. Our results indicate the domestic cat Y chromosome has retained most X-degenerate genes that were present on the ancestral eutherian Y chromosome. Transcriptional analysis revealed that most feline X-degenerate genes have retained housekeeping functions shared by their X chromosome partners and have not been specialized for testis-specific functions. Physical mapping data indicate that the cat SRY gene is present as multiple functional copies and that very little of the felid Y chromosome may be single copy. X-Y gene divergence time estimates obtained using Bayesian methods confirm an early origin of Stratum 1 genes prior to the origin of therian mammals. We observed no statistical difference in the ages of Stratum 2 and Stratum 3 gene pairs, suggesting that eutherian and marsupial Stratum 2 genes may have been independently retained in each lineage.

The horse Y chromosome as an informative marker for tracing sire lines.

Analysis of the Y chromosome is the best-established way to reconstruct paternal family history in humans. Here, we applied fine-scaled Y-chromosomal haplotyping in horses with biallelic markers and demonstrate the potential of our approach to address the ancestry of sire lines. We de novo assembled a draft reference of the male-specific region of the Y chromosome from Illumina short reads and then screened 5.8 million basepairs for variants in 130 specimens from intensively selected and rural breeds and nine Przewalski's horses. Among domestic horses we confirmed the predominance of a young'crown haplogroup' in Central European and North American breeds. Within the crown, we distinguished 58 haplotypes based on 211 variants, forming three major haplogroups. In addition to two previously characterised haplogroups, one observed in Arabian/Coldblooded and the other in Turkoman/Thoroughbred horses, we uncovered a third haplogroup containing Iberian lines and a North African Barb Horse. In a genealogical showcase, we distinguished the patrilines of the three English Thoroughbred founder stallions and resolved a historic controversy over the parentage of the horse 'Galopin', born in 1872. We observed two nearly instantaneous radiations in the history of Central and Northern European Y-chromosomal lineages that both occurred after domestication 5,500 years ago.

FISH for mapping single copy genes.

During the past two decades fluorescent in-situ hybridization (FISH) has become a standard technique to directly localize, orient, and order genes in the genomes of a wide range of species. Despite the availability of a variety of probes, probe labeling and signal-detection systems, and advanced image analysis software, the core procedures used to carry out FISH remain the same. A detailed overview of these procedures, including target preparation (metaphase/interphase chromosomes and DNA fibers), probe labeling, in-situ hybridization, signal detection, and imaging, is here provided in a stepwise manner.

Potential applications of equine genomics in dissecting diseases and fertility.

Following the recent development of high-resolution gene maps and generation of several basic tools and resources to use them in analyzing traits that are economically important to horse owners, genome analysis in horses is witnessing a shift towards developing an ability to analyze complex traits. The likelihood of this happening in the very near future is great, mainly because of the recent availability of the whole genome sequence in the horse. The latter has triggered the development of novel tools like SNP-chip and expression arrays that will permit rapid genome-wide analysis. While these tools will be used for a range of multi-factorial disease traits, attempts are underway to develop focused tools that can target reproduction, fertility and sex determination. For this, a catalog of sex and reproduction related (SRR) genes is being developed in horses. A recently developed dense map of the horse Y chromosome will provide genes that are expressed exclusively in males and, therefore, have an impact on stallion fertility. Overall, these advances in equine genome analysis hold promise for improved diagnosis and treatment of various conditions in horses.

Horse Y chromosome assembly displays unique evolutionary features and putative stallion fertility genes.

Dynamic evolutionary processes and complex structure make the Y chromosome among the most diverse and least understood regions in mammalian genomes. Here, we present an annotated assembly of the male specific region of the horse Y chromosome (eMSY), representing the first comprehensive Y assembly in odd-toed ungulates. The eMSY comprises single-copy, equine specific multi-copy, PAR transposed, and novel ampliconic sequence classes. The eMSY gene density approaches that of autosomes with the highest number of retained X-Y gametologs recorded in eutherians, in addition to novel Y-born and transposed genes. Horse, donkey and mule testis RNAseq reveals several candidate genes for stallion fertility. A novel testis-expressed XY ampliconic sequence class, ETSTY7, is shared with the parasite Parascaris genome, providing evidence for eukaryotic horizontal transfer and inter-chromosomal mobility. Our study highlights the dynamic nature of the Y and provides a reference sequence for improved understanding of equine male development and fertility.

Characterization of the equine glycogen debranching enzyme gene (AGL): Genomic and cDNA structure, localization, polymorphism and expression.

Glycogen debranching enzyme (AGL) is a multifunctional enzyme acting in the glycogen degradation pathway. In humans, the AGL activity deficiency causes a type III glycogen storage disease (Cori-Forbes disease). One particularity of AGL gene expression lies in the multiple alternative splicing in its 5' region. The AGL gene was localized on ECA5q14-q15. The sequence of the equine cDNA was determined to be 7.5 kb in length with an open reading frame of 4602 bp. The gene is 69 kb long and contains 35 exons. The equine AGL gene has an ubiquitous expression and presents five tissue-dependent cDNA variants arising from alternative splicing of the first exons. The equine skeletal muscle and heart contain four out of six variants previously described in humans and the equine liver express three of these four human variants. We identified a new alternative splicing variant expressed in equine skeletal and heart muscles. All these mRNA variants most probably encode only two different protein isoforms of 1533 and 1377 amino-acids. Four SNPs were detected in the mRNA. The equine in silico promoter sequence reveals a structure similar to those of other mammalian species. The disposition of the transcription factor biding sites does not correlate to the transcription start sites of tissue-specific variants.

Sequence analysis of a 212 kb defensin gene cluster on ECA 27q17.

Defensins are a family of evolutionary ancient antimicrobial peptides consisting of three sub-families: alpha-, beta- and theta-defensins. This investigation was focused on the genomic characterization of equine beta-defensins and the investigation of the potential clustering of beta-defensin genes in the equine genome. Six genomic BAC clones were isolated from the CHORI-241 library and one of these was mapped by FISH to ECA 27q17. This location was confirmed by RH-mapping. The contiguous 212 kb sequence of this clone was determined. Sequence analysis revealed the identification of ten pseudogenes and nine genes, six of which were highly homologous to human beta-defensin DEFB4. Clustering of the beta-defensin genes was confirmed and the order of the genes on the analyzed BAC was related to the corresponding defensin cluster on HSA 8. The knowledge about the sequence and the genomic structure of the equine beta-defensin genes will improve the classification of different paralogous defensin genes and is a prerequisite for subsequent functional studies. Additionally, the first alpha-defensin-like sequence outside the groups of primates, lagomorphs and rodents (glires) was identified.

Polymorphism identification, RH mapping, and association analysis with the anxiety trait of the equine serotonin transporter (SLC6A4) gene.

Equine anxiety trait is considered an important temperament in various situations, including riding, training, and daily care. This study examined the polymorphism of the equine serotonin transporter (SLC6A4) gene as a candidate genetic element influencing equine anxiety trait. The sequence of the coding region of this gene was highly homologous with those of other mammals, and four single nucleotide polymorphisms were found by comparing the sequences of ten genetically unrelated thoroughbred horses. Radiation hybrid mapping revealed that this gene was located 26.92 cR from neurofibromin 1 on ECA 11. Using two-year-old thoroughbred horses (n=67), the association of these polymorphisms with the anxiety trait was examined, but no significant association was identified between each haplotype of the serotonin transporter gene and the anxiety score.

Chromosome Aberrations and Fertility Disorders in Domestic Animals.

The association between chromosomal abnormalities and reduced fertility in domestic animals is well recorded and has been studied for decades. Chromosome aberrations directly affect meiosis, gametogenesis, and the viability of zygotes and embryos. In some instances, balanced structural rearrangements can be transmitted, causing fertility problems in subsequent generations. Here, we aim to give a comprehensive overview of the current status and future prospects of clinical cytogenetics of animal reproduction by focusing on the advances in molecular cytogenetics during the genomics era. We describe how advancing knowledge about animal genomes has improved our understanding of connections between gross structural or molecular chromosome variations and reproductive disorders. Further, we expand on a key area of reproduction genetics: cytogenetics of animal gametes and embryos. Finally, we describe how traditional cytogenetics is interfacing with advanced genomics approaches, such as array technologies and next-generation sequencing, and speculate about the future prospects.