Publications by authors named "Bhanu P Chowdhary"

45 Publications

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

Genome Res 2020 Dec 18;30(12):1716-1726. Epub 2020 Nov 18.

Whitehead Institute, Cambridge, Massachusetts 02142, USA.

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 (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.
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http://dx.doi.org/10.1101/gr.269902.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706723PMC
December 2020

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

Sci Rep 2019 04 15;9(1):6095. Epub 2019 Apr 15.

Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, 1210, Austria.

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.
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http://dx.doi.org/10.1038/s41598-019-42640-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465346PMC
April 2019

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

Nat Commun 2018 07 27;9(1):2945. Epub 2018 Jul 27.

Texas A&M University, College Station, TX, 77843, USA.

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.
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http://dx.doi.org/10.1038/s41467-018-05290-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063916PMC
July 2018

Chromosome Aberrations and Fertility Disorders in Domestic Animals.

Annu Rev Anim Biosci 2016 ;4:15-43

New Research Complex, Qatar University, Al Tarfa, Doha 2713, Qatar; email:

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.
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http://dx.doi.org/10.1146/annurev-animal-021815-111239DOI Listing
December 2016

The Eutherian Pseudoautosomal Region.

Cytogenet Genome Res 2015 6;147(2-3):81-94. Epub 2016 Jan 6.

Department of Veterinary Integrative Biosciences, CVM, Texas A&M University, College Station, Tex., USA.

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.
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http://dx.doi.org/10.1159/000443157DOI Listing
August 2016

Copy number variation in the horse genome.

PLoS Genet 2014 Oct 23;10(10):e1004712. Epub 2014 Oct 23.

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, Texas, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1004712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207638PMC
October 2014

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

Physiol Genomics 2014 May 11;46(10):339-47. Epub 2014 Mar 11.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas;

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.
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http://dx.doi.org/10.1152/physiolgenomics.00180.2013DOI Listing
May 2014

No bull: upholding community standards in public sharing of biological datasets.

Proc Natl Acad Sci U S A 2013 Nov 30;110(46):E4277. Epub 2013 Oct 30.

Whitehead Institute, Cambridge, MA 02142.

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http://dx.doi.org/10.1073/pnas.1315122110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831999PMC
November 2013

Age-related changes following in vitro stimulation with Rhodococcus equi of peripheral blood leukocytes from neonatal foals.

PLoS One 2013 17;8(5):e62879. Epub 2013 May 17.

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, United States of America.

Rhodococcus equi is an intracellular bacterium primarily known as an equine pathogen that infects young foals causing a pyogranulomatuous pneumonia. The molecular mechanisms mediating the immune response of foals to R. equi are not fully elucidated. Hence, global genomic high-throughput tools like gene expression microarrays might identify age-related gene expression signatures and molecular pathways that contribute to the immune mechanisms underlying the inherent susceptibility of foals to disease caused by R. equi. The objectives of this study were 2-fold: 1) to compare the expression profiles at specific ages of blood leukocytes from foals stimulated with virulent R. equi with those of unstimulated leukocytes; and, 2) to characterize the age-related changes in the gene expression profile associated with blood leukocytes in response to stimulation with virulent R. equi. Peripheral blood leukocytes were obtained from 6 foals within 24 hours (h) of birth (day 1) and 2, 4, and 8 weeks after birth. The samples were split, such that half were stimulated with live virulent R. equi, and the other half served as unstimulated control. RNA was extracted and the generated cDNA was labeled with fluorescent dyes for microarray hybridizations using an equine microarray. Our findings suggest that there is age-related differential expression of genes involved in host immune response and immunity. We found induction of genes critical for host immunity against pathogens (MHC class II) only at the later time-points (compared to birth). While it appears that foals up to 8-weeks of age are able to initiate a protective inflammatory response against the bacteria, relatively decreased expression of various other immune-related genes points toward inherent diminished immune responses closer to birth. These genes and pathways may contribute to disease susceptibility in foals if infected early in life, and might thus be targeted for developing preventative or therapeutic strategies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062879PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656898PMC
December 2013

Cellular and humoral immunity in chronic equine laminitis.

Vet Immunol Immunopathol 2013 Jun 13;153(3-4):217-26. Epub 2013 Mar 13.

Veterinary Integrative Biosciences, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843-4458, USA.

Chronic equine laminitis causes persistent pain and lameness in affected animals and often necessitates euthanasia when pain management strategies become ineffective. Published studies as well as anecdotal reports suggest that this chronic inflammatory disease is associated with systemic alterations in immune responsiveness, perhaps involving an autoimmune component. We investigated this broad hypothesis by measuring a variety of immune indicators in healthy control horses (CON) and horses with chronic laminitis (LMN). We found that white blood cells from LMN horses produced more IFNγ than did cells from CON horses when stimulated in vitro with polyinosinic-polycytidylic acid [poly(I:C)], possibly due to an elevated number of circulating monocytes. No differences between groups were observed in plasma concentrations of IgG, IgA, IgM, IgE, or rheumatoid factor. Laminar tissue from LMN horses expressed elevated levels of keratinocyte damage-related genes as well as inflammatory cytokines and chemokines, which corresponded with a modest amount of neutrophil infiltration as shown by histological staining of fixed tissue and accumulation of neutrophil elastase protein. Taken together, our results do not support the hypothesis of an autoimmune component in chronic laminitis, although the strong induction of neutrophil chemokines and the presence of tissue neutrophils suggests that this cell type is likely involved in perpetuating the inflammation and tissue damage associated with this disease.
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http://dx.doi.org/10.1016/j.vetimm.2013.03.001DOI Listing
June 2013

Stallion sperm transcriptome comprises functionally coherent coding and regulatory RNAs as revealed by microarray analysis and RNA-seq.

PLoS One 2013 11;8(2):e56535. Epub 2013 Feb 11.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, United States of America.

Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analyzing sperm and testes RNA on a 21,000-element equine whole-genome oligoarray and by RNA-seq. Microarray analysis revealed 6,761 transcripts in the sperm, of which 165 were sperm-enriched, and 155 were differentially expressed between the sperm and testes. Next, 70 million raw reads were generated by RNA-seq of which 50% could be aligned with the horse reference genome. A total of 19,257 sequence tags were mapped to all horse chromosomes and the mitochondrial genome. The highest density of mapped transcripts was in gene-rich ECA11, 12 and 13, and the lowest in gene-poor ECA9 and X; 7 gene transcripts originated from ECAY. Structural annotation aligned sperm transcripts with 4,504 known horse and/or human genes, rRNAs and 82 miRNAs, whereas 13,354 sequence tags remained anonymous. The data were aligned with selected equine gene models to identify additional exons and splice variants. Gene Ontology annotations showed that sperm transcripts were associated with molecular processes (chemoattractant-activated signal transduction, ion transport) and cellular components (membranes and vesicles) related to known sperm functions at fertilization, while some messenger and micro RNAs might be critical for early development. The findings suggest that the rich repertoire of coding and non-coding RNAs in stallion sperm is not a random remnant from spermatogenesis in testes but a selectively retained and functionally coherent collection of RNAs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056535PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3569414PMC
July 2013

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

PLoS Genet 2012 20;8(12):e1003139. Epub 2012 Dec 20.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, USA.

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.
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http://dx.doi.org/10.1371/journal.pgen.1003139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527208PMC
May 2013

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

BMC Vet Res 2012 Nov 27;8:231. Epub 2012 Nov 27.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843-4458, USA.

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.
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http://dx.doi.org/10.1186/1746-6148-8-231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538718PMC
November 2012

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

BMC Vet Res 2012 Sep 27;8:179. Epub 2012 Sep 27.

Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX 77845-4458, USA.

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.
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http://dx.doi.org/10.1186/1746-6148-8-179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511297PMC
September 2012

A gene catalogue of the euchromatic male-specific region of the horse Y chromosome: comparison with human and other mammals.

PLoS One 2011 25;6(7):e21374. Epub 2011 Jul 25.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, United States of America.

Studies of the Y chromosome in primates, rodents and carnivores provide compelling evidence that the male specific region of Y (MSY) contains functional genes, many of which have specialized roles in spermatogenesis and male-fertility. Little similarity, however, has been found between the gene content and sequence of MSY in different species. This hinders the discovery of species-specific male fertility genes and limits our understanding about MSY evolution in mammals. Here, a detailed MSY gene catalogue was developed for the horse--an odd-toed ungulate. Using direct cDNA selection from horse testis, and sequence analysis of Y-specific BAC clones, 37 horse MSY genes/transcripts were identified. The genes were mapped to the MSY BAC contig map, characterized for copy number, analyzed for transcriptional profiles by RT-PCR, examined for the presence of ORFs, and compared to other mammalian orthologs. We demonstrate that the horse MSY harbors 20 X-degenerate genes with known orthologs in other eutherian species. The remaining 17 genes are acquired or novel and have so far been identified only in the horse or donkey Y chromosomes. Notably, 3 transcripts were found in the heterochromatic part of the Y. We show that despite substantial differences between the sequence, gene content and organization of horse and other mammalian Y chromosomes, the functions of MSY genes are predominantly related to testis and spermatogenesis. Altogether, 10 multicopy genes with testis-specific expression were identified in the horse MSY, and considered likely candidate genes for stallion fertility. The findings establish an important foundation for the study of Y-linked genetic factors governing fertility in stallions, and improve our knowledge about the evolutionary processes that have shaped Y chromosomes in different mammalian lineages.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021374PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143126PMC
December 2011

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

Dig Dis Sci 2011 Sep 16;56(9):2595-604. Epub 2011 Mar 16.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843-4458, USA.

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.
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http://dx.doi.org/10.1007/s10620-011-1652-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139012PMC
September 2011

Total RNA isolation from stallion sperm and testis biopsies.

Theriogenology 2010 Oct 7;74(6):1099-1106, 1106e1-2. Epub 2010 Jul 7.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX77843, USA.

Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200 x g for 30 min through a 40% Equipure silanized silica particle solution. Sperm RNA isolation was more efficient with TRIzol reagent than with a spin-column based method; it resulted in 2 microg of total RNA per 100 x 10(6) sperm. To evaluate RNA quantity and quality, we used a NanoDrop spectrophotometer and Agilent Bioanalyzer. A protocol for reverse transcriptase PCR with equine primers for PRM2 and PTPRC genes was developed to determine sperm RNA contamination with genomic DNA or RNA from somatic cells. By these methods, hybridization- and sequencing-quality RNA was isolated from 11 samples of stallion sperm. Stallion testis biopsy with a 14 gauge 22 mm deep biopsy needle yielded approximately 12 microg of good quality total RNA, and could serve as an alternative to excision surgery for sample procurement. Compared to RNA isolation from testis, the sperm required advanced processing and RNA quality control. The described methodologies provided a foundation to establish functional genomic studies of stallion fertility.
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http://dx.doi.org/10.1016/j.theriogenology.2010.04.023DOI Listing
October 2010

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

Genomics 2009 Apr 5;93(4):299-304. Epub 2008 Nov 5.

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Mail Stop 4458, College Station, TX 77843-4458, USA.

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.
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http://dx.doi.org/10.1016/j.ygeno.2008.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2656592PMC
April 2009

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

Genomics 2008 Nov 28;92(5):329-38. Epub 2008 Aug 28.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843-4458, USA.

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.
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http://dx.doi.org/10.1016/j.ygeno.2008.06.012DOI Listing
November 2008

FISH for mapping single copy genes.

Methods Mol Biol 2008 ;422:31-49

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA.

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.
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http://dx.doi.org/10.1007/978-1-59745-581-7_3DOI Listing
August 2008

Potential applications of equine genomics in dissecting diseases and fertility.

Anim Reprod Sci 2008 Sep 29;107(3-4):208-18. Epub 2008 Apr 29.

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4458, USA.

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.
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http://dx.doi.org/10.1016/j.anireprosci.2008.04.010DOI Listing
September 2008

The horse genome derby: racing from map to whole genome sequence.

Chromosome Res 2008 ;16(1):109-27

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 77843-4458, USA.

The map of the horse genome has undergone unprecedented expansion during the past six years. Beginning from a modest collection of approximately 300 mapped markers scattered on the 31 pairs of autosomes and the X chromosome in 2001, today the horse genome is among the best-mapped in domestic animals. Presently, high-resolution linearly ordered gene maps are available for all autosomes as well as the X and the Y chromosome. The approximately 4350 mapped markers distributed over the approximately 2.68 Gbp long equine genome provide on average 1 marker every 620 kb. Among the most remarkable developments in equine genome analysis is the availability of the assembled sequence (EquCab2) of the female horse genome and the generation approximately 1.5 million single nucleotide polymorphisms (SNPs) from diverse breeds. This has triggered the creation of new tools and resources like the 60K SNP-chip and whole genome expression microarrays that hold promise to study the equine genome and transcriptome in ways not previously envisaged. As a result of these developments it is anticipated that, during coming years, the genetics underlying important monogenic traits will be analyzed with improved accuracy and speed. Of larger interest will be the prospects of dissecting the genetic component of various complex/multigenic traits that are of vital significance for equine health and welfare. The number of investigations recently initiated to study a multitude of such traits hold promise for improved diagnostics, prevention and therapeutic approaches for horses.
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http://dx.doi.org/10.1007/s10577-008-1204-zDOI Listing
May 2008

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

Gene 2007 Dec 23;404(1-2):1-9. Epub 2007 Aug 23.

Institut National de la Recherche Agronomique, UR339, Centre de Recherches de Jouy, Laboratoire de Génétique biochimique et de Cytogénétique, 78350 Jouy-en-Josas, France.

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.
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http://dx.doi.org/10.1016/j.gene.2007.07.034DOI Listing
December 2007

High-resolution gene maps of horse chromosomes 14 and 21: additional insights into evolution and rearrangements of HSA5 homologs in mammals.

Genomics 2007 Jan 17;89(1):89-112. Epub 2006 Aug 17.

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.

High-resolution physically ordered gene maps for equine homologs of human chromosome 5 (HSA5), viz., horse chromosomes 14 and 21 (ECA14 and ECA21), were generated by adding 179 new loci (131 gene-specific and 48 microsatellites) to the existing maps of the two chromosomes. The loci were mapped primarily by genotyping on a 5000-rad horse x hamster radiation hybrid panel, of which 28 were mapped by fluorescence in situ hybridization. The approximately fivefold increase in the number of mapped markers on the two chromosomes improves the average resolution of the map to 1 marker/0.9 Mb. The improved resolution is vital for rapid chromosomal localization of traits of interest on these chromosomes and for facilitating candidate gene searches. The comparative gene mapping data on ECA14 and ECA21 finely align the chromosomes to sequence/gene maps of a range of evolutionarily distantly related species. It also demonstrates that compared to ECA14, the ECA21 segment corresponding to HSA5 is a more conserved region because of preserved gene order in a larger number of and more diverse species. Further, comparison of ECA14 and the distal three-quarters region of ECA21 with corresponding chromosomal segments in 50 species belonging to 11 mammalian orders provides a broad overview of the evolution of these segments in individual orders from the putative ancestral chromosomal configuration. Of particular interest is the identification and precise demarcation of equid/Perissodactyl-specific features that for the first time clearly distinguish the origins of ECA14 and ECA21 from similar-looking status in the Cetartiodactyls.
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http://dx.doi.org/10.1016/j.ygeno.2006.06.012DOI Listing
January 2007

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

J Vet Med Sci 2006 Jun;68(6):619-21

Laboratory of Veterinary Ethology, The University of Tokyo, Japan.

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.
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http://dx.doi.org/10.1292/jvms.68.619DOI Listing
June 2006

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

Gene 2006 Jul 5;376(2):192-8. Epub 2006 Apr 5.

Institute of Animal Breeding and Husbandry, Christian Albrecht University of Kiel, D-24098 Kiel, Germany.

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.
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http://dx.doi.org/10.1016/j.gene.2006.03.006DOI Listing
July 2006

A human-horse comparative map based on equine BAC end sequences.

Genomics 2006 Jun 17;87(6):772-6. Epub 2006 Apr 17.

Institute of Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559 Hannover, Germany.

In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E
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http://dx.doi.org/10.1016/j.ygeno.2006.03.002DOI Listing
June 2006

Novel gene acquisition on carnivore Y chromosomes.

PLoS Genet 2006 Mar 31;2(3):e43. Epub 2006 Mar 31.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.0020043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1420679PMC
March 2006

Evolutionary movement of centromeres in horse, donkey, and zebra.

Genomics 2006 Jun 18;87(6):777-82. Epub 2006 Jan 18.

Department of Genetics and Microbiology, University of Bari, Via Amendola 165/A, 70126 Bari, Italy.

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years). These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.
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http://dx.doi.org/10.1016/j.ygeno.2005.11.012DOI Listing
June 2006

A high-resolution physical map of equine homologs of HSA19 shows divergent evolution compared with other mammals.

Mamm Genome 2005 Aug 14;16(8):631-49. Epub 2005 Sep 14.

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, 77843, USA.

A high-resolution (1 marker/700 kb) physically ordered radiation hybrid (RH) and comparative map of 122 loci on equine homologs of human Chromosome 19 (HSA19) shows a variant evolution of these segments in equids/Perissodactyls compared with other mammals. The segments include parts of both the long and the short arm of horse Chromosome 7 (ECA7), the proximal part of ECA21, and the entire short arm of ECA10. The map includes 93 new markers, of which 89 (64 gene-specific and 25 microsatellite) were genotyped on a 5000-rad horse x hamster RH panel, and 4 were mapped exclusively by FISH. The orientation and alignment of the map was strengthened by 21 new FISH localizations, of which 15 represent genes. The approximately sevenfold-improved map resolution attained in this study will prove extremely useful for candidate gene discovery in the targeted equine chromosomal regions. The highlight of the comparative map is the fine definition of homology between the four equine chromosomal segments and corresponding HSA19 regions specified by physical coordinates (bp) in the human genome sequence. Of particular interest are the regions on ECA7 and ECA21 that correspond to the short arm of HSA19-a genomic rearrangement discovered to date only in equids/Perissodactyls as evidenced through comparative Zoo-FISH analysis of the evolution of ancestral HSA19 segments in eight mammalian orders involving about 50 species.
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http://dx.doi.org/10.1007/s00335-005-0023-1DOI Listing
August 2005