Publications by authors named "Terje Raudsepp"

75 Publications

An 8.22 Mb Assembly and Annotation of the Alpaca () Y Chromosome.

Genes (Basel) 2021 Jan 16;12(1). Epub 2021 Jan 16.

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

The unique evolutionary dynamics and complex structure make the Y chromosome the most diverse and least understood region in the mammalian genome, despite its undisputable role in sex determination, development, and male fertility. Here we present the first contig-level annotated draft assembly for the alpaca () Y chromosome based on hybrid assembly of short- and long-read sequence data of flow-sorted Y. The latter was also used for cDNA selection providing Y-enriched testis transcriptome for annotation. The final assembly of 8.22 Mb comprised 4.5 Mb of male specific Y (MSY) and 3.7 Mb of the pseudoautosomal region. In MSY, we annotated 15 X-degenerate genes and two novel transcripts, but no transposed sequences. Two MSY genes, and are multicopy. The pseudoautosomal boundary is located between and Comparative analysis shows that the small and cytogenetically distinct alpaca Y shares most of MSY sequences with the larger dromedary and Bactrian camel Y chromosomes. Most of alpaca X-degenerate genes are also shared with other mammalian MSYs, though is Y-specific only in alpaca/camels and the horse. The partial alpaca Y assembly is a starting point for further expansion and will have applications in the study of camelid populations and male biology.
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http://dx.doi.org/10.3390/genes12010105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830431PMC
January 2021

HER2 Overexpression and Amplification in Feline Pulmonary Carcinoma.

Vet Pathol 2021 Jan 19:300985820988147. Epub 2021 Jan 19.

University of Bologna, Bologna, Italy.

HER2 is overexpressed, amplified, and mutated in a subset of human lung cancer. The aim of this study was to investigate HER2 protein overexpression and gene amplification in feline pulmonary carcinomas. Thirteen pulmonary carcinomas were selected and TTF-1 and HER2 expression was evaluated by immunohistochemistry. Fluorescence in situ hybridization (FISH) was performed with a probe and a BAC probe for the feline chromosome E1p1.12-p1.11 region. Twelve adenocarcinomas and 1 squamous cell carcinoma were diagnosed. TTF-1 was positive in 7 carcinomas (58%). HER2 was overexpressed in 2 (15%), equivocal in 5 (38%), and negative in 6 cases (46%). FISH analysis of was indeterminate in 2 cases. Three pulmonary carcinomas (27%) had amplification and 8 cases were not amplified (73%). The significant correlation between HER2 protein overexpression and gene amplification are promising preliminary data, but study of additional cases is needed to confirm HER2 as a target for possible innovative treatments.
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http://dx.doi.org/10.1177/0300985820988147DOI Listing
January 2021

Two Novel Cases of Autosomal Translocations in the Horse: Warmblood Family Segregating t(4;30) and a Cloned Arabian with a de novo t(12;25).

Cytogenet Genome Res 2020 Dec 16:1-10. Epub 2020 Dec 16.

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

We report 2 novel autosomal translocations in the horse. In Case 1, a breeding stallion with a balanced t(4p;30) had produced normal foals and those with congenital abnormalities. Of his 9 phenotypically normal offspring, 4 had normal karyotypes, 4 had balanced t(4p;30), and 1 carried an unbalanced translocation with tertiary trisomy of 4p. We argue that unbalanced forms of t(4p;30) are more tolerated and result in viable congenital abnormalities, without causing embryonic death like all other known equine autosomal translocations. In Case 2, two stallions produced by somatic cell nuclear transfer from the same donor were karyotyped because of fertility issues. A balanced translocation t(12q;25) was found in one, but not in the other clone. The findings underscore the importance of routine cytogenetic screening of breeding animals and animals produced by assisted reproductive technologies. These cases will contribute to molecular studies of translocation breakpoints and their genetic consequences in the horse.
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http://dx.doi.org/10.1159/000512206DOI Listing
December 2020

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

Novel Complex Unbalanced Dicentric X-Autosome Rearrangement in a Thoroughbred Mare with a Mild Effect on the Phenotype.

Cytogenet Genome Res 2020 5;160(10):597-609. Epub 2020 Nov 5.

Molecular Cytogenetics Laboratory, College of Veterinary Medicine and Biomedical Sciences,Texas A&M University, College Station, Texas, USA,

Complex structural X chromosome abnormalities are rare in humans and animals, and not recurrent. Yet, each case provides a fascinating opportunity to evaluate X chromosome content and functional status in relation to the effect on the phenotype. Here, we report the first equine case of a complex unbalanced X-autosome rearrangement in a healthy but short in stature Thoroughbred mare. Studies of about 200 cells by chromosome banding and FISH revealed an abnormal 2n = 63,X,der(X;16) karyotype with a large dicentric derivative chromosome (der). The der was comprised of normal Xp material, a palindromic duplication of Xq12q21, and a translocation of chromosome 16 to the inverted Xq12q21 segment by the centromere, whereas the distal Xq22q29 was deleted from the der. Microsatellite genotyping determined a paternal origin of the der. While there was no option to experimentally investigate the status of X chromosome inactivation (XCI), the observed mild phenotype of this case suggested the following scenario to retain an almost normal genetic balance: active normal X, inactivated X-portion of the der, but without XCI spreading into the translocated chromosome 16. Cases like this present unique resources to acquire information about species-specific features of X regulation and the role of X-linked genes in development, health, and disease.
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http://dx.doi.org/10.1159/000511236DOI Listing
January 2021

Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse.

Sci Rep 2020 08 7;10(1):13314. Epub 2020 Aug 7.

Department of Comparative Biomedical Sciences, The Royal Veterinary College, University of London, London, UK.

The first 8 weeks of pregnancy is a critical time, with the majority of pregnancy losses occurring during this period. Abnormal chromosome number (aneuploidy) is a common finding in human miscarriage, yet is rarely reported in domestic animals. Equine early pregnancy loss (EPL) has no diagnosis in over 80% of cases. The aim of this study was to characterise aneuploidies associated with equine EPL. Genomic DNA from clinical cases of spontaneous miscarriage (EPLs; 14-65 days of gestation) and healthy control placentae (various gestational ages) were assessed using a high density genotyping array. Aneuploidy was detected in 12/55 EPLs (21.8%), and 0/15 healthy control placentae. Whole genome sequencing (30X) and digital droplet PCR (ddPCR) validated results. The majority of these aneuploidies have never been reported in live born equines, supporting their embryonic/fetal lethality. Aneuploidies were detected in both placental and fetal compartments. Rodents are currently used to study how maternal ageing impacts aneuploidy risk, however the differences in reproductive biology is a limitation of this model. We present the first evidence of aneuploidy in naturally occurring equine EPLs at a similar rate to human miscarriage. We therefore suggest the horse as an alternative to rodent models to study mechanisms resulting in aneuploid pregnancies.
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http://dx.doi.org/10.1038/s41598-020-69967-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415156PMC
August 2020

Genetics of Equine Reproductive Diseases.

Authors:
Terje Raudsepp

Vet Clin North Am Equine Pract 2020 Aug 10;36(2):395-409. Epub 2020 Jun 10.

Department of Veterinary Integrative Biosciences, Molecular Cytogenetics Laboratory, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, Veterinary Research Building Room 306, 588 Raymond Stotzer Parkway, College Station, TX 77843-4458, USA. Electronic address:

Reproductive disorders are genetically heterogeneous and complex; available genetic tests are limited to chromosome analysis and 1 susceptibility gene. Cytogenetic analysis should be the first test to confirm or rule out chromosomal aberrations. No causative genes/mutations are known. The only available genetic test for stallion subfertility is based on a susceptibility gene FKBP6. The ongoing progress in equine genomics will improve the status of genetic testing. However, because subfertile phenotypes do not facilitate collection of large numbers of samples or pedigrees, and clinical causes of many cases remain unknown, further progress requires constructive cross-talk between geneticists, clinicians, breeders, and owners.
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http://dx.doi.org/10.1016/j.cveq.2020.03.013DOI Listing
August 2020

Cytogenetic Mapping of 35 New Markers in the Alpaca ().

Genes (Basel) 2020 05 8;11(5). Epub 2020 May 8.

Animal Science, University of Minnesota, St. Paul, MN 55108, USA.

Alpaca is a camelid species of broad economic, biological and biomedical interest, and an essential part of the cultural and historical heritage of Peru. Recently, efforts have been made to improve knowledge of the alpaca genome, and its genetics and cytogenetics, to develop molecular tools for selection and breeding. Here, we report cytogenetic mapping of 35 new markers to 19 alpaca autosomes and the X chromosome. Twenty-eight markers represent alpaca SNPs, of which 17 are located inside or near protein-coding genes, two are in ncRNA genes and nine are intergenic. The remaining seven markers correspond to candidate genes for fiber characteristics (, coat color () and development (). The results take the tally of cytogenetically mapped markers in alpaca to 281, covering all 36 autosomes and the sex chromosomes. The new map assignments overall agree with human-camelid conserved synteny data, except for mapping to VPA3, suggesting a hitherto unknown homology with HSA14. The findings validate, refine and correct the current alpaca assembly by anchoring unassigned sequence scaffolds, and ordering and orienting assigned scaffolds. The study contributes to the improvement in the alpaca reference genome and advances camelid molecular cytogenetics.
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http://dx.doi.org/10.3390/genes11050522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7288448PMC
May 2020

Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.

Sci Rep 2020 05 4;10(1):7411. Epub 2020 May 4.

Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas, USA.

Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1 mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations.
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http://dx.doi.org/10.1038/s41598-020-62723-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198616PMC
May 2020

Characterization of A Homozygous Deletion of Steroid Hormone Biosynthesis Genes in Horse Chromosome 29 as A Risk Factor for Disorders of Sex Development and Reproduction.

Genes (Basel) 2020 02 27;11(3). Epub 2020 Feb 27.

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

Disorders of sex development (DSD) and reproduction are not uncommon among horses, though knowledge about their molecular causes is sparse. Here we characterized a ~200 kb homozygous deletion in chromosome 29 at 29.7-29.9 Mb. The region contains genes which function as ketosteroid reductases in steroid hormone biosynthesis, including androgens and estrogens. Mutations in genes are associated with human DSDs. Deletion boundaries, sequence properties and gene content were studied by PCR and whole genome sequencing of select deletion homozygotes and control animals. Deletion analysis by PCR in 940 horses, including 622 with DSDs and reproductive problems and 318 phenotypically normal controls, detected 67 deletion homozygotes of which 79% were developmentally or reproductively abnormal. Altogether, 8-9% of all abnormal horses were homozygous for the deletion, with the highest incidence (9.4%) among cryptorchids. The deletion was found in ~4% of our phenotypically normal cohort, ~1% of global warmblood horses and ponies, and ~7% of draught breeds of general horse population as retrieved from published data. Based on the abnormal phenotype of the carriers, the functionally relevant gene content, and the low incidence in general population, we consider the deletion in chromosome 29 as a risk factor for equine DSDs and reproductive disorders.
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http://dx.doi.org/10.3390/genes11030251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140900PMC
February 2020

The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins.

Nat Genet 2020 01 6;52(1):106-117. Epub 2020 Jan 6.

Molecular Biology Department, Genentech, Inc., South San Francisco, CA, USA.

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the 'venom-ome' and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 'venom-ome-specific toxins' (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.
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http://dx.doi.org/10.1038/s41588-019-0559-8DOI Listing
January 2020

Azoospermia and Y Chromosome-Autosome Translocation in a Friesian Stallion.

J Equine Vet Sci 2019 11 11;82:102781. Epub 2019 Jul 11.

Comparative Theriogenology, Department of Veterinary Clinical Science, College of Veterinary Medicine, Center for Reproductive Biology, Washington State University, Pullman, WA.

This case report describes spermatogenic arrest and azoospermia in a stallion with a unique Y chromosome-autosome translocation. Clinical diagnosis of azoospermia was based on history of infertility and evaluation of ejaculates collected for artificial insemination. Clinical and ultrasonographic evaluation of the external and internal genitalia did not reveal any abnormalities except for smaller than normal testicular size. Azoospermia of testicular origin was confirmed by determining alkaline phosphatase concentration in semen. Histological evaluation of testicular tissue after castration confirmed early spermatogenic arrest. Cytogenetic evaluation showed the presence of translocation between the Y chromosome and chromosome 13. To the authors' knowledge, this is the first case of azoospermia with a cytogenetically detected Y chromosome abnormality, suggesting that the horse Y chromosome may carry sequences critical for normal spermatogenesis.
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http://dx.doi.org/10.1016/j.jevs.2019.07.002DOI Listing
November 2019

Population Genetic Analysis of the Estonian Native Horse Suggests Diverse and Distinct Genetics, Ancient Origin and Contribution from Unique Patrilines.

Genes (Basel) 2019 08 20;10(8). Epub 2019 Aug 20.

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

The Estonian Native Horse (ENH) is a medium-size pony found mainly in the western islands of Estonia and is well-adapted to the harsh northern climate and poor pastures. The ancestry of the ENH is debated, including alleged claims about direct descendance from the extinct Tarpan. Here we conducted a detailed analysis of the genetic makeup and relationships of the ENH based on the genotypes of 15 autosomal short tandem repeats (STRs), 18 Y chromosomal single nucleotide polymorphisms (SNPs), mitochondrial D-loop sequence and lateral gait allele in . The study encompassed 2890 horses of 61 breeds, including 33 ENHs. We show that the expected and observed genetic diversities of the ENH are among the highest within 52 global breeds, and the highest among 8 related Northern European ponies. The genetically closest breeds to the ENH are the Finn Horse, and the geographically more distant primitive Hucul and Konik. ENH matrilines are diverse and relate to draught and Pontic-Caspian breeds. ENH patrilines relate to draught breeds, and to a unique haplogroup not described before. None of the 33 ENHs carried the "gait" mutation, but the mutation was found in 2 Huculs. The study demonstrates that the ENH is a genetically distinct and diverse breed of ancient origin with no notable pressure of selective breeding.
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http://dx.doi.org/10.3390/genes10080629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722507PMC
August 2019

Chromosome-Level Alpaca Reference Genome Improves Genomic Insight Into the Biology of New World Camelids.

Front Genet 2019 21;10:586. Epub 2019 Jun 21.

Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, United States.

The development of high-quality chromosomally assigned reference genomes constitutes a key feature for understanding genome architecture of a species and is critical for the discovery of the genetic blueprints of traits of biological significance. South American camelids serve people in extreme environments and are important fiber and companion animals worldwide. Despite this, the alpaca reference genome lags far behind those available for other domestic species. Here we produced a chromosome-level improved reference assembly for the alpaca genome using the DNA of the same female Huacaya alpaca as in previous assemblies. We generated 190X Illumina short-read, 8X Pacific Biosciences long-read and 60X Dovetail Chicago chromatin interaction scaffolding data for the assembly, used testis and skin RNAseq data for annotation, and cytogenetic map data for chromosomal assignments. The new assembly contains 90% of the alpaca genome in just 103 scaffolds and 76% of all scaffolds are mapped to the 36 pairs of the alpaca autosomes and the X chromosome. Preliminary annotation of the assembly predicted 22,462 coding genes and 29,337 isoforms. Comparative analysis of selected regions of the alpaca genome, such as the major histocompatibility complex (MHC), the region involved in the (MCS) and candidate genes for high-altitude adaptations, reveal unique features of the alpaca genome. The alpaca reference genome presents a significant improvement in completeness, contiguity and accuracy over and is an important tool for the advancement of genomics research in all New World camelids.
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http://dx.doi.org/10.3389/fgene.2019.00586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598621PMC
June 2019

Chromosomal Localization of Candidate Genes for Fiber Growth and Color in Alpaca ().

Front Genet 2019 19;10:583. Epub 2019 Jun 19.

Department of Animal Science, University of Minnesota, Minneapolis, MN, United States.

The alpaca () is an economically important and cultural signature species in Peru. Thus, molecular genomic information about the genes underlying the traits of interest, such as fiber properties and color, is critical for improved breeding and management schemes. Current knowledge about the alpaca genome, particularly the chromosomal location of such genes of interest is limited and lags far behind other livestock species. The main objective of this work was to localize alpaca candidate genes for fiber growth and color using fluorescence hybridization (FISH). We report the mapping of candidate genes for fiber growth , , , , , and to chromosomes 16, 17, 4, 16, 1, and 16, respectively. Likewise, we report the mapping of candidate genes for fiber color , , , , and to chromosomes 9, 19, 16, 1, and 14, respectively. In addition, since clusters with five other keratin genes (, , , , and ) in scaffold 450 (Vic.Pac 2.0.2), the entire gene cluster was assigned to chromosome 16. Similarly, mapping to chromosome 19, anchored scaffold 34 with 8 genes, viz., , , , , , , , and to chromosome 19. These results are concordant with known conserved synteny blocks between camelids and humans, cattle and pigs.
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http://dx.doi.org/10.3389/fgene.2019.00583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593342PMC
June 2019

An Autosomal Translocation 73,XY,t(12;20)(q11;q11) in an Infertile Male Llama () With Teratozoospermia.

Front Genet 2019 16;10:344. Epub 2019 Apr 16.

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

Structural chromosome abnormalities, such as translocations and inversions occasionally occur in all livestock species and are typically associated with reproductive and developmental disorders. Curiously, only a few structural chromosome aberrations have been reported in camelids, and most involved sex chromosomes. This can be attributed to a high diploid number (2n = 74) and complex chromosome morphology, which makes unambiguous identification of camelid chromosomes difficult. Additionally, molecular tools for camelid cytogenetics are sparse and have become available only recently. Here we present a case report about an infertile male llama with teratozoospermia and abnormal chromosome number 2n = 73,XY. This llama carries an autosomal translocation of chromosomes 12 and 20, which is the likely cause of defective spermatogenesis and infertility in this individual. Our analysis underlines the power of molecular cytogenetics methods over conventional banding-based chromosome analysis for explicit identification of normal and aberrant chromosomes in camelid karyotypes. This is the first case of a translocation and the first autosomal aberration reported in any camelid species. It is proof of principle that, like in other mammalian species, structural chromosome abnormalities contribute to reproductive disorders in camelids.
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http://dx.doi.org/10.3389/fgene.2019.00344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476961PMC
April 2019

Comparative FISH-Mapping of , , and in New and Old World Camelids and Association Analysis With Coat Color Phenotypes in the Dromedary ().

Front Genet 2019 16;10:340. Epub 2019 Apr 16.

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

Melanocortin 1 receptor (), the agouti signaling protein (), and tyrosinase related protein 1 () are among the major regulators of pigmentation in mammals. Recently, and sequence variants were associated with white and black/dark brown coat colors, respectively, in the dromedary. Here we confirmed this association by independent sequencing and mutation discovery of and coding regions and by TaqMan genotyping in 188 dromedaries from Saudi Arabia and United States, including 38 black, 53 white, and 97 beige/brown/red animals. We showed that heterozygosity for a missense mutation c.901C > T in is sufficient for the white coat color suggesting a possible dominant negative effect. Likewise, we confirmed that the majority of black dromedaries were homozygous for a frameshift mutation in exon 2, except for 4 animals, which were heterozygous. In search for additional mutations underlying the black color, we identified another frameshift mutation in exon 4 and 6 new variants in including a significantly associated SNP in 3'UTR. In pursuit of sequence variants that may modify dromedary wild-type color from dark-reddish brown to light beige, we identified 4 SNPs and one insertion in non-coding regions. However, none of these were associated with variations in wild-type colors. Finally, the three genes were cytogenetically mapped in New World (alpaca) and Old World (dromedary and Bactrian camel) camelids. The was assigned to chr21, to chr19 and to chr4 in all 3 species confirming extensive conservation of camelid karyotypes. Notably, while the locations of and were in agreement with human-camelid comparative map, mapping identified a new evolutionary conserved synteny segment between camelid chromosome 21 and HSA16. The findings contribute to coat color genomics and the development of molecular tests in camelids and toward the chromosome level reference assemblies of camelid genomes.
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http://dx.doi.org/10.3389/fgene.2019.00340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477024PMC
April 2019

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

Evolutionary conservation of Y Chromosome ampliconic gene families despite extensive structural variation.

Genome Res 2018 12 31;28(12):1841-1851. Epub 2018 Oct 31.

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

Despite claims that the mammalian Y Chromosome is on a path to extinction, comparative sequence analysis of primate Y Chromosomes has shown the decay of the ancestral single-copy genes has all but ceased in this eutherian lineage. The suite of single-copy Y-linked genes is highly conserved among the majority of eutherian Y Chromosomes due to strong purifying selection to retain dosage-sensitive genes. In contrast, the ampliconic regions of the Y Chromosome, which contain testis-specific genes that encode the majority of the transcripts on eutherian Y Chromosomes, are rapidly evolving and are thought to undergo species-specific turnover. However, ampliconic genes are known from only a handful of species, limiting insights into their long-term evolutionary dynamics. We used a clone-based sequencing approach employing both long- and short-read sequencing technologies to assemble ∼2.4 Mb of representative ampliconic sequence dispersed across the domestic cat Y Chromosome, and identified the major ampliconic gene families and repeat units. We analyzed fluorescence in situ hybridization, qPCR, and whole-genome sequence data from 20 cat species and revealed that ampliconic gene families are conserved across the cat family Felidae but show high transcript diversity, copy number variation, and structural rearrangement. Our analysis of ampliconic gene evolution unveils a complex pattern of long-term gene content stability despite extensive structural variation on a nonrecombining background.
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http://dx.doi.org/10.1101/gr.237586.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280758PMC
December 2018

Comparative Chromosome Painting in Genets (Carnivora, Viverridae, Genetta), the Only Known Feliforms with a Highly Rearranged Karyotype.

Cytogenet Genome Res 2018 Aug 8. Epub 2018 Aug 8.

Mammalian carnivores have been extensively studied by cross-species chromosome painting, which indicated a high degree of karyotypic conservatism in the cat-like suborder Feliformia relative to the ancestral carnivore karyotype (ACK). The first exception to this high degree of karyotypic conservation in feliforms was recently confirmed in genets, mesocarnivores belonging to the basal family Viverridae. Here, we present a comparative analysis of the chromosome rearrangements among 2 subspecies of the small-spotted genet Genetta genetta (the Iberian nominate and the Arabian grantii) and the panther genet G. maculata, the 2 most common and widespread genets, using whole-chromosome paints from the domestic cat (Felis catus). The chromosome homology maps and the presence of numerous interstitial telomeric sites in both genet species strengthen the hypothesis that a highly rearranged karyotype compared to the ACK may occur throughout Genetta. The karyotype of G. maculata appears to have undergone more rearrangements than that of G. genetta, which is an older lineage. Notably, we identified a tandem fusion distinguishing G. g. genetta and G. g.grantii. As G. g. grantii is morphologically and genetically distinctive, and tandem fusions have been associated with substantial postzygotic isolation in mammals, this cytogenetic finding flags the subspecies for future taxonomic investigations.
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http://dx.doi.org/10.1159/000491868DOI Listing
August 2018

Pathology in Practice.

J Am Vet Med Assoc 2018 Aug;253(4):427-429

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http://dx.doi.org/10.2460/javma.253.4.427DOI Listing
August 2018

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

Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis.

Stem Cell Res Ther 2018 07 4;9(1):181. Epub 2018 Jul 4.

Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.

Background: Skin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium.

Methods: Conditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium.

Results: Conditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers.

Conclusions: Our results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation.
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http://dx.doi.org/10.1186/s13287-018-0918-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032561PMC
July 2018

Construction of two whole genome radiation hybrid panels for dromedary (Camelus dromedarius): 5000 and 15000.

Sci Rep 2018 01 31;8(1):1982. Epub 2018 Jan 31.

Animal Production and Health Laboratory, Joint FAO/IAEA Division, International Atomic Energy Agency, Vienna, Austria.

The availability of genomic resources including linkage information for camelids has been very limited. Here, we describe the construction of a set of two radiation hybrid (RH) panels (5000 and 15000) for the dromedary (Camelus dromedarius) as a permanent genetic resource for camel genome researchers worldwide. For the 5000 panel, a total of 245 female camel-hamster radiation hybrid clones were collected, of which 186 were screened with 44 custom designed marker loci distributed throughout camel genome. The overall mean retention frequency (RF) of the final set of 93 hybrids was 47.7%. For the 15000 panel, 238 male dromedary-hamster radiation hybrid clones were collected, of which 93 were tested using 44 PCR markers. The final set of 90 clones had a mean RF of 39.9%. This 15000 panel is an important high-resolution complement to the main 5000 panel and an indispensable tool for resolving complex genomic regions. This valuable genetic resource of dromedary RH panels is expected to be instrumental for constructing a high resolution camel genome map. Construction of the set of RH panels is essential step toward chromosome level reference quality genome assembly that is critical for advancing camelid genomics and the development of custom genomic tools.
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http://dx.doi.org/10.1038/s41598-018-20223-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792482PMC
January 2018

retrogene on CFA12 is responsible for chondrodystrophy and intervertebral disc disease in dogs.

Proc Natl Acad Sci U S A 2017 10 11;114(43):11476-11481. Epub 2017 Oct 11.

Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616;

Chondrodystrophy in dogs is defined by dysplastic, shortened long bones and premature degeneration and calcification of intervertebral discs. Independent genome-wide association analyses for skeletal dysplasia (short limbs) within a single breed ( = 0.01) and intervertebral disc disease (IVDD) across breeds ( = 4.0 × 10) both identified a significant association to the same region on CFA12. Whole genome sequencing identified a highly expressed retrogene within this shared region. The retrogene segregated with limb length and had an odds ratio of 51.23 (95% CI = 46.69, 56.20) for IVDD. Long bone length in dogs is a unique example of multiple disease-causing retrocopies of the same parental gene in a mammalian species. FGF signaling abnormalities have been associated with skeletal dysplasia in humans, and our findings present opportunities for both selective elimination of a medically and financially devastating disease in dogs and further understanding of the ever-growing complexity of retrogene biology.
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http://dx.doi.org/10.1073/pnas.1709082114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664524PMC
October 2017

Validation of a candidate causative mutation for white spotting in donkeys.

Anim Genet 2017 Feb 9;48(1):124-125. Epub 2016 Sep 9.

School of Life and Environmental Sciences, Faculty of Veterinary Science, University of Sydney, Camperdown, NSW, 2006, Australia.

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http://dx.doi.org/10.1111/age.12494DOI Listing
February 2017

Large Deletions at the SHOX Locus in the Pseudoautosomal Region Are Associated with Skeletal Atavism in Shetland Ponies.

G3 (Bethesda) 2016 07 7;6(7):2213-23. Epub 2016 Jul 7.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Sweden 751 23

Skeletal atavism in Shetland ponies is a heritable disorder characterized by abnormal growth of the ulna and fibula that extend the carpal and tarsal joints, respectively. This causes abnormal skeletal structure and impaired movements, and affected foals are usually killed. In order to identify the causal mutation we subjected six confirmed Swedish cases and a DNA pool consisting of 21 control individuals to whole genome resequencing. We screened for polymorphisms where the cases and the control pool were fixed for opposite alleles and observed this signature for only 25 SNPs, most of which were scattered on genome assembly unassigned scaffolds. Read depth analysis at these loci revealed homozygosity or compound heterozygosity for two partially overlapping large deletions in the pseudoautosomal region (PAR) of chromosome X/Y in cases but not in the control pool. One of these deletions removes the entire coding region of the SHOX gene and both deletions remove parts of the CRLF2 gene located downstream of SHOX. The horse reference assembly of the PAR is highly fragmented, and in order to characterize this region we sequenced bacterial artificial chromosome (BAC) clones by single-molecule real-time (SMRT) sequencing technology. This considerably improved the assembly and enabled size estimations of the two deletions to 160-180 kb and 60-80 kb, respectively. Complete association between the presence of these deletions and disease status was verified in eight other affected horses. The result of the present study is consistent with previous studies in humans showing crucial importance of SHOX for normal skeletal development.
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http://dx.doi.org/10.1534/g3.116.029645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938674PMC
July 2016

Comparative Cytogenetics of the Congo African Grey Parrot (Psittacus erithacus).

Cytogenet Genome Res 2015 20;147(2-3):144-53. Epub 2016 Feb 20.

Department of Veterinary Integrative Biosciences, Schubot Exotic Bird Health Center, CVM, Texas A&M University, College Station, Tex., USA.

The Congo African grey parrot (Psittacus erithacus, PER) is an endemic species of Central Africa, valued for its intelligence and listed as vulnerable due to poaching and habitat destruction. Improved knowledge about the P. erithacus genome is needed to address key biological questions and conservation of this species. The P. erithacus genome was studied using conventional and molecular cytogenetic approaches including Zoo-FISH. P. erithacus has a 'typical' parrot karyotype with 2n = 62-64 and 8 pairs of macrochromosomes. A distinct feature was a sharp macro-microchromosome boundary. Telomeric sequences were present at all chromosome ends and interstitially in PER2q, the latter coinciding with a C-band. NORs mapped to 4 pairs of microchromosomes which is in contrast to a single NOR in ancestral type avian karyotypes. Zoo-FISH with chicken macrochromosomes GGA1-9 and Z revealed patterns of conserved synteny similar to many other avian groups, though neighboring synteny combinations of GGA6/7, 8/9, and 1/4 were distinctive only to parrots. Overall, P. erithacus shared more Zoo-FISH patterns with neotropical macaws than Australian species such as cockatiel and budgerigar. The observations suggest that Psittaciformes karyotypes have undergone more extensive evolutionary rearrangements compared to the majority of other avian genomes.
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http://dx.doi.org/10.1159/000444136DOI Listing
August 2016

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