Generation of a de novo transcriptome from equine lamellar tissue.

BACKGROUND: Laminitis, the structural failure of interdigitated tissue that suspends the distal skeleton within the hoof capsule, is a devastating disease that is the second leading cause of both lameness and euthanasia in the horse. Current transcriptomic research focuses on the expression of known genes. However, as this tissue is quite unique and equine gene annotation is largely derived from computational predictions, there are likely yet uncharacterized transcripts that may be involved in the etiology of laminitis. In order to create a novel annotation resource, we performed whole transcriptome sequencing of sagittal lamellar sections from one control and two laminitis affected horses. RESULTS: Whole transcriptome sequencing of the three samples resulted in 113 million reads. Overall, 88 % of the reads mapped to the equCab2 reference genome, allowing for the identification of 119,430 SNPs. The de novo assembly generated around 75,000 transcripts, of which 36,000 corresponded to known annotations. Annotated transcript models are hosted in a public data repository and thus can be easily accessed or loaded into genome browsers. RT-PCR of 12 selected assemblies confirmed structure and expression in lamellar tissue. CONCLUSIONS: Transcriptome sequencing represents a powerful tool to expand on equine annotation and identify novel targets for further laminitis research.

Detection of two equine trisomies using SNP-CGH.

Chromosomal aberrations in the horse are known to cause congenital abnormalities, embryonic loss, and infertility. While diagnosed mainly by karyotyping and FISH in the horse, the use of SNP array comparative genome hybridization (SNP-CGH) is becoming increasingly common in human diagnostics. Normalized probe intensities and allelic ratios are used to detect changes in copy number genome-wide. Two horses with suspected chromosomal abnormalities and six horses with FISH-confirmed aberrant karyotypes were chosen for genotyping on the Equine SNP50 array. Karyotyping of the first horse indicated mosaicism for an additional small, acrocentric chromosome, although the identity of the chromosome was unclear. The second case displayed a similar phenotype to human disease caused by a gene deletion and so was chosen for SNP-CGH due to the ability to detect changes at higher resolutions than those achieved with conventional karyotyping. The results of SNP-CGH analysis for the six horses with known chromosomal aberrations agreed completely with previous karyotype and FISH analysis. The first undiagnosed case showed a pattern of altered allelic ratios without a noticeable shift in overall intensity for chromosome 27, consistent with a mosaic trisomy. The second case displayed a more drastic change in both values for chromosome 30, consistent with a complete trisomy. These results indicate that SNP-CGH is a viable method for detection of chromosomal aneuploidies in the horse.

Novel variants in the KIT and PAX3 genes in horses with white-spotted coat colour phenotypes.

Variants in the EDNRB, KIT, MITF, PAX3 and TRPM1 genes are known to cause white spotting phenotypes in horses, which can range from the common white markings up to completely white horses. In this study, we investigated these candidate genes in 169 horses with white spotting phenotypes not explained by the previously described variants. We identified a novel missense variant, PAX3:p.Pro32Arg, in Appaloosa horses with a splashed white phenotype in addition to their leopard complex spotting patterns. We also found three novel variants in the KIT gene. The splice site variant c.1346+1G>A occurred in a Swiss Warmblood horse with a pronounced depigmentation phenotype. The missense variant p.Tyr441Cys was present in several part-bred Arabians with sabino-like depigmentation phenotypes. Finally, we provide evidence suggesting that the common and widely distributed KIT:p.Arg682His variant has a very subtle white-increasing effect, which is much less pronounced than the effect of the other described KIT variants. We termed the new KIT variants W18-W20 to provide a simple and unambiguous nomenclature for future genetic testing applications.

Transcriptome diversity and differential expression in supporting limb laminitis.

Laminitis results in impaired tissue integrity and Inflammation of the epidermal and dermal lamellae connecting the hoof capsule to the underlying distal phalanx and causes loss-of-use, poor quality of life and euthanasia in horses. Historically, studies to better understand the etiology of laminitis by documenting changes in gene expression were hampered by the paucity of gene annotation specific to hoof tissues. Next-generation sequencing enables improvements to annotation by incorporating equine- and hoof-specific transcripts. Here we characterize the hoof lamellar tissue transcriptome of naturally occurring supporting limb laminitis (SLL) using archived lamellar tissue from Thoroughbred racehorses consisting of 13 SLL hospital cases and seven age-matched control horses. This was achieved using: 1) Applied transcriptome annotation by long-read sequencing to document transcript diversity and 2) short-read RNA sequencing to document changes in gene expression correlating to the developmental and acute stages of naturally occurring SLL. 1.99Gbp of long-read transcriptome sequencing deeply documented 5067 unique loci, while short read RNA-seq under very stringent quality filters described 66 differentially expressed loci. Functional analysis of these loci revealed alterations in cell replication and growth, stress response and leukocyte recruitment and activation pathways. Differential expression of the Ezrin and TIMP3 genes suggests they may have utility as biomarkers for laminitis disease, while NR1D1 and genes relevant to the inflammasome are promising targets for novel pharmacological treatments.

Whole genome detection of sequence and structural polymorphism in six diverse horses.

The domesticated horse has played a unique role in human history, serving not just as a source of animal protein, but also as a catalyst for long-distance migration and military conquest. As a result, the horse developed unique physiological adaptations to meet the demands of both their climatic environment and their relationship with man. Completed in 2009, the first domesticated horse reference genome assembly (EquCab 2.0) produced most of the publicly available genetic variations annotations in this species. Yet, there are around 400 geographically and physiologically diverse breeds of horse. To enrich the current collection of genetic variants in the horse, we sequenced whole genomes from six horses of six different breeds: an American Miniature, a Percheron, an Arabian, a Mangalarga Marchador, a Native Mongolian Chakouyi, and a Tennessee Walking Horse, and mapped them to EquCab3.0 genome. Aside from extreme contrasts in body size, these breeds originate from diverse global locations and each possess unique adaptive physiology. A total of 1.3 billion reads were generated for the six horses with coverage between 15x to 24x per horse. After applying rigorous filtration, we identified and functionally annotated 17,514,723 Single Nucleotide Polymorphisms (SNPs), and 1,923,693 Insertions/Deletions (INDELs), as well as an average of 1,540 Copy Number Variations (CNVs) and 3,321 Structural Variations (SVs) per horse. Our results revealed putative functional variants including genes associated with size variation like LCORL gene (found in all horses), ZFAT in the Arabian, American Miniature and Percheron horses and ANKRD1 in the Native Mongolian Chakouyi horse. We detected a copy number variation in the Latherin gene that may be the result of evolutionary selection impacting thermoregulation by sweating, an important component of athleticism and heat tolerance. The newly discovered variants were formatted into user-friendly browser tracks and will provide a foundational database for future studies of the genetic underpinnings of diverse phenotypes within the horse.

Genome Diversity and the Origin of the Arabian Horse.

The Arabian horse, one of the world's oldest breeds of any domesticated animal, is characterized by natural beauty, graceful movement, athletic endurance, and, as a result of its development in the arid Middle East, the ability to thrive in a hot, dry environment. Here we studied 378 Arabian horses from 12 countries using equine single nucleotide polymorphism (SNP) arrays and whole-genome re-sequencing to examine hypotheses about genomic diversity, population structure, and the relationship of the Arabian to other horse breeds. We identified a high degree of genetic variation and complex ancestry in Arabian horses from the Middle East region. Also, contrary to popular belief, we could detect no significant genomic contribution of the Arabian breed to the Thoroughbred racehorse, including Y chromosome ancestry. However, we found strong evidence for recent interbreeding of Thoroughbreds with Arabians used for flat-racing competitions. Genetic signatures suggestive of selective sweeps across the Arabian breed contain candidate genes for combating oxidative damage during exercise, and within the "Straight Egyptian" subgroup, for facial morphology. Overall, our data support an origin of the Arabian horse in the Middle East, no evidence for reduced global genetic diversity across the breed, and unique genetic adaptations for both physiology and conformation.

Use of principle component analysis to quantitatively score the equine metabolic syndrome phenotype in an Arabian horse population.

Equine metabolic syndrome (EMS), like human metabolic syndrome, comprises a collection of clinical signs related to obesity, insulin dysregulation and susceptibility to secondary inflammatory disease. Although the secondary conditions resulting from EMS can be life-threatening, diagnosis is not straightforward and often complicated by the presence of other concurrent conditions like pituitary pars intermedia dysfunction (PPID). In order to better characterize EMS, we sought to describe the variation within, and correlations between, typical physical and endocrine parameters for EMS. Utilizing an unsupervised statistical approach, we evaluated a population of Arabian horses using a physical examination including body measurements, as well as blood plasma insulin, leptin, ACTH, glucose, and lipid values. We investigated the relationships among these variables using principle component analysis (PCA), hierarchical clustering, and linear regression. Owner-assigned assessments of body condition were one full score (on a nine-point scale) lower than scores assigned by researchers, indicating differing perception of healthy equine body weight. Rotated PCA defined two factor scores explaining a total of 46.3% of variation within the dataset. Hierarchical clustering using these two factors revealed three groups corresponding well to traditional diagnostic categories of "Healthy", "PPID-suspect", and "EMS-suspect" based on the characteristics of each group. Proxies estimating up to 93.4% of the composite "EMS-suspect" and "PPID-suspect" scores were created using a reduced set of commonly used diagnostic variables, to facilitate application of these quantitative scores to horses of the Arabian breed in the field. Use of breed-specific, comprehensive physical and endocrinological variables combined in a single quantitative score may improve detection of horses at-risk for developing EMS, particularly in those lacking severe clinical signs. Quantification of EMS without the use of predetermined reference ranges provides an advantageous approach for future studies utilizing genomic or metabolomics approaches to improve understanding of the etiology behind this troubling condition.

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

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 (5000RAD and 15000RAD) for the dromedary (Camelus dromedarius) as a permanent genetic resource for camel genome researchers worldwide. For the 5000RAD 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 15000RAD 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 15000RAD panel is an important high-resolution complement to the main 5000RAD 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.

A Frameshift Mutation in KIT is Associated with White Spotting in the Arabian Camel.

While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five kITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation.

Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse.

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ(2)=1022.00, p<<0.0005), and CSNB, testing 43 horses (χ(2)=43, p<<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.