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Skeletal Size and Shape Diversity in the Horse
Abstracts Vol 29, No 5 (2009)
4. Walsh DM, McGowan CM, McGowan TW, Lamb SV, Schanbacher BJ, Place NJ. Equine Cushing’s Disease/Equine Metabolic Syndrome: A Practitioner Field Study. Proceedings of the Fourth International Equine Conference on Laminitis and Diseases of the Foot 2007:1-24.
31866 Skeletal Size and Shape Diversity in the Horse
323
Hair bulb samples are collected from every measured horse to serve as a source of genomic DNA. These will enable eventual candidate gene screens and, using the EquineSNP50 chip (Illumina), genome-wide associations with skeletal traits.
RESULTS
The domestication of the horse occurred by intense artificial selection in response to divergent needs for the horse in labor, war, and sport. This resulted in a variety of skeletal sizes and conformations. For example, the streamlined skeleton of the Thoroughbred is optimal for speed; in comparison, the Shire, the ‘‘great horse’’ of the Crusades, can easily carry the combined weight of a Medieval knight and all the plate armor of both man and horse. Despite the influence of the horse on human history, there has been little investigation of horse morphometrics; as of yet skeletal variation in body size, shape, and bone thickness have not been quantified, nor have breed-associated traits such as the dish face, the Roman nose, and feathering on the legs. To fill this gap in knowledge we report here preliminary findings that for the first time quantify variation in skeletal size and shape both within and across a diverse cross section of horse breeds. Our long term goal is to use our morphometric traits in a whole genome association scan to identify genes under selection for and contributing to skeletal variation in the horse.
As a preliminary dataset, we have collected measurements from 96 horses of 19 different breeds, with a median of 4 horses measured per breed. Notably our dataset includes 9 American Miniatures, 8 Clydesdales, 20 Paso Finos, 4 Percherons, 11 Shires, 8 Standardbreds, and 5 Thoroughbreds. One horse each has been measured from the Cleveland Bay, Westfalen Horse, and Dales Pony breeds. The height at the withers ranges from a 9.1hh American Miniature mare to an 18.3hh Shire mare. Other traits with large variation include the fore coronet circumference, which ranges from 9 inches in an American Miniature gelding to 23.25 inches in a Shire stallion, and the barrel girth behind the shoulder, which ranges from 46.25 inches in an American Miniature mare to 93 inches in a Percheron mare. To assay variation within and among breeds, we have performed PC analysis on the dataset. All 35 measurements load onto PC1 in the same direction, thus PC1 is capturing variation in overall skeletal size. 81% of variance in the dataset is captured by PC1. Subsequent PCs are independent of (orthogonal to) PC1. Limb and neck lengths load onto PC2 with an opposite sign compared to bone circumference measures (e.g. coronet, cannon and muzzle circumference), indicating this PC captures variation in bone thickness. In ‘‘PC-space,’’ individuals cluster by breed. For example, all measured Shires and Percherons have high PC1 (size) and PC2 (thickness) values. In contrast, American Miniatures have extremely low PC1 values as a breed. Thoroughbreds, Standardbreds, and Quarter Horses have intermediate values.
MATERIALS AND METHODS
DISCUSSION
Our skeletal morphometric assay for the horse spans the entire body; we collect 35 measurements from the head, neck, body, and limbs. We also capture factor level subjective measures for body condition score, variation in the Roman nose to the dish face, the degree of feathering, and variation in bite from the parrot mouth to the underbite. The 35 measurements capture bone lengths and circumferences as quantitative variables, including height at the withers, the neck length and circumference, body girth behind the shoulder, cannon length, and coronet circumference for example. These traits are then subjected to principal components (PC) analysis to detect underlying correlations and define traits for size and shape.
Principal components analysis of measurements from a sample of horses of different breeds has shown consistent trends in several quantified traits, including skeletal size (PC1) and shape (PC2). Skeletal development and structure critically impacts a number of orthopedic conditions in the horse, ranging from OCD in the young, rapidlygrowing individual to arthritis in the aged athlete. This preliminary data, therefore, has several implications regarding overall skeletal structure and function in the horse, as well as the relative significance of individual traits in determining a horse’s quality of athletic performance or labor efficiency. For example, the Dales Pony, which stands about 14hh, ranks fourth highest for PC2 (just after the
E.T. Chu,*1 J.J. Allen,1 C.L. Streeter,1 N.B. Sutter,2 and S.A. Brooks3, 1Cornell University, Ithaca, NY, USA, 2 Department of Clinical Sciences, Cornell University, Ithaca, NY, USA, 3Department of Animal Science, Cornell University, Ithaca, NY, USA
INTRODUCTION
Abstracts Vol 29, No 5 (2009)
324
Percherons of our dataset). This compact pony was bred to transport lead between the moors and the coast of England. In comparison, the Thoroughbreds of our dataset range between 15 and 17hh and have a very low PC2 value. The Thoroughbred has been bred for increased speed, reduced bone mass and increased stride length. This effective selection for a fine-boned skeleton may increase fracture risk under high performance stress. Additional sample collection is critically needed to expand this data set and identify all skeletal variation present in the horse. Our ultimate goal, to screen for and identify genes responsible for skeletal variation in the horse, will enable the identification of loci affecting various traits including conformation, disease susceptibility, and athletic performance. In particular, genes affecting skeletal growth in the horse could affect the length and density of the bone. By identifying skeletal trait-associated polymorphisms and assessing their frequency in fracture-prone and non-fracture-prone individuals, we could appreciably improve the quality of life of many performance horse breeds by predicting fracture risk. Furthermore, as body size genes affect fundamental processes such as cellular growth, they could be examined for connections to the abnormal cell growth seen in cancer. Ultimately, our findings could be used to significantly decrease the frequency of detrimental alleles at identified disease loci in the horse through the use of genetic screening and breeding programs. Keywords: Morphometrics; Skeletal growth
REFERENCES
MATERIALS AND METHODS Primers for the last exon of the NMES1 gene were designed as a PCR assay for screening a Chori 241 equine BAC library for a BAC containing the genomic NMES1 sequence. The identified clone containing the BAC was grown in culture, and DNA was extracted with the Qiagen Large Construct kit. We used direct sequencing of the BAC DNA by designing primer assays for primer walking to obtain the genomic sequence. Fluorescent in situ hybridization was used to map the BAC containing the NMES1 gene to an equine chromosome.
RESULTS The equine gene was fully sequenced from a CHORI-241 BAC clone. The BAC was mapped to ECA 1q23 using fluorescent in situ hybridization. From the genomic sequence, we obtained the putative amino acid sequence. The encoded protein is highly conserved among mammals and preliminary antibody tests suggested specific binding in the testis to spermatogonia and primary spermatocytes, but not to more differentiated cell types.
DISCUSSION NMES1 is a newly described gene that encodes a protein expressed in epithelial tissue, with abundant expression in testis. Our initial BLAT search for the NMES1 gene in the equine genome identified only a region that appeared to be orthologous to the third exon of the human, mouse, and bovine sequences. We designed PCR primers to amplify the conserved equine sequence and screened an equine CHORI-241 BAC library. After locating a BAC containing
1. Sutter NB, Mosher DS, Gray MM, Ostrander EA. Morphometrics within dog breeds are highly reproducible and dispute Rensch’s rule. Mamm Genome 2007;19(10-12):713-723.
31619 Initial Characterization of a Gene Abundantly Expressed in Stallion Testis J.E. Shields,* K.J. Kochan, J. Jeong, C.A. Abbey, T. Raudsepp, and P.K. Riggs, Texas A&M University, College Station, TX, USA
INTRODUCTION We recently identified a gene (NMES1) that is expressed more abundantly in testis than any other tissue examined. Initial results suggested NMES1 may play a role in cellular differentiation. We sequenced and carried out preliminary characterization of this novel gene in the horse to investigate its function and potential role in stallion spermatogenesis.
Figure 1. Fluorescent in situ hybridization mapping of NMES1 to ECA 1q23.
4. Walsh DM, McGowan CM, McGowan TW, Lamb SV, Schanbacher BJ, Place NJ. Equine Cushing’s Disease/Equine Metabolic Syndrome: A Practitioner Field Study. Proceedings of the Fourth International Equine Conference on Laminitis and Diseases of the Foot 2007:1-24.
31866 Skeletal Size and Shape Diversity in the Horse
323
Hair bulb samples are collected from every measured horse to serve as a source of genomic DNA. These will enable eventual candidate gene screens and, using the EquineSNP50 chip (Illumina), genome-wide associations with skeletal traits.
RESULTS
The domestication of the horse occurred by intense artificial selection in response to divergent needs for the horse in labor, war, and sport. This resulted in a variety of skeletal sizes and conformations. For example, the streamlined skeleton of the Thoroughbred is optimal for speed; in comparison, the Shire, the ‘‘great horse’’ of the Crusades, can easily carry the combined weight of a Medieval knight and all the plate armor of both man and horse. Despite the influence of the horse on human history, there has been little investigation of horse morphometrics; as of yet skeletal variation in body size, shape, and bone thickness have not been quantified, nor have breed-associated traits such as the dish face, the Roman nose, and feathering on the legs. To fill this gap in knowledge we report here preliminary findings that for the first time quantify variation in skeletal size and shape both within and across a diverse cross section of horse breeds. Our long term goal is to use our morphometric traits in a whole genome association scan to identify genes under selection for and contributing to skeletal variation in the horse.
As a preliminary dataset, we have collected measurements from 96 horses of 19 different breeds, with a median of 4 horses measured per breed. Notably our dataset includes 9 American Miniatures, 8 Clydesdales, 20 Paso Finos, 4 Percherons, 11 Shires, 8 Standardbreds, and 5 Thoroughbreds. One horse each has been measured from the Cleveland Bay, Westfalen Horse, and Dales Pony breeds. The height at the withers ranges from a 9.1hh American Miniature mare to an 18.3hh Shire mare. Other traits with large variation include the fore coronet circumference, which ranges from 9 inches in an American Miniature gelding to 23.25 inches in a Shire stallion, and the barrel girth behind the shoulder, which ranges from 46.25 inches in an American Miniature mare to 93 inches in a Percheron mare. To assay variation within and among breeds, we have performed PC analysis on the dataset. All 35 measurements load onto PC1 in the same direction, thus PC1 is capturing variation in overall skeletal size. 81% of variance in the dataset is captured by PC1. Subsequent PCs are independent of (orthogonal to) PC1. Limb and neck lengths load onto PC2 with an opposite sign compared to bone circumference measures (e.g. coronet, cannon and muzzle circumference), indicating this PC captures variation in bone thickness. In ‘‘PC-space,’’ individuals cluster by breed. For example, all measured Shires and Percherons have high PC1 (size) and PC2 (thickness) values. In contrast, American Miniatures have extremely low PC1 values as a breed. Thoroughbreds, Standardbreds, and Quarter Horses have intermediate values.
MATERIALS AND METHODS
DISCUSSION
Our skeletal morphometric assay for the horse spans the entire body; we collect 35 measurements from the head, neck, body, and limbs. We also capture factor level subjective measures for body condition score, variation in the Roman nose to the dish face, the degree of feathering, and variation in bite from the parrot mouth to the underbite. The 35 measurements capture bone lengths and circumferences as quantitative variables, including height at the withers, the neck length and circumference, body girth behind the shoulder, cannon length, and coronet circumference for example. These traits are then subjected to principal components (PC) analysis to detect underlying correlations and define traits for size and shape.
Principal components analysis of measurements from a sample of horses of different breeds has shown consistent trends in several quantified traits, including skeletal size (PC1) and shape (PC2). Skeletal development and structure critically impacts a number of orthopedic conditions in the horse, ranging from OCD in the young, rapidlygrowing individual to arthritis in the aged athlete. This preliminary data, therefore, has several implications regarding overall skeletal structure and function in the horse, as well as the relative significance of individual traits in determining a horse’s quality of athletic performance or labor efficiency. For example, the Dales Pony, which stands about 14hh, ranks fourth highest for PC2 (just after the
E.T. Chu,*1 J.J. Allen,1 C.L. Streeter,1 N.B. Sutter,2 and S.A. Brooks3, 1Cornell University, Ithaca, NY, USA, 2 Department of Clinical Sciences, Cornell University, Ithaca, NY, USA, 3Department of Animal Science, Cornell University, Ithaca, NY, USA
INTRODUCTION
Abstracts Vol 29, No 5 (2009)
324
Percherons of our dataset). This compact pony was bred to transport lead between the moors and the coast of England. In comparison, the Thoroughbreds of our dataset range between 15 and 17hh and have a very low PC2 value. The Thoroughbred has been bred for increased speed, reduced bone mass and increased stride length. This effective selection for a fine-boned skeleton may increase fracture risk under high performance stress. Additional sample collection is critically needed to expand this data set and identify all skeletal variation present in the horse. Our ultimate goal, to screen for and identify genes responsible for skeletal variation in the horse, will enable the identification of loci affecting various traits including conformation, disease susceptibility, and athletic performance. In particular, genes affecting skeletal growth in the horse could affect the length and density of the bone. By identifying skeletal trait-associated polymorphisms and assessing their frequency in fracture-prone and non-fracture-prone individuals, we could appreciably improve the quality of life of many performance horse breeds by predicting fracture risk. Furthermore, as body size genes affect fundamental processes such as cellular growth, they could be examined for connections to the abnormal cell growth seen in cancer. Ultimately, our findings could be used to significantly decrease the frequency of detrimental alleles at identified disease loci in the horse through the use of genetic screening and breeding programs. Keywords: Morphometrics; Skeletal growth
REFERENCES
MATERIALS AND METHODS Primers for the last exon of the NMES1 gene were designed as a PCR assay for screening a Chori 241 equine BAC library for a BAC containing the genomic NMES1 sequence. The identified clone containing the BAC was grown in culture, and DNA was extracted with the Qiagen Large Construct kit. We used direct sequencing of the BAC DNA by designing primer assays for primer walking to obtain the genomic sequence. Fluorescent in situ hybridization was used to map the BAC containing the NMES1 gene to an equine chromosome.
RESULTS The equine gene was fully sequenced from a CHORI-241 BAC clone. The BAC was mapped to ECA 1q23 using fluorescent in situ hybridization. From the genomic sequence, we obtained the putative amino acid sequence. The encoded protein is highly conserved among mammals and preliminary antibody tests suggested specific binding in the testis to spermatogonia and primary spermatocytes, but not to more differentiated cell types.
DISCUSSION NMES1 is a newly described gene that encodes a protein expressed in epithelial tissue, with abundant expression in testis. Our initial BLAT search for the NMES1 gene in the equine genome identified only a region that appeared to be orthologous to the third exon of the human, mouse, and bovine sequences. We designed PCR primers to amplify the conserved equine sequence and screened an equine CHORI-241 BAC library. After locating a BAC containing
1. Sutter NB, Mosher DS, Gray MM, Ostrander EA. Morphometrics within dog breeds are highly reproducible and dispute Rensch’s rule. Mamm Genome 2007;19(10-12):713-723.
31619 Initial Characterization of a Gene Abundantly Expressed in Stallion Testis J.E. Shields,* K.J. Kochan, J. Jeong, C.A. Abbey, T. Raudsepp, and P.K. Riggs, Texas A&M University, College Station, TX, USA
INTRODUCTION We recently identified a gene (NMES1) that is expressed more abundantly in testis than any other tissue examined. Initial results suggested NMES1 may play a role in cellular differentiation. We sequenced and carried out preliminary characterization of this novel gene in the horse to investigate its function and potential role in stallion spermatogenesis.
Figure 1. Fluorescent in situ hybridization mapping of NMES1 to ECA 1q23.