Progress 10/01/03 to 09/30/08
Outputs
OUTPUTS: During the course of this project, a whole genome sequence for the horse was completed based on work done at the Broad Institute under the auspices of the National Human Genome Research Institute at the request of the horse geome research community. The scientists at Kentucky spearheaded this request and provided DNA samples for sequencing and corrected or validated the sequence analysis using other genetics approaches. This is the most significant output for the project. However, applications have been reported on a yearly basis. The following are actions with outputs during the past year: Investigate equine laminitis using qRT-PCR. Discovered genetic basis for junctional epidermolysis bullosa in American Saddlebred horses. Discovered genetic basis for champagne dilution hair color modification in horses. Identified chromosome region responsible for lordosis in American Saddlebred horses. Investigate the genetics of dwarfism in Miniature horses. Reported inversion that appears to be responsible for Tobiano hair color patterns. Reported chromosomal translocations that resulted in reduced fertility of mares. Investigate maturation and health of articular cartilage. Investigate Tendon and Ligament Maturation and Repair. PARTICIPANTS: During the project the following students completed graduate studies while working in connection with the program: Mike Mienaltowski, Barbara Murphy, Samantha Brooks, Stephen Coleman,Christine Mains, Catherine Wagner,Michelle Mousel. The current students are currently working on graduate degress in the program: Deborah Cook, John Eberth, Rose Magee, Rebakah Cosden, Jennifer Janes, Lauren Detlefsen, Kadie Vanderman and Wenying Zhu. TARGET AUDIENCES: Horse owners, horses breeders, veterinarians, animal sicence academics, veterinary academics and other scientists for the target audiences for this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Over the entire course of this project, new genetic tools have been created and applied to investigate issues of importance to horse breeders. Applications have been reported each year for the project. During the past year, the following applications have become available. Diagnostic tests are available for junctional epidermolysis bullosa in Saddlebred horses, and for the Tobiano and Champagne, dilution hair color in horses. The cytogenetic work identifying translocations was of immediate benefit to the horse owners who were attempting to use affected mares as breeding stock. For a broader application, the work demonstrated the importance of considering translocations in cases of reduced fertility. Work on gene expression in association with laminitis and arthritis will lead to prognostic tests and therapeutic treatments.
Publications
- Bellone, R.R., Brooks, S.A., Sandmeyer, L., Murphy, B.A., Forsyth, G., Archer, S., Bailey, E., and Grahn, B. (2008), Differential gene expression of TRPM1, the likely cause of congenital stationary night blindness (CSNB) and coat spotting pattern (LP) in Appaloosa horses (Equus caballus), Genetics, 179(4): 1861-1870. (doi: 10.1534/genetics.108.088807).
- Brooks, S.A., Lear, T.L., Adelson, D.A., and Bailey, E. (2008). A chromosome inversion near the KIT gene and the tobiano spotting pattern in horses, Cytogenetics and Genome Research, 119: 225-230.
- Cook, D., Brooks, S.A., Bellone, R., and Bailey, E. (2008), Missense Mutation in Exon 2 of SLC36A1 Responsible for Champagne Dilution in Horses, PLOS Genetics, 4(9): e1000195. doi:10.1371/journal.pgen.1000195.
- Graves, K.T., Henney, P.J., and Ennis, R.B. (2008). Partial deletion of the LAMA3 gene is responsible for hereditary junctional epidermolysis bullosa in the American Saddlebred Horse, Animal Genetics, Nov 11, ahead of print PMID: 19016681.
- Huang, L., Zhu, W., Saunders, C.P., MacLeod, J.N., Zhou, M., Stromberg, A.J., and Bathke, A. (2008). A novel application of quantile regression for identification of cartilage biomarkers in equine microarray data, BMC Bioinformatics, 9: 300.
- Katepalli, M., Adams, A.A., Lear, T.L., and Horohov, D.W. (2008). The effect of age and telomere length on immune function in the horse, Developmental and Comparative Immunology, 32: 1409-1415.
- Lear, T.L., and Bailey, E. (2008). Equine clinical cytogenetics: The past and future, Cytogenetic and Genome Research, 120: 42-49.
- Lear, T.L., Lundquist, J., Zent, W.W., Fishback, W.D., Jr., and Clark, A. (2008). Three autosomal chromosome translocations associated with repeated early embryonic loss (REEL) in the domestic horse (Equus caballus), Cytogenetic and Genome Research, 120: 117-122.
- Mienaltowski, M.J., Huang, L., Stomberg, A.J., and MacLeod, J.N. (2008). Differential gene expression associated with postnatal articular cartilage maturation, BMC Musculoskeletal Disorders, 9: 149.
- Perelygin, A.A., Zharkikh, A.A., Astakhova, N.M., Lear, T.L., and Brinton, M.A. (2008). Concerted evolution of vertebrate CCR2 and CCR5 genes and the origin of a recombinant equine CCR5/2 gene, Journal of Heredity, 99: 500-511.
- Raudsepp, T., Gustafson-Seabury, A., Durkin, K., Wagner, M.L., Goh, G., Seabury, C.M., Brinkmeyer-Langford, C., Lee, E.-J., Agarwala, R., Stallknecht-Rice, E., Schaffer, A.A., Tozaki, T., Yasue, H., Penedo, M.C., Lyons, L.A., Khazanehdari, K.A., Binns, M.M., MacLeod, J.N., Distl, O., Guerin, G., Leeb, T., Mickelson, J.R., and Chowdhary, B.P. (2008). A 4103 marker integrated map of the horse genome, Cytogenetic and Genome Research, 122: 28-36.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Objective: 1. Enhance and integrate genetic and physical maps of agriculturally important animals for cross species comparisons and sequence annotation. The whole genome sequence of the horse was completed by the Broad Institute (MA) in 2006 however the assembly of the sequences was conducted during 2007. A first assembly was reported based on computer alignments but without consideration of mapping, BAC contig construction or fish mapped markers. A major activity in our program was to support the second assembly by fish mapping clones at the request of the Broad Institute in support of determining an accurate second assembly. Objective: 2. Facilitate integration of genomic, transcriptional, proteomic and metabolomic approaches toward better understanding of biological mechanisms underlying economically important traits. The availability of the horse genome map and the horse genome sequence made ITG possible to use the information for investigation of problems and hereditary
traits in horses. Below is a list of activities conducted during the past year in connection with this objective: Investigate equine laminitis based on gene expression of potential therapeutic targets. Investigate genetics of epidermolysis bullosa in American Saddlebred horses. Investigate genetics of lordosis in American Saddlebred horses. Investigate the genetics of dwarfism in miniature horses. Reported inversion that appears to be responsible for tobiano hair color patterns. Reported on the genetics of dominant white among different horse breeds. Identified chromosomal translocations that resulted in reduced fertility of mares. Investigate articular cartilage maturation. Investigate the repair of articular cartilage lesions. Investigate glucocorticoid efficacy and safety in synovial joints. Objective: 3. Facilitate and implement bioinformatic tools to extract, analyze, store and disseminate information. The completed horse genome sequence makes it possible to make predictions
about the organization and function of horse genes based on previous work with the human genome. One of the primary computational tools, in this regard, is a program called Compass, developed at the University of Illinois for comparison of genomic information between species. We used gene expression information to test in silico mapping of ESTs using the program Compass.
PARTICIPANTS: Principal Investigators: Ernest Bailey, Teri Lear, James N. MacLeod, Kathryn Graves, Bruce Webb Collaborative activities: During the year we have had productive collaborations with scienstists at the University of Tampa, Colorado State University, Pennsylvania State University, University of Berne, Switzerland, University College Dublin, Ireland, University of Tokyo, Japan, and The University of Queensland, Australia. Training: Post doctoral student: Samantha Brooks. Graduate Students: Deborah Cook, John Eberth, Mike Mienaltowski, Stephen Coleman, Rebekah Cosden, Lauren Detlefsen, Jennifer James, Wenying Zhu.
TARGET AUDIENCES: Scientists conducting research on all aspects of horse biology, especially veterinary scientists and animal scientists; horse owners and breeders; news media serving the horse industry
Impacts
Diagnostic tests can be provided for epidermolysis bullosa in Saddlebred horses and for the Tobiano and White hair color in horses. The cytogenetic work identifying translocations was of immediate benefit to the horse owners who were attempting to use affected mares as breeding stock. For a broader application, the work demonstrated the importance of considering translocations in cases of reduced fertility. Work on gene expression in association with laminitis and arthritis will lead to prognostic tests and therapeutic treatments. The work on objectives 1 and 3 make genomic research more productive.
Publications
- Coleman, S.J., Gong, G., Faile, D.P., Chowdhary, B.P., Bailey, E., Liu, L., and MacLeod, J.N. (2007). Evaluation of COMPASS as a Comparative Mapping Tool for ESTs Using Horse Radiation Hybrid Maps, Animal Genetics, 38: 294-302.
- Haase, B., Brooks, S.A., Schlumbaum, A., Azor, P,J,, Bailey, E., Alaeddine, F., Mevissen, M., Burger, D., Poncet, P.-A., Rieder, S., and Leeb, T. (2007). Allelic Heterogeneity at the Equine KIT Locus in Dominant White (W) Horses, PLoS Genetics, 3(11), e195 doi:10.1371/journal.pgen.0030195.
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Progress 01/01/06 to 12/31/06
Outputs
Gene mapping studies were focused on physical mapping genes in support of RH and the horse genome sequence. In addition, genes playing a role in several diseases were mapped using fluorescence in situ hybridization (FISH) including resistance markers to West Nile Virus and night blindness in horses. Clinical cytogenetic studies also led to discovery of several novel chromosome rearrangements affecting fertility of mares. A chromosome rearrangement was also found associated with the color pattern called Tobiano and the DNA sequence changes associated with the rearrangement were also identified. The gene in question also influences mast cell development and investigations are underway to determine if the genetic change influences mast cell function as well as coat color patterns. In other studies related to color patterns, OCA2 and TRPM1 were investigated as the causes of appaloosa pattern. Results were inconclusive and did not implicate one gene or the other.
Impacts
The FISH mapping supported the assembly of the horse genome sequence and will resolve questions regarding alignment and location of chromosome segments. The investigations on influence of genes on horse diseases will result in future tests to improve diagnostics for veterinarians and horse owners. The information about Tobiano will result in a test for breeders seeking to produce horses with these patterns. The test will also make it possible to evaluate and understand the immune function of horses with this pattern.
Publications
- Perelygin, A.A., Lear, T.L., Zharkikh, A.A. and Brinton, M.A. 2006. Comparative analysis of vertebrate EIF2AK2 (PKR) sequences and assignment of equine gene to ECA15q24-125 and the bovine gene to BTA11q12-q15. Genet. Sel. Evol. 38:551-563.
- Bellone, R., Lear, T.L., Adelson, D. and Bailey, E. 2006. Comparative mapping of oculocutaneous albinism type II (OCA2) and transient receptor potential cation channel, subfamily M member 1 (TRPM1) and two equine microsatellites, ASB08 and 1CA43, among four equid species by fluorescence in situ hybridization. Cytogenet. Genome Res. 114:93A.
- Perrocheau, M., Boutreux, V., Chadi, S., Mata, X., Decaunes, P., Raudsepp, T., Durkin, K., Incarnato, D., Iannuzzi, L., Lear, T.L., Hirota, K., Hasegawa, T., Zhu, B., de Jong, P., Cribiu, E.P., Chowdhary, B.P. and Guerin, G. 2006. Construction of a medium density equine gene map. Anim. Genet. 37:145-155.
- Bellone, R., Lawson, S., Creeley, N., Archer, S. and Bailey, E. 2006. Analysis of a SNP found in exon 7 of equine OCA2 and its exclusion as a cause of appaloosa spotting. Anim. Genet. 37:525.
- Bailey, E. 2006. Moving horse genomics across disciplinary lines. In: Nutritional Biotechnology in the Feed and Food Industry. Lyons, T.P., Jaques, K.A. and Hower, J.M. (Eds.). Nottingham University Press, Notthingham, UK. pp. 293-300.
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Progress 01/01/05 to 12/31/05
Outputs
Objective: 1. Enhance and integrate genetic and physical maps of agriculturally important animals for cross species comparisons and sequence annotation. a) To improve the horse gene maps gene were FISH mapped to resolve discrepancies and to expand the scope of the map. Genes of interest include those that show discrepancies in gene order when comparing the linkage and RH maps. Other genes of interest are those that may function in the immune response or be responsible for genetic traits or diseases of horses. b) Studies were conducted to map genes on ECA1 that may be responsible for appaloosa coat color pattern and night blindness. Two candidate genes on ECA1, OCA2 and TRPM1 were evaluated and a SNP that produces a change in the amino acid sequences of OCA2 was identified. Work is underway to determine what impact that change has on the eye. c) Studies were conducted on genes on ECA3 responsible for the sabino1 and tobiano coat color patterns in horses. The sabino1
color pattern was shown to be caused by a mutation in an intron that alters exon splicing. Meanwhile, a major chromosome inversion was identified associated with the tobiano color pattern. Work is underway to identify DNA sequences associated with the inversion. d) Agreement between the RH and linkage maps was investigated in two ways. First, the program Carthagene was used to integrate data from the two maps to create a single comprehensive map. Discrepancies were apparent in the analysis and generated testable hypotheses resolved using FISH techniques described above. Second, ESTs were artificially mapped using predictions from the RH map and the program Compass. Then the ESTs were actually mapped on the RH panel and the predictions compared to the actual results. Objective: 3. Facilitate and implement bioinformatic tools to extract, analyze, store and disseminate information. Develop an integrated map for linkage and RH data. The results are represented on the Horse Gene Map viewer
(http://www.vgl.ucdavis.edu/equine/caballus/)
Impacts
The improved quality and resolution of the horse gene map makes it feasible to investigate hereditary traits in horses with the objective of mapping them to a chromosome region and possibly even identifying the gene responsible. During the past year we have demonstrated the feasibility of various approaches (candidate gene, genome scan, investigation of mRNA splice variants) to shed light on the genetics of coat color patterns in the horse. These genes may also have an impact on health traits in the horse as well.
Publications
- Brooks, S.A. and Bailey, E. 2005. Exon skipping in the KIT gene causes the Sabino spotting pattern in horses. Mamm. Genome 16: 893-902.
- Luis C., Cothran, E.G., Oom, M.M. and Bailey, E. 2005. Major histocompatibility complex locus DRA polymorphism in the endangered Sorraia horse and related breeds. J. Anim. Breed. Genet. 122: 69-72.
- Penedo M.C.T., Millon, L.V., Bernoco, D., Bailey, E., Binns, M., Cholewinski, G., Ellis, N., Flynn, J., Gralak, B., Guthrie, A., Lindgren, G., Lyons, L.A., Tozaki, T., Roed, K. and Swinburne, J. 2005. International equine gene mapping workshop report: A comprehensive linkage map constructed with data from new markers and by merging four mapping resources. Cytogenet. Genome Res. 111:5-15.
- Perelygin, A.A., Lear, T.L., Zharkikh, A.A. and Brinton, M.A. 2005. Structure of equine 2-5 oligoadenylate synthetase gene family. Cytogenet. Genome Res. 111: 51-56.
- Tallmadge, R.L., Lear, T.L. and Antczak, D.F. 2005. Genomic characterization of MHC class I genes of the horse. Immunogenetics 57:763-774.
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Progress 01/01/04 to 12/31/04
Outputs
Lear: Genes were cytogenetically mapped using FISH in collaboration with a mapping project at INRA, Jouy-en-Josas, France with Gerard Guerin (manuscript in preparation), OCA clones in collaboration with Georgia State group (manuscript in press), immune response genes in collaboration with Doug Antczak and Bettina Wagner at Cornell University and also several clones in collaboration with Rebecca Terry at the University of Tampa (manuscript in preparation). All together, 30 genes were FISH mapped during 2004 in my laboratory. In addition, work was completed on a comparative map between horse and other members of the perissodactyla group, the rhinoceriodae. Bailey: Forty-seven equine TCRB variable genes were identified from the T cell receptor beta chain mRNA of two horses using anchored PCR. The 47 equine variable genes were divided into 19 families based on 75% nucleotide identity and each family had between one and six members. Humans have 40-48 functional TCRBV
genes, grouped into 21-23 families. The equine equivalents of human families TCRBV4, TCRBV13, TCRBV18, TCRBV19, and TCRBV24 were not found in this study. Sequences similar, but not identical, to eight of the variable genes and four of the joining genes determined in this study were published by Schrenzel and co-workers in 1994. The vast number of unique, high quality equine T cell receptor beta chain clones sequenced, 469, ensures a high level of confidence in the sequence information contained in this study. Two diversity genes and eight joining genes were identified from the T cell receptor beta chain mRNA of two horses using anchored PCR. An attempt was made to use the information in a study of T-cell responses to superantigen stimulation, however, the diversity of the genes made a cloning and sequencing approach impractical. During 2004 we conducted studies demonstrating that one form of sabino spotting pattern in horses, she designated sabino 1, is caused by deletion of exon 17
in the KIT gene. This was shown associated with a SNP in intron 6 that is likely to play a role in splicing of the KIT gene. This gene is distributed widely among horse breeds. However, it does not explain all types of sabino patterns and the phenotype clearly has a heterogeneous genetic origin. The work is submitted for publication. Domenico Bernoco, Cecilia Penedo and Lee Millon conducted analyses on the Workshop Phase III linkage map based on amalgamation of previously published data from other laboratories (Workshop, Animal Health Trust, Swedish Agricultural University and UC, Davis.) plus new markers, many of which were discovered by T. Tozaki and mapped at UC DAvis. Bailey was a minor participant in analyses related to that important accomplishment (to be published in 2005). Cothran: We have tested over 160 msats on our affected and unaffected panels for the Degenerative Suspensory Ligment Disease (DSLD) and have identified about 7 regions that were are now looking at in greater
detail. At this point none of the regions have been confirmed or fully rejected but hope to get to that point during this calender year.
Impacts
The cytogenetic mapping conducted by Lear expanded the comparative map and provided ancillary information on genetic systems important in equine health. The information on the T-cell receptor genes developed by Wagner provided useful information for designing and conducting immunology experiments for horses. The coat color genetics work by Brooks identifies a novel genetic mechanism for spotting patterns in horses that will certainly have a commercial application for horse breeders. More importantly, this information provide a basis for creating more specific hypotheses regarding gene action during development as well as to address the potential health associated aspects of coat color in horses. The work on DSLD should led to development of a diagnostic tool that breeders can use to avoid this condition.
Publications
- Wagner, B., Miller, D.M., Lear, T.L., Antczak, D.F. 2004. The immunoglobulin constant heavy chain gene region of the horse contains seven IGHG genes. Journal of Immunology 173:3230-3242.
- Takahashi, T., Yawata, M., Raudsepp, T., Lear, T.L., Antczak, D.F., Kasahara, M. 2004. Natural killer cell receptors in the horse: evidence for the existence of multiple transcribed LY49 genes. European Journal of Immunology 34:773-784.
- Terry, R.B., Archer, S., Brooks, S., Bernoco, D. and Bailey, E. 2004. Linkage mapping the appaloosa coat color gene (LP) of the horse. Animal Genetics 35: 134-137.
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