Investigations of candidate genes for appaloosa coat color in horses

Appaloosa horses are characterized by eight different spotting patterns. All of these patterns are controlled by a single dominant gene, designated as Leopard Complex, Lp. Each pattern is characterized by patches of white spotting on the coat of the animal. These white areas lack pigment. Therefore the genes involved with the production of melanocytes during development are good candidate genes for Lp. In mice, mutations in the KIT, MGF, and MITF genes, produce white spotting patterns similar to those produced by Lp. KIT, MGF, and MITF all play critical roles in melanocyte migration, differentiation, proliferation and survival. The hypothesis of this study was that one of these genes encodes the appaloosa coat color gene, Lp.; Each gene was investigated as a candidate gene by performing a linkage analysis with markers located on the same chromosome as the candidate gene. KIT is mapped to ECA3q21. The three known polymorphisms within KIT were uninformative and could not be used to test for linkage directly to KIT. Linkage was investigated by analyzing two point and multi-point LOD scores for linkage of Lp with GC, ALB, ES, LEX57, UCDEQ437, LEX007, and ASB23. Linkage was rejected over this entire chromosome region excluding KIT as the gene for Lp.; MGF maps to ECA28q13. Since there are no known equine polymorphisms of MGF to test for linkage to Lp directly, microsatellite markers mapped to this chromosome, UM003, HTG30, NYEQ54, IGF1, and TKY18, were used to test for linkage. TKY18 is the only marker of the 5 in which the physical map position is known. This marker is mapped to ECA28q14 which is close to MGF. TKY18 provided evidence against linkage with Lp. Further support to determine MGF was not Lp was sought by trying to identify polymorphism with in the gene itself. No polymorphisms were detected over the 5705 bases evaluated between solid and appaloosa spotted horses. Therefore from this data we conclude that it is unlikely that MGF is Lp.; MITF maps to ECA16q14–q16. No sequence information was available for this gene in the horse and no polymorphisms were known. Sequence information was generated for the coding region of this gene along with intron one to try to identify a SNP within this region to test for linkage of MITF with Lp. Of the 2204 bases evaluated no informative polymorphisms were found when comparing solid colored horses to Appaloosas. Again microsatellite markers that map to this chromosome region were used to test for linkage, specifically HTG03, AHT38, COR011, and LEX059. Linkage was rejected for both HTG03 and AHT38. These markers flank MITF, thus Lp does not map to this region and MITF is not the gene for Lp.; This study has ruled out three candidate genes, KIT, MGF, and MITF, as the gene for Lp. This study added to the resolution of the Equine genome physical map. This study also generated equine sequence information for MITF and MGF that could have value in future comparative studies. The sequence information also could be used in the future to test for polymorphisms in other spotting patterns. Sets of microsatellite primers now are available to perform a genome scan as the next step in the identification of other candidate genes for