The Molecular Genetical Investigation On Formation Mechanism Of Depigmentation Phenotype In Mongolian White Horses

The research of horse’s coat color gene is one of the important contents in the field of coat color genetics. The coat color is not only important basis for identification both in breeds and individual, but also be used to diagnosis certain diseases, is also the one of important traits to develop breeding plan. The horse coat color research has not only application meaning but also special economic and culture value. Therefore the researches on depigmentation of coat have become an important part of horse coat color study, which are trying to find the real mechanism. Mongolian horses are ones of the oldest horse breeds in the world. Not only have basic color such as bay, chestnut and black, also have spot, gray and fade, which were belongs to rare phenotypes coat color. Mongolian horses are the best models for researching horse color genetic. The coat color completely faded horses are very rare, but in West Uzhumchin of Shiligol League in Inner Mongolia discovered a larger number of the coat color completely faded horses, called Mongolian white horses.We divided the Mongolian white horse into two types, consist character of dominant white color horses and the grey coat individual.With the publication of the horse genome sequence, different genes that control different phenotype were discovered gradually. According to the data, the formation of horse’s fade color phenotype is controlled by several genes; However, there is a large difference between varieties. Gene mutations in the sequences can lead to body appears different properties, sometimes even if gene sequences are same, also have different phenotype. In recent years with the arrival of Post Genome Era, scientists have proposed many new experiment methods, methylation modified of epigenetics can explain the same gene sequences and different biological phenomenon.Therefore it become a hotspot in the field of molecular biology.This study choose Mongolian white horses as the research objects. Firstlly, take a genotyping to choice individual according to restriction of polymorphic loci of MC1R、 ASIP、MATP、SBI and STX-17five candidate genes, made the molecular biology identification for coat color, to make sure whether the Mongolian horses phenotype accordance with the announced coat color gene polymorphism; Secondly by using real time quantitative PCR, as β-actin gene as housekeeping gene, to determining the expression quantity of six colors candidate gene (MCIR, ASIP. KIT. STX-17, MATP, EDNRB)of Mongolian white horse in the skin; meanwhile, using the high throughput sequencing technologies to research polymorphism of MC1R,ASIP, KIT, STX-17, MATP and EDNRB five genes all coding region; In addition, use Western blotting method for protein in the skin to further validation KIT gene’s expression of Mongolian white horses; at last, analyzed the methylation variance research of different color Mongolian horse’s KIT gene’s promoter area;Through the above research, provides important theoretical basis and clues to the formation of Mongolian white horses fade phenotype, obtained the main research results are as follows:1、The experimental research object will serve as the17Mongolian horses (colored hair and faded hair) on molecular biology coat color identification, and the results show that, Mongolian horses’ color phenotype and candidate genes are certain relevance. we can through testing MC1R, ASIP, SB1, STX-17and MATP genotype to judge Mongolian horses coat type preliminary, and suggest we can not only consider a particular gene type, but a comprehensive analysis before make the final conclusion.2、we tested the color candidate gene’s quantitative expression in17Mongolian horses’(colored hair and faded hair) skin, the results show that color candidate genes, such as:MC1R, ASIP, KIT, STX-17, MATP and EDNRB, have a-higher expression in the bay individual than faded hair, it suggest that the phenotype of Mongolian horses’ depigmentation relative with expression content of coalt color candidate genes in their skin tissue, not be caused different express of certain gene, It is the result of several genes, but its mutual mechanism still need further research.3、After amplification comparative analysis on exon and portion non coding region of MC1R, ASIP,KIT, STX-17, MATP and EDNRB, we found that MATP gene’s5’ regulation region have a lack of68bp in Mongolian white horses which consist character of dominant white horses.4、After the protein analysis on the quantitative expression of KIT both in color hair and faded hair individual, we found that:KIT protein’s expression in Mongolian white horses skin was obviously lower than the colored hair individual, grey coat individuals a little higher than Mongolian white horses.5、Analysis above six gene’s5’regulation region use epigenetics analysis software, we found that KIT gene’s5’control area is consisted593bp CpG island, then we measured the methylation pattern of bay and depigmentation coat Mongolia horses’ KIT gene’s5’ control region in skin DNA, we found that in this region have no different methylation pattern between bay and depigmengtation color horses.