Screening And Identification Of Candidate Genes And Proteins For Horn Trait Of Yak

The yak?Bos grunniens?is a special and important bovine species that lives on the Qinghai-Tibetan plateau and its adjacent areas at altitudes of more than 3,000 m.Yak can provide the basic resources?food,transport,fuel?that are necessary for Tibetans and other nomadic pastoralists in the high altitude environment.The large and stout horns are the key external properties of the yak,and serve a tremendous diversity of functions,such as mate selection,self-defense,and so on.In modern husbandry systems,the presence of horns increases the risk of injuries to handlers and animals,especially as the housing densities have increased.The polled yak is easy to manage,and more and more people like to feeding polled yak.Thus understanding the molecular mechanism and breeding polled yak is a promising alternative.1.Based on the previous conducted Genome-wide association study on yak Polled trait in yak,we selected 207 kb interval?Reference: Bos mutus;scaffold526₁:1,116,000-1,323,000?for targeted resequencing.A total of 2,617 SNPs and 330 Indels were identified,39 of which were synonymous mutations,14 of which were missense mutations,and one Indel was located in coding region.The interval contained three protein-coding genes: SYNJ1,GCFC1 and C1H21orf62.Further analysis identified that 1,076 SNPs were significantly associated with polled phenotype?P<0.05?,31 of which were co-segregated with phenotype.Two SNPs 1235483G>A and 1235468G>A of C1H21orf62 gene were identified to be associated with Polled trait on yak.The two SNPs were missense mutations,resulting in Methionine?MeT?and Isoleucine?Ile?to Valine?Val?,respectively.2.PCR analysis was used to validate the genetic variation in 398 horned yaks and 369 polled yaks on SNPs 1235483G>A and 1235468G>A.The results showed that the two loci were complete linkage,most of horned individuals?97.2%?were GG-GG genotype,whereas 2.8% were heterozygous?GA-GA?.In polled polulations,82.7% were heterozygous?GA-GA?,3% were homozygous?AA-AA?,whereas 14.3% polled yaks were GG-GG genotype.It must be conclude that the two SNPs were significantly associated with Polled trait,but not the causative mutations.Also,we cannot rule out possible allelic heterogeneity of significantly associated mutations.3.The real-time quantitative PCR of three candidate genes?SYNJ1,GCFC1 and C1H21orf62?on the eight tissues?horn bud,frontal skin of horned yak,polled yak frontal skin,heart,liver,spleen,lungs and kidney?of horned fetal yak and polled fetal yak were conducted.The results showed that SYNJ1 gene highly expressed in heart,liver and spleen.GCFC1 gene expressed at a lower level in all the tissues.C1H21orf62 gene expressed at a highest level in liver,followed by horn bud,frontal skin of horned yak and polled yak frontal skin.Whereas no significant difference was detected among horn bud,frontal skin of horned yak and polled yak frontal skin.The genetic mechanism on Polled trait of yak is complex,the candidate genes regulating horn formation is still unidentified.4.Then,a differential proteomic analysis was completed comparing the horn bud and polledskin of yak fetuses using iTRAQ technology coupled with 2D LC-MS/MS.100 differentially abundant proteins were identified.Of these,29 were up-regulated and 71 were down-regulated in polled skin compared to the horn bud.Bioinformatic analyses showed that the majority of up-regulated DAPs were related to metabolic activities,while most of the down-regulated DAPs were involved in cellular developmental processes and organization of subcellular components.We also found that some important proteins associated with cell adhesion,cell motility,keratinocyte differentiation,cytoskeleton organization,osteoblast differentiation and fatty acid metabolism.Histological sections of horn bud displayed multiple layers of vacuolated keratinocytes,Immunofluorescence staining showed that FABP5 were highly expressed in epidermis,and the expression in horn buds was higher than that of polled skin and frontal skin,it must be conclude that FABP5 is related to vacuolated keratinocytes.The study will provide basic data for understanding of development of horn bud.