Characterization of a major locus affecting coat color reddening in cattle
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Date
2014-10-02
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Abstract
In cattle, differences in base coat color are attributed to the enzymatic activity specified by the melanocortin-1 receptor (MC1R) locus with alleles coding for black (ED), red (e), and wild-type (E+). These alleles have a presumed dominance model of ED > E+ > e. In Nellore-Angus F2 cattle, some EDE+ heterozygotes have displayed various degrees of red pigmentation when predicted to have a black background. This variation has been associated with a major locus on Bos taurus chromosome (BTA) 6 interacting with MC1R. The objective of this study is to identify the causative mutation on BTA6 and characterize the effect of the mutation on expression of genes in the melanocyte pigmentation pathway.
This region of BTA6 coincides with a cluster of tyrosine kinase receptor genes including PDGFRA, KIT, and KDR. We hypothesize that a structural variant in or near PDGFRA, KIT or KDR causes this novel reddening phenotype in cattle. First, the critical interval containing the reddening locus was refined to BTA6:70,714,167-71,404,818 using imputed and phased HD SNP data in our F2 Nellore-Angus mapping population. Then SNP and indels from whole-genome sequencing of the founders of the mapping population were used to impute the region to sequence scale to find variants concordant with recessive inheritance of the reddening alleles from Nellore. Next, skin biopsies from biological triplicates were collected for each genotypic combination of the extension locus (EDED, EDE+, E+E+) and the reddening locus (NN, NA, AA). A TRI Reagent isolation method and RNeasy mini kit was used to extract total RNA (RIN > 6.5). Primers for realtime RT-qPCR were designed for each of the tyrosine kinase receptor genes (PDGFRA, KIT or KDR), MC1R and ASIP.
The relative expression of each gene was calculated and no significant differences in expression of the candidate genes (PDGFRA, KIT, and KDR) categorized by genotype at MC1R and reddening were found; however, long-range regulatory effects on PDGFRA, KIT or KDR still cannot be ruled out. Among individuals genotyped as heterozygous EDE+ at the extension locus, those that were homozygous Nellore at reddening exhibited significantly (P < 0.05) lower levels of expression for MC1R than those that were homozygous Angus at reddening. Reexamination of HD SNP data identified CHIC2 as another positional candidate.