Asip and MC1R cDNA polymorphism in alpaca

Agouti signaling protein (Asip) and melanocortin 1 receptor (MC1R) represent key players in coat color determination in mammals. To investigate the role of those genes in coat color variation in alpaca, mRNA purified from skin biopsies of different colored animals were reverse-transcribed into single-stranded cDNAs and the coding sequences (CDS) amplified by PCR, cloned and sequenced. Moreover, beside CDSs, the 5’& 3’untraslated regions (UTRs) of both genes were also characterized by RACE experiments. In fact, those regulatory regions can potentially host polymorphisms able to significantly alter Asip and MC1R gene expression levels. In this study, we described for the first time, seven single point mutations in the CDS of Asip, of which three were found to be silent mutations (18A>C, 102G>A, 290C>A) and four were missense (amino acid changing) mutations (T4S, Y51S, C98T, H118R). MC1R analysis unveiled a total of ten mutations in the CDS, among those one was 4bp (ACTT) frameshift mutation, four were silent mutations (126C>T, 354T>C, 618G>A, 933G>A) and five were missense mutations (T28A, T31M, M87V, S126G, and R301C). Further analysis of the Asip 3’UTR (223bp) showed three mutations, among those one was transversion (412C>A) and two were transition mutation (440A>G and 479T>C) located 10, 38 & 77 nt downstream from the stop codon, respectively. Similarly, three transition mutation (959C>T, 1120C>T and 1352G>A) were also identified in the 3’UTR (602 nt) of MC1R located 166 & 398 nt downstream from the stop codon, respectively. Two different 5’UTRs were characterized for MC1R (294 and 153 nt). Genotyping assays and semi quantitative RT-PCR analysis of Asip and MC1R transcripts to evaluate expression dependent coat color variation are currently under progress.