Asip and MCIR cDNA polymorphism in alpaca

Agouti signalling protein (Asip) and Melanocortin 1 Receptor (MC1R) represent key players in coat colour determination in mammals. To investigate the role of these genes in coat colour variation in alpaca, mRNA purified from skin biopsies of different coloured animals were reverse-transcribed into single-stranded cDNAs and the coding sequences (CDS) amplified by PCR, cloned and sequenced. Moreover, and in addition to CDS, the 5’and 3’untranslated regions (UTRs) of both genes were also characterised by RACE experiments. In fact, these 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 and four were missense (amino acid changing) mutations. MC1R analysis unveiled a total of ten mutations in the CDS and among those one was 4 bp frameshift mutation, four were silent mutations and five were missense mutations. Further analysis of the Asip 3’UTR (223 nt) showed three mutations, including one transversion and two transition mutations located at 10, 38 and 77 nt downstream from the stop codon, respectively. Similarly, three transition mutation were also identified in the 3’UTR (602 nt) of MC1R located 5,166 and 398 nt downstream from the stop codon. In contrast, two different 5’UTRs were characterised for MC1R (294 and 153 nt). Genotyping assays and semiquantitative RT-PCR analysis of Asip and MC1R transcripts to evaluate expression-dependent coat colour variation are currently in progress.