Segregation Analysis and Molecular Characterization of Black and Brown Phenotypes in Alpaca

Principal investigator:

Carlo Renieri, DVM
University of Camerino, Italy

In order to characterize white, black and brown coat color in alpaca, an experimental trial was made in Peruvian INIA Quinsachata Experimental Station. One hundred forty five offspring born from the following matings : 4 white rams x 36 white dams, 4 white rams x 39 pigmented dams, 9 pigmented rams x 70 pigmented dams and, among the last mating, 4 black rams x 25 black dams, 2 black dams x 20 brown dams, and 3 brown dams x 25 brown dams. The inheritance of white is defined by a single gene; it is independent and completely dominant on pigmented animals, without any modifying effect and without difference in segregation on black and brown patterns. Differently, the relation of dominance of black over brown does not seem fully supported by clear cut evidences. ASP (Agouti) is a well studied gene which encodes for a small secreted factor (agouti signalling protein) that influence functions as a paracrine regulator of hair pigmentation. Mutually exclusive binding of the melanocortin-1-receptor (MC1R) by the agouti signalling protein or the α-melanocyte stimulating hormone (α-MSH) signals the hair-bulb melanocytes to synthesize either pheomelanin (yellow-red pigments) or eumelanin (dark pigments), respectively. Loss of function mutations of ASP produce black color, while gain of function mutations are responsible for reddish brown color. We observed 17 single nucleotide polymorphisms (SNPs) in total and an in-frame 57 bp deletion at the position g.3866-3923 in the exon-4 (cystine rich domain). In which: a synonymous SNP g.102G>A was observed in the exon-2; 6 SNPs (g.458T>C, g.589T>A, g.594G>A, g.681T>C, g.1000G>A, g.1143C>T and g.1164T>C) were observed in the intron-2; 5 SNPs (g.1556C>A, g.2360A>G, g.3193C>A, g.3263T>C and g.3277T>G) were observed in the intron-3; 3 SNPs were observed in the exon-4, among those g.3819C>A was synonyms and g.3820C>T and g.3881G>A were observed to be non synonymous. Apart from these, analysis of the 3UTR showed one transversion and two transition mutations located at 10 (g.3955C>A), 38 (g.3983A>G) and 77 bp (g.4022T>C) downstream to the stop codon. The SNPs : g.3836C>T (ahT), g.3881G>A (aH), and an in-frame 57 bp deletion (g.3866_3923del57) (aΔ57bp) are predicted to independently cause functional changes to agouti protein with complete or partial loss of function. The aΔ57bp would result in a 114 amino acid containing agouti protein, which lacks 19 amino acids (p.C109_R127del19) from the cysteine (C) rich domain, which is critical in agouti function. The ahT would predict a change of arginine (R) to C, which would disrupt the highly conserved region of the protein. The aH changes the R to histidine (H) in the cystine-rich domain, which disrupt the highly conserved Arg-Phe-Phe (R-F-F) motif in the protein, respectively. Functional analysis suggests that aH may produces a non functional agouti and the ahT may produces agouti protein with minimal/partial activity. These three alleles are validated for black phenotype by half sib segregation analysis

Publication resulting from this work:

A., APAZA N., LA MANNA V., GONZALES M.L., HUANCA T., RENIERI C., 2011. Inheritance of white, black and brown coat colours in alpaca (Vicuna pacos L.). Small Ruminant Research, 99,16-19