Background

A large number of coat colour phenotypes have been described in different mammalian species. This diversity is due to the presence, distribution and biochemical activity of the melanocytes in which two types of melanin pigments (eumelanins and pheomelanins, that produce black/brown and red/yellow colours, respectively) are synthesized. Extension and Agouti are the main loci that affect the relative amount of eumelanin and pheomelanin production in these cells [1]. These loci show epistatic interactions in different mammals. Dominant alleles at the Extension locus induce black pigmentation, whereas recessive alleles extend the production of pheomelanins, determining red/yellow/pale pigmentation. Mutations at the Agouti locus have, in general, opposite models of action, i.e. dominant alleles determine pheomelanic phenotypes, whereas recessive alleles cause black coat colour with a few exceptions.

The Extension locus encodes the melanocortin 1 receptor (MC1R), a seven transmembrane domains protein belonging to the G protein coupled receptors [2] that binds the α melanocyte-stimulating hormone (αMSH) inducing eumelanin synthesis. Agouti, instead, encodes the agouti signaling protein (ASIP), a paracrine signalling molecule that affects pigmentation acting as antagonist of MC1R, blocking αMSH-receptor interaction and causing a pigment-type switching from eumelanins to pheomelanins [3, 4].

Mutations of the MC1R gene affecting coat colour have been described in several mammals, such as mice [2], humans [5], guinea pigs [6], cattle [7–9], pigs [10], horses [11], sheep [12], dogs [13, 14], foxes [15], bears [16], felids [17], rabbits [18], and pocket mice [19], in which gain of function mutations produce black/dark coat colour, whereas loss of function mutations cause red/yellow or white coat colour.

In goats, a large number of alleles at the Agouti locus, accounting for a broad variability on coat colour, has been predicted by classical crossbreeding studies in several breeds [1, 20–24]. From these studies, the Extension locus does not seem to play a major role on coat colour variability in goats. The existence of a dominant EDblack allele and a recessive e red allele has been suggested in few breeds [1, 25]. In other goat populations, epistatic effects of Agouti alleles might mask and confound the action of the Extension locus. On the other hand, the wild type E+ allele, the most common form supposed at this locus, should make the phenotypic effects of the different Agouti alleles possible, as observed in other species [1]. In Boer goats, Wu et al. [9]. Sequences were obtained from 48 random goats across six breeds (Girgentana, 10; Maltese, 10; Derivata di Siria, 10; Murciano-Granadina, 6; Camosciata delle Alpi, 6; Saanen, 6). In addition, sequencing of the MC1R gene was carried out from the Maltese buck, the red Maltese-like goat and the three F1 animals obtained crossing these two goats. PCR was performed using a TGradient thermal cycler (Biometra, Goettingen, Germany) or a PT-100 thermal cycler (MJ Research, Watertown, MA, USA) in a volume of 20 μL containing 10–100 ng DNA template, 1 U DNA EuroTaq DNA polymerase (EuroClone Ltd., Paington, Devon, UK), 1× PCR Buffer, 2.5 mM dNTPs, 10 pmol of each primer and optimised MgCl2concentrations (from 2.0 to 2.5 mM). PCR profile was as follows: 5 min at 95°C; 35 amplification cycles of 30 s at 95°C, 30 s at 60/65°C, 30 s at 72°C; 5 min at 72°C. For the MC1R fragments sequencing 3–5 μL of PCR product was treated with 2 μL of ExoSAP-IT® (USB Corporation, Cleveland, Ohio, USA) following the manufacturer's protocol. Cycle sequencing of the PCR products was obtained with the Big Dye v3.1 kit (Applied Biosystems, Foster City, CA, USA) and sequencing reactions, after a few purification steps using EDTA 0.125 M, Ethanol 100% and Ethanol 70%, were loaded on an ABI3100 Avant sequencer (Applied Biosystem). All sequences were visually inspected, edited, assembled, and aligned with the help of the BioEdit software v. 7.0.5.2 http://www.mbio.ncsu.edu/BioEdit/bioedit.html and the CodonCode Aligner software http://www.codoncode.com/aligner.

SNP genoty**

To analyse the five point mutations found by sequencing, four different PCR-RFLP methods were established using primer pairs 2-ch7, E1-2, and A-2 and four different restriction endonucleases (Additional file 1). PCR was performed as described above and in Additional file 1. Both c.183C>T and c.242C>T SNPs disrupt/create a GGCC restriction site for Hae III endonuclease; therefore, we could genotype our samples for both mutations, using the same 2-ch7 primer pair, amplifying a 169 bp fragment and performing the same PCR-RFLP reaction. The nonsense mutation (c.673C>T) was analysed using primer pair E1-2, that amplifies a 267 bp fragment, and Xba I restriction enzyme (TCTAGA). The c.748T>G mutation was analysed amplifying a fragment of 123 bp, using primer pair A-2 with a forward primer that creates an artificial restriction site (ACGT) for Tai I endonuclease. The same PCR product was subjected to a further PCR-RFLP reaction with Hae III to investigate the c.801C>G point mutation. Additional file 2 reports the electrophoretic patterns of the investigated mutations.

Sequence analysis and statistics

In silico functional analysis of missense mutations was obtained using PANTHER [31] whose predictions have been experimentally validated [48]. PANTHER estimates the likelihood of a particular non-synonymous (amino-acid changing) coding SNP to cause a functional impact on the protein. It calculates the substitution position-specific evolutionary conservation (subPSEC) score based on an alignment of evolutionarily related proteins [31–33]. The probability that a given variant will cause a deleterious effect on protein function is estimated by Pdeleterious, such that a subPSEC score of -3 corresponds to a Pdeleterious of 0.5 [48]. The subPSEC score is the negative logarithm of the probability ratio of the wild-type and mutant amino acids at a particular position. PANTHER subPSEC scores are continuous values from 0 (neutral) to about -10 (most likely to be deleterious). For the analysed animals, haplotypes including the five SNPs within the goat MC1R gene were inferred using the PHASE program v. 2.1 [49].

A median-joining network [50] for these haplotypes was constructed using Network v. 4.510 [51], including the sheep MC1R coding sequence (GenBank accession number: Y13965) [12]. When appropriate, association between SNPs and coat colours was tested using 2 × 2 contingency tables with Fisher exact test implemented in the procedure FREQ of SAS version 8.02 (SAS Institute Inc. Cary, NC, USA).