## 3 Molecular Characterization of Coat Color Affecting Genes in Rabbits
### Mutations in the Agouti Signaling Protein (ASIP) Gene and the Agouti Locus  
The agouti signaling protein (_ASIP_) gene, structurally constituted by three coding exons, encodes for a small protein that regulates the eumelanin to pheomelanin synthesis switch in the melanocytes [51]. The wild type agouti coat color that is characterized by banded-hairs is obtained by the alternative and mutually exclusive binding of MC1R by the \(\alpha\)-melanocyte-stimulating hormone or by ASIP [52; 53]. Mutations in the _ASIP_ gene or in the regulatory motifs controlling _ASIP_ expression that disrupt the protein structure or reduce the expression level and protein synthesis are usually recessive and impair the ASIP regulation activity on MC1R. This altered interaction between the two proteins, in turn, determines the production of eumelanin and the absence of pheomelanin, resulting in a non-agouti black coat color (e.g., [54; 55; 56; 57; 58]). Classical genetic studies have described three alleles at the rabbit _Agouti_ locus [30; 31]. The wild type allele (\(A\) or \(A^{av}\), as it resembles the murine \(A^{av}\) light-bellied agouti allele [59]) produces a greyish-brown or grey dorsal fur with a lighter (almost white) underside with a particular distribution of pheomelanin and eumelanin in the different types of rabbit hairs [30; 31]. The wild type allele is dominant over the other two alleles described in rabbits at this locus: \(a^{t}\) (black and tan pattern) and \(a\) (non-agouti or self). The rabbit \(a^{t}\) allele is characterized by a unique haplotype in the coding region determined by three missense mutations (p.L55M, pK77R and p.L89P), one of which (p.L55M; located in a conserved position of the basic amino-terminal domain of the protein) was reported only in Tan rabbits that are homozygous for the \(a^{t}\) allele [60]. The \(a^{t}\) haplotype extends for a large chromosome region spanning the _ASIP_ gene and includes the putative causative mutation determined by a 11 kb deletion encompassing the hair cycle-specific _ASIP_ promoter [61]. Rabbits homozygous for the \(a^{t}\) allele have non-agouti (not banded) hairs on the dorsal surface of the body, whereas the belly and other regions of the body (i.e.,  
Figure 1: A detail of the 2D structure of the rabbit MC1R protein with indicated the amino acids that are eliminated by the three in-frame deletions that cause the \(E^{D}\) (and/or \(E^{S}\); see the text), \(e^{d}\), and \(e\) alleles at the _Extension_ locus (modified from [42]). eye-circles, line of jowl, inside the ears, undertail, and feet pads) have whitish color [30; 31]. The \(a^{t}\) allele is dominant over the \(a\) allele. Homozygous rabbits for the \(a\) allele are self-colored (i.e., they are completely black). The \(a\) allele is determined by a frameshift mutation (c_5_6 insA) caused by one nucleotide insertion in the coding region of exon 2 [60]. The resulting shift of the reading frame determines the production of a truncated and non-functional protein of only 21 amino acids. The homozygous c_5_6 insA mutated genotype has been observed in all rabbit breeds with black coat color or variation of black (e.g., blue or silver) that also carried a wild type \(MC1R\) allele [41; 60]: Alaska, Blanc de Hotot, Champagne d'Argent, English Spot, Havana, Mini Silver, Russian, Silver, and Vienna Blue. Other breeds (Californian, Checkered Giant, Checkered Small, and Dutch) that have black coat color patterns but that also carry the dominant allele at the _Extension_ locus [41] are fixed for or have high frequency of the \(ASIP\) c_5_6 insA mutated allele. The non-agouti allele has been also identified in other breeds (New Zealand White, Thuringian, and White Vienna) for which mutations in other loci that have epistatic effects over the _Agouti_ locus (_Albino, Extension_, and _Vienrese White_, respectively) determine their coat color [30; 31; 62; 63]. The non-agouti allele has been also identified in Giant Grey rabbits but in heterozygous condition with the wild type allele [60].