## 3 Molecular Characterization of Coat Color Affecting Genes in Rabbits
### Mutations in the Melanocortin 1 Receptor (MC1R) Gene and the Extension Locus  
In many species, mutations in the single-exon melancocortin 1 receptor (_MC1R_) gene determine the multi-allelic series at the _Extension_ (\(E\)) locus (e.g., [32; 33; 34; 35; 36]). _MC1R_ encodes for a seven transmembrane G-protein coupled receptor that is localized within the membrane of the melanocytes. The melanocytes are specialized cells that produce melanin of different types that are the final molecular products of the biological pathway that lead to the pigmentation. This locus is epistatic over the _Agouti_ locus. Therefore, the types of produced melanin are directly related to the allelic structure at the _Extension_ and _Agouti_ loci. Classical genetic studies carried out in rabbits have suggested that the _Extension_ series in this species is determined by five alleles [30; 31; 37; 38; 39; 40]. Allele \(E^{D}\) determines the dominant black coat color and is caused by one in-frame deletion of 6 bp (c.280_285 del6 or D6 allele), which eliminates two amino acids in the second transmembrane domain [41]. The black color of the spots of the Checkered Giant breed is due to this allele. Allele \(E^{S}\) (steel) is considered a weaker version of \(E^{D}\), even if it could be probably determined by the same mutation causing \(E^{D}\), as another _MC1R_ variant associated to this allele has not been described so far [41]. The steel coat color effect associated to this allele could be due to the involvement of modifier genes or heterozygous combinations at the _Extension_ locus. Allele \(E\) or \(E^{+}\) is the wild type allele that determines the normal grey or the normal extension of black. Two wild type haplotypes have been described at the _Extension_ locus that can determine the same phenotypic effect [41]. Another in-frame deletion of 6 bp flanked by a G \(>\) A transition in 5' (c.[124G \(>\) A;125_130 del6]) is the causative composite mutation of the \(e^{l}\) allele, which produces the Japanese brinding coat color phenotype [42]. This allele determines a mosaic distribution of black and yellow pigmentation and could be an interesting model of alternative fixation of the production of eumelanin and pheomelanin in specifically devoted melanocytes. Epigenetic regulations might be involved in determining this unique coat color phenotype [42]. Two breeds have the characteristic coat color determined by the homozygous state of the \(e^{l}\) allele: Japanese and Rhinelander (knows also as Tricolor) breeds. Allele \(e^{l}\) is recessive over all other alleles described above but is dominant or partially dominant over allele \(e\), which determines the non-extension of black and the yellow/red (with white belly) coat color [39; 42]. Allele \(e\) is caused by a 30 nucleotides in-frame deletion (c.304_333 del30; A30 allele), which eliminates 10 amino acids of the first extracellular loop of the transmembrane MC1R protein [41]. The modified protein is not functional; thus, the melanocytes of the homozygous _e/e_ rabbits can only produce pheomelanin. Allele \(e\) is fixed in all rabbit breeds that have a yellow/red characterizing coat color (e.g., Burgundy Fawn, Gold Saxony, New Zealand Red, and Thuringian [41]). The allele series at this locus in rabbit is probably unique if compared to those of all other domestic animals. In rabbit, all mutant alleles (dominant and recessive, if related to the wild type forms) are caused by in-frame deletions that eliminate a few amino acids in the translated protein (Figure 1). In dog, cattle, sheep, goat, pig, horse, donkey, chicken,and in several other species that have been investigated at the \(MC1R\) gene, variants affecting the function of this gene are due to missense mutations, frameshift mutations, non-sense mutations, or structural mutations that alter the function of the protein or completely disrupt it (e.g., [32; 33; 34; 35; 36; 43; 44; 45; 46; 47; 48]).