Technical note: Advantages and limitations of authenticating Palmera goat dairy products by pyrosequencing the melanocortin 1 receptor (MC1R) gene.

Inferring the breed of origin of dairy products can be achieved through molecular analysis of genetic markers with a population-specific pattern of segregation. The goal of the current work was to generate such markers in goats by resequencing several pigmentation genes [melanocortin 1 receptor (MC1R), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), tyrosinase (TYR), and tyrosinase-related protein 2 (TYRP2)]. This experiment revealed 10 single nucleotide polymorphisms (SNP), including 5 missense mutations and 1 nonsense mutation. These markers were genotyped in 560 goats from 18 breeds originally from Italy, the Iberian Peninsula, the Canary Islands, and North Africa. Although the majority of SNP segregated at moderate frequencies in all populations (including 2 additional markers that were used as a source of information), we identified a c.764G>A SNP in MC1R that displayed highly divergent allelic frequencies in the Palmera breed compared with the Majorera and Tinerfeña breeds from the Canary Islands. Thus, we optimized a pyrosequencing-based technique that allowed us to estimate, very accurately, the allele frequencies of this marker in complex DNA mixtures from different individuals. Once validated, we applied this method to generating breed-specific DNA profiles that made it possible to detect fraudulent cheeses in which Palmero cheese was manufactured with milk from Majorera goats. One limitation of this approach, however, is that it cannot be used to detect illegal manufacturing where Palmero dairy products are produced by mixing milk from Palmera and Majorera goats, because the c.764G>A SNP segregates in both breeds.

Badaoui B ，Manunza A ，Castelló A ，D'Andrea M ，Pilla F ，Capote J ，Jordana J ，Ferrando A ，Martínez A ，Cabrera B ，Delgado JV ，Landi V ，Gómez M ，Pons A ，El Ouni M ，Vidal O ，Amills M ... -  《-》

A melanocortin 1 receptor (MC1R) gene polymorphism is useful for authentication of Massese sheep dairy products.

Massese is an Italian sheep breed, with black or grey coat colour, mainly reared in the Tuscany and Emilia Romagna regions. Recently, the emerging interests in this breed have resulted in the production of Pecorino cheese obtained with only Massese milk. In order to be profitable, this marketing link between Massese breed and its products should be defended against fraudsters who could include milk of other sheep breeds or cow milk in Massese labelled productions. To identify the genetic factors affecting coat colour in sheep, we have recently analysed the melanocortin 1 receptor (MC1R) gene and identified several single nucleotide polymorphisms (SNPs). In this work, as a first step to set up a DNA based protocol for authentication of Massese dairy products, we further investigated the presence and distribution of one of these SNPs (c.-31G>A) in 143 Massese sheep and in another 13 sheep breeds (for a total of 351 animals). The Massese breed was fixed for allele c.-31A, whereas in all other breeds allele c.-31 G was the most frequent or with frequency of 0·50. At the same nucleotide position the cattle MC1R gene carries the G nucleotide. Using these data we developed a method to detect adulterating milk (from other sheep breeds or from cow) in Massese dairy products based on the analysis of the c.-31G>A SNP. We first tested the sensitivity of the protocol and then applied it to analyse DNA extracted from ricotta and Pecorino cheese obtained with only Massese milk or obtained with unrestricted sheep and cattle milk. To our knowledge, this system represents the first one that can be used for breed authentication of a sheep production and that, at the same time, can reveal frauds derived from the admixture of milk of an unreported species.

Fontanesi L ，Beretti F ，Dall'Olio S ，Portolano B ，Matassino D ，Russo V ... -  《-》

Missense and nonsense mutations in melanocortin 1 receptor (MC1R) gene of different goat breeds: association with red and black coat colour phenotypes but with unexpected evidences.

Agouti and Extension loci control the relative amount of eumelanin and pheomelanin production in melanocytes that, in turn, affects pigmentation of skin and hair. The Extension locus encodes the melanocortin 1 receptor (MC1R) whose permanent activation, caused by functional mutations, results in black coat colour, whereas other inactivating mutations cause red coat colour in different mammals. The whole coding region of the MC1R gene was sequenced in goats of six different breeds showing different coat colours (Girgentana, white cream with usually small red spots in the face; Maltese, white with black cheeks and ears; Derivata di Siria, solid red; Murciano-Granadina, solid black or solid brown; Camosciata delle Alpi, brown with black stripes; Saanen, white; F1 goats and the parental animals). Five single nucleotide polymorphisms (SNPs) were identified: one nonsense mutation (p.Q225X), three missense mutations (p.A81V, p.F250V, and p.C267W), and one silent mutation. The stop codon at position 225 should cause the production of a shorter MC1R protein whose functionality may be altered. These SNPs were investigated in a larger sample of animals belonging to the six breeds. The Girgentana breed was almost fixed for the p.225X allele. However, there was not complete association between the presence of red spots in the face and the presence of this allele in homozygous condition. The same allele was identified in the Derivata di Siria breed. However, its frequency was only 33%, despite the fact that these animals are completely red. The p.267W allele was present in all Murciano-Granadina black goats, whereas it was never identified in the brown ones. Moreover, the same substitution was present in almost all Maltese goats providing evidence of association between this mutation and black coat colour. According to the results obtained in the investigated goat breeds, MC1R mutations may determine eumelanic and pheomelanic phenotypes. However, they are probably not the only factors. In particular, the surprising not complete association of the nonsense mutation (p.Q225X) with red coat colour raises a few hypotheses on the determination of pheomelanic phenotypes in goats that should be further investigated.

Fontanesi L ，Beretti F ，Riggio V ，Dall'Olio S ，González EG ，Finocchiaro R ，Davoli R ，Russo V ，Portolano B ... -  《BMC GENETICS》

Strong phylogeographic relationships among three goat breeds from the Canary Islands.

We partially sequenced the mitochondrial D-loop region in 47 individuals from eleven Spanish and foreign goat breeds. Phylogenetic analysis of these sequences allowed us to identify a particular D-loop haplotype shared by individuals from the Palmera, Majorera and Tinerfeña Canarian breeds. Genotyping of 281 goats from 17 different breeds by PCR-Hpall RFLP evidenced that the geographical distribution of this haplotype is restricted to the Canary Islands. This ancestral mitochondrial haplotype might originate in the domestic goat herds brought by the native Canarian inhabitants approximately 3000 years ago. Although we observed other miscellaneous D-loop haplotypes in the Palmera, Majorera and Tinerfeña breeds, any of them allowed us to group individuals from these three populations in a single cluster, a feature that suggests that these haplotypes might have diverse origins. The remarkable degree of phylogeographic structure of the Canary goat breeds with regard to other Spanish and foreign populations might be attributed to the isolation of these breeds in the Canary Islands for approximately 2500 years, without exposure to the migratory movements and commercial trading events that probably affected the genesis of most domestic goat breeds worldwide. The Canarian D-loop haplotype can be efficiently genotyped by using DNA isolated from milk and cheese samples, which paves the way for the future establishment of a Canary breed identity test for these dairy products.

Amills M ，Capote J ，Tomàs A ，Kelly L ，Obexer-Ruff G ，Angiolillo A ，Sanchez A ... -  《JOURNAL OF DAIRY RESEARCH》

The demographic history and adaptation of Canarian goat breeds to environmental conditions through the use of genome-wide SNP data.

The presence of goats in the Canary Islands dates back to the late 1st millennium BC, which coincides with the colonization by the Amazigh settlers. However, the exact geographic origin of Canarian goats is uncertain since the Amazigh peoples were distributed over a wide spatial range. Nowadays, three Canarian breeds (Palmera, Majorera and Tinerfeña) are officially recognized, along with two distinct South and North Tinerfeña ecotypes, with the South Tinerfeña and Majorera goats thriving in arid and dry semi-desertic environments and the Palmera and North Tinerfeña goats are adapted to humid and temperate areas that are influenced by trade winds. Genotypes for 224 Canarian goats were generated using the Illumina Goat single nucleotide polymorphism (SNP)50 BeadChip. By merging these data with the genotypes from 1007 individuals of African and Southern European ancestry, our aim was to ascertain the geographic origin of the Canarian goats and identify genes associated with adaptation to diverse environmental conditions. The diversity indices of the Canarian breeds align with most of those of the analyzed local breeds from Africa and Europe, except for the Palmera goats that showed lower levels of genetic variation. The Canarian breeds demonstrate a significant genetic differentiation compared to other populations, which indicates a history of prolonged geographic isolation. Moreover, the phylogenetic reconstruction indicated that the ancestry of the Canarian goats is fundamentally North African rather than West African. The ADMIXTURE and the TreeMix analyses showed no evidence of gene flow between Canarian goats and other continental breeds. The analysis of runs of homozygosity (ROH) identified 13 ROH islands while the window-based F method detected 25 genomic regions under selection. Major signals of selection were found on Capra hircus (CHI) chromosomes 6, 7, and 10 using various comparisons and methods. This genome-wide analysis sheds new light on the evolutionary history of the four breeds that inhabit the Canary Islands. Our findings suggest a North African origin of the Canarian goats. In addition, within the genomic regions highlighted by the ROH and F approaches, several genes related to body size and heat tolerance were identified.

Senczuk G ，Macrì M ，Di Civita M ，Mastrangelo S ，Del Rosario Fresno M ，Capote J ，Pilla F ，Delgado JV ，Amills M ，Martínez A ... -  《-》