New Insights into the Melanophilin (MLPH) Gene Affecting Coat Color Dilution in Rabbits.

Coat color dilution corresponds to a specific pigmentation phenotype that leads to a dilution of wild type pigments. It affects both eumelanin and pheomelanin containing melanosomes. The mode of inheritance of the dilution phenotype is autosomal recessive. Candidate gene approaches focused on the melanophilin (MLPH) gene highlighted two variants associated with the dilution phenotype in rabbits: The c.111-5C>A variant that is located in an acceptor splice site or the c.585delG variant, a frameshift mutation. On the transcript level, the skipping of two exons has been reported as the molecular mechanism responsible for the coat color dilution. To clarify, which of the two variants represents the causal variant, (i) we analyzed their allelic segregation by genotyping Castor and Chinchilla populations, and (ii) we evaluated their functional effects on the stability of MLPH transcripts in skin samples of animals with diluted or wild type coat color. Firstly, we showed that the c.585delG variant showed perfect association with the dilution phenotype in contrast to the intronic c.111-5C>A variant. Secondly, we identified three different MLPH isoforms including the wild type isoform, the exon-skipping isoform and a retained intron isoform. Thirdly, we observed a drastic and significant decrease of MLPH transcript levels in rabbits with a coat color dilution (p-values ranging from 10-03 to 10-06). Together, our results bring new insights into the coat color dilution trait.

Genome-Wide Identification of the Mutation Underlying Fleece Variation and Discriminating Ancestral Hairy Species from Modern Woolly Sheep.

The composition and structure of fleece variation observed in mammals is a consequence of a strong selective pressure for fiber production after domestication. In sheep, fleece variation discriminates ancestral species carrying a long and hairy fleece from modern domestic sheep (Ovis aries) owning a short and woolly fleece. Here, we report that the "woolly" allele results from the insertion of an antisense EIF2S2 retrogene (called asEIF2S2) into the 3' UTR of the IRF2BP2 gene leading to an abnormal IRF2BP2 transcript. We provide evidence that this chimeric IRF2BP2/asEIF2S2 messenger 1) targets the genuine sense EIF2S2 RNA and 2) creates a long endogenous double-stranded RNA which alters the expression of both EIF2S2 and IRF2BP2 mRNA. This represents a unique example of a phenotype arising via a RNA-RNA hybrid, itself generated through a retroposition mechanism. Our results bring new insights on the sheep population history thanks to the identification of the molecular origin of an evolutionary phenotypic variation.

Genetic Diversity and Population Structure in South African, French and Argentinian Angora Goats from Genome-Wide SNP Data.

The Angora goat populations in Argentina (AR), France (FR) and South Africa (SA) have been kept geographically and genetically distinct. Due to country-specific selection and breeding strategies, there is a need to characterize the populations on a genetic level. In this study we analysed genetic variability of Angora goats from three distinct geographical regions using the standardized 50k Goat SNP Chip. A total of 104 goats (AR: 30; FR: 26; SA: 48) were genotyped. Heterozygosity values as well as inbreeding coefficients across all autosomes per population were calculated. Diversity, as measured by expected heterozygosity (HE) ranged from 0.371 in the SA population to 0.397 in the AR population. The SA goats were the only population with a positive average inbreeding coefficient value of 0.009. After merging the three datasets, standard QC and LD-pruning, 15 105 SNPs remained for further analyses. Principal component and clustering analyses were used to visualize individual relationships within and between populations. All SA Angora goats were separated from the others and formed a well-defined, unique cluster, while outliers were identified in the FR and AR breeds. Apparent admixture between the AR and FR populations was observed, while both these populations showed signs of having some common ancestry with the SA goats. LD averaged over adjacent loci within the three populations per chromosome were calculated. The highest LD values estimated across populations were observed in the shorter intervals across populations. The Ne for the Angora breed was estimated to be 149 animals ten generations ago indicating a declining trend. Results confirmed that geographic isolation and different selection strategies caused genetic distinctiveness between the populations.

Levels and Patterns of Genetic Diversity and Population Structure in Domestic Rabbits.

Over thousands of years humans changed the genetic and phenotypic composition of several organisms and in the process transformed wild species into domesticated forms. From this close association, domestic animals emerged as important models in biomedical and fundamental research, in addition to their intrinsic economical and cultural value. The domestic rabbit is no exception but few studies have investigated the impact of domestication on its genetic variability. In order to study patterns of genetic structure in domestic rabbits and to quantify the genetic diversity lost with the domestication process, we genotyped 45 microsatellites for 471 individuals belonging to 16 breeds and 13 wild localities. We found that both the initial domestication and the subsequent process of breed formation, when averaged across breeds, culminated in losses of ~20% of genetic diversity present in the ancestral wild population and domestic rabbits as a whole, respectively. Despite the short time elapsed since breed diversification we uncovered a well-defined structure in domestic rabbits where the FST between breeds was 22%. However, we failed to detect deeper levels of structure, probably consequence of a recent and single geographic origin of domestication together with a non-bifurcating process of breed formation, which were often derived from crosses between two or more breeds. Finally, we found evidence for intrabreed stratification that is associated with demographic and selective causes such as formation of strains, colour morphs within the same breed, or country/breeder of origin. These additional layers of population structure within breeds should be taken into account in future mapping studies.

LIPH expression in skin and hair follicles of normal coat and Rex rabbits.

Natural mutations in the LIPH gene were shown to be responsible for hair growth defects in humans and for the rex short hair phenotype in rabbits. In this species, we identified a single nucleotide deletion in LIPH (1362delA) introducing a stop codon in the C-terminal region of the protein. We investigated the expression of LIPH between normal coat and rex rabbits during critical fetal stages of hair follicle genesis, in adults and during hair follicle cycles. Transcripts were three times less expressed in both fetal and adult stages of the rex rabbits than in normal rabbits. In addition, the hair growth cycle phases affected the regulation of the transcription level in the normal and mutant phenotypes differently. LIPH mRNA and protein levels were higher in the outer root sheath (ORS) than in the inner root sheath (IRS), with a very weak signal in the IRS of rex rabbits. In vitro transfection shows that the mutant protein has a reduced lipase activity compared to the wild type form. Our results contribute to the characterization of the LIPH mode of action and confirm the crucial role of LIPH in hair production.

A deletion in exon 9 of the LIPH gene is responsible for the rex hair coat phenotype in rabbits (Oryctolagus cuniculus).

The fur of common rabbits is constituted of 3 types of hair differing in length and diameter while that of rex animals is essentially made up of amazingly soft down-hair. Rex short hair coat phenotypes in rabbits were shown to be controlled by three distinct loci. We focused on the "r1" mutation which segregates at a simple autosomal-recessive locus in our rabbit strains. A positional candidate gene approach was used to identify the rex gene and the corresponding mutation. The gene was primo-localized within a 40 cM region on rabbit chromosome 14 by genome scanning families of 187 rabbits in an experimental mating scheme. Then, fine mapping refined the region to 0.5 cM (Z = 78) by genotyping an additional 359 offspring for 94 microsatellites present or newly generated within the first defined interval. Comparative mapping pointed out a candidate gene in this 700 kb region, namely LIPH (Lipase Member H). In humans, several mutations in this major gene cause alopecia, hair loss phenotypes. The rabbit gene structure was established and a deletion of a single nucleotide was found in LIPH exon 9 of rex rabbits (1362delA). This mutation results in a frameshift and introduces a premature stop codon potentially shortening the protein by 19 amino acids. The association between this deletion and the rex phenotype was complete, as determined by its presence in our rabbit families and among a panel of 60 rex and its absence in all 60 non-rex rabbits. This strongly suggests that this deletion, in a homozygous state, is responsible for the rex phenotype in rabbits.

A composite six bp in-frame deletion in the melanocortin 1 receptor (MC1R) gene is associated with the Japanese brindling coat colour in rabbits (Oryctolagus cuniculus).

BACKGROUND: In the domestic rabbit (Oryctolagus cuniculus), classical genetic studies have identified five alleles at the Extension locus: ED (dominant black), ES (steel, weaker version of ED), E (wild type, normal extension of black), eJ(Japanese brindling, mosaic distribution of black and yellow) and e (non-extension of black, yellow/red with white belly). Sequencing almost the complete coding sequence (CDS) of the rabbit MC1R gene, we recently identified two in-frame deletions associated with dominant black (c.280_285del6; alleles ED or ES) and recessive red (c.304_333del30; allele e) coat colours. It remained to characterize the eJallele whose phenotypic effect is similar to the Orange and Sex-linked yellow loci of cat and Syrian hamster. RESULTS: We sequenced the whole CDS in 25 rabbits of different coat colours including 10 Japanese and 10 Rhinelander (tricolour) rabbits and identified another 6 bp-in frame deletion flanked by a G > A transition in 5' (c.[124G>A;125_130del6]) that was present in all animals with Japanese brindling coat colour and pattern. These mutations eliminate two amino acids in the first transmembrane domain and, in addition, cause an amino acid substitution at position 44 of the wild type sequence. Genotyping 371 rabbits of 31 breeds with different coat colour this allele (eJ) was present in homozygous state in Japanese, Rhinelander and Dutch tricolour rabbits only (except one albino rabbit). Rabbits with eJ/eJ genotype were non fixed at the non-agouti mutation we previously identified in the ASIP gene. Segregation in F1 and F2 families confirmed the order of dominance already determined by classical genetic experiments with a possible dose effect evident comparing eJ/eJ and eJ/e animals. MC1R mRNA was expressed in black hair skin regions only. CONCLUSIONS: The c.[124A;125_130del6] allele may be responsible for a MC1R variant determining eumelanin production in the black areas. However, the mechanism determining the presence of both red and black hairs in the same animal seems more complex. Expression analyses of the c.[124A;125_130del6] allele suggest that MC1R transcription may be regulated epigenetically in rabbits with the Japanese brindling phenotype. Further studies are needed to clarify this issue.

Characterization of the rabbit agouti signaling protein (ASIP) gene: transcripts and phylogenetic analyses and identification of the causative mutation of the nonagouti black coat colour.

The agouti locus encodes the agouti signalling protein (ASIP) which is involved in determining the switch from eumelanin to pheomelanin synthesis in melanocytes. In the domestic rabbit (Oryctolagus cuniculus) early studies indicated three alleles at this locus: A, light-bellied agouti (wild type); a(t), black and tan; a, black nonagouti. We characterized the rabbit ASIP gene and identified the causative mutation (an insertion in exon 2) of the black nonagouti allele whose frequency was evaluated in 31 breeds. Phylogenetic analysis of ASIP sequences from Oryctolagus and 9 other species of the family Leporidae placed Oryctolagus as sister species to Pentalagus and Bunolagus. Transcription analysis in wild type agouti rabbits revealed the presence of two major transcripts with different 5'-untranslated regions having ventral or dorsal skin specific expression. ASIP gene transcripts were also detected in all examined rabbit tissues distinguishing the rabbit expression pattern from what was observed in wild type mice.

Quantitative trait loci linked to PRNP gene controlling health and production traits in INRA 401 sheep.

In this study, the potential association of PrP genotypes with health and productive traits was investigated. Data were recorded on animals of the INRA 401 breed from the Bourges-La Sapinière INRA experimental farm. The population consisted of 30 rams and 852 ewes, which produced 1310 lambs. The animals were categorized into three PrP genotype classes: ARR homozygous, ARR heterozygous, and animals without any ARR allele. Two analyses differing in the approach considered were carried out. Firstly, the potential association of the PrP genotype with disease (Salmonella resistance) and production (wool and carcass) traits was studied. The data used included 1042, 1043 and 1013 genotyped animals for the Salmonella resistance, wool and carcass traits, respectively. The different traits were analyzed using an animal model, where the PrP genotype effect was included as a fixed effect. Association analyses do not indicate any evidence of an effect of PrP genotypes on traits studied in this breed. Secondly, a quantitative trait loci (QTL) detection approach using the PRNP gene as a marker was applied on ovine chromosome 13. Interval mapping was used. Evidence for one QTL affecting mean fiber diameter was found at 25 cM from the PRNP gene. However, a linkage between PRNP and this QTL does not imply unfavorable linkage disequilibrium for PRNP selection purposes.

Relationship between MT1 melatonin receptor gene polymorphism and seasonal physiological responses in Ile-de-France ewes.

The gene encoding the MT1 melatonin receptor in sheep has a restriction fragment length polymorphism (RFLP) site to the MnlI enzyme whose incidence is associated to the expression of seasonality in several breeds. The aim of this study was to examine the relationship between this genetic marker and the physiological effects of MT1 receptor gene polymorphism on several seasonal functions in Ile-de-France ewes. The study was performed using 12 pairs of half-sib adult Ile-de-France ewes. Within each pair, ewes were selected on the basis of their genotype at the MnlI RFLP site: group +/+ and -/- (presence and absence of MnlI restriction site, respectively). No difference in the dates of the beginning, the end or the length of the breeding season was observed between groups during the two-year study. The seasonal changes in prolactin secretion were not different between groups. Similarly, wool growth rate and primary follicle activity, measured for one year, varied with the time of the year in the same way in the two groups. Our study therefore failed to show any relationship between MT1 polymorphism and reproductive seasonality in Ile-de-France ewes. This suggests that the influence of this polymorphism on the regulation of seasonal function is dependent upon the breed and/or environmental conditions. The MT1 polymorphism can explain only a small part of the genetic variability of seasonal functions and the implication of other genes must be investigated.

Genetic variability of the pattern of night melatonin blood levels in relation to coat changes development in rabbits.

To assess the genetic variability in both the nocturnal increase pattern of melatonin concentration and photoresponsiveness in coat changes, an experiment on 422 Rex rabbits (from 23 males) raised under a constant light programme from birth was performed. The animals were sampled at 12 weeks of age, according to 4 periods over a year. Blood samples were taken 7 times during the dark phase and up to 1 h after the lighting began. Maturity of the fur was assessed at pelting. Heritability estimates of blood melatonin concentration (0.42, 0.17 and 0.11 at mid-night, 13 and 15 h after lights-out respectively) and strong genetic correlations between fur maturity and melatonin levels at the end of the dark phase (-0.64) indicates that (i) the variability of the nocturnal pattern of melatonin levels is under genetic control and (ii) the duration of the nocturnal melatonin increase is a genetic component of photoresponsiveness in coat changes.