The mutation causing the black-and-tan pigmentation phenotype of Mangalitza pigs maps to the porcine ASIP locus but does not affect its coding sequence

The gene for agouti signaling protein (ASIP) is centrally involved in the expression of coat color traits in animals. The Mangalitza pig breed is characterized by a black-and-tan phenotype with black dorsal pigmentation and yellow or white ventral pigmentation. We investigated a Mangalitza x Piétrain cross and observed a coat color segregation pattern in the F2 generation that can be explained by virtue of two alleles at the MC1R locus and two alleles at the ASIP locus. Complete linkage of the black-and-tan phenotype to microsatellite alleles at the ASIP locus on SSC 17q21 was observed. Corroborated by the knowledge of similar mouse coat color mutants, it seems therefore conceivable that the black-and-tan pigmentation of Mangalitza pigs is caused by an ASIP allele a(t), which is recessive to the wild-type allele A. Toward positional cloning of the a(t) mutation, a 200-kb genomic BAC/PAC contig of this chromosomal region has been constructed and subsequently sequenced. Full-length ASIP cDNAs obtained by RACE differed in their 5' untranslated regions, whereas they shared a common open reading frame. Comparative sequencing of all ASIP exons and ASIP cDNAs between Mangalitza and Piétrain pigs did not reveal any differences associated with the coat color phenotype. Relative qRT-PCR analyses showed different dorsoventral skin expression intensities of the five ASIP transcripts in black-and-tan Mangalitza. The a(t) mutation is therefore probably a regulatory ASIP mutation that alters its dorsoventral expression pattern.

Genomewide association for a dominant pigmentation gene in sheep

Most published genomewide association studies (GWAS) in sheep have investigated recessively inherited monogenic traits. The objective here was to assess the feasibility of performing GWAS for a dominant trait for which the genetic basis was already known. A total of 42 Manchega and Rasa Aragonesa sheep that segregate solid black or white coat pigmentation were genotyped using the SNP50 BeadChip. Previous analysis in Manchegas demonstrated a complete association between the pigmentation trait and alleles of the MC1R gene, setting an a priori expectation for GWAS. Multiple methods were used to identify and quantify the strength of population substructure between black and white animals, before allelic association testing was performed for 49 034 SNPs. Following correction for substructure, GWAS identified the most strongly associated SNP (s26449) was also the closest to the MC1R gene. The finding was strongly supported by the permutation tree-based random forest (RF) analysis. Importantly, GWAS identified unlinked SNP with only slightly lower p-values than for s26449. Random forest analysis indicated these were false positives, suggesting interpretation based on both approaches was beneficial. The results indicate that a combined analytical approach can be successful in studies where a modest number of animals are available and substantial population stratification exists.

In search of epigenetic marks in testes and sperm cells of differentially fed boars

In search of transmittable epigenetic marks we investigated gene expression in testes and sperm cells of differentially fed F0 boars from a three generation pig feeding experiment that showed phenotypic differences in the F2 generation. RNA samples from 8 testes of boars that received either a diet enriched in methylating micronutrients or a control diet were analyzed by microarray analysis. We found moderate differential expression between testes of differentially fed boars with a high FDR of 0.82 indicating that most of the differentially expressed genes were false positives. Nevertheless, we performed a pathway analysis and found disparate pathway maps of development_A2B receptor: action via G-protein alpha s, cell adhesion_Tight junctions and cell adhesion_Endothelial cell contacts by junctional mechanisms which show inconclusive relation to epigenetic inheritance. Four RNA samples from sperm cells of these differentially fed boars were analyzed by RNA-Seq methodology. We found no differential gene expression in sperm cells of the two groups (adjusted P-value>0.05). Nevertheless, we also explored gene expression in sperm by a pathway analysis showing that genes were enriched for the pathway maps of bacterial infections in cystic fibrosis (CF) airways, glycolysis and gluconeogenesis p.3 and cell cycle_Initiation of mitosis. Again, these pathway maps are miscellaneous without an obvious relationship to epigenetic inheritance. It is concluded that the methylating micronutrients moderately if at all affects RNA expression in testes of differentially fed boars. Furthermore, gene expression in sperm cells is not significantly affected by extensive supplementation of methylating micronutrients and thus RNA molecules could not be established as the epigenetic mark in this feeding experiment.

A 19-Mb de novo deletion on BTA 22 including MITF leads to microphthalmia and the absence of pigmentation in a Holstein calf.

Mutations in MITF lead to a large variety of phenotypes in human, mice and other species. They mostly affect pigmentation and hearing, whereas in mice, they may additionally cause microphthalmia and osteopetrosis. In this study, we report a single case of a Holstein calf with lack of pigmentation and microphthalmia born to healthy parents. Mendelian analysis of high-density SNP genotypes reveals a large number of parentage errors showing missing paternal alleles in the offspring, indicating a deletion encompassing 19 Mb on BTA 22. The genomic deletion affects the paternal allele and includes MITF and 131 other annotated genes. As the calf shows only one copy of the BTA 22 segment, the observed phenotype is probably caused by haploinsufficiency of the genes in that genomic region. Both the observed lack of skin pigmentation and reduced eye size can most likely be explained by a lack of MITF function.

Genomic amplification of the caprine EDNRA locus might lead to a dose dependent loss of pigmentation

The South African Boer goat displays a characteristic white spotting phenotype, in which the pigment is limited to the head. Exploiting the existing phenotype variation within the breed, we mapped the locus causing this white spotting phenotype to chromosome 17 by genome wide association. Subsequent whole genome sequencing identified a 1 Mb copy number variant (CNV) harboring 5 genes including EDNRA. The analysis of 358 Boer goats revealed 3 alleles with one, two, and three copies of this CNV. The copy number is correlated with the degree of white spotting in goats. We propose a hypothesis that ectopic overexpression of a mutant EDNRA scavenges EDN3 required for EDNRB signaling and normal melanocyte development and thus likely lead to an absence of melanocytes in the non-pigmented body areas of Boer goats. Our findings demonstrate the value of domestic animals as reservoir of unique mutants and for identifying a precisely defined functional CNV.