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
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Bern Open Repository and Information System (BORIS) 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 Cord Dro¨ gemu¨ ller,1,2 Alexander Giese,1 Fla´via Martins-Wess,1 Sabine Wiedemann,3 Leif Andersson,4 Bertram Brenig,5 Ruedi Fries,3 Tosso Leeb1,2 1Institute of Animal Breeding and Genetics, University of Veterinary Medicine Hannover, 30559 Hannover, Germany 2Institute of Genetics, Vetsuisse Faculty, University of Berne, 3012 Berne, Switzerland 3Chair of Animal Breeding and Molecular Genetics, Technical University of Munich, 85354 Freising-Weihenstephan, Germany 4Swedish University of Agricultural Sciences, Uppsala, Sweden 5Institute of Veterinary Medicine, University of Go¨ ttingen, 37073 Go¨ ttingen, Germany Received: 29 July 2005 / Accepted: 13 September 2005 the coat color phenotype. Relative qRT-PCR analy- Abstract ses showed different dorsoventral skin expression The gene for agouti signaling protein (ASIP) is cen- intensities of the five ASIP transcripts in black-and- trally involved in the expression of coat color traits tan Mangalitza. The at mutation is therefore proba- in animals. The Mangalitza pig breed is character- bly a regulatory ASIP mutation that alters its dor- ized by a black-and-tan phenotype with black dorsal soventral expression pattern. pigmentation and yellow or white ventral pigmen- tation. We investigated a Mangalitza · Pie´train 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 Introduction locus. Complete linkage of the black-and-tan phe- notype to microsatellite alleles at the ASIP locus on Agouti signaling protein (ASIP) acts as an antagonist SSC 17q21 was observed. Corroborated by the to a-melanocortin stimulating hormone (a-MSH) at knowledge of similar mouse coat color mutants, it the melanocortin receptor 1 (MC1R) (Lu et al. 1994). seems therefore conceivable that the black-and-tan MC1R activation, following binding of a-MSH, leads pigmentation of Mangalitza pigs is caused by an to the production of black eumelanin in hair folli- ASIP allele at, which is recessive to the wild-type cles. In contrast, the binding of the high-affinity allele A. Toward positional cloning of the at muta- antagonist ASIP blocks MC1R signaling and leads to tion, a 200-kb genomic BAC/PAC contig of this the production of red pheomelanin. The MC1R gene chromosomal region has been constructed and sub- corresponds to the classical Extension (E) coat color sequently sequenced. Full-length ASIP cDNAs ob- locus in mammals. In addition to binding to the tained by RACE differed in their 5¢ untranslated MC1R, ASIP also acts as antagonist at the melano- regions, whereas they shared a common open read- cortin receptor 4 (MC4R), where it regulates food ing frame. Comparative sequencing of all ASIP exons intake and lipid metabolism (Harris et al. 2002). and ASIP cDNAs between Mangalitza and Pie´train Inhibition of the MC4R by ectopically expressed pigs did not reveal any differences associated with high levels of ASIP in the agouti lethal yellow mouse leads to pronounced obesity (Carroll et al. 2004). A similar obese phenotype results from the targeted disruption of the Mc4r gene in knockout mice Nucleotide sequence data reported are available in the EMBL (Huszar et al. 1997). Therefore, the ASIP gene can be database under accession numbers AJ427478, AJ634673À considered a candidate locus for feed intake and fat AJ634677, and AM050718. deposition in pigs. Correspondence to: Tosso Leeb, Institute of Genetics, Vetsuisse The ASIP locus is central in the expression of Faculty University of Berne, Bremgartenstr. 109a, 3012 Berne, Switzerland; E-mail: [email protected] coat color traits in animals. In mice, genetic analyses 58 DOI: 10.1007/s00335-005-0104-1 Volume 17, 58À66 (2006) Ó Springer Science+Business Media, Inc. 2006 C. DRO¨ GEMU¨ LLER ET AL.: PORCINE AGOUTI 59 Fig. 1. Coat color phenotypes in the resource family. (A) Black-and-tan F2 piglet. The stripes were visible only in juvenile animals; they disappeared in adult animals. (B) Gray F2 piglet. Similar to the black-and-tan animals, the stripes of these animals also vanished in adult animals. (C) Black-spotted F2 piglet. The hypothesized ASIP and MC1R genotypes for the three different phenotypes are indicated. showed that Mcr1 alleles are epistatic to Asip alleles al. 1998a). We have previously characterized the (Siracusa 1994). Among the variety of coat color genomic organization and SNPs of the coding exons phenotypes in the domestic pig, only two breeds, of the porcine ASIP gene as well as the downstream Duroc and Mangalitza, are characterized by general flanking gene AHCY (Leeb et al. 2000; Leeb and or regional pheomelanin expression. While porcine Rohrer 2002). MC1R alleles have been previously characterized at The initial aim of the study was to identify the the molecular level (Kijas et al. 1998b, 2001), so far gene underlying the black-and-tan phenotype in pigs no functional mutation of the porcine ASIP gene has using a Mangalitza · Pie´train F2 resource population. been reported. In pigs a recessively inherited MC1R In this article we report complete linkage between loss-of-function mutation that is responsible for ASIP and this phenotype. We also provide an ex- generalized pheomelanin production has been iden- tended analysis of the genomic organization of the tified in the red-colored Duroc breed (Kijas et al. porcine ASIP locus. A mutation scan of the complete 1998b). The ‘‘swallow-bellied’’ Mangalitza pig breed exonic and partial intronic ASIP sequence did not is characterized by black dorsal pigmentation and uncover the causative mutation for the porcine at yellow or white ventral pigmentation and a sharp allele. However, expression analyses of five different lateral line of demarcation. Similar phenotypes in ASIP transcripts in black-and-tan Mangalitza black-and-tan or white-bellied agouti mice are asso- showed differences in dorsal and ventral expression ciated with the at or AW alleles at the Asip locus, and suggested the existence of a regulatory ASIP which are recessive to the wild-type allele A. The at mutation. and AW alleles in the mouse arose from 6-kb or 0.6- kb insertions, respectively, into important regula- Material and methods tory regions of the murine Asip intron 1 (Bultman et al. 1994). It still remains a fundamental question Resource population construction. Two Mangalitza in developmental biology how adjacent regions of boars were mated to 20 Pie´train sows establishing an the vertebrate body acquire differences in their F1 generation consisting of 7 boars and 37 sows. F1 appearance or morphology. intercross resulted in 1492 piglets. Coat color phe- ASIP cDNAs have been cloned from several notypes of all animals were classified as black-and- mammalian species including human, mouse, cat- tan, gray, or black-spotted, respectively (Fig. 1; Wi- tle, dog, and fox (Bultman et al. 1992; Girardot et al. edemann et al. 2000). DNA samples of all founder 2005; Kerns et al. 2004; Kwon et al. 1994; Vage et al. animals, 76 F2 black-and-tan piglets, and their F1 1997). In all analyzed mammalian species the ASIP parents were used for genotyping. The Mangalitza cDNA is approximately 700 bp long and encodes a founders were postulated to be homozygous for the protein of 131À133 amino acids. The murine Asip ASIP*at allele and homozygous for the wild-type gene consists of three coding exons termed exons MC1R*E+ allele, respectively. The Pie´train founders 2À4 and four 5¢-untranslated exons termed exon 1A, were assumed to be homozygous for the wild-type exon 1AÕ, exon 1B, and exon 1C, respectively (Bult- ASIP*A allele and for the breed-specific MC1R*EP man et al. 1992). Contiguous porcine genomic se- allele (Kijas et al. 1998b). quences of the 5¢-untranslated exons, which are separated by large introns from the coding part of the Microsatellite genotyping. A polymorphic gene, have not been available in the public databases dinucleotide repeat (AC)21 denoted AgCA was de- so far. The chromosomal location of the porcine rived from a porcine genomic BAC clone containing ASIP gene has been assigned to SSC 17q21 (Kijas et the porcine ASIP gene (Kijas et al. 1998a). The 60 C. DRO¨ GEMU¨ LLER ET AL.: PORCINE AGOUTI primers AgCA-F (5¢-CCC TTA GGT GGA AAT TAG nih.gov/) and the RepeatMasker searching tool for CTA GAG-3¢) and AgCA-R (5¢-CTC ATA TCA CCC repetitive elements (A.F.A. Smit and P. Green, CTG AGA GGT AG-3¢) were used for microsatellite http://repeatmasker.genome.washington.edu/). fragment analyses in the resource population using Single copy sequences were used to design primer an ABI 377 sequencer with Genescan 3.1 and Geno- pairs for the chromosome walking using the programs typer 2.5 software (Applied Biosystems, Foster City, GeneFisher and Primer3 (http://bibiserv.techfak.uni- CA) (Wiedemann et al. 2000). In addition, linkage bielefeld.de/cgi-bin/gf_submit?mode=START and mapping of the ASIP gene-associated microsatellite http://www-genome.wi.mit.edu/cgi-bin/primer/prim AgCA was performed within the Wild Boar · Large er3_www.cgi). PAC and BAC DNA was restricted White pedigree (Ellegren et al. 1994). PCR was carried with different enzymes and separated on 0.8% agarose out in a total volume of 20 ll containing 25 ng gels. DNA fragments were excised and cloned into the genomic DNA, 1.0 mM MgCl2, 50 mM KCl, l0 mM polylinker of pGEM-4Z (Promega, Mannheim, Ger- Tris-HCl, pH 8.3, 200 pM dNTPs, 0.5 U AmpliTaq many).