The aim of this study was to ascertain the role of the Agouti signaling
peptide (ASIP) gene coding region in the Agouti locus variation within
wild-type coat colour in cattle. We determined the Extension genotype
in 241 individuals from six Spanish and three French brown cattle
breeds representative of wild-type coat variation. Polymerase chain reaction–
single-strand conformation polymorphism (PCR-SSCP) analysis was
carried out in individuals of each Extension genotypes within the same
breed in an attempt to identify variants in the three coding exons of the
ASIP gene. No SSCP variants were found. Results were confirmed by
sequencing the coding exons of the ASIP gene in 20 individuals. Our
results suggest that the ASIP coding region does not play a central role
in coat colour variation in cattle

Arranz, J.J. (J.)

Fernández, Itziar

Goyache, Félix

Gómez, Enrique

Royo, L.J. (Luis)

Álvarez, Isabel

The aim of this study was to ascertain the role of the Agouti signaling
peptide (ASIP) gene coding region in the Agouti locus variation within
wild-type coat colour in cattle. We determined the Extension genotype
in 241 individuals from six Spanish and three French brown cattle
breeds representative of wild-type coat variation. Polymerase chain reaction–
single-strand conformation polymorphism (PCR-SSCP) analysis was
carried out in individuals of each Extension genotypes within the same
breed in an attempt to identify variants in the three coding exons of the
ASIP gene. No SSCP variants were found. Results were confirmed by
sequencing the coding exons of the ASIP gene in 20 individuals. Our
results suggest that the ASIP coding region does not play a central role
in coat colour variation in cattle

Royo Martín, Luis J.

Arranz, J. J.

Repositorio Institucional de Asturias (RIA)

SHORT COMMUNICATIONThe coding sequence of the ASIP gene is identical in ninewild-type coloured cattle breedsL.J. Royo1, I. A´lvarez1, I. Ferna´ndez1, J.J. Arranz2, E. Go´mez1 & F. Goyache1 SERIDA-CENSYRA-Somio´, C/Camino de los Claveles, Gijo´n, Asturias, Spain2 Departamento de Produccio´n Animal I, Universidad de Leo´n, Leo´n, SpainIntroductionCoat colour in mammals basically depends on the rel-ative amount of the two basic types of tyrosine-derived melanin: eumelanin (black/brown) and pha-eomelanin (yellow/red) which are controlled, in turn,by the Extension (E) and Agouti (A) loci (Searle1968). Mutations at either locus may commit the mel-anocyte to exclusive synthesis of a single pigment. Ina variety of mammals, dominant alleles at the E locusact to produce a uniform black coat colour (Klunglandet al. 1995; Jackson 1997), whereas recessive alleles atthis locus extend the amount of red/yellow pigment.The wild-type state in many species involves synthesisof both melanin types. Conversely, dominant allelesat the A locus cause a yellow coat whereas homozyg-osity for the recessive allele would be associated witha uniform black coat (Jackson 1997). Molecular stud-ies (Cone et al. 1996; Robbins et al. 1993) revealedthat E locus encodes a melanocyte-stimulating hor-mone receptor, also known as the melanocortin-1receptor (MC1R) which is involved in eumelanin andphaeomelanin production while the Agouti signalingpeptide (encoded in the ASIP locus) acts as an antag-onist of melanocyte-stimulating hormone (MSH) bybinding to MC1R and thereby preventing the MC1R–MSH interaction, resulting in phaeomelanin synthesis(Bultman et al. 1992). In mice, the E locus is epistaticto A locus (Bateman & Sombre 1961; Wolff et al.1978), but in fox it has been demonstrated that theE locus is not epistatic to the A locus (Adalsteinssonet al. 1987; Vage et al. 1997).As the E locus is responsible for most of the vari-ation in cattle coat colour, many studies have exam-ined the MC1R locus in cattle. Initial studiesidentified the dominant black ED (L99P) and therecessive red e (310delG) alleles (Klungland et al.1995; Joerg et al. 1996). Any coding sequence with-out these mutations was considered as the wild-typeallele (E+) responsible for most combinations of redor reddish brown and black coat variants (Olson1998). Recent studies have identified two more mis-sense mutations in the base E locus sequence: alleleE1 (ARGI218-219ins) found in Brown Swiss andsome light brown-coated French cattle breeds(Graphodatskaya et al. 2000; Rouzaud et al. 2000;CorrespondenceLuis J. Royo, CENSYRA-SERIDA-Somio´,C/Camino de los Claveles 604, E-33203, Gijo´n,Asturias, Spain. Tel: +34 985 19 53 00;Fax: +34 985 19 53 10;E-mail: ljroyo@serida.orgReceived: 22 June 2004;accepted: 13 May 2005SummaryThe aim of this study was to ascertain the role of the Agouti signalingpeptide (ASIP) gene coding region in the Agouti locus variation withinwild-type coat colour in cattle. We determined the Extension genotypein 241 individuals from six Spanish and three French brown cattlebreeds representative of wild-type coat variation. Polymerase chain reac-tion–single-strand conformation polymorphism (PCR-SSCP) analysis wascarried out in individuals of each Extension genotypes within the samebreed in an attempt to identify variants in the three coding exons of theASIP gene. No SSCP variants were found. Results were confirmed bysequencing the coding exons of the ASIP gene in 20 individuals. Ourresults suggest that the ASIP coding region does not play a central rolein coat colour variation in cattle.J. Anim. Breed. Genet. ISSN 0931-2668J. Anim. Breed. Genet. 122 (2005) 357–360 ª 2005 Blackwell Verlag, Berlin 357Kriegesmann et al. 2001) and allele E2 (R223W) alsofound in Brown Swiss cattle and many Italian breeds(Graphodatskaya et al. 2000; Maudet & Taberlet2002). All three E+, E1 and E2 alleles, present in cat-tle breeds that are wild type in colouration and thuswould allow the Agouti variation. The E+, E1 and E2alleles are hormonally activated receptors but both,the recessive red allele, e, and the dominant blackallele ED, have been characterized at the cell level asunresponsive to increased intracellular cAMP stimu-lation by a wide range of alpha-MSH concentrations(Graphodatskaya et al. 2002).In contrast with the E locus (Adalsteisson et al.1995), the A locus in cattle has not been com-pletely studied. Mutants at the A locus modify theexpression of the wild-type pattern. Reviewing theliterature Olson (1998) describes the following A al-leles: Abp (patterned blackish agouti) modifying thewild-type to nearly entirely black and not influ-enced by the sex; aw (white-bellied agouti) remov-ing red pigment and a part of the black pigmentwhile causing a more uniform distribution of blackpigmentation especially across the sides of the ani-mal; and ai (fawn) removing red and black pigmen-tation and, particularly red along the underline andalong the back (dorsal strip) resulting in tan tofawn colour. Adalsteisson et al. (1995) also proposedthe existence of a wild allele of Agouti (A+) thatallows the expression of the combination of redand black pigmentation of the wild-type coat col-our, and a recessive black (a), which, when homo-zygous, would modify the wild-type coat to solidblack. Girardot et al. (2005) found no evidence ofcoding region sequence variation in ten animalsbelonging to eight cattle breeds, most of themshowing the red recessive or black dominant coatcolour. This limited sampling do not permit toascertain the possible influence of the coding regionsequence of the ASIP gene on the whole wild typecoat colour variation in cattle, especially for therecessive black coat pattern.The objective of this work is to ascertain the roleof the ASIP gene coding region in the A locus vari-ation of wild-type coat colouration in cattle. To dothis, we will study a sample of individuals showingmost of phenotypic variation of this coat type des-cribed in the literature. Individuals within breedswill be first genotyped for the E locus. Animals carri-ying e or ED alleles will be excluded from subse-quent analysis. Homozygotes showing the sameExtension genotype and different phenotypes will beselected as candidates to search for allelic variants inthe ASIP coding region.Materials and methodsA total of 241 animals belonging to six Spanish(Asturiana de los Valles, Asturiana de la Montan˜a,Negra Serrana, Parda Alpina, Sayaguesa andTudanca) and three French cattle breeds (Parthenais,Tarantaise and Normande) were sampled. The ninepopulations were representative of the wild-typephenotype in coat colour. DNA was isolated fromsemen or blood using standard procedures (Sam-brook et al. 1989). A list of chosen breeds, numberof individuals and coat characteristics are detailed inTable 1. Polymerase chain reaction–restriction frag-ment-length polymorphism (PCR-RFLP) assays toidentify E locus genotypes were conducted usingprimers and PCR protocols described by Graphodats-kaya et al. (2000). E1 allele (ARGI218-219ins) wassimply identified by electrophoresis in acrylamidegels based on its size. E2 allele (R223W) was identi-fied by means of the HapII enzyme (Amersham Bio-sciences, Buckinghamshire, UK). Dominant ED allele(L99P) was diagnosed using the enzyme AciITable 1 Description of breeds, wild-type Extension genotypes and coat patternsBreed N Coat type Coat variationExtension genotypeE+/E+ E+/E1 E+/E2 E1/E1 E1/E2 E2/E2Asturiana de los Valles 64 Jersey type From fawn to mahogany 1 3 9 – 12 32Asturiana de la Montan˜a 35 Jersey type From fawn to mahogany 1 – 2 – 4 28Parthenais 16 Jersey type From fawn to mahogany 4 8 – 4 – –Tarantaise 16 Jersey type From fawn to mahogany 1 – – 14 – 1Parda Alpina 18 Brown Swiss type From brownish grey to light grey 9 6 – 2 1 –Negra Serrana 22 Black Black with different degrees of brown dorsal stripe 17 1 – 1 – –Sayaguesa 18 Black Black with different degrees of brown dorsal stripe 3 – 3 – 1 –Tudanca 36 Grey Dark grey in dams and blackish in sires – – – – – 36Normande 16 Brown High frequency of brindle individuals 16 – – – – –ASIP coding region in cattle L. J. Royo et al.358 J. Anim. Breed. Genet. 122 (2005) 357–360 ª 2005 Blackwell Verlag, Berlin(NewEngland Biolabs, Hertfordshire, UK). Presence ofthe recessive e allele (310delG) was detected withHapII. Primers were designed based on the publishedsequence X99691, except Exon2-forward and Exon4-reverse which are based on human–murine sequences(Table 2). PCR-single-strand conformation poly-morphism (SSCP) analysis was carried out in at leastone individual for each Extension genotypes withinthe same breed to identify variants existing into thethree coding exons of the ASIP gene (Table 2). Fivedifferent electrophoretic protocols were used: (i) 0.5 XMutation Detection Enhancement gels (MDE; Camb-rex BioScience, Rockland, ME, USA), 6C, 20 W,3.5 h; (ii) 0.7 X MDE, 16C, 20 W, 3.5 h; (iii) Acryl:-Bis (37.5:1) 12%, 5% Glycerol, 4C, 400 V, 12 h; (iv)Acryl:Bis (37.5:1) 12%, 5% glycerol, 20C, 400 V,12 h; and (v) Acryl:Bis (100:1) 12%, 5% glycerol,4C, 400 V, 20 h). The PCR products for the three ex-ons were sequenced by fluorescent dye-terminatorcycle sequencing on an automated sequencer (AlfEx-pressII; Amersham Biosciences).Results and discussionA total of 241 individuals were genotyped for the Elocus. Individuals showing e (seven Asturiana de losValles and one Asturiana de la Montan˜a) or ED (11Sayaguesa and three Negra Serrana) alleles wereexcluded from subsequent analysis and they are notdetailed in Table 1. After the five assays of PCR-SSCP, no SSCP variants were found in any of thethree coding exons. Results were confirmed bysequencing both strands of the PCR fragments con-taining the coding sequence of ASIP gene in 20 indi-viduals: one for each breed/homozygous extensiongenotype (E+/E+, E1/E1 and E2/E2) and two addi-tional (randomly chosen) individuals for each of theblack patterned breeds (Sayaguesa and Negra Serr-ana). The obtained sequences are available in theGenbank data base with the following accessionnumbers: AY348953, AY348954, AY352659. TwoSayaguesa individuals were found to be heterozy-gous at nt163(G-A) in intron 2. This polymorphismis located one nucleotide after the donor (5¢) splicesite (GT) of intron 2. Notice that the most frequentnucleotide in the donor splice site third position isadenine (62%) (Lewin 2000). Then it is consideredunlikely to affect the ASIP mRNA.The phenotypic coat colour variation in cattle cannot be explained solely by the action of the Exten-sion locus (Rouzaud et al. 2000; Jo¨rg et al. 2002).Earlier studies suggest that the ASIP gene is respon-sible for the agouti variation (Bultman et al. 1992;Mountjoy et al. 1992; Robbins et al. 1993; Jackson1997). However, evidence of the relationshipbetween ASIP gene and colour has been obtained inmice and other laboratory rodents (Bultman et al.1992; Miltenberger et al. 2002). In domestic animalsthis task has not been clearly stated so far. Riederet al. (2001) found only one allelic variant in theASIP gene (recessive black) in 22 different horsebreeds that showed considerable variation in coatcoloration. In pigs only one mutation has beenfound in the sequence of the exon 3 (E68K) and itfailed to show an association with coat colour vari-ation (Ferna´ndez 2003). Three mutations have beendescribed in the untranslated region or introns ofthe agouti gene, obviously without affecting coatcolour in pigs (Leeb et al. 2000). Even in humans,Dinulescu & Cone (2000) reported that the agoutigene does not appear to play a role in human pig-mentation. Taking into account these findings andour results, the ASIP coding region does not play acentral role in coat colour variation in cattle.AcknowledgementsThis paper was partially funded by a grant No.PC-REC01-01 and Dismed S.L. 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The coding sequence of the ASIP gene is identical in nine wild-type coloured cattle breeds

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