doi: 10.1111/age.12104
A frameshift mutation in the melanophilin gene causes the dilute coat colour in rabbit (Oryctolagus cuniculus) breeds L. Fontanesi*†, E. Scotti*, D. Allain‡§ and S. Dall’Olio* *Division of Animal Sciences, Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Viale Fanin 46, 40127, Bologna, Italy. †Centre for Genome Biology, University of Bologna, 40126, Bologna, Italy. ‡INRA, UR631, SAGA, CS52627, 31326, Castanet eres, France. Tolosan, France. §INRA, UE 1372, GenESI, Le Magneraud, BP52, 17700, Surg
Summary
In rabbit, the dilute locus is determined by a recessive mutated allele (d) that causes the dilution of both eumelanic and pheomelanic pigmentations. In mice, similar phenotypes are determined by mutations in the myosin VA, Rab27a and melanophilin (MLPH) genes. In this study, we investigated the rabbit MLPH gene and showed that a mutation in this gene appears responsible for the dilute coat colour in this species. Checkered Giant F1 families segregating for black and grey (diluted or blue) coat colour were first genotyped for a complex indel in intron 1 of the MLPH gene that was completely associated with the coat colour phenotype (h = 0.00; LOD = 4.82). Then, we sequenced 6357 bp of the MLPH gene in 18 rabbits of different coat colours, including blue animals. A total of 165 polymorphisms were identified: 137 were in non-coding regions and 28 were in coding exons. One of them was a frameshift deletion in exon 5. Genotyping the half-sib families confirmed the complete cosegregation of this mutation with the blue coat colour. The mutation was analysed in 198 rabbits of 23 breeds. All Blue Vienna and all other blue/grey/ash rabbits in other breeds (Californian, Castor Rex, Checkered Giant, English Spot, Fairy Marburg and Fairy Pearly) were homozygous for this deletion. The identification of MLPH as the responsible gene for the dilute locus in rabbit provides a natural animal model for human Griscelli syndrome type 3 and a new mutant to study the role of this gene on pigmentation. Keywords Blue Vienna, breed, Checkered Giant, dilute locus, dilution, pigmentation, polymorphism
Introduction Different coat colours and colour patterns in the European rabbit (Oryctolagus cuniculus) have been selected through the domestication process of this species and, later, by fancy breeders, and then fixed in particular strains/breeds that often are named after their colour. These phenotypic traits have been the matter of pioneering genetics studies that identified several loci affecting coat colour and colour patterns (Castle 1905, 1907; Punnett 1912, 1915). Early comparative genetic analyses across mammals have established homology for several rabbit coat colour loci (Robinson 1958; Searle 1968). However, the number of loci affecting this phenotypic trait is larger than previously Address for correspondence L. Fontanesi, Division of Animal Sciences, Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Viale Fanin 46, 40127 Bologna, Italy. E-mail: [email protected] Accepted for publication 26 September 2013
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expected as, at present, more than 300 loci have been shown to affect coat colour in mice (Lamoureux et al. 2010). These loci correspond to genes that are involved in the development, distribution, morphology and structure of the melanocytes and that constitute or regulate their enzymatic machinery. Melanocytes are specialized cells that contain lysosome-related organelles (the melanosomes) by which two types of melanins, eumelanins (black/brown pigments) and pheomelanins (yellow/red pigments) are synthesized. The agouti and extension loci play a key role in mammalian pigmentation, as they regulate the production and relative amount of these two melanin types. These loci encode the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes (Bultman et al. 1992; Robbins et al. 1993). Analysing the rabbit ASIP gene, we identified the causative mutation of the recessive black non-agouti allele (Fontanesi et al. 2010a). Our investigations of the rabbit MC1R gene identified mutations associated with several extension alleles, including the dominant black, the Japanese brindling and the recessive red alleles (Fontanesi et al. 2006, 2010b).
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MLPH and dilute locus in rabbits In rabbit, the dilution of both eumelanic and pheomelanic pigmentations is caused by the dilute locus (Castle et al. 1909). As a result of the recessive mutated allele (d), the black is diluted to grey (termed blue by fancy breeders), which characterizes a few breeds, such as Blue Vienna (Fig. 1; Castle 1930; Robinson 1958; Fox 1994). In mice, similar phenotypes are determined by mutations in the myosin VA (Myo5a; dilute locus), Rab27a (ashen locus) and melanophilin (MLPH; leaden locus) genes (Mercer et al. 1991; Wilson et al. 2000; Matesic et al. 2001) that encode proteins that constitute the melanosome transport complex (Barral & Seabra 2004). In these mutants, mature melanosomes cluster in the perinuclear area of the cell, rather than at the periphery. Subsequently, pigment granules are released unevenly into the developing hair shaft causing a decrease in the amount of light absorbability, and hence, a phenotype in which coat colour is lightened (Marks & Seabra 2001; Barral & Seabra 2004). In humans, mutations in the same genes are responsible for Griscelli syndrome (GS) type 1, 2 and 3 (Pastural et al. 1997; Menasche et al. 2000, 2003). Griscelli syndrome type 3 is restricted to a hypopigmentation defect that occurs also in GS1, together with neurological dysfunctions and impairments, and in GS2, together with immunological defects and immunodeficiency (Van Gele et al. 2009). We first investigated the rabbit homologous gene of the mouse dilute locus and excluded MYO5A as the determinant of the dilute locus in rabbit (Fontanesi et al. 2012). After that, the strongest candidate gene could be MLPH, as defective humans and mice for this gene do not show other apparent alterations, similar to what might happen in rabbits. Mutations in this gene causing a dilutelike coat colour phenotype have been identified in cat (Ishida et al. 2006), dog (Philipp et al. 2005; Dr€ ogem€ uller et al. 2007; Welle et al. 2009) and American mink (Cirera et al. 2013). In chicken and Japanese quail, MLPH mutations are responsible for the lavender plumage colour (Vaez et al. 2008; Bed’hom et al. 2012), that was referred to be the avian homologous of the
leaden locus in mice (Brumbaugh et al. 1972). Here we investigated the MLPH gene in rabbits and showed that it causes the dilute phenotype in this species.
Materials and methods Animals Three half-sib families were produced by crossing a spotted blue Checkered Giant buck (homozygous for the dilute locus recessive d allele) with three unrelated spotted black Checkered Giant does. Of these does, two were expected to be heterozygous for the mutated dilute allele (genotype D/d at the dilute locus) as they were obtained by crossing blackspotted bucks with other blue-spotted does that were not possible to sample. The third doe was expected to be homozygous for the normal dominant dilute allele (genotype D/D), according to genealogical data. From the first two crossings, a total of 16 F1 rabbits (eight + eight) were produced (Fig. 2). The other crossing produced 10 F1 rabbits. Pictures were taken of all these animals after weaning. Resequencing of the MLPH gene was obtained from 18 rabbits of different coat colour: one Belgian Hare, two Burgundy Fawn, one Champagne d’Argent, four Checkered Giant, two Giant Grey, two Rhinelander, one Vienna Blue and five F1 rabbits of the two half-sib families (two blue rabbits and three black rabbits). Of the four Checkered Giant rabbits, three were the parental animals of the F1 families (the spotted blue buck, a putative heterozygous doe and the putative homozygous normal doe) and one was another spotted black buck of a different line. In addition, another 198 rabbits of 23 breeds having different coat colours (Table 1) were used for genotyping MLPH polymorphisms. One of these breeds (Castor Rex) included two lines selected for fur production: one line having a ‘normal’ brown-black coat colour with a yellow agouti band and the other line derived from the previous one selecting the recessive ash/blue coat colour phenotype. Genomic DNA was extracted from blood (for the rabbits used for sequencing and the F1 animals) using the Wizard® Genomic DNA Purification kit (Promega Corp.) or from hair roots (for the rabbits used for genotyping) using the protocol already described by Fontanesi et al. (2007).
Resequencing of the rabbit MLPH gene and identification of polymorphisms
Figure 1 Blue Vienna rabbits.
PCR primer pairs were designed on the rabbit MLPH gene sequence (Ensembl accession number ENSOCUG00000016496, reported in the scaffold GL018840 of the oryCun2.0 genome assembly of the Oryctolagus cuniculus provided by the Broad Institute within the Mammalian Genome Project, http://www.broadinstitute.org/science/ projects/mammals-models/rabbit/rabbit-genome-sequencingproject) to amplify all recognized coding sequences, portions
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Figure 2 Segregation of the blue and black coat colours in Checkered Giant F1 families and genotype of the rabbits for the g.549853delG mutation identified in exon 5 of the melanophilin gene. The wild-type allele is indicated with ‘G’ and the mutated allele is indicated with ‘del’. The genotype at the dilute locus of the parental animals is reported (black does = D/d; blue buck = d/d).
of intronic regions downstream or upstream to recognized exons, 5′- and 3′-untranslated regions and other conserved sequences detected aligning the rabbit and human MLPH genes (Table S1 and Fig. S1). Genomic DNA used for PCR amplification and sequencing was from the rabbits already described above. PCR was carried out using a PTC-100 thermal cycler (MJ Research) in a 20-ll reaction volume containing ~50 ng genomic DNA, 1 U DNA EuroTaq DNA polymerase (EuroClone Ltd.), 19 Euro Taq PCR buffer, 2.5 mM dNTPs, 10 pmol of each primer and 1.5–3.0 mM of MgCl2 (Table S1). PCR profile was as follows: 5 min at 95 °C; 35 amplification cycles of 30 s at 95 °C, 30 s at 57– 60 °C, 30 s at 72 °C; 5 min at 72 °C (Table S1). The amplified fragments were sequenced after treatment with 1 ll of ExoSAP-ITâ (USB Corporation) for 15 min at 37 °C. Cycle sequencing of the treated PCR products was produced using the same PCR primers and the Big Dye version 3.1 kit (Applied Biosystems). Sequencing reactions, after precipitation with EDTA in Ethanol 100% and Ethanol 70%, were loaded on an ABI 3100 Avant capillary sequencer (Applied Biosystems). Sequences were aligned, and polymorphisms were detected using CODONCODE ALIGNER (http://www.codoncode.com/aligner) with the genomic rabbit MLPH sequence used as reference. All sequence chromatograms were visually inspected. Nomenclature of identified polymorphisms was based on the system coordinates of the
unassembled scaffold (Scaffold GL018840) in the OryCun2.0 genome version containing the rabbit MLPH gene (http://www.ensembl.org/Oryctolagus_cuniculus/ Info/). In silico functional analysis of the identified missense mutations was carried out using PANTHER (Thomas et al. 2003). Genotyping of MLPH polymorphisms and data analysis PCR-RFLP was used to genotype the g.549853delG polymorphism in the half-sib families and in the 198 rabbits of 23 different breeds. Briefly, the amplified product obtained using a mismatched forward primer that inserted an artificial restriction site for DdeI when the produced amplicons containing the deletion were digested with this restriction enzyme (Table S1). Digestion reaction was carried out overnight at 37 °C in a 25-ll reaction volume including 5 ll of PCR product, 19 restriction enzyme buffer and 2 U of DdeI (MBI Fermentas). The complex indel in intron 1 was analysed by fragment analysis in the half-sib families and in a subset of the rabbits (n = 100) of different breeds (Table S2). The resulting DNA fragments were electrophoresed on 10% 29:1 polyacrylamide/bis acrylamide gel and visualized with 19 GelRed Nucleic Acid Gel Stain (Biotium, Inc.). Calculation of the LOD score between coat colour phenotypes and MLPH geno-
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MLPH and dilute locus in rabbits Table 1 Breeds and animals genotyped for the melanophilin (MLPH) g.549853delG mutation.
Breed (No. of animals) Alaska (3) Belgian Hare (1) Blue Vienna (40) Burgundy Fawn (9) Californian (22) Castor Rex (16) Champagne d’Argent (15) Checkered Giant (20) Coloured Dwarf (5) Dutch (6) English Spot (9) Fairy Marburg (1) Fairy Pearly (2) Giant Chinchilla (12) Giant Grey (9) Giant White (4) Lop (2) New Zealand White (6) Rhinelander (7) Silver (5) Tan (1) Thuringian (1) White Vienna (2) Total (198)
MLPH:g.549853delG genotype2,3
Coat colour and [proposed genotype at the dilute locus]1
G/G
Self-black [D/-] Reddish laced with black [D/-] Dark blue [d/d] Fawn [D/-] White with black (20) [D/-] or blue markings (2*) [d/d] Brown-black with yellow agouti band (8) [D/-] or ash/blue coloured with very light yellow agouti band (8*) [d/d] Silver as surface colour and black as under-colour [D/-] White with black (19) [D/-] or blue markings (1*) [d/d] Bristle white (1) [?]; hare-grey (2) [D/-]; Havana (1) [D/-] chinchilla (1) [D/-] With black markings [D/-] White with black (6) [D/-] or blue (3*) [d/d] markings Grey-light blue [d/d] Pearling grey [d/d] Chinchilla [D/-] Wild grey [D/-] White albino [?] Wild grey [D/-] White albino [?] White with black and yellow markings [D/-] Black with silvering [D/-] Black fire [D/-] Shaded yellow/brown [D/-] White-blue eyes [D/-]
G/del
del/del
3 1 9 18 8
2 -
40 2* 8*
15 16 3 6 4 12 9 4 2 6 7 5 1 1 2 132
3 2 2 9
1* 3* 1 2 57
1
In breeds for which animals of different coat colour have been sampled, the number of the rabbits showing the distinctive phenotypes is indicated with rounded brackets and linked with the genotyped animals with an asterisk (*). Squared brackets contain the proposed genotype at the dilute locus, when reported or deduced from the literature (Castle et al. 1909; Castle 1930; Robinson 1958; Fox 1994). 2 Genotypes obtained by PCR-RFLP for the MLPH:g.549853delG mutation. The normal allele is indicated with ‘G’, the mutated allele is indicated with ‘del’. 3 The number of animals with the different genotypes is reported.
types in the half-sibling families was obtained with the LODS program (Linkage Utility Programs, Rockefeller University).
Results and discussion Coat colour segregation in the two F1 families obtained by crossing a blue-spotted Checkered Giant buck (genotype d/d at the dilute locus) with two black-spotted Checkered Giant does (genotype D/d at the dilute locus) did not deviate from the expected 1:1 Mendelian ratio (seven black and nine blue rabbits; Fig. 2). All F1 rabbits obtained by crossing a blackspotted doe, with a putative D/D genotype, with the same blue-spotted Checkered Giant buck had black coat colour. These data confirmed the recessive mode of inheritance of the blue against the black coat colour as already reported from early genetic studies in rabbit (Castle et al. 1909; Castle 1930). Spotted inheritance is controlled by another locus (Fontanesi et al. 2010c). As a first step, to identify polymorphisms in the rabbit MLPH gene that could help to evaluate if this gene would be a putative causative gene of the dilute phenotype by segregation analysis, we first amplified a few regions in
the parental animals of the F1 families. Using primer pair 1a (Table S1), a three-allele complex indel locus (that was subsequently characterized by sequencing), derived by the duplication, presence or absence of a 29-bp sequence in intron 1 (with a few other nucleotide differences), was identified (Fig. S2). The two D/d does had genotype 1/2 (amplified fragments: 443 bp, allele 1; 386 bp, allele 2) and 2/3 (amplified fragments: 386 bp, allele 2; 359 bp, allele 3), whereas the blue buck (d/d) was homozygous for allele 2 (Fig. S2). This polymorphic site was then analysed in all F1 rabbits resulting in a complete cosegregation between the two coat colours and genotypes at this variable region (h = 0.00; LOD = 4.82): all blue rabbits were homozygous for the same allele that was homozygous in the blue-spotted buck, whereas the black F1 animals were either 1/2 or 2/3 (Fig. S2 reports a few F1 animals with their gel electrophoresis patterns at this polymorphic site). This marker made it possible to show that the most likely gene affecting the dilute coat colour in rabbit is the MLPH gene, as already reported in cat (Ishida et al. 2006) and dog (Philipp et al. 2005; Dr€ ogem€ uller et al. 2007). However, sequence analysis of the three alleles could not identify an obvious mutational event that might directly affect
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Fontanesi et al. functionality of the MLPH gene or protein. This polymorphism was due to intronic differences that did not affect the coding region of exon 2 (Fig. S2). This complex indel was genotyped in 100 rabbits of different breeds with different coat colour (Table S2). All blue rabbits were homozygous 2/2 but not all 2/2 rabbits had a diluted coat colour, indicating that allele 2 in the F1 families was in linkage disequilibrium with the true causative mutation. For example, a few Champagne d’Argent rabbits were homozygous 2/2 but their background colour was black, and all these animals were completely black early in their life (Table S2). Genotype 2/2 also was observed in one Alaska, one Californian, one Checkered Giant, one Giant Grey and one Rhinelander that did not have any diluted coat colour (Table S2). To identify the causative mutation of the dilute phenotype, we amplified and resequenced a total of 6357 bp of the rabbit MLPH gene (that corresponds to a sum of discontinuous gene fragments; sum is referred to the reference OryCun2.0 gene sequence ENSOCUG00000016496; Fig. S1) in animals with different coat colours (including parental animals of the F1 families). Sequenced regions encompassed non-coding regions (5′-flanking region including the putative 5′- UTR, the putative 3′-UTR and intronic regions mostly adjacent to coding exons) and all 15 exons identified in Ensembl (Fig. S1), except two putative close exons, exons 6 and 7 of this assembled sequence (considering the order of the reported coding regions; positions from 550665 and 550880 of the scaffold GL018840), that we were not able to amplify, despite four different primer pairs and their combinations (forward with a pair and reverse with another pair) that were tested. BLASTN analysis of these two putative exons did not show any matches with any other sequences. Taken together, this evidence might suggest that (i) the putative exons 6 and 7, automatically deduced in the reference sequence, do not exist in the investigated rabbits (even if it could be present in the Thorbecke rabbit that originated the OryCun2.0 genome version), or that (ii) assembling errors have included this region within the rabbit MLPH gene. Obtained sequences have been deposited in EMBL/GenBank databases under the accession numbers HG380502 and HG380503 (indicated below as haplotype 1 and haplotype 2 respectively). Comparing the sequences obtained in different rabbits, a large number of polymorphisms (n = 165) was identified (Table S3). On average, one polymorphism was present every ~39 bp. Most of these polymorphisms were in noncoding regions (5′-flanking including 5′-UTR, n = 8; the putative 3′-UTR, n = 16; and intronic regions: n = 113) and 28 were in coding exons (one every ~51 bp). Of these latter polymorphic sites, 15 were synonymous substitutions and 12 produced potential amino acid substitutions located in exon 1 (p.Val18Ile), exon 2 (p.Gln71Arg or p.Gln71Trp, depending on the combination with the previous SNP, Table S3; and p.Thr87Ala), exon 3 (p.Gln146His), exon 5
(p.Trp204Arg), exon 8 (p.Gly307Ala and p.Val311Ala), exon 9 (p.Lys349Arg), exon 10 (p.Ser393Gly) and exon 12 (p.Asp481Ser). PANTHER analysis (Table S3) indicated that none of these amino acid substitutions could have a deleterious effect. The only substitution that could modify the protein function was the p.Glu71Trp amino acid change (subPSEC = 4.370; Pdeleterious = 0.797). However, none of these polymorphisms occurred only in blue rabbits. Most of the identified polymorphisms defined two frequent haplotypes (referred as haplotype 1 and haplotype 2 in Table S3; clearly identified because several animals were homozygous at the polymorphic positions), not fixed in any breed sequenced. A few other minor haplotypes, resulting from putative multiple recombination events between the two major haplotypes, were identified (data not shown). These haplotypes differed from the sequence reported in OryCun2.0, derived from a partially inbred albino Thorbecke strain by several recombination events (Table S3). Among the 28 polymorphisms identified in the coding region, the remaining and most interesting was a deletion of one nucleotide in exon 5 (g.549853delG) that causes a shift in the reading frame that determines a completely different protein from the beginning of this exon (Fig. 3) until the second half of exon 8 in which a stop codon would be introduced (data not shown). By deducing the putative functional domains of the MLPH protein identified by PFAM, the mutated protein would maintain the N terminus FYVE zinc finger domain but would lose the Rab effector MyRIP/melanophilin C-terminus domain. This polymorphism was the only one that was homozygous in the sequenced blue rabbits; it was heterozygous in the d allele carrier black rabbits and was absent in all other sequenced animals. This mutation occurred in a new haplotype that differed from haplotype 2 just at this position (Table S3). Genotyping the half-sib families confirmed the complete cosegregation of this frameshift mutation with the blue coat colour in the F1 rabbits (Fig. 2). This mutation was analysed in 198 rabbits of different breeds (Table 1). All Blue Vienna rabbits were homozygous for this deletion. Homozygous subjects for the deleted nucleotide were also spotted blue rabbits of the Californian, Checkered Giant and English Spot breeds. In these three breeds, a few spotted black animals were heterozygous for the g.549853delG mutation, further confirming the recessive behaviour of this allele and the fact that this allele is segregating in several rabbit breeds. Two lines of Castor Rex were sampled. All rabbits of the ‘normal line’ with brown-black coat colour were homozygous for the wild-type allele, whereas all rabbits of the ash/blue coloured line, as expected, were homozygous for the g.549853delG mutation. Animals of two other rare fancy breeds, Fairy Marburg and Fairy Pearly having grey-light blue and pearling grey coat colours respectively, were homozygous for the MLPH frameshift mutation. Again, their genotype could be expected according to their coat colour. All Champagne d’Argent rabbits
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MLPH and dilute locus in rabbits
wild type allele
dilute allele
Figure 3 Frameshift deletion (g.549853delG) identified in exon 5 of the rabbit melanophilin gene. The deleted nucleotide is indicated in red in the wild-type allele sequence. Underlined nucleotides are other two polymorphic positions identified in exon 5 (g.549876G > A and g.549877T > C; Table S3).
were homozygous for the wild-type allele (not carriers for the frameshift mutation) even if some of them were homozygous 2/2 at the intron 1 complex indel. In addition, heterozygous 1/2 rabbits at this polymorphic locus resulted in homozygous wild type at the g.549853delG position (Table S2). This means that recombination events created different haplotypes at the MLPH locus, as already suggested, that were present in rabbits with black or nondiluted coat colours. Considering the genotyped 198 rabbits of different breed and the occurrence of homozygous g.549853delG animals only in blue (diluted) rabbits, across breed association between the genotype at this mutation and coat colour (diluted or not diluted) was highly significant (P = 3.88E-51; Fisher exact test), further supporting a causative role of the deletion in exon 5. Summarizing, even if we could not formally exclude the presence of other mutations in the linkage disequilibrium with the frameshift mutation in exon 5, all these data strongly argue for the g.549853delG deletion being a causative mutation of the recessive d allele at the dilute locus in rabbits. In particular, only this mutation distinguished haplotype 2 (one of the wild-type haplotypes) from the d
haplotype (Table S3), suggesting that the presence of other mutations in the MLPH gene causing this phenotype is highly unlikely. In addition, the disrupting mutation in exon 5 is upstream to the two non-sequenced putative exons 6 and 7 (not confirmed by genomic amplification and BLASTN analysis). Therefore, even if other mutations could be present in downstream exons/regions, the effect of the g.549853delG deletion is upstream to all other eventually occurring downstream mutational events, making the frameshift mutation in exon 5 the first causative mutation. The identification of the MLPH gene as the responsible gene for the dilute coat colour locus in rabbit provides another natural animal model for the human Griscelli syndrome type 3, in addition to other farm animal species, for example the cat and the dog (Philipp et al. 2005; Ishida et al. 2006; Dr€ ogem€ uller et al. 2007). This human syndrome seems restricted to hypopigmentation only, but just a few studies have reported its effects (Menasche et al. 2003; Al-Idrissi et al. 2010). It could be interesting to investigate blue rabbits to further evaluate the role of the MLPH gene on pigmentation, hair morphology and formation.
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Fontanesi et al. Combining results obtained at the dilute locus with information on the ASIP and MC1R alleles that we previously obtained (Fontanesi et al. 2006, 2010a,b), it is possible to have a first picture of the genetic structure of coat colour loci in several rabbit breeds, characterized by different coat colours. Moreover, it is also possible to predict the effects of combinations of different mutations at these loci. For example, Blue Vienna rabbits have their specific coat colour because they are fixed for the black non-agouti frameshift mutation (Fontanesi et al. 2010a) and for the dilute recessive d allele determined by a frameshift mutation at the MLPH gene. The same genotypic structure is present in English Spot rabbits with blue markings (Fontanesi et al. 2010a). Blue markings in Checkered Giant and Californian are derived from the dilution of the black coat colour caused by the g.549853delG on a black background derived by the dominant black allele at the MC1R gene and/or by the recessive non-agouti allele at the agouti locus (Fontanesi et al. 2006, 2010a). There is no apparent difference between black markings derived by the extension and agouti loci; therefore, it seems that there is no apparent difference on the effect of the dilution on the two black-determining genotypes. However, fancy breeders suggest the existence of two types of dilution: one that gives a darker grey (or blue) and another one that gives a lighter grey (or blue). It could be possible that mutations in other loci modify the degree of dilution derived by the frameshift mutation of the MLPH gene.
Acknowledgements We thank Associazione Nazionale Coniglicoltori Italiani (ANCI) for its help during the collection of samples, Renate Regitz for providing several rabbit samples and INRA GenEsi unit (UE 1372, Le Magneraud, BP52, 17700 Surgeres) for providing rabbit samples from the two Castor Rex lines. This work was supported by University of Bologna funds (FAGenomicH project and RFO funds) and has been carried out in the framework of the COST Action ‘A Collaborative European Network on Rabbit Genome Biology – RGB-Net’ TD1101 (2011–2015).
Conflict of interests The authors have declared no potential conflicts.
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MLPH and dilute locus in rabbits coat color phenotype in the domestic cat. Genomics 88, 698– 705. Lamoureux M.L., Delmas V., Larue L. & Bennett D.C. (2010) The Colors of Mice – A Model Genetic Network. Wiley-Blackwell, West Sussex, UK. Marks M.S. & Seabra M.C. (2001) The melanosome: membrane dynamics in black and white. Nature Reviews Molecular Cell Biology 2, 738–48. Matesic L.E., Yip R., Reuss A.E., Swing D.A., O’Sullivan T.N., Fletcher C.F., Copeland N.G. & Jenkins N.A. (2001) Mutations in Mlph, encoding a member of the Rab effector family, cause the melanosome transport defects observed in leaden mice. Proceedings of the National Academy of Sciences of the United States of America 98, 10238–43. Menasche G., Pastural E., Feldmann J. et al. (2000) Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nature Genetics 25, 173–6. Menasche G., Ho C.H., Sanal O., Feldmann J., Tezcan I., Ersoy F., Houdusse A., Fischer A. & de Saint Basile G. (2003) Griscelli syndrome restricted to hypopigmentation results from a melanophilin defect (GS3) or a MYO5A F-exon deletion (GS1). The Journal of Clinical Investigation 112, 450–6. Mercer J.A., Seperack P.K., Strobel M.C., Copeland N.G. & Jenkins N.A. (1991) Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 349, 709–13. Pastural E., Barrat F.J., Dufourcq-Lagelouse R., Certain S., Sanal O., Jabado N., Seger R., Griscelli C., Fischer A. & De Saint Basile G. (1997) Griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin-Va gene. Nature Genetics 16, 289–92. Philipp U., Hamann H., Mecklenburg L., Nishino S., Mignot E., G€ unzel-Apel A.R., Schmutz S.M. & Leeb T. (2005) Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs. BMC Genetics 6, 34. Punnett R.C. (1912) Inheritance of coat colour in rabbits. Journal of Genetics 2, 221–38. Punnett R.C. (1915) Further experiments on the inheritance of coat colour in rabbits. Journal of Genetics 5, 38–50. Robbins L.S., Nadeau J.H., Johnson K.R., Kelly M.A., Roselli-Rehfuss L., Baack E., Mountjoy K.G. & Cone R.D. (1993) Pigmentation phenotypes of variant Extension locus alleles result from point mutations that alter MSH receptor function. Cell 72, 827–34.
Robinson R. (1958) Genetic studies of the rabbit. Bibliographia Genetica 17, 229–558. Searle A.G. (1968) Comparative Genetics of Coat Colour in Mammals. Logos Press, London, UK. Thomas P.D., Campbell M.J., Kejariwal A., Mi H., Karlak B., Daverman R., Diemer K., Muruganujan A. & Narechania A. (2003) PANTHER: a library of protein families and subfamilies indexed by function. Genome Research 13, 2129–41. Vaez M., Follett S.A., Bed’hom B., Gourichon D., Tixier-Boichard M. & Burke T. (2008) A single point-mutation within the melanophilin gene causes the lavender plumage colour dilution phenotype in the chicken. BMC Genetics 9, 7. Van Gele M., Dynoodt P. & Lambert J. (2009) Griscelli syndrome: a model system to study vesicular trafficking. Pigment Cell & Melanoma Research 22, 268–82. Welle M., Philipp U., R€ ufenacht S. et al. (2009) MLPH genotype melanin phenotype correlation in dilute dogs. Journal of Heredity 100, S75–9. Wilson S.M., Yip R., Swing D.A., O’Sullivan T.N., Zhang Y., Novak E.K., Swank R.T., Russell L.B., Copeland N.G. & Jenkins N.A. (2000) A mutation in Rab27a causes the vesicle transport defects observed in ashen mice. Proceedings of the National Academy of Sciences of the United States of America 97, 7933–8.
Supporting information Additional supporting information may be found in the online version of this article. Figure S1 Schematic representation of the rabbit melanophilin gene with sequenced regions and positions of the genotyped polymorphisms. Figure S2 Fragment analysis, segregation (partial F1 families) and sequence of the complex indel genotyped indel in intron 1 of the rabbit melanophilin gene. Table S1 PCR primers, PCR conditions and use of the obtained fragments. Table S2 Rabbits genotyped for the complex indel in intron 1 of the melanophilin gene. Table S3 Polymorphisms identified in the rabbit melanophilin gene.
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A frameshift mutation in the melanophilin gene causes the dilute coat colour in rabbit (Oryctolagus cuniculus) breeds L. Fontanesi*†, E. Scotti*, D. Allain‡§ and S. Dall’Olio* *Division of Animal Sciences, Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Viale Fanin 46, 40127, Bologna, Italy. †Centre for Genome Biology, University of Bologna, 40126, Bologna, Italy. ‡INRA, UR631, SAGA, CS52627, 31326, Castanet eres, France. Tolosan, France. §INRA, UE 1372, GenESI, Le Magneraud, BP52, 17700, Surg
Summary
In rabbit, the dilute locus is determined by a recessive mutated allele (d) that causes the dilution of both eumelanic and pheomelanic pigmentations. In mice, similar phenotypes are determined by mutations in the myosin VA, Rab27a and melanophilin (MLPH) genes. In this study, we investigated the rabbit MLPH gene and showed that a mutation in this gene appears responsible for the dilute coat colour in this species. Checkered Giant F1 families segregating for black and grey (diluted or blue) coat colour were first genotyped for a complex indel in intron 1 of the MLPH gene that was completely associated with the coat colour phenotype (h = 0.00; LOD = 4.82). Then, we sequenced 6357 bp of the MLPH gene in 18 rabbits of different coat colours, including blue animals. A total of 165 polymorphisms were identified: 137 were in non-coding regions and 28 were in coding exons. One of them was a frameshift deletion in exon 5. Genotyping the half-sib families confirmed the complete cosegregation of this mutation with the blue coat colour. The mutation was analysed in 198 rabbits of 23 breeds. All Blue Vienna and all other blue/grey/ash rabbits in other breeds (Californian, Castor Rex, Checkered Giant, English Spot, Fairy Marburg and Fairy Pearly) were homozygous for this deletion. The identification of MLPH as the responsible gene for the dilute locus in rabbit provides a natural animal model for human Griscelli syndrome type 3 and a new mutant to study the role of this gene on pigmentation. Keywords Blue Vienna, breed, Checkered Giant, dilute locus, dilution, pigmentation, polymorphism
Introduction Different coat colours and colour patterns in the European rabbit (Oryctolagus cuniculus) have been selected through the domestication process of this species and, later, by fancy breeders, and then fixed in particular strains/breeds that often are named after their colour. These phenotypic traits have been the matter of pioneering genetics studies that identified several loci affecting coat colour and colour patterns (Castle 1905, 1907; Punnett 1912, 1915). Early comparative genetic analyses across mammals have established homology for several rabbit coat colour loci (Robinson 1958; Searle 1968). However, the number of loci affecting this phenotypic trait is larger than previously Address for correspondence L. Fontanesi, Division of Animal Sciences, Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Viale Fanin 46, 40127 Bologna, Italy. E-mail: [email protected] Accepted for publication 26 September 2013
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expected as, at present, more than 300 loci have been shown to affect coat colour in mice (Lamoureux et al. 2010). These loci correspond to genes that are involved in the development, distribution, morphology and structure of the melanocytes and that constitute or regulate their enzymatic machinery. Melanocytes are specialized cells that contain lysosome-related organelles (the melanosomes) by which two types of melanins, eumelanins (black/brown pigments) and pheomelanins (yellow/red pigments) are synthesized. The agouti and extension loci play a key role in mammalian pigmentation, as they regulate the production and relative amount of these two melanin types. These loci encode the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes (Bultman et al. 1992; Robbins et al. 1993). Analysing the rabbit ASIP gene, we identified the causative mutation of the recessive black non-agouti allele (Fontanesi et al. 2010a). Our investigations of the rabbit MC1R gene identified mutations associated with several extension alleles, including the dominant black, the Japanese brindling and the recessive red alleles (Fontanesi et al. 2006, 2010b).
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MLPH and dilute locus in rabbits In rabbit, the dilution of both eumelanic and pheomelanic pigmentations is caused by the dilute locus (Castle et al. 1909). As a result of the recessive mutated allele (d), the black is diluted to grey (termed blue by fancy breeders), which characterizes a few breeds, such as Blue Vienna (Fig. 1; Castle 1930; Robinson 1958; Fox 1994). In mice, similar phenotypes are determined by mutations in the myosin VA (Myo5a; dilute locus), Rab27a (ashen locus) and melanophilin (MLPH; leaden locus) genes (Mercer et al. 1991; Wilson et al. 2000; Matesic et al. 2001) that encode proteins that constitute the melanosome transport complex (Barral & Seabra 2004). In these mutants, mature melanosomes cluster in the perinuclear area of the cell, rather than at the periphery. Subsequently, pigment granules are released unevenly into the developing hair shaft causing a decrease in the amount of light absorbability, and hence, a phenotype in which coat colour is lightened (Marks & Seabra 2001; Barral & Seabra 2004). In humans, mutations in the same genes are responsible for Griscelli syndrome (GS) type 1, 2 and 3 (Pastural et al. 1997; Menasche et al. 2000, 2003). Griscelli syndrome type 3 is restricted to a hypopigmentation defect that occurs also in GS1, together with neurological dysfunctions and impairments, and in GS2, together with immunological defects and immunodeficiency (Van Gele et al. 2009). We first investigated the rabbit homologous gene of the mouse dilute locus and excluded MYO5A as the determinant of the dilute locus in rabbit (Fontanesi et al. 2012). After that, the strongest candidate gene could be MLPH, as defective humans and mice for this gene do not show other apparent alterations, similar to what might happen in rabbits. Mutations in this gene causing a dilutelike coat colour phenotype have been identified in cat (Ishida et al. 2006), dog (Philipp et al. 2005; Dr€ ogem€ uller et al. 2007; Welle et al. 2009) and American mink (Cirera et al. 2013). In chicken and Japanese quail, MLPH mutations are responsible for the lavender plumage colour (Vaez et al. 2008; Bed’hom et al. 2012), that was referred to be the avian homologous of the
leaden locus in mice (Brumbaugh et al. 1972). Here we investigated the MLPH gene in rabbits and showed that it causes the dilute phenotype in this species.
Materials and methods Animals Three half-sib families were produced by crossing a spotted blue Checkered Giant buck (homozygous for the dilute locus recessive d allele) with three unrelated spotted black Checkered Giant does. Of these does, two were expected to be heterozygous for the mutated dilute allele (genotype D/d at the dilute locus) as they were obtained by crossing blackspotted bucks with other blue-spotted does that were not possible to sample. The third doe was expected to be homozygous for the normal dominant dilute allele (genotype D/D), according to genealogical data. From the first two crossings, a total of 16 F1 rabbits (eight + eight) were produced (Fig. 2). The other crossing produced 10 F1 rabbits. Pictures were taken of all these animals after weaning. Resequencing of the MLPH gene was obtained from 18 rabbits of different coat colour: one Belgian Hare, two Burgundy Fawn, one Champagne d’Argent, four Checkered Giant, two Giant Grey, two Rhinelander, one Vienna Blue and five F1 rabbits of the two half-sib families (two blue rabbits and three black rabbits). Of the four Checkered Giant rabbits, three were the parental animals of the F1 families (the spotted blue buck, a putative heterozygous doe and the putative homozygous normal doe) and one was another spotted black buck of a different line. In addition, another 198 rabbits of 23 breeds having different coat colours (Table 1) were used for genotyping MLPH polymorphisms. One of these breeds (Castor Rex) included two lines selected for fur production: one line having a ‘normal’ brown-black coat colour with a yellow agouti band and the other line derived from the previous one selecting the recessive ash/blue coat colour phenotype. Genomic DNA was extracted from blood (for the rabbits used for sequencing and the F1 animals) using the Wizard® Genomic DNA Purification kit (Promega Corp.) or from hair roots (for the rabbits used for genotyping) using the protocol already described by Fontanesi et al. (2007).
Resequencing of the rabbit MLPH gene and identification of polymorphisms
Figure 1 Blue Vienna rabbits.
PCR primer pairs were designed on the rabbit MLPH gene sequence (Ensembl accession number ENSOCUG00000016496, reported in the scaffold GL018840 of the oryCun2.0 genome assembly of the Oryctolagus cuniculus provided by the Broad Institute within the Mammalian Genome Project, http://www.broadinstitute.org/science/ projects/mammals-models/rabbit/rabbit-genome-sequencingproject) to amplify all recognized coding sequences, portions
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Figure 2 Segregation of the blue and black coat colours in Checkered Giant F1 families and genotype of the rabbits for the g.549853delG mutation identified in exon 5 of the melanophilin gene. The wild-type allele is indicated with ‘G’ and the mutated allele is indicated with ‘del’. The genotype at the dilute locus of the parental animals is reported (black does = D/d; blue buck = d/d).
of intronic regions downstream or upstream to recognized exons, 5′- and 3′-untranslated regions and other conserved sequences detected aligning the rabbit and human MLPH genes (Table S1 and Fig. S1). Genomic DNA used for PCR amplification and sequencing was from the rabbits already described above. PCR was carried out using a PTC-100 thermal cycler (MJ Research) in a 20-ll reaction volume containing ~50 ng genomic DNA, 1 U DNA EuroTaq DNA polymerase (EuroClone Ltd.), 19 Euro Taq PCR buffer, 2.5 mM dNTPs, 10 pmol of each primer and 1.5–3.0 mM of MgCl2 (Table S1). PCR profile was as follows: 5 min at 95 °C; 35 amplification cycles of 30 s at 95 °C, 30 s at 57– 60 °C, 30 s at 72 °C; 5 min at 72 °C (Table S1). The amplified fragments were sequenced after treatment with 1 ll of ExoSAP-ITâ (USB Corporation) for 15 min at 37 °C. Cycle sequencing of the treated PCR products was produced using the same PCR primers and the Big Dye version 3.1 kit (Applied Biosystems). Sequencing reactions, after precipitation with EDTA in Ethanol 100% and Ethanol 70%, were loaded on an ABI 3100 Avant capillary sequencer (Applied Biosystems). Sequences were aligned, and polymorphisms were detected using CODONCODE ALIGNER (http://www.codoncode.com/aligner) with the genomic rabbit MLPH sequence used as reference. All sequence chromatograms were visually inspected. Nomenclature of identified polymorphisms was based on the system coordinates of the
unassembled scaffold (Scaffold GL018840) in the OryCun2.0 genome version containing the rabbit MLPH gene (http://www.ensembl.org/Oryctolagus_cuniculus/ Info/). In silico functional analysis of the identified missense mutations was carried out using PANTHER (Thomas et al. 2003). Genotyping of MLPH polymorphisms and data analysis PCR-RFLP was used to genotype the g.549853delG polymorphism in the half-sib families and in the 198 rabbits of 23 different breeds. Briefly, the amplified product obtained using a mismatched forward primer that inserted an artificial restriction site for DdeI when the produced amplicons containing the deletion were digested with this restriction enzyme (Table S1). Digestion reaction was carried out overnight at 37 °C in a 25-ll reaction volume including 5 ll of PCR product, 19 restriction enzyme buffer and 2 U of DdeI (MBI Fermentas). The complex indel in intron 1 was analysed by fragment analysis in the half-sib families and in a subset of the rabbits (n = 100) of different breeds (Table S2). The resulting DNA fragments were electrophoresed on 10% 29:1 polyacrylamide/bis acrylamide gel and visualized with 19 GelRed Nucleic Acid Gel Stain (Biotium, Inc.). Calculation of the LOD score between coat colour phenotypes and MLPH geno-
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MLPH and dilute locus in rabbits Table 1 Breeds and animals genotyped for the melanophilin (MLPH) g.549853delG mutation.
Breed (No. of animals) Alaska (3) Belgian Hare (1) Blue Vienna (40) Burgundy Fawn (9) Californian (22) Castor Rex (16) Champagne d’Argent (15) Checkered Giant (20) Coloured Dwarf (5) Dutch (6) English Spot (9) Fairy Marburg (1) Fairy Pearly (2) Giant Chinchilla (12) Giant Grey (9) Giant White (4) Lop (2) New Zealand White (6) Rhinelander (7) Silver (5) Tan (1) Thuringian (1) White Vienna (2) Total (198)
MLPH:g.549853delG genotype2,3
Coat colour and [proposed genotype at the dilute locus]1
G/G
Self-black [D/-] Reddish laced with black [D/-] Dark blue [d/d] Fawn [D/-] White with black (20) [D/-] or blue markings (2*) [d/d] Brown-black with yellow agouti band (8) [D/-] or ash/blue coloured with very light yellow agouti band (8*) [d/d] Silver as surface colour and black as under-colour [D/-] White with black (19) [D/-] or blue markings (1*) [d/d] Bristle white (1) [?]; hare-grey (2) [D/-]; Havana (1) [D/-] chinchilla (1) [D/-] With black markings [D/-] White with black (6) [D/-] or blue (3*) [d/d] markings Grey-light blue [d/d] Pearling grey [d/d] Chinchilla [D/-] Wild grey [D/-] White albino [?] Wild grey [D/-] White albino [?] White with black and yellow markings [D/-] Black with silvering [D/-] Black fire [D/-] Shaded yellow/brown [D/-] White-blue eyes [D/-]
G/del
del/del
3 1 9 18 8
2 -
40 2* 8*
15 16 3 6 4 12 9 4 2 6 7 5 1 1 2 132
3 2 2 9
1* 3* 1 2 57
1
In breeds for which animals of different coat colour have been sampled, the number of the rabbits showing the distinctive phenotypes is indicated with rounded brackets and linked with the genotyped animals with an asterisk (*). Squared brackets contain the proposed genotype at the dilute locus, when reported or deduced from the literature (Castle et al. 1909; Castle 1930; Robinson 1958; Fox 1994). 2 Genotypes obtained by PCR-RFLP for the MLPH:g.549853delG mutation. The normal allele is indicated with ‘G’, the mutated allele is indicated with ‘del’. 3 The number of animals with the different genotypes is reported.
types in the half-sibling families was obtained with the LODS program (Linkage Utility Programs, Rockefeller University).
Results and discussion Coat colour segregation in the two F1 families obtained by crossing a blue-spotted Checkered Giant buck (genotype d/d at the dilute locus) with two black-spotted Checkered Giant does (genotype D/d at the dilute locus) did not deviate from the expected 1:1 Mendelian ratio (seven black and nine blue rabbits; Fig. 2). All F1 rabbits obtained by crossing a blackspotted doe, with a putative D/D genotype, with the same blue-spotted Checkered Giant buck had black coat colour. These data confirmed the recessive mode of inheritance of the blue against the black coat colour as already reported from early genetic studies in rabbit (Castle et al. 1909; Castle 1930). Spotted inheritance is controlled by another locus (Fontanesi et al. 2010c). As a first step, to identify polymorphisms in the rabbit MLPH gene that could help to evaluate if this gene would be a putative causative gene of the dilute phenotype by segregation analysis, we first amplified a few regions in
the parental animals of the F1 families. Using primer pair 1a (Table S1), a three-allele complex indel locus (that was subsequently characterized by sequencing), derived by the duplication, presence or absence of a 29-bp sequence in intron 1 (with a few other nucleotide differences), was identified (Fig. S2). The two D/d does had genotype 1/2 (amplified fragments: 443 bp, allele 1; 386 bp, allele 2) and 2/3 (amplified fragments: 386 bp, allele 2; 359 bp, allele 3), whereas the blue buck (d/d) was homozygous for allele 2 (Fig. S2). This polymorphic site was then analysed in all F1 rabbits resulting in a complete cosegregation between the two coat colours and genotypes at this variable region (h = 0.00; LOD = 4.82): all blue rabbits were homozygous for the same allele that was homozygous in the blue-spotted buck, whereas the black F1 animals were either 1/2 or 2/3 (Fig. S2 reports a few F1 animals with their gel electrophoresis patterns at this polymorphic site). This marker made it possible to show that the most likely gene affecting the dilute coat colour in rabbit is the MLPH gene, as already reported in cat (Ishida et al. 2006) and dog (Philipp et al. 2005; Dr€ ogem€ uller et al. 2007). However, sequence analysis of the three alleles could not identify an obvious mutational event that might directly affect
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Fontanesi et al. functionality of the MLPH gene or protein. This polymorphism was due to intronic differences that did not affect the coding region of exon 2 (Fig. S2). This complex indel was genotyped in 100 rabbits of different breeds with different coat colour (Table S2). All blue rabbits were homozygous 2/2 but not all 2/2 rabbits had a diluted coat colour, indicating that allele 2 in the F1 families was in linkage disequilibrium with the true causative mutation. For example, a few Champagne d’Argent rabbits were homozygous 2/2 but their background colour was black, and all these animals were completely black early in their life (Table S2). Genotype 2/2 also was observed in one Alaska, one Californian, one Checkered Giant, one Giant Grey and one Rhinelander that did not have any diluted coat colour (Table S2). To identify the causative mutation of the dilute phenotype, we amplified and resequenced a total of 6357 bp of the rabbit MLPH gene (that corresponds to a sum of discontinuous gene fragments; sum is referred to the reference OryCun2.0 gene sequence ENSOCUG00000016496; Fig. S1) in animals with different coat colours (including parental animals of the F1 families). Sequenced regions encompassed non-coding regions (5′-flanking region including the putative 5′- UTR, the putative 3′-UTR and intronic regions mostly adjacent to coding exons) and all 15 exons identified in Ensembl (Fig. S1), except two putative close exons, exons 6 and 7 of this assembled sequence (considering the order of the reported coding regions; positions from 550665 and 550880 of the scaffold GL018840), that we were not able to amplify, despite four different primer pairs and their combinations (forward with a pair and reverse with another pair) that were tested. BLASTN analysis of these two putative exons did not show any matches with any other sequences. Taken together, this evidence might suggest that (i) the putative exons 6 and 7, automatically deduced in the reference sequence, do not exist in the investigated rabbits (even if it could be present in the Thorbecke rabbit that originated the OryCun2.0 genome version), or that (ii) assembling errors have included this region within the rabbit MLPH gene. Obtained sequences have been deposited in EMBL/GenBank databases under the accession numbers HG380502 and HG380503 (indicated below as haplotype 1 and haplotype 2 respectively). Comparing the sequences obtained in different rabbits, a large number of polymorphisms (n = 165) was identified (Table S3). On average, one polymorphism was present every ~39 bp. Most of these polymorphisms were in noncoding regions (5′-flanking including 5′-UTR, n = 8; the putative 3′-UTR, n = 16; and intronic regions: n = 113) and 28 were in coding exons (one every ~51 bp). Of these latter polymorphic sites, 15 were synonymous substitutions and 12 produced potential amino acid substitutions located in exon 1 (p.Val18Ile), exon 2 (p.Gln71Arg or p.Gln71Trp, depending on the combination with the previous SNP, Table S3; and p.Thr87Ala), exon 3 (p.Gln146His), exon 5
(p.Trp204Arg), exon 8 (p.Gly307Ala and p.Val311Ala), exon 9 (p.Lys349Arg), exon 10 (p.Ser393Gly) and exon 12 (p.Asp481Ser). PANTHER analysis (Table S3) indicated that none of these amino acid substitutions could have a deleterious effect. The only substitution that could modify the protein function was the p.Glu71Trp amino acid change (subPSEC = 4.370; Pdeleterious = 0.797). However, none of these polymorphisms occurred only in blue rabbits. Most of the identified polymorphisms defined two frequent haplotypes (referred as haplotype 1 and haplotype 2 in Table S3; clearly identified because several animals were homozygous at the polymorphic positions), not fixed in any breed sequenced. A few other minor haplotypes, resulting from putative multiple recombination events between the two major haplotypes, were identified (data not shown). These haplotypes differed from the sequence reported in OryCun2.0, derived from a partially inbred albino Thorbecke strain by several recombination events (Table S3). Among the 28 polymorphisms identified in the coding region, the remaining and most interesting was a deletion of one nucleotide in exon 5 (g.549853delG) that causes a shift in the reading frame that determines a completely different protein from the beginning of this exon (Fig. 3) until the second half of exon 8 in which a stop codon would be introduced (data not shown). By deducing the putative functional domains of the MLPH protein identified by PFAM, the mutated protein would maintain the N terminus FYVE zinc finger domain but would lose the Rab effector MyRIP/melanophilin C-terminus domain. This polymorphism was the only one that was homozygous in the sequenced blue rabbits; it was heterozygous in the d allele carrier black rabbits and was absent in all other sequenced animals. This mutation occurred in a new haplotype that differed from haplotype 2 just at this position (Table S3). Genotyping the half-sib families confirmed the complete cosegregation of this frameshift mutation with the blue coat colour in the F1 rabbits (Fig. 2). This mutation was analysed in 198 rabbits of different breeds (Table 1). All Blue Vienna rabbits were homozygous for this deletion. Homozygous subjects for the deleted nucleotide were also spotted blue rabbits of the Californian, Checkered Giant and English Spot breeds. In these three breeds, a few spotted black animals were heterozygous for the g.549853delG mutation, further confirming the recessive behaviour of this allele and the fact that this allele is segregating in several rabbit breeds. Two lines of Castor Rex were sampled. All rabbits of the ‘normal line’ with brown-black coat colour were homozygous for the wild-type allele, whereas all rabbits of the ash/blue coloured line, as expected, were homozygous for the g.549853delG mutation. Animals of two other rare fancy breeds, Fairy Marburg and Fairy Pearly having grey-light blue and pearling grey coat colours respectively, were homozygous for the MLPH frameshift mutation. Again, their genotype could be expected according to their coat colour. All Champagne d’Argent rabbits
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MLPH and dilute locus in rabbits
wild type allele
dilute allele
Figure 3 Frameshift deletion (g.549853delG) identified in exon 5 of the rabbit melanophilin gene. The deleted nucleotide is indicated in red in the wild-type allele sequence. Underlined nucleotides are other two polymorphic positions identified in exon 5 (g.549876G > A and g.549877T > C; Table S3).
were homozygous for the wild-type allele (not carriers for the frameshift mutation) even if some of them were homozygous 2/2 at the intron 1 complex indel. In addition, heterozygous 1/2 rabbits at this polymorphic locus resulted in homozygous wild type at the g.549853delG position (Table S2). This means that recombination events created different haplotypes at the MLPH locus, as already suggested, that were present in rabbits with black or nondiluted coat colours. Considering the genotyped 198 rabbits of different breed and the occurrence of homozygous g.549853delG animals only in blue (diluted) rabbits, across breed association between the genotype at this mutation and coat colour (diluted or not diluted) was highly significant (P = 3.88E-51; Fisher exact test), further supporting a causative role of the deletion in exon 5. Summarizing, even if we could not formally exclude the presence of other mutations in the linkage disequilibrium with the frameshift mutation in exon 5, all these data strongly argue for the g.549853delG deletion being a causative mutation of the recessive d allele at the dilute locus in rabbits. In particular, only this mutation distinguished haplotype 2 (one of the wild-type haplotypes) from the d
haplotype (Table S3), suggesting that the presence of other mutations in the MLPH gene causing this phenotype is highly unlikely. In addition, the disrupting mutation in exon 5 is upstream to the two non-sequenced putative exons 6 and 7 (not confirmed by genomic amplification and BLASTN analysis). Therefore, even if other mutations could be present in downstream exons/regions, the effect of the g.549853delG deletion is upstream to all other eventually occurring downstream mutational events, making the frameshift mutation in exon 5 the first causative mutation. The identification of the MLPH gene as the responsible gene for the dilute coat colour locus in rabbit provides another natural animal model for the human Griscelli syndrome type 3, in addition to other farm animal species, for example the cat and the dog (Philipp et al. 2005; Ishida et al. 2006; Dr€ ogem€ uller et al. 2007). This human syndrome seems restricted to hypopigmentation only, but just a few studies have reported its effects (Menasche et al. 2003; Al-Idrissi et al. 2010). It could be interesting to investigate blue rabbits to further evaluate the role of the MLPH gene on pigmentation, hair morphology and formation.
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Fontanesi et al. Combining results obtained at the dilute locus with information on the ASIP and MC1R alleles that we previously obtained (Fontanesi et al. 2006, 2010a,b), it is possible to have a first picture of the genetic structure of coat colour loci in several rabbit breeds, characterized by different coat colours. Moreover, it is also possible to predict the effects of combinations of different mutations at these loci. For example, Blue Vienna rabbits have their specific coat colour because they are fixed for the black non-agouti frameshift mutation (Fontanesi et al. 2010a) and for the dilute recessive d allele determined by a frameshift mutation at the MLPH gene. The same genotypic structure is present in English Spot rabbits with blue markings (Fontanesi et al. 2010a). Blue markings in Checkered Giant and Californian are derived from the dilution of the black coat colour caused by the g.549853delG on a black background derived by the dominant black allele at the MC1R gene and/or by the recessive non-agouti allele at the agouti locus (Fontanesi et al. 2006, 2010a). There is no apparent difference between black markings derived by the extension and agouti loci; therefore, it seems that there is no apparent difference on the effect of the dilution on the two black-determining genotypes. However, fancy breeders suggest the existence of two types of dilution: one that gives a darker grey (or blue) and another one that gives a lighter grey (or blue). It could be possible that mutations in other loci modify the degree of dilution derived by the frameshift mutation of the MLPH gene.
Acknowledgements We thank Associazione Nazionale Coniglicoltori Italiani (ANCI) for its help during the collection of samples, Renate Regitz for providing several rabbit samples and INRA GenEsi unit (UE 1372, Le Magneraud, BP52, 17700 Surgeres) for providing rabbit samples from the two Castor Rex lines. This work was supported by University of Bologna funds (FAGenomicH project and RFO funds) and has been carried out in the framework of the COST Action ‘A Collaborative European Network on Rabbit Genome Biology – RGB-Net’ TD1101 (2011–2015).
Conflict of interests The authors have declared no potential conflicts.
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Supporting information Additional supporting information may be found in the online version of this article. Figure S1 Schematic representation of the rabbit melanophilin gene with sequenced regions and positions of the genotyped polymorphisms. Figure S2 Fragment analysis, segregation (partial F1 families) and sequence of the complex indel genotyped indel in intron 1 of the rabbit melanophilin gene. Table S1 PCR primers, PCR conditions and use of the obtained fragments. Table S2 Rabbits genotyped for the complex indel in intron 1 of the melanophilin gene. Table S3 Polymorphisms identified in the rabbit melanophilin gene.
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