Journal of Heredity Advance Access originally published online on August 31, 2005
Journal of Heredity 2005 96(5):607-613; doi:10.1093/jhered/esi096
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Brief Communication |
Independent Nonframeshift Deletions in the MC1R Gene Are Not Associated with Melanistic Coat Coloration in Three Mustelid Lineages
From Taikyu High School, 1985 Yuasa-cho, Arida-gun, Wakayama 643-0004, Japan (Hosoda); Laboratory of Animal Cell Technology, Faculty of Life Science and Technology, Fukuyama University, Higashimura-cho, Aza, Sanzo, 985 Fukuyama, 729-0292, Japan (Sato); Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan (Shimada and Suzuki); and Department of Zoology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 (Campbell)
Address correspondence to Dr. Hitoshi Suzuki at the address above, or e-mail: htsuzuki{at}ees.hokudai.ac.jp.
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Abstract |
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Sequence variation within the 5' flanking (about 240 bp) and exon regions (426 bp) of the melanocortin-1 receptor (MC1R) gene was examined to determine the potential role of this protein in the melanistic coat coloration of 17 mustelid species in four genera: Gulo (wolverines), Martes (martens), Mustela (weasels), and Meles (badgers). Members of the genera Mustela and Meles, together with Martes flavigula and Martes pennanti, were shown to have intact gene sequences. However, several "in frame" deletions of the MC1R gene region implicated in melanism of other species were detected within members of the genera Martes and Gulo. For instance, Gulo gulo possessed a 15 bp deletion in the second transmembrane domain coding region, while Martes americana, Martes melampus, Martes zibellina, and Martes martes shared a 45 bp deletion overlapping this area. In addition, Martes foina was found to possess a 10 bp insertion followed closely by a 28 bp deletion immediately downstream of the deletion found in other martens. Notably, none of these indels was associated with a melanistic phenotype. Phylogenetic analysis revealed that each of these nonrandomly distributed deletions arose independently during the evolution of this family. Specific indel-neighboring motifs appear to largely account for the biased and repeated occurrence of deletion events in the Martes/Gulo clade.
Mammals display conspicuous variation in pelage coloration. Not surprisingly, several genes have been linked to the differential expression of pigments within the melanocytes of a broad range of species (Eizirik et al. 2003; Rieder et al. 2001). One such locus, encoding the transmembrane melanocortin-1 receptor (MC1R), is thought to play a major role in red-yellow (phaeomelanin) and black-brown (eumelanin) melanization (MacDougall-Shackleton et al. 2003). Indeed, numerous amino acid substitutions within the coding region of this gene have been reported to alter the coat coloration of laboratory mice (Robbins et al. 1993), pocket mice (Nachman et al. 2003), horses (Johansson et al. 1994; Marklund et al. 1996; Rieder et al. 2001), cattle (Joerg et al. 1996; Klungland et al. 1995), foxes (Våge et al. 1997), cats (Eizirik et al. 2003), dogs (Everts et al. 2000; Newton et al. 2000; but see Kerns et al. 2003), and several species of birds (Mundy et al. 2004). These MC1R gene polymorphisms are classified as either loss-of-function or gain-of-function mutations, each resulting in red-yellow or black-brown coat colorations, respectively (Robbins et al. 1993). For example, point mutations in the second (Glu92Lys) and third (Cys125Arg) transmembrane domains are associated with melanism in mice (Robbins et al. 1993) and red foxes (Våge et al. 1997), respectively. Similarly Eizirik et al. (2003) suggested that 15 bp (codons 100–105) and 24 bp (codons 95–102) deletions within this region of the gene are responsible for intraspecific gain-of-function mutations in jaguars and jaguarundies, respectively. Notably, an eight amino acid deletion identical to that found in jaguarundies has also been linked to the melanic phenotype of golden-headed lion tamarins (Mundy and Kelly 2003).
Within the family Mustelidae, weasels, minks, and martens exhibit remarkable intra- and interspecific pelage color variations (Anderson 1970; Thomas 1897). For example, two distinct populations of Japanese marten (Martes melampus) are easily identified by coat coloration: a yellow phenotype more common to the Japanese islands and a dark brown phenotype particular to populations in the Tsushima and Shikoku Islands and the Kii peninsula (Hosoda and Oshima 1993). In addition to these color variants, most weasels undergo seasonal molts that involve dramatic alterations in color, though this trait tends to be limited to northern populations within each species (Nowak 1999). Although numerous studies have been completed on the genetic systems that affect coat coloration [see Rees (2003) for a review], less attention has been focused on the evolution of melanistic pelage traits (Eizirik et al. 2003; Mundy and Kelly 2003). In addition, almost nothing is known regarding the genetic basis of seasonal changes in pelage coloration common to several north-temperate mammalian groups.
Here we examined sequence variations in a portion of the MC1R gene in 17 mustelid species as an initial step for such genetic investigation. Evolution of the MC1R gene within each of these lineages was inferred by mapping coat coloration and genetic traits onto a molecular phylogenetic tree constructed from mitochondrial (Hosoda et al. 2000) and nuclear (Sato et al. 2003) gene sequences.
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Materials and Methods |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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Amplification and Sequencing of DNA
Seventeen mustelid species (36 individuals) were examined in this study (Table 1). We designed primers for the 5' upstream and exon regions of the MC1R gene using dog (Everts et al. 2000) and fox (Våge et al. 1997) sequences. Base pair numbers in primer names refer to nucleotide positions of the genomic canine MC1R sequence in GenBank (AF064455; Everts et al. 2000). In the 5' flanking region, primers used for the first round of amplification were 5'MC1R-70 (5'-AAACGTACGTCTAACCTGAGCAA-3') and 3'MC1R-469 (5'-GCTCACCAGCCCCAGGCTGAGGAA-3'). The second primer pair, for polymerase chain reaction (PCR), was 5'MC1R-70 and 3'MC1R-339 (5'-GTTGGGAATGGACACCTCCAGGCA-3'). For exon amplification, primers used in the first PCR were 5'MC1R-302 (5'-GATGAGCTGAGCGGGACGCCTG-3') and 3'MC1R-772 (5'-GGTATCGCAGCGCGTAGAAGATG-3'). The primers for the second PCR were 5'MC1R-322 (5'-CTGCGAGTGAGGACCCCTTTCTG-3') and 3'MC1R-772. Following an initial denaturation cycle (94°C for 3 min), cycling conditions were 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C (30 cycles), followed by a final 5 min extension period at 72°C. Products of the second PCR were sequenced directly according to the manufacturer's instructions with a Big Dye Terminator Cycle Sequencing Kit on an ABI 3100 Genetic Analyzer. Sequences obtained in this study were deposited in international DNA databases (Table 1).
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Data Analyses
Maximum parsimony (MP; Swofford and Olsen 1990) phylogenetic trees were constructed using PAUP* version 4.0b10 (Swofford 2001) from a concatenated dataset incorporating published sequences of the mitochondrial cytochrome b (cyt b; 1140 bp) and nuclear interphotoreceptor retinoid binding protein (IRBP; 1185 bp) genes of the 17 mustelid species (Hosoda et al. 2000; Sato et al. 2003, 2004). The Eurasian badger (Meles meles) was employed as an outgroup on the basis of previous phylogenetic hypotheses (Sato et al. 2003, 2004) and the analysis was conducted using 100 heuristic tree-bisection reconnection searches in which the input order of taxa was randomized and based on the following character weighting: equally weighted nucleotide substitutions (IRBP), transversions only at the third codon positions, and all nucleotide substitutions at the first and second codon positions (cyt b). Bootstrap proportions (BS; Felsenstein 1985) were obtained by generating 1000 heuristic replicates with PAUP, each consisting of 100 heuristic tree-bisection reconnection searches in which the input order of taxa was randomized. Finally, synonymous and nonsynonymous nucleotide changes along the MC1R exon were mapped onto the tree with the aid of MacClade version 4 (Maddison and Maddison 2000). Although the cyt b/IRBP tree supported a monophyletic relationship between Mustela altaica and Mustela nivalis with moderate bootstrap values (75%), this relationship was not parsimonious after accounting for the MC1R gene sequences of each species. Consequently the topology was modified slightly (see dashed lines of Figure 1) to minimize the number of substitution events required.
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The number of synonymous substitutions per synonymous sites (dS) and nonsynonymous substitutions per nonsynonymous sites (dN) among coding region sequences were computed with MEGA2 (Kumar et al. 2001) using a modified Nei-Gojobori method (Nei and Gojobori 1986; Zhang et al. 1998) with the Jukes and Cantor (1969) model. The 10 bp insertion in the Martes foina MC1R sequence was excluded from this analysis.
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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Nucleotide sequences of the upstream flanking (238–241 bp) and the 5' exon region (382–427 bp, 127–142 codons) of the MC1R gene were determined from 17 mustelid species (Table 1). Unfortunately we were unable to obtain sequences upstream of the MC1R gene for the yellow-throated marten (Martes flavigula). Among the mustelids examined, intraspecific variations were found in some species, but not in others. For instance, gene sequences for the stone marten (M. foina) from Primorye, Russia (n = 2) and south China (n = 1) differed at three nucleotide positions: 29, 73, and 229 (site numbers counted from A of the initiation codon, ATG, in the coding region). The MC1R sequences of three sables (Martes zibellina) from Hokkaido possessed three variable sites, one in the upstream region (site –180) and two in the coding region (sites 104 and 203), while the coding sequences of the two M. flavigula specimens exhibited one base substitution (site 363). In contrast, the five individual pine martens (Martes martes) possessed matching sequences. Similarly the 11 Japanese martens showed no variation along the region of the MC1R gene examined, despite exhibiting seasonal differences in coat color (yellow, n = 6; dark brown, n = 5).
Comparative analyses among sequences of the 17 mustelid species revealed remarkable divergence in both the upstream and coding regions of the MC1R gene with respect to both nucleotide substitutions and insertion/deletion (indel) events (Table 1 and Figure 1). The sequence alignment revealed that, excluding indels, 18.6% of sites (45 of 242 bp) were variable in the 5' flanking region. Unexpectedly a high percentage of variable sites (68 of 427 bp, 15.9%) were observed in the exon sequence, even in regions encoding functional domains. Excluding indels, 29 nucleotide substitutions were identified among the eight species of the Martes/Gulo clade (Figure 1). Notably substitutions at the first (8 sites) and second (15 sites) codon positions occurred more frequently than the third position (6 sites), leading to amino acid substitutions at 23 residues. Remarkably all but one intraspecific nucleotide variation identified within this clade was found to be nonsynonymous (white bars of Figure 1). Among the eight species of the Mustela clade, 31 nucleotide substitutions were detected, with 12, 8, and 11 substitutions at the first, second, and third codon positions, respectively, leading to amino acid substitutions at 21 residues (Figure 1). Considering the large proportion (74% to 79%) of nucleotide substitutions leading to amino acid changes, nonsynonymous substitutions appear to dominate the history of the MC1R gene in mustelids. However, while the mean dN/dS ratios were 1.47 and 0.97 in the Martes/Gulo and Mustela lineages, respectively, we could not detect a clear trend for positive selection (dS < dN) at the P < 0.05 level. These results suggest that the MC1R coding region of mustelids has been subjected to either diversifying selection, supporting the previous notion based on human MC1R gene sequences (Rana et al. 1999), or relaxation of functional constraints in the MC1R gene.
Numerous amino acid substitutions have been associated with changes in MC1R function in a broad range of mammal species. For instance, Glu92Lys is thought to induce black coat coloration in mice (Robbins et al. 1993). We found an identical substitution at the corresponding site (codon 94) in the six derived species of Mustela (Figure 1). However, none of the six individuals examined showed signs of melanism (Table 1), suggesting the substitution Glu94Lys in mustelids is unlikely to lead to the same functional change as that of the murine rodent. Similarly M. martes possessed a single substitution at codon 125 (Cys125Arg) that is known to cause a functional change in red fox coloration (Våge et al. 1997). However, there seems to be no substantial similarity in coat colors of M. martes with the variant color (silver) of Vulpes vulpes (Våge et al. 1997). Two additional substitutions implicated in the dominant melanistic phenotypes of cattle and pigs (Leu99Pro) (Kijas et al. 1998; Klungland et al. 1995) and sheep (Met73Thr) (Våge et al. 1999) were detected in our American mink (Mustela vison) and badger (M. meles) sequences, respectively. Again, neither of these mustelids showed signs of melanism (Table 1). In contrast, amino acid changes that may be associated with a loss-of-function mutation in the MC1R gene were observed in one M. zibellina specimen. This homozygous individual (TH047HS645), with a rare light yellow coat color, was found to have two substitutions (Cys35Phe and Asn68Ser) compared to one of the wild-type individuals (TH043HS641), while the second, gray brown sable (TH053HS805), was heterozygous at site 68 (photographs comparing the rare type with the common dark brown phenotype are available upon request).
In addition to the high rate of nonsynonymous substitutions within the Martes/Gulo clade, our MC1R sequence alignment revealed that at least four independent indel events occurred in the coding region of this gene during the evolution of this group (Figure 1). The first, a 15 bp deletion found in the second transmembrane domain (codons 94–98) of the Gulo gulo MC1R, presumably followed the divergence of wolverines from the Martes lineage (Figure 2). A second 45 bp in-frame deletion overlapping this region (codons 98–112) occurred before the radiation of Martes americana, M. martes, M. melampus, and M. zibellina (Figure 2). Finally, two indels were detected in the stone marten MC1R gene sequence: a 10 bp duplication immediately downstream of that found in the four other marten species, followed closely by a 28 bp deletion (Figure 2). Interestingly, these indels all occur in a gene region implicated to be involved in melanism of jaguarundies and golden-headed lion tamarins (codons 95–102) (Eizirik et al. 2003; Mundy and Kelly 2003) and jaguars (codons 101–105) (Eizirik et al. 2003). However, none of the mustelid MC1R indels appear to represent melanistic gain-of function mutations. Consequently our data suggest that deletions near the end of the second transmembrane domain are not always associated with phenotypic changes in coat coloration.
Our multiple alignment (Figure 2) of the MC1R exon further suggests that certain nucleotide motifs are associated with the relatively high incidence of indels that we detected. Thus it is reasonable to predict that the independent deletion events observed in the three mustelid lineages are related to specific nucleotide arrangements within the MC1R gene sequences. We observed three notable features associated with the observed deletion events. First, two of the three deletion events were associated with hexanucleotide direct repeats at both ends of each deletion (Figure 2). A similar pattern was found in the MC1R gene of jaguarundies (Eizirik et al. 2003), supporting the hypothesis of polymerase slippage due to slipped-strand mispairing (Nishizawa and Nishizawa 2002; Taylor et al. 2004). The second feature relates to the deletion of a repeated triplet at the 3' end of each deletion event, while the third is the presence of short palindromic regions near the middle of each deleted segment. Notably one or two of these possible structural prerequisites favoring a deletion event are not found in the corresponding sequence region of the weasel lineages (Figure 2). Thus the consistent patterns around the deleted regions may provide useful insight into the molecular basis of deletion events that have occurred during the evolution of mammalian genomes.
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Acknowledgments |
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We thank Kay Fuhrmann, Sang-Hoon Han, Daniel J. Harrison, Mitsuhiro Hayashida, Sigeki Watanabe, Alexei P. Kryukov, Yoshitaka Obara, Kimiyuki Tsuchiya, and Ya-Ping Zhang for their help in collecting specimens. This study was supported by a grant-in-aid for scientific research (to H.S.) from the Japanese Society for the Promotion of Sciences (JSPS). Additional financial support (to T.H.) was provided by a Sasakawa Grant for Science Fellows (SGSF).
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Footnotes |
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Corresponding Editor: C. Scott Baker
Received December 1, 2004
Accepted July 12, 2005
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