The role of MC1R gene in buffalo coat color

SCIENCE CHINA Life Sciences • RESEARCH PAPER •

February 2010 Vol.53 No.2: 267–272 doi: 10.1007/s11427-010-0026-3

The role of MC1R gene in buffalo coat color MIAO YongWang1,2†, WU GuiSheng3†, WANG Lei2, LI DaLin4, TANG ShouKun5, LIANG JianPing2, MAO HuaMing2, LUO HuaiRong3 & ZHANG YaPing1,6* 1

Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091, China; 2 Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China; 3 Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China; 4 Yunnan Institute of Buffalo Science and Technology, Kunming 650021, China; 5 Animal Husbandry and Veterinary Station of Luxi city, Luxi 678400, China; 6 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China Received September 21, 2009; accepted October 12, 2009

Melanocortin-1 receptor (MC1R) plays a major role in pigmentation in many species. To investigate if the MC1R gene is associated with coat color in water buffalo, the coding region of MC1R gene of 216 buffalo samples was sequenced, which included 49 black river buffalo (Murrah and Nili-Ravi), 136 swamp buffalo (Dehong, Diandongnan, Dechang, Guizhou, and Xilin) with white and gray body, and 31 hybrid offspring of river buffalo Nili-Ravi (or Murrah) and swamp buffalo. Among the three variation sites found, SNP684 was synonymous, while SNP310 and SNP384 were nonsynonymous, leading to p.S104G and p.I128M changes, respectively. Only Individuals carrying homozygote EBR/EBR were black. The genotype and phenotype analysis of the hybrid offspring of black river buffalo and gray swamp buffalo further revealed that the river buffalo type allele EBR or the allele carrying the amino acid p.104S was important for the full function of MC1R. The in silico functional analysis showed that the amino acid substitutions p.G104S and p.M128I had significant impact on the function of MC1R. Above results indicate that the allele EBR or the allele carrying the amino acid p.104S was associated with the black coat color in buffalo. Buffalo, coat color, MC1R

Citation:

Miao Y W, Wu G S, Wang L, et al. The role of MC1R gene in buffalo coat color. Sci China Life Sci, 2010, 53: 267–272, doi: 10.1007/s11427-010-0026-3

The domestic water buffalo (Bubalis bubalis) is a member of family Bovidae, and is widely distributed throughout Asia, Southern Europe, South America and North Africa, used as draft, meat and dairy animals. The domestic water buffalo contains two major types, the river buffalo and swamp buffalo. The swamp buffalo is usually gray or dark gray in body color with white stockings and a light band or “collar” below the neck. The less common body color in swamp buffalo is solid white hair covering a pink skin (Figure 1). The most common body color in river buffalo is black, with brown or white patches sometimes (Figure 1). A

*Corresponding author (email: [email protected]; [email protected]) †Contributed equally to this work © Science China Press and Springer-Verlag Berlin Heidelberg 2010

previous observation indicated that the white body color might be conditioned by a single gene which is completely dominant to gray, and is partially dominant or epistatic to black, while black is completely dominant or epistatic to gray [1]. Until now, there are no genes reported to correlate with the coat color in buffalo, although the genetic mechanism of coat color has been extensively investigated in other animals [2–9]. As a member of the same family, the molecular genetics of coat color in cattle (Bos primigenius) was much clearer than that of water buffalo. In cattle, the coat color types were mainly regulated by E- and A-locus alleles, which encode MC1R and agouti proteins, respectively [2,10–12]. There are 4 major E-alleles found in cattle regulating diflife.scichina.com

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ferent pigmentation situations. The coat color in animals carrying wild type E+ alleles is dependent on other loci regulating the pigmentation [13], while ED, e, and EL alleles give black, red (e/e homozygote), and fawn to brown or grey coat colours, respectively [11,13,14]. However, in Yak (Bos grunniens), another member of the same bovidae family, the MC1R was reported to have no association with Yak coat color [15]. In the present study, we investigated if the polymorphism of MC1R gene is associated with coat color in buffalo.

1 1.1

Materials and methods Materials

A total of 216 ear or blood tissue samples with clear genetic background were collected from the black river buffalo Murrah (n=20) and Nili-Ravi (n=29), the swamp buffalo Dehong (n=62), Diandongnan (n=36), Guizhou (n=15), Dechang (n=13) and Xilin (n=10) with white and gray body. The hybrid offspring (n=31) of river buffalo Nili-Ravi (or Murrah) and swamp buffalo were also included in this study (Table 1). 1.2

Extracting genomic DNA

DNA was isolated from the samples with the regular phenol/chloroform method and stored at −20°C.

1.3

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PCR amplification and sequencing

The complete coding region of MC1R gene was amplified using forward primer 74U18: 5′-GGGCAACCGCACATC CAG-3′ and reverse primer 1292L20: 5′-GGTCTAGCCG ATCCTCTTTG-3′, in 25 µL reaction mixture containing 50-100 ng genomic DNA, 1.5 mmol/L MgCl2, 100 µmol/L dNTP, 0.5 µmol/L of each primer, and 1 U Taq polymerase (TaKaRa). Amplification was carried out with denaturing at 95°C for 3 min, followed by 35 cycles at 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min, and ended with extension incubation at 72°C for 10 min. PCR products were purified on spin columns (Watson BioTechnologies) and sequenced directly with two PCR primers and two inner primers MC-1R2-F: 5′-CATCCCTG ACGGGCTCTTTCTC-3′ and MC1R2-R: 5′-AGCACCTCT TGGAGCGTCTTCC-3′ [16]. The amplification primers were designed based on cattle MC1R gene sequence (GenBank accession no. AF445641) with Oligo 6 software (Molecular Biology Insights, Inc., Cascade, Colo.). 1.4

Sequences analysis and detection of SNPs

Sequences were examined and edited using the DNASTAR software (DNAstar Inc.) and submitted to GenBank (accession numbers: GQ359864-GQ359954 and GU121238GU121363). The position and number of SNPs as well as corresponding haplotypes were exported with Mega version 4.0 [17].

Figure 1 Buffalos with various coat color and corresponding genotypes. A, Gray swamp buffalo; B, black river buffalo (Nili-Ravi); C, white swamp buffalo; D, F1 black brown hybrids of black river buffalo with gray swamp buffalo; E, F1 hybrids of black river buffalo with white swamp buffalo; F, G and H, multi-generation hybrids (Fn) of river buffalo and gray swamp buffalo.


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Gene function prediction

The functional effects of the coding variants were estimated by PANTHER software [18,19], which uses a multiple alignment of a family of protein sequences with known functional information, arguing that the functional effects are correlated with the probabilities of the amino acids occurring at different positions in the protein family. The likely functional effect was quantified with a substitution position-specific evolutionary conservation (subPSEC) score, which was further expressed as Pdeleterious, the probability of a nonsynominous SNP being deleterious. Smaller (more negative) subPSEC scores or Pdeleterious indicate a higher likelihood of being deleterious.

2 2.1

Results Sequence Characterization of the MC1R Gene

The obtained coding sequences of river and swamp buffalo MC1R gene are 954 bp long and encode 317 amino acids. The sequences have 97% identity and the same length with wild type homologous cattle sequences, while the yak MC1R gene sequence was reported to be of 99% identity with cattle [15] (Figure 2). It is noteworthy that the anoa was thought to be a different subgenus in bovidae family, and its MC1R gene sequence has 100% identity with swamp buffalo [20] (Figure 2).


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Among the 216 individuals sequenced, a total of three variation sites (SNP 310, SNP384, and SNP618) were found in this study (Figure 2) (For convenience, a swamp buffalo sequence characterized by c.310G, c.384G and c.618G was used as reference sequence here. Mutations were scored in this study based on this reference sequence). SNP310 (c.310G>A) and SNP384 (c.384G>T) were nonsynonymous variations and caused p.G104S and p.M128I changes, respectively, while SNP618 (c.618G>C) was synonymous variation. The river buffalo differs from swamp buffalo in MC1R gene sequence by two nonsynonymous substitutions, c.310G>A (p.G104S) and c.384C>T (p.M128I), whereas the synonymous SNP618 (c.618G>C) was detected within the populations of swamp buffalo only. Comparison with swamp buffalo shows there was no polymorphism found in river buffalo. All the MC1R gene sequences from river buffalo were holding nucleotides c.310A (p.104S) and c.384T (p.128I) and could be assigned as allele EBR. Though the synonymous SNP618 (c.618G>C) existed in swamp buffalo populations, there was no polymorphism found at the protein level in swamp buffalo. Therefore all the MC1R gene sequences in swamp buffalo carrying c.310G (p.104G) and c.384G (p.128M) were defined as allele EBS. Thus, all the river buffalo and swamp buffalo are homozygotes with genotype EBR/EBR and EBS/EBS, respectively. As expected, the F1 hybrids crossed between river buffalo and swamp buffalo were heterozygotes with genotype EBR/EBS. While in multi-generation hybrid offspring (Fn) of river buffalo

Figure 2 Sequence variations of the MC1R alelles in buffalo. Alleles with nucleotide and amino acid information found in various buffalo types, such as river buffalo, swamp buffalo and multi-generation hybrid offspring of river buffalo and swamp buffalo (Fn hybrids), and other species within Bovidae family, such as anoa, yak and cattle are given. Dots (•) denote identity with the swamp buffalo MC1R allele EBS, which was used as a reference sequence. Numbering was scored relative to the first codon of the buffalo MC1R allele EBS. Amino acid substitutions are listed below the nucleotide information, followed by their structural domain information. Four known cattle MC1R alleles and three yak MC1R alleles were also included to demonstrate the divergences between the buffalo, yak and cattle sequences. The amino acid substitutions found in this study are boxed.

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and swamp buffalo, an individual that was homozygote carrying a new allele derived from the recombination between the two loci SNP310 and SNP384 was found. The intermediate type new allele carrying c.310A (p.104S) and c.384G (p.128M) was assigned as allele EBRS. The other two Fn individuals were both heterozygotes with genotype EBR/ EBRS and EBS/ EBRS, respectively.

which indicates the functional importance of this change. The subPSEC score and Pdeleterious of the variation p.M128I are −1.89112 and 0.24808, respectively, which indicates a less important functional impact on this change than on p.G104S change. The allele EBR with combined changes of both p.G104S and p.M128I might have more robust functional impact on MC1R.

2.2

2.3

Influence of SNPs on the function of MC1R

Both the amino acid substitutions p.G104S and p.M128I found in the present study are from nonpolar to polar changes, which are located in the first extracellular loop region and the third transmembrane region, respectively (Figure 2). These changes should have potential to change the conformation of MC1R, or/and change the ligandbinding activity directly [21]. To predict the functional impact of the coding variations found in MC1R gene, we compared the specific sites at which the variants occurred in evolutionarily related proteins and generated a likelihood score of functional impairment. The subPSEC scores are the negative logarithm of the probability ratio of the wild-type and mutant amino acids at a particular position and are continuous values from 0 (neutral) to about −10 (most likely to be deleterious). The subPSEC score and Pdeleterious of the variation p.G104S are −3.88051 and 0.70693, respectively, Table 1

The phenotype and genotype of coat color

As shown in Table 1, all the 49 black river buffalo individuals, Nili-Ravi and Murrah, are homozygotes with genotype EBR/EBR at MC1R locus (p.104S and p.128I) (Table 1, Figure 2). Whereas all the swamp breeds (58 White and 78 Gray), Diandongnan, Guizhou, Dechang, Dehong and Xilin, in spite of white and gray in coat color phenotype, have the genotype EBS/EBS, carrying the amino acids p.104G and p.128M. To determine if the river buffalo type allele EBR is also associated with the black coat color，the phenotype and genotype of the F1 hybrid offspring of gray swamp buffalo and river buffalo are determined. As expected, F1 individuals are all the heterozygote with genotype EBR/EBS and present black brown coat color (Figure 1), which indicates that the allele EBR is associated with black coat color. Further investigation of the genotype and phenotype of multi-generation hybrids (Fn) of swamp and river buffalo revealed

The sample information and the relationship between the buffalo coat color and the polymorphism of the melanocortin-1 receptor gene

Breeds or populations

Coat Color

Location

Dehong

White

Yunnan

Gray Xilin

White

Guizhou

White

Diandongnan

White

Guangxi

Gray Guizhou

Gray Yunnan

Gray Dechang

Gray

Sichuan

sample size

Haplotypesa)

Genotypes BS

BS

GGG GGC ATC AGC E /E

35

40

30

35

27

40

14

27

8

11

5

8

2

3

1

2

5

9

1

5

10

18

2

10

10

3

17

10

26

42

10

26

13

20

6

13

BR

E /E

Nili-Ravi

Black

Yunnan

29

58

29

Murrah

Black

Yunnan

20

40

20

F1 Hybridsb)

Black Brwon

Sichuan

9

9

F1 Hybridsb)

Black Brwon White or white gray

Yunnan

14

9

Yunnan

5

Yunnan

F1 Hybridc) Fn Multi-generation hybridsd) Total

Dark Brwon

BR

BR

E /EBS EBR/EBRS EBS/EBRS EBRS/EBRS

9

9

14

14

5

5

5

3

1

1

4

216

210

127

4

5

91

136

49

28

1

1

1

1

1

1

a) The haplotypes were defined based on the nucleotide differences appearing at the three variation sites, SNP310, SNP384, and SNP618 e.g. the haplotype ATC represents the sequence with the nucleotides at positions 310, 384, and 618 were A, T, and C, respectively; b) hybrid offspring of gray swamp buffalo and black river buffalo; c) hybrid offspring of white swamp buffalo and black river buffalo; d) Multi-generation hybrid offspring (Fn) of black river buffalo and gray swamp buffalo.


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that the Fn individuals with genotype EBS/EBRS, EBRS/EBRS or EBR/ EBRS have coat color varying from brown to dark brown, respectively (Table 1, Figure 1). Thus, the river type allele EBR, especially the amino acid p.104S in allele EBR, is important for the normal function of MC1R.

3

Discussion

The characterization of MC1R gene in buffalo showed that compared with yak, the buffalo is less closely related with cattle, which is consistent with their phylogentic status [22,23]. As a member of different subgenera, the close relationship of anoa with swamp buffalo was quite unexpected. The synonymous variation sites of MC1R gene were more than the nonsynominous variation sites among different bovine species, indicating a functional conservation of MC1R within the Bovidae family (Figure 2). The allele EBRS found in the multi-generation hybrid offspring of river buffalo and swamp buffalo indicates a recombination event between two closely adjacent loci SNP310 and SNP384. A previous study identified the MC1Re allele with high frequency of 20% in river buffalo [24]. In this study, we failed to find the MC1Re allele either in river buffalo or swamp buffalo. In fact, none of any other mutation events presented in cattle and yak was found in buffalo sequenced here. As we have shown that the buffalo MC1R gene sequences are quite different from cattle homologues. Therefore, according to the MC1R alleles found in cattle to infer their role in coat color in buffalo might not be appropriate. In this study, the synonymous substitution c.618C>G occurs only within Chinese breeds or swamp buffalo, such as Dehong, Xilin and so on, and also occurs in individuals with the same color, such as white and gray. Therefore, the results of this study support that c.618C>G is not associated with either breeds or coat color. The melanocortin 1 receptor (MC1R) was found to play a major role in pigmentation in many species through regulating the synthesis of two major pigment components, Eumelanin and phaeomelanin [2–7,9,25,26]. Stimulation of MC1R by endogenous agonist α-melanocyte-stimulating hormone (α-MSH) leads to production of eumelanin through activating tyrosinase, and gives black coat color. Under the low level of tyrosinase without MC1R stimulation, either caused by a non-functional MC1R or inhibited by antagonist agouti protein, leads to the dilution of eumelanin and gives light coat color, such as brown or red [12,27,28]. The white and gray swamp buffalo carry the same genotype EBS/EBS in this study, which indicates that the MC1R has no association with white and gray coat color in buffalo. The white and gray coat color might be regulated by other loci that were on the upstream of the signal pathway controlling the production of eumelanin and phaeomelanin, such as the migration and cellular genesis of melanocytes. This is similar with the pigmentation regulation


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mechanism in cattle [13]. The F1 hybrid offspring of white swamp buffalo with black river buffalo were either white or white gray with genotype EBR/EBS, which is consistent with previous observation that the white body color is partially dominant or epistatic to black and was regulated by different loci other than MC1R (Figure 1 and Table 1) [1]. Only individuals that are the homozygote with genotype EBR/EBR (containing amino acid p.104S and p.128I) are black. The F1 hybrid offspring of black river buffalo with gray swamp buffalo have black brown coat color and are carrying one copy EBR allele. A previous observation suggested that black is completely dominant or epistatic to gray [1]. Our observation showed that the black component is slightly diluted in the F1 individuals, which carry the black brown coat color. The swamp type allele EBS might impair the function of MC1R in a certain degree, leading to the diminished production of eumelanin and the production of phaeomelanin. The Fn individuals have black brown coat color and carry either one copy of EBR allele or alleles containing amino acid substitution p.G104S, confirming that only one copy of allele containing p.104S cannot maintain the full function of MC1R. The in silico functional analysis showed that the substitutions p.G104S and p.M128I have significant impact on the functional changes, which supports the linkage of allele EBR, especially the substitution p.G104S with black coat color. In this study, we showed that the allele EBR or the SNP104 of MC1R gene was associated with black coat color in buffalo. Except MC1R gene, many other genes involved in the biosynthesis of melanin, especially the genes coding for proteins essential for pigmentation and the biology of melanocytes, participate in the regulation of coat color [7,8]. Thus, further efforts to investigate the variation of other candidate genes and the functional assay of the variants found are necessary to reveal the molecular mechanism of coat color in buffalo. This work was supported by the National Natural Science Foundation of China (Grant Nos. 30660024 and 30621092), the National Major Projects for Transgenic organisms (Grant No. 2008ZX08009), Natural Science Foundation of Yunnan Province, China (Grant Nos. 2007C0003Z and 2006C0034M) and the National Hi-New Technology Research and Development Plan of China (Grant No. 2008AA101001).

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