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GENETICS |
National Livestock Breeding Center, Nishigo, Fukushima 961-8511, Japan
1 Corresponding author: e0kobays{at}nlbc.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: microsatellite marker • mapping • recessive white chicken • tyrosinase gene • avian retroviral sequence
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In humans and mice, the C locus has been genetically defined as the structural tyrosinase gene locus. In chickens, 3 alleles of albinism at the C locus have been reported. These types are red-eye white (cre), recessive white (c), and autosomal albino (ca; Brumbaugh et al., 1983; Smith, 1990). These 3 alleles all correspond to a phenotype of all-white plumage, whereas the wild type (C+) has strong pigmentation. A previous study using 2-dimensional gel electrophoresis suggested that the C locus is the structural locus for tyrosinase in the fowl (Oetting et al., 1985). The tyrosinase gene in chickens has been cloned by Mochii et al. (1992). Tobita-Teramoto et al. (2000) reported that a 6-nucleotide deletion at a Cu-binding site of the tyrosinase gene leads to the albino chicken (ca).
Some genes at the classical locus that are related to plumage color have already been identified by linkage analysis and sequence analysis. The Extension (E) locus affecting plumage color, black (eumelanin) and red (phaeomelanin), has been assigned to chicken chromosome 11 by linkage analysis using an intercross between the Red Jungle Fowl and the White Leghorn chicken (Kerje et al., 2003). This same report presented evidence that the gene of the classical E locus is the melanocortin 1 receptor gene. The dominant white locus influencing white plumage has been mapped to chicken E22C19W28 (Ruyter-Spira et al., 1996). Linkage analysis and sequence analysis has revealed that dominant white chickens are produced by the PMEL17 gene, which is the premelanosomal protein gene, located on chicken E22C19W28 (Kerje et al., 2004). The Dun and Smokey phenotypes have also been found to be caused by mutations in the PMEL17 gene (Kerje et al., 2004). Although there is a report that the insertion of retroviral sequence into tyrosinase gene associates completely the recessive white plumage (Chung et al., 2006), the C locus in chickens has been not correctly mapped to a chromosome by microsatellite markers covering the whole genome. The aim of the current study was to detect the candidate region and the gene of the recessive white in chicken by linkage analysis using an intercross between the Japanese Game and the White Plymouth Rock breeds and to examine the cause of the recessive white.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Japanese Game (colored plumage) and the White Plymouth Rock chicken (recessive white) lines are maintained at the Hyogo station, National Livestock Breeding Center. An F2 resource family was created by crossing 1 Japanese Game male and 7 White Plymouth Rock females. Two hundred sixty-five F2 chickens were produced by intercrossing 8 F1 males with 57 F1 females, with 5 to 12 full-sib females selected to mate with each male. Plumage color was observed in the F2 chickens at 9 wk of age. Samples from the White Plymouth Rock and Aizudidori (Japanese native chicken with the colored plumage, such as the Japanese Game) for the gene expression were provided by the Hyogo station National Livestock Breeding Center and Fukushima Prefecture Agricultural Institute (Fukushima, Japan), respectively.
Genotyping and Mapping
Microsatellite markers [498 USDA microsatellite Kits 1-2, 3, and 4 supplied by the Poultry Subcommittee of the National Animal Genome Research Program (USDA, Washington, DC), 666 ABR microsatellite markers (National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan), and 2 original markers created from the information from the UCSC Genome Browser supplied by the Genome Bioinformatics Group of the University of California Santa Cruz] covering the autosomal chromosomes and the Z chromosome were used to genotype the 8 grandparents, 57 F1 parents, and 265 F2 offspring. GeneScan 3.1.2 and Genotyper 2.5 software (Applied Bio-systems, Foster City, CA) were used to determine these alleles. Linkage analyses were performed using the program Map Manager QTX b18 (Manly et al., 2001) and the CRI-MAP version 2.4 (Green et al., 1994). The mapping of the recessive white was conducted with the QTL Express software program (Seaton et al., 2002).
Sequence Analysis
Genomic DNA was isolated from blood samples by a phenol-chloroform method, and the DNA concentration was adjusted to 20 ng/µL. The 5 tyrosinase exons were amplified by PCR from genomic DNA from the Japanese Game and the White Plymouth Rock. Five pairs of primers were produced based on the chicken tyrosinase gene information (UCSC Genome Browser on Chicken; alignment of AB023291 and chromosome 1:179545508 to 179595094; Table 1
). The PCR was performed in a total volume of 15 µL containing 20 ng of genomic DNA, 6.25 pmol of each primer, 0.2 mM each deoxynucleoside tri-phosphate (dNTP), 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.375 U of Taq polymerase. The PCR conditions were as follows: 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 5 min. The PCR products were sequenced by direct sequencing using the ABI PRISM 3730 DNA Sequencer and Sequencing Analysis 3.4 (Applied Biosystems). A primer set designed for exon 5 failed to detect the PCR products in the White Plymouth Rock. Therefore, a new 5' primer was designed to cover a 30-bp region upstream of exon 5.
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shows primer sets used to analyze intron 4 in the Japanese Game and the White Plymouth Rock. The intron 4 was segregated into 8 primer sets based on the tyrosinase gene information, because the full-length sequence of the intron 4 (6 kb) was not amplified in long-range PCR. The PCR for intron 4 was performed under the same conditions as for the tyrosinase exons. These products were directly sequenced in both breeds. For the primer sets that failed to amplify in the first PCR reaction, long-range PCR was performed in a total volume of 20 µL containing 100 ng of genomic DNA, 6.25 pmol of each primer, 0.1 mM each dNTP, 10 mM Tris · HCl (pH 8.0), 50 mM KCl, 0.05 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.25% Tween 20, 0.25% Nonidet P-40, 25% glycerol, 2.5 mM MgCl2, and 0.5 U of LA Taq polymerase (Takara, Otsu, Japan). The PCR conditions were as follows: 94°C for 1 min followed by 40 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 3 min, and a final extension at 72°C for 5 min. This fragment was cloned using the TOPO XL cloning kit (Invitrogen, Carlsbard, CA) and sequenced. Segregation of the Retroviral Sequence in the Tyrosinase Gene in F2 Chickens
Two hundred sixty-five F2 chickens were tested to identify homozygous carriers of the retroviral insertion. The PCR for 2 primer pairs was carried out in a 15-µL reaction volume containing 100 ng of genomic DNA, 6.25 pmol of each primer, 0.1 mM each dNTP, 10 mM Tris · HCl (pH 8.0), 50 mM KCl, 0.05 mM EDTA, 0.5 mM DTT, 0.25% Tween 20, 0.25% Nonidet P-40, 25% glycerol, 2.5 mM MgCl2, and 0.5 U of LA Taq polymerase (Takara). The PCR conditions were as follows: 94°C for 1 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, and a final extension at 72°C for 5 min. The allele types of the PCR products were determined by electrophoresis.
Reverse Transcription PCR and Sequencing of the Tyrosinase Gene
Total RNA was isolated from the skins of White Plymouth Rock and the colored chicken (Aizu-didori), using the Trizol reagent (Invitrogen). To ascertain whether the purified cDNA was synthesized, 5 µg of the DNase I (Takara)-treated total RNA was reverse transcribed with or without SuperScript III reverse transcription (Invitrogen). To investigate differences in the expression of the tyrosinase gene, reverse transcription PCR (RT-PCR) for each primer (Table 2) was performed in a total volume of 20 µL containing 6.25 pmol of each primer, 0.1 mM each dNTP, 10 mM Tris · HCl (pH 8.0), 50 mM KCl, 0.05 mM EDTA, 0.5 mM DTT, 0.25% Tween 20, 0.25% Nonidet P-40, 25% glycerol, 2.5 mM MgCl2, and 0.5 U of LA Taq polymerase (Takara). The PCR products were cloned into the pCR2.1 vector (Invitrogen) and sequenced with the ABI PRISM 3730 DNA Sequencer and Sequencing Analysis 3.4.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Table 3 shows plumage color patterns in the F2 chickens. The segregation ratio into white plumage and colored plumage in the F2 chickens did not different significantly from the expected 3:1 ratio.
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A total of 1,164 microsatellite markers (USDA markers and ABR markers) were initially tested for their information contents in the Japanese Game and the White Plymouth Rock. Markers at which the parental breeds shared alleles could not be used in the analysis of the F2 generation. A total of 225 informative markers were used to genotype the F2 resource family. Linkage analysis was performed in 240 F2 chickens, which were identified in genotypes of all the 225 markers. These markers were mapped to 26 linkage groups on 19 autosomal chromosomes (chromosomes 1 to 11, 13 to 15, 19, 20, and 26 to 28) and 2 linkage groups on the Z chromosome (Table 4). Chromosome 1 was segregated into 4 linkage groups (chromosome 1a, 1b, 1c, and 1d), chromosome 2 into 2 linkage groups (chromosome 2a and 2b), chromosome 3 into 3 linkage groups (chromosome 3a, 3b, and 3c), and chromosome 7 into 2 linkage groups (chromosome 7a and 7b). The linkage groups encompassed 1,724.9 cM, with average marker spacing for 7.7 cM.
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shows the genotypes of the marker used to determine gene order around the C locus. Based on the recombination between the markers, the most likely gene order and genetic distance were determined as follows: 163386361-(4.0 cM)-ABR631-(7.7 cM)-ADL0195-(7.1 cM)-C locus-(4.8 cM)-180893258. The C locus was mapped to the position between the ADL0195 and 180893258 markers. Furthermore, the logarithm of odds support for the assignment to chromosome 1 in the recessive white was very convincing (score = 128.0) for the position between the markers ADL0195 and 180893258 (data not shown). The physical distance between the markers ADL0195 and 180893258 was about 5.8 Mbp according to the whole genome sequence for the chicken (UCSC Genome Browser).
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There was a robust candidate gene, the tyrosinase gene, located between the ADL0195 and 180893258 markers. The tyrosinase gene, which is associated with the recessive white and albino phenotypes, was examined and compared between the Japanese Game (colored chicken) and the White Plymouth Rock (recessive white). Sequence analysis of the 5 tyrosinase exons using genomic DNA revealed some silent mutations (data not shown). Intron 4 of the tyrosinase gene was examined using original primer sets, because the PCR product for exon 5 of the tyrosinase gene was not easily obtained from the White Plymouth Rock chicken. The sequence orders of intron 4 in both breeds were somewhat different from that of Red Jungle Fowl, the sequence for which has been published previously. However, there was a remarkable difference between the recessive white chicken and the colored chicken in intron 4. A 7.5-kb retroviral insertion sequence was detected at the 3' end of intron 4 in the tyrosinase gene of the recessive white chicken (Figure 2) and showed 99% homology to the sequence of the endogenous avian leukosis virus ev-1.
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Table 3 shows the association between plumage color and the presence of the retroviral insertion sequence in F2 chickens. The white plumage F2 chickens were all homozygous carriers of the retroviral sequence in intron 4. No homozygous carriers of the retroviral sequence were observed in the colored F2 chickens. Various plumage colors were distributed in heterozygous carriers and non-carriers of the retroviral sequence in colored chickens. In these F2 chickens, sex differences were observed in black, brown, and barred plumage.
Tyrosinase Gene Expression and Detection of Splicing Variants Using RT-PCR
To investigate tyrosinase gene expression, cDNA obtained from the skin of recessive white chickens and colored chickens was amplified by RT-PCR. The PCR products from exon 1 to exon 4 were detected in both breeds (Figure 3). However, expression of exon 4 to exon 5 of the tyrosinase gene was substantially reduced in recessive white chickens compared with the colored chickens. The complete transcript, comprising exon 1 to exon 5, was not observed in the recessive white chickens. The presence of splicing variants was examined to assess the influence of the retroviral insertion sequence. Two aberrant RT-PCR products, where exon 3 to intron 4 were amplified, were detected in recessive white chickens; in 1 variant, exon 4 is followed by the first 772 bp of intron 4, and in the other variant, exon 4 is followed by a 360-bp region of intron 4 resulting from splicing at 1 bp to 1,278 bp of intron 4 (Figure 4). Furthermore, a new variant sequence was amplified by RT-PCR from the retroviral sequence 3' long terminal repeat to exon 5 in the White Plymouth Rock chickens (Figure 5). This sequence is the same as that of the tyrosinase genome DNA but was not detected in the cDNA synthesized from the colored chickens or the cDNA synthesized without reverse transcription in the recessive white chicken (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study has also presented further evidence that the gene corresponding to the recessive white plumage in chickens was the tyrosinase gene. The tyrosinase gene is located in the region between the ADL0195 and 180893258 markers. In humans and mice, the C locus has been genetically defined as the tyrosinase gene. More specifically, the association between albinism, a congenital lack of pigment in the hair, skin, and eyes, and the tyrosinase gene has been studied not only in humans and mice but also in other domestic animals. For example, in albino cattle, a single nucleotide insertion in tyrosinase gene exon 2, which causes a frame shift resulting in a premature stop codon at residue 316, was detected (Schmutz et al., 2004). Similarly, sequence analysis in albino cats revealed a cytosine deletion in the tyrosinase gene at position 975 in exon 2, which induces a premature stop codon 9 residues downstream from the mutation (Imes et al., 2006). In an albino chicken line (ca) with white plumage and pink eyes, a deletion of 6 nucleotides at 817 bp, which was located within the Cu A domain of the tyrosinase sequence, was observed (Tobita-Teramoto et al., 2000). Thus, previous studies in chickens have demonstrated that the albino phenotype is a result of gene mutations in the tyrosinase coding region. The recessive white chicken (c) has white plumage and black eyes. In the present study, sequence analysis of the 5 exons of the tyrosinase gene involved genomic DNA and revealed some silent mutations between the recessive white and colored chickens (data not shown). Therefore, the recessive white in chicken is not caused by differences in the tyrosinase gene expression or the changed form of the tyrosinase enzyme based on these mutations. The current results revealed that the recessive white mutation in chickens is not due to amino acid substitutions, deletions, or insertions in the coding region.
The white plumage F2 chickens exhibited homozygous alleles containing the retroviral sequence, in agreement with a recent report (Chung et al., 2006), whereas the colored F2 chickens were divided into 5 groups based on plumage color pattern. Remarkable sex differences were found in some plumage color groups. Two colored loci are known to be sex-linked: the silver (S) locus and the barred (B) locus. The S allele inhibits the expression of phaeomelanin. Kerje et al. (2003) estimated that the segregation of the silver locus accounts for the higher frequency of females with black plumage. In the current study, the frequency of black chickens was 14% among females and 0% among males. It is likely that the black plumage for the females is due to the S allele. The presence of the B allele was observed in barred plumage F2 chickens; 6% of the females and 30% of the males showed the barred color. However, the rate of segregation between males and females was different from that reported by Kerje et al. (2003). This segregation appears to be due to differences in the ratio between B/W and b+/W females. Thus, the plumage colors and the sex differences were not affected by the insertion of the retroviral sequence in colored F2 chickens.
The effect of the retroviral sequence insertion into the tyrosinase gene on plumage color was investigated. Association between the inserted retroviral sequence, ev21, and the sex-linked dominant feathering mutation (K) was described in a previous report (Bacon et al., 1988). The feathering gene has been located between markers ADL0022 and MCW0331 on the Z chromosome but has not yet been identified (Hamoen et al., 2001). The tyrosinase cDNA from exon 1 to exon 4 showed the same expression in the recessive white chicken and in the colored chicken, but the full-length tyrosinase transcript, from exon 1 to exon 5, was not detected in the recessive white chicken. Although there was limited expression of cDNA for exon 4 to exon 5, expression of the normal transcript including exon 5 was inhibited by the presence of the 7.5-kb retroviral sequence in intron 4. The 3' rapid amplification of the cDNA end was performed to find short sequences from intron 4 (from 124 to 140 bp) before terminating with a poly A tail as reported by Chung et al. (2006), but no transcripts with a poly A tail were found in the current study. However, aberrant transcripts from exon 3 to intron 4 and a retroviral insertion sequence at 3' long terminal repeat to exon 5 were detected in the recessive white chicken. These results suggest that the retroviral sequence insertion in the tyrosinase gene leads to the generation of various aberrant splicing patterns and the loss of tyrosinase function. However, in the recessive white chicken, the full-length tyrosinase transcript may be only weakly expressed. It is likely that expression of the normal transcript is not sufficient for the synthesis of melanin in pigment cells of the feather. Unlike the albino chicken, the subtle expression of the normal tyrosinase may result in the black eye phenotype in the recessive white chicken. In conclusion, it was confirmed that the recessive white gene was located on the region between the ADL0195 and 180893258 markers on chromosome 1 by linkage analysis using an F2 resource family and that a retroviral sequence insertion in the tyrosinase gene was the cause of the recessive white chicken phenotype.
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ACKNOWLEDGMENTS |
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Received for publication March 8, 2007. Accepted for publication June 25, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Brumbaugh, J. A., T. W. Barger, and W. S. Oetting. 1983. A "new" allele at the C pigment locus in the fowl. J. Hered. 74:331–336.
Chung, C. M., J. L. Coville, G. Coquerelle, D. Gourichon, A. Oulmouden, and M. Tixier-Boichard. 2006. Complete association between a retroviral insertion in the tyrosinase gene and the recessive white mutation in chickens. BMC Genomics 7:19.[Medline]
Green, P., K. Fallis, and S. Crooks. 1994. Documentation for CRI-MAP version 2.4. Washington Univ. Sch. Med., St. Louis, MO.
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Imes, D. L., L. A. Geary, R. A. Grahn, and L. A. Lyons. 2006. Albinism in the domestic cat (Felis catus) is associated with a tyrosinase (TYR) mutation. Anim. Genet. 37:175–178.[Web of Science][Medline]
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= homozygous for the White Plymouth Rock allele at the marker loci; 
= heterozygous or homozygous for the Japanese Game allele at the markers. W and C indicate white plumage and colored plumage, respectively.
