Download Mapping of the Recessive White Locus and

GENETICS
Mapping of the Recessive White Locus and Analysis
of the Tyrosinase Gene in Chickens
S. Sato, T. Otake, C. Suzuki, J. Saburi, and E. Kobayashi1
National Livestock Breeding Center, Nishigo, Fukushima 961-8511, Japan
ABSTRACT An F2 chicken population of 265 individuals, obtained from an intercross between the Japanese
Game (colored plumage) and the White Plymouth Rock
(the recessive white) and genotyped for microsatellite
markers, was used for determining the locus of the gene
responsible for the recessive white plumage phenotype
in chickens. Two hundred twenty-five markers were
mapped in 28 linkage groups. Linkage analysis revealed
that the recessive white gene was mapped to chromosome
1. Detailed analysis using additional markers uncovered
a significant linkage between 2 new markers, mapped
to the flanking region of the tyrosinase gene, which is
associated with skin and plumage color. The sequence of
the tyrosinase gene was investigated in recessive white
chickens and colored chickens. There were no obvious
differences in the tyrosinase gene exons between the re-
cessive white chicken and the colored chicken. However,
sequence analysis of tyrosinase intron 4 in the recessive
white chicken revealed a presence of an insertion of an
avian retroviral sequence. The White Plymouth Rock and
the F2 generation with white plumage were identified as
homozygous carriers of the retroviral sequence. Expression of the normal transcript containing exon 5 was substantially decreased in the recessive white chicken compared with the colored chicken. Some abnormal tyrosinase gene transcripts were expressed in the skin of the
White Plymouth Rock: reverse transcription PCR products amplified from exon 3 to intron 4 and from retroviral
sequence 3′ long terminal repeat to exon 5. Based on these
results, it was confirmed that an avian retroviral sequence
insertion in the tyrosinase gene was the cause of recessive
white phenotype in chickens.
Key words: microsatellite marker, mapping, recessive white chicken, tyrosinase gene, avian retroviral sequence
2007 Poultry Science 86:2126–2133
INTRODUCTION
In mammals and birds, skin, coat, and feather color
are determined mainly by 2 melanins, eumelanin and
phaeomelanin. In the synthesis pathways of both of these
melanins, tyrosinase-induced oxidation of Tyr, which is
the first biochemical step, leads to production of dihydroxyphenylalanine and then, via oxidation of dihydroxyphenylalanine, the production of dopaquinone
(Lerner and Fitzpatrick, 1950). In addition, tyrosinase is
associated with formation of eumelanin, which is produced by the dehydrogenation of 5,6-dihydroxyindole2-carbonic acid in a subsequent reaction (Korner and Pawelek, 1982). Thus, tyrosinase is an essential enzyme in
melanin synthesis in pigment cells.
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,
©2007 Poultry Science Association Inc.
Received March 8, 2007.
Accepted June 25, 2007.
1
Corresponding author: [email protected]
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
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MAPPING OF RECESSIVE WHITE GENE
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.
MATERIALS AND METHODS
Birds
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 Aizu-didori
(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 Biosystems, 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/␮L. The 5 tyrosinase exons were
amplified by PCR from genomic DNA from the Japanese
2127
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 ␮L containing 20 ng of genomic DNA, 6.25
pmol of each primer, 0.2 mM each deoxynucleoside triphosphate (dNTP), 10 mM TrisⴢHCl (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.
An attempt was made to investigate the sequence differences in tyrosinase gene intron 4, because PCR products for tyrosinase gene exon 5 were not easily obtained
from the White Plymouth Rock. Table 1 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 ␮L containing 100 ng
of genomic DNA, 6.25 pmol of each primer, 0.1 mM each
dNTP, 10 mM TrisⴢHCl (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-␮L reaction
volume containing 100 ng of genomic DNA, 6.25 pmol
of each primer, 0.1 mM each dNTP, 10 mM TrisⴢHCl (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.
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SATO ET AL.
Table 1. Sequences of PCR primers for genomic DNA
Primer name
Sequence (5′ → 3′)
tyr-ex1-f
tyr-ex1-r
tyr-ex2-f
tyr-ex2-r
tyr-ex3-f
tyr-ex3-r
tyr-ex4-f
tyr-ex4-r
tyr-ex5-f
tyr-ex5-r1
tyr-ex5-f21
tyr-int4-1F
tyr-int4-1R
tyr-int4-2F
tyr-int4-2R
tyr-int4-3F
tyr-int4-3R
tyr-int4-4F
tyr-int4-4R
tyr-int4-5F
tyr-int4-5R
tyr-int4-6F
tyr-int4-6R
tyr-int4-7F
tyr-int4-7R
tyr-int4-8F
tyr-int4-8R
tyr-int4-af2,3
tyr-int4-br2
ALV-f3-comp3
GTGGTGAAGCATTCCCAGTT
CTCTCCAGATAGCGCACCTC
TCCTTCACTTCTTCCCCAAA
TTAAAGGCTCCAATCCCAGA
GCATTAGCTACATGGCAGGA
TCGCAGTTTTGAAGCGTAAG
AGCCCTGAAGTCCTTTGCTT
CTTGCAGGCATGATTCGTAA
AAGGACTGGTGGAATTTGCTT
CTTGGGTGGCATGGTAACTT
GGTACTGCATTGCATCATGT
AATTACATGGTTCCCTTTATC
TATTACCAATGACAGCAACCTT
GTGCTTGCTCTGTGGTTATTT
AAGCAGTCTTTTGTGGGAGTAA
AAACAACGGTTCAAATGGTCCTA
GCAGGCTTTCTTGCTTCACTTCC
TTAAGAGGAACATAAAAGTGAACA
TTCTGACGTGTGTTTTTGATTC
TCTGCTTTCTGGGCGGCTGATTTC
TTAAACATGCCTTCCTCCAGC
ATGATAACTTTATGATAGAG
GTCTCTCACAGAATCATAGA
GAAGATTTAGATTTAGTGGT
TTGAGCTGAAAGATGCTCTTG
GAAATGTTTTCTAACTAAGAGA
GATAACACTGGAAGGCTCAG
AAACTTTAAGGACTGGTGGAATTTGCTTAG
TTTCTGTGAGTAAGGGTTGTATTTCTGGAG
TAATTCAATCAGCTATCACACG
Fragment
size (bp)
1,064
372
436
579
585
467
877
790
1,177
599
576
706
659
450
336, 7,8334
726
Description
Forward exon 1
Reverse exon 1
Forward exon 2
Reverse exon 2
Forward exon 3
Reverse exon 3
Forward exon 4
Reverse exon 4
Forward exon 5
Reverse exon 5
Forward exon 5 for the White Plymouth Rock
Forward for intron 4 group 1
Reverse for intron 4 group 1
Forward for intron 4 group 2
Reverse for intron 4 group 2
Forward for intron 4 group 3
Reverse for intron 4 group 3
Forward for intron 4 group 4
Reverse for intron 4 group 4
Forward for intron 4 group 5
Reverse for intron 4 group 5
Forward for intron 4 group 6
Reverse for intron 4 group 6
Forward for intron 4 group 7
Reverse for intron 4 group 7
Forward for intron 4 group 8
Reverse for intron 4 group 8
Forward for intron 4 group 9 and retroviral sequence
Reverse for intron 4 group 9 and retroviral sequence
Reverse primer in retroviral sequence
1
Tyr-ex5-f2 and tyr-ex5-r are the primer sets used to detect the exon 5 of the White Plymouth Rock.
Tyr-int4-af and tyr-int4-br are the primer sets used to detect the full length of the retroviral sequence.
3
Tyr-int4-af and ALV-f3-comp are the primer sets used for simple detection of the retroviral sequence.
4
The 336- and 7,833-bp products were detected in the Japanese Game and the White Plymouth Rock, respectively.
2
Reverse Transcription PCR and
Sequencing of the Tyrosinase Gene
plumage in the F2 chickens did not different significantly
from the expected 3:1 ratio.
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 ␮g 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 ␮L containing 6.25 pmol of each primer, 0.1 mM
each dNTP, 10 mM TrisⴢHCl (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.
Linkage Analysis and Mapping
RESULTS
Plumage Color in F2 Chickens
Table 3 shows plumage color patterns in the F2 chickens. The segregation ratio into white plumage and colored
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.
Linkage analysis revealed that the recessive white gene
was mapped to chromosome 1d. To examine the precise
location of the C locus, the F2 resource family was geno-
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MAPPING OF RECESSIVE WHITE GENE
Table 2. Sequences of the reverse transcription PCR primers for cDNA
Primer name
Sequence (5′ → 3′)
Description
tyr-ex1-f21,2
tyr-ex4-f23
tyr-ex4-r21
tyr-ex5-r22,3
TCCATGCACTGAAGCATAAC
AGCGGTGGTTAAGAAGACAC
TTCTTGCAGATACTCATAGTC
CACTTTCTGTGAGTAAGGGT
Forward exon 1
Forward exon 4
Reverse exon 4
Reverse exon 5
1
Tyr-ex1-f2 and tyr-ex4-r2 are the primer sets used to detect the product from exon 1 to exon 4 (product
size = 1,388 bp).
2
Tyr-ex1-f2 and tyr-ex5-r2 are the primer sets used to detect the product from exon 1 to exon 5 (product
size = 1,588 bp).
3
Tyr-ex4-f2 and tyr-ex5-r2 are the primer sets used to detect the product from exon 4 to exon 5 (product
size = 346 bp).
typed for 2 new markers (169333023 and 180893258). Figure 1 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).
Sequence Analysis and Detection
of Retroviral Sequence Insertion
in the Tyrosinase Gene
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.
Segregation of the Retroviral Sequence
in the Tyrosinase Gene in F2 Chickens
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 noncarriers of the retroviral sequence in colored chickens. In
Figure 1. Genotyping carried out to determine gene order around the C locus and the microsatellite markers on chromosome 1. 䊐 = homozygous
for the White Plymouth Rock allele at the marker loci; 䊏 = heterozygous or homozygous for the Japanese Game allele at the markers. W and C
indicate white plumage and colored plumage, respectively.
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SATO ET AL.
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).
Figure 2. The PCR products with or without the insertion sequence
in intron 4 of recessive white and colored chickens. Lanes 1 to 3 =
colored chicken; lanes 4 to 6 = recessive white chicken. M1 and M2
show the 100-bp ladder marker and 1-kb ladder marker, respectively.
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
Figure 3. The reverse transcription PCR products of the tyrosinase
gene in colored (lanes 1, 3, and 5) and recessive white chickens (lanes
2, 4, and 6). M indicates the DNA marker of the 100-bp ladder. Lanes
1 and 2 = products from exon 1 to exon 4; lanes 3 and 4 = products
from exon 1 to exon 5; lanes 5 and 6 = products from exon 4 to exon 5.
DISCUSSION
The detailed locus associated with the recessive white
was detected on the telomere of the long arm of chromosome 1 by genotyping of the F2 resource family from an
intercross between the Japanese Game and the White
Plymouth Rock breeds. In previous studies, the detailed
position of the recessive white gene had not been determined, even though the classical C locus has been located
on chromosome 1 in chickens and has been genetically
defined as the structural tyrosinase gene locus in humans
and mice. In the current study, several large chromosomes were divided into several linkage groups, because
the number of informative markers was limited, even
though many markers were tested at the beginning.
Therefore, the genome was not fully covered, and the
markers were not uniformly mapped on chromosome.
However, the number in the F2 population, the marker
number, and spacing were sufficient for the recessive
white position to be between the ADL0195 and 180893258
markers. There was no correlation between sex and the
white plumage. Therefore, a detailed analysis of the Z
chromosome was not performed. In addition, the sexlinked albino (Sal) has been mapped to the Z chromosome.
Tyrosinase activity was detected in sex-linked albino
chickens (Sal) as well as in the wild type (C+/C+), whereas
there is no tyrosinase activity seen in the recessive white
or albino chicken (Oetting et al., 1985). No significant
linkage was detected in any linkage groups on the other
chromosomes. The dominant white mutation in chickens
has a white plumage phenotype and is caused by mutations of the PMEL17 where there is a 9-bp insertion in
exon 10 and a 12-bp deletion in exon 6 (Kerje et al., 2004).
In current study, microsatellite markers were not mapped
on E22C19W28, but the dominant white allele was not
observed in the Japanese Game and the White Plymouth
Rock parents of the F2 resource family (data not shown).
This is the first report to provide detailed linkage analysis
in the recessive white chicken with microsatellite markers
covering the whole genome.
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MAPPING OF RECESSIVE WHITE GENE
Figure 4. Splicing variants of the tyrosinase gene in recessive white chickens obtained by reverse transcription PCR. Two fragments were
amplified by primer sets from exon 3 to intron 4; (a) is followed by intron 4 of 772 bp, and (b) is followed by 360 bp behind splicing 1 bp to 1,278
bp of intron 4. Arrows show the primers.
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
Table 3. Plumage color and retroviral sequence insertion distributions
in the Japanese Game × the White Plymouth Rock F2 chickens
Retroviral insertion
Plumage pattern
Females
White
Black
Barred
Black with brown
Brown with black
Brown
Total for females
Males
White
Black
Barred
Black with brown
Brown with black
Brown
Total for males
Total across sex
spots
spots
spots
spots
+/+
+/−
−/−
Total
35
0
0
0
0
0
35
0
15
6
8
23
26
78
0
5
3
0
11
13
32
35
20
9
8
34
39
145
32
0
0
0
0
0
32
67
0
0
26
21
6
6
59
137
0
0
10
6
8
5
29
61
32
0
36
27
14
11
120
265
Table 4. Number of microsatellite markers, chromosome linkage group,
map length, and the first marker on each chromosome used for a whole
genome scan of the Japanese Game and the White Plymouth Rock
chickens
Chromosome
1a
1b
1c
1d
2a
2b
3a
3b
3c
4
5
6
7a
7b
8
9
10
11
13
14
15
19
20
26
27
28
z-a
z-b
Total
Markers
(n)
Map
length (cM)
12
7
22
2
2
39
7
2
8
24
18
8
4
7
9
11
6
4
3
4
6
2
4
4
2
3
3
2
60.0
77.2
188.9
7.4
1.4
280.5
90.3
4.4
116.1
167.7
133.3
72.3
27.3
56.9
110.2
49.4
59.3
26.7
28.3
54.7
32.1
1.3
14.1
7.8
22.6
28.8
4.9
1
225
1,724.9
First
markers
ABR649
HUJ0001
MCW0313
ABR631
LEI0163
LEI0141
LEI0265
LEI0118
LEI0043
ABR357
LEI0133
ABR11
LEI0064
MCW0199
LEI0044
ABR377
MCW0309
ADL0287
ADL0254
LEI0098
MCW0031
MCW0304
LEI0080
MCW0285
MCW0328
LEI0251
MCW0269
LEI0229
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SATO ET AL.
Figure 5. Other splicing variants of the tyrosinase gene in recessive white chickens obtained by reverse transcription PCR (RT-PCR). This
fragment was amplified by a primer set from 3′ long terminal repeat (LTR) of the retroviral insertion sequence to exon 5. Arrows show the primers.
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
MAPPING OF RECESSIVE WHITE GENE
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.
ACKNOWLEDGMENTS
We thank Tetsuo Kunueda, Okayama University,
Okayama, Japan, for assistance and excellent advice and
Taeko Sato, Fukushima Prefecture Agriculture Institute,
Fukushima, Japan, for providing us with the colored
chicken samples.
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