Complete association between a retroviral insertion in the tyrosinase gene and the recessive white mutation in chickens

Abstract

Background: In chickens, three mutant alleles have been reported at the C locus, including the albino mutation, and the recessive white mutation, which is characterized by white plumage and pigmented eyes. The albino mutation was found to be a 6 bp deletion in the tyrosinase (TYR) gene. The present work describes an approach to identify the structural rearrangement in the TYR gene associated with the recessive white mutation.

Results: Molecular analysis of the chicken TYR gene has revealed a major structural difference (Restriction Fragment Length Polymorphism, RFLP) in the genomic DNA of the recessive white chicken. A major size difference of 7.7 kb was found in intron 4 of the TYR gene by long-range PCR. Molecular cloning and sequencing results showed the insertion of a complete avian retroviral sequence of the Avian Leukosis Virus (ALV) family. Several aberrant transcripts of the tyrosinase gene were found in 10 week old recessive white chickens but not in the homozygous wild type colored chicken. We established a rapid genotyping diagnostic test based on the discovery of this retroviral insertion. It shows that all homozygous carriers of this insertion had a white plumage in various chicken strains. Furthermore, it was possible to distinguish heterozygous carriers from homozygous normal chickens in a segregating line.

Conclusion: In this study, we conclude that the insertion of a complete avian retroviral sequence in intron 4 of the tyrosinase gene is diagnostic of the recessive white mutation in chickens. This insertion causes aberrant transcripts lacking exon 5, and we propose that this insertion is the causal mutation for the recessive white allele in the chicken.

Figure 1

Comparison of plumage color in full sib chickens differing for their genotype at the C locus. On the left, a chicken carrying the wild type allele at the C locus exhibits a colored plumage as determined by other feather color loci. Here the animal carries the wild type allele at the Extension locus, the wild type allele at the Columbian locus and the silver allele at the Silver locus. On the right, a recessive white chicken, full sib from the previous one, exhibits full white plumage.

Figure 2

RFLP banding pattern revealed by hybridization with a total chicken tyrosinase cDNA probe after HindIII digestion of genomic DNA from recessive white mutant and wild type chickens. Lane 1, 2, 3, 4, 5: recessive white chicken genomic DNA. Lane 6, 7, 8, 9, and 10: wild type (colored chicken) genomic DNA. The 4.1 kb band was only found in homozygous recessive white chickens while the 2.7 kb band was only found in the wild type chickens.

Figure 3

RFLP banding pattern revealed by hybridization with a partial tyrosinase cDNA probe. The partial cDNA probe included the total exon 5 region of chicken tyrosinase cDNA. This probe identified exactly the same restriction pattern as the total chicken tyrosinase cDNA probe. A simple HindIII digestion yielded 4.1 kb and 2.7 kb fragments in carriers of the recessive white or wild type allele at the C locus, respectively. A double digestion with BamHI+HindIII enzymes yielded a 2.7 kb fragment in chickens carrying the normal allele C*N and a 1.8 kb fragment in chickens carrying the recessive white allele C*C.

Figure 4

Long-range PCR amplification of intron 4 in recessive white and wild type chickens. The size of intron 4 of the TYR gene is 13.7 kb in the recessive white (left) and 6.0 kb in the wild type chicken (right).

Figure 5

Comparative analysis of intron 4 sequence of the TYR gene of a recessive white chicken, a colored chicken and the Red Jungle Fowl. I: A complete ALV sequence was found in the recessive white chicken, inserted in the reverse orientation as compared to the usual ALV sequence numbered from 1 to 7525. II: A and A' or B and B' sequences respectively have up to 90% identity in the Red Jungle Fowl intron 4 sequence, and both A and A' sequences end by a polyA repeat (45 bp). Both of the recessive white and the wild type colored INRA chickens do not have B and A' sequences in their intron 4. The recessive white intron 4 "A" region contains the polyA sequence but not the "A" region of the wild type colored chicken intron 4. III: This region contains a stretch of N signals (chromosome 1: position 179546417–1795546627) in the published Red Jungle Fowl genome sequence that we were able to replace by a 1 kb informative sequence in both the recessive white and the wild type colored INRA chicken.

Figure 6

Explanation of the RFLP results obtained for the TYR gene in recessive white chickens according to the structure of intron 4. A 2.7 kb HindIII fragment was obtained in the wild type chicken after a single HindIII digestion and hybridization with an exon 5 probe. A 4.1 kb HindIII band and a 1.8 kb BamHI-HindIII band were obtained after HindIII single digestion and BamHI-HindIII double digestion in the recessive white mutation.

Figure 7

Principle of the PCR diagnostic test for the molecular identification of the insertional mutation in intron 4 of the chicken TYR gene. Two fragments may be obtained: 481 bp between Diag05-nor-up and Diagnostic05-dw for normal C*N allele, and 345 bp between Diag05-cc-up and Diagnostic05-dw for the C*C allele.

Figure 8

Diagnostic genotyping test result. Only one band either 481 bp or 345 bp was found in homozygous colored or homozygous recessive white chickens, respectively. In heterozygous chickens, both 481 bp and 345 bp bands were detected by the diagnostic genotyping test.

Figure 9

Shorter 3'UTR are isolated by RACE from recessive white skin samples. The major 3'UTR isolated in the recessive white was 345 bp and the major 3'UTR isolated in the heterozygous colored chicken was 675 bp.

Figure 10

Nucleotide and deduced amino acid sequences of 7 major transcripts belonging to 3 types of recessive white chickens that were isolated by RACE and compared with the published Tyrosinase cDNA sequence. All of these 7 transcripts had normal exon 1 to exon 4 sequences but lacked the exon 5 sequence. A short sequence (from 124 bp to 140 bp) of intron 4 replaced the exon 5 sequence followed by a polyA tail. The exon 4 sequence is shown in grey color. Three putative polyadenylation signals ATATAAA, AGATAAA and ATATAAA in the intron 4 sequence are shown in boxes. For each transcript, the proposed polyadenylation signal is shown in the red box. The predicted amino acid sequence is shown below the nucleotide sequence.

Figure 11

RT-PCR detection of aberrant and normal-sized transcripts of the TYR gene. The major transcript in the recessive white genotype was the truncated transcript (lane 1, 2) and the major transcript in the heterozygous colored chicken was the normal sized transcript (lane 5, 6). There was no aberrant transcript in the homozygous black plumage chickens (lane 3, 4).