- Research article
- Open Access
Two new structural mutations in the 5′ region of the ASIP gene cause diluted feather color phenotypes in Japanese quail
© The Author(s) 2019
- Received: 28 November 2018
- Accepted: 3 April 2019
- Published: 15 April 2019
In quail, two feather colour phenotypes i.e. fawn-2/beige and yellow are associated with the ASIP locus. The aim of our study was to characterize the structural modifications within this locus that explain the yellow mutation (large deletion) and the fawn-2/beige mutation (assumed to be caused by a different structural modification).
For the yellow phenotype, we identified a complex mutation that involves a 141,162-bp long deletion. For the fawn-2/beige phenotype, we identified a 71-kb tandem duplication that comprises one unchanged copy of ASIP and one copy present in the ITCH-ASIP fusion gene, which leads to a transcript coding for a normal ASIP protein. Although this agrees with previous reports that reported an increased level of ASIP transcripts in the skin of mutant animals, we show that in the skin from fawn-2/beige embryos, this level is higher than expected with a simple duplication of the ASIP gene. Thus, we hypothesize that the 5′ region of the ITCH-ASIP fusion gene leads to a higher transcription level than the 5′ region of the ASIP gene.
We were able to conclude that the fawn-2 and beige phenotypes are caused by the same allele at the ASIP locus. Both of the associated mutations fawn-2/beige and yellow lead to the formation of a fusion gene, which encodes a transcript for the ASIP protein. In both cases, transcription of ASIP depends on the promoter of a different gene, which includes alternative up-regulating sequences. However, we cannot exclude the possibility that the loss of the 5′ region of the ASIP gene itself has additional impacts, especially for the fawn-2/beige mutation. In addition, in several other species including mammals, the existence of other dominant gain-of-function structural modifications that are localized upstream of the ASIP coding sequences has been reported, which supports our hypothesis that repressors in the 5′ region of ASIP are absent in the fawn-2/beige mutant.
The pigmentation of hair in mammals and of feathers in birds is mainly determined by the relative distribution of two types of melanin, i.e. eumelanin (black/brown) and pheomelanin (yellow/red). The agouti (ASIP) gene codes for the agouti-signaling protein (ASIP), which is an antagonist of the α-MSH hormone (melanocyte-stimulating hormone) for the melanocortin-1 receptor (MC1R) and leads to decreased eumelanin synthesis in favor of pheomelanin synthesis in melanocytes .
In general, (recessive) loss-of-function mutations in the ASIP gene, which either impair protein function or reduce its transcription, lead to increased production of eumelanin and to darker coat color as observed in mice , rabbits  or quails . Conversely, (dominant) gain-of-function mutations that display ubiquitous and constitutive expression of ASIP, such as the lethal yellow allele in mice, cause a yellow pigmentation . Several studies have shown that structural modifications in the ASIP gene are responsible for lighter coat colors in various species: (1) a large deletion in the lethal yellow allele in mice [5–7], (2) an insertion in the Abr allele in Normande cattle  and in the viable yellow allele in mice [9, 10], (3) a tandem duplication in the dominant white|tan allele in sheep , (4) an inverted duplication in the ASIP gene in mice , and (5) an insertion or an inverted duplication in the white allele in Alpaca .
An allelic series of variants at the autosomal yellow (Y) locus, which involves the ASIP gene on quail chromosome 20, has been identified in Japanese quail (Coturnix japonica): recessive black and yellow alleles, three alleles (fawn, fawn-2 and beige) that result in three similar phenotypes and the wild-type allele (WT) . The aim of our work was to characterize precisely the molecular basis of two of these variants that cause light plumage colours: yellow  and fawn-2/beige [16, 17].
The yellow allele is dominant over the wild-type allele, and homozygous carriers for the yellow allele are lethal. Heterozygotes have wheat-straw yellow-coloured feathers . In the literature, the genetic basis of the yellow phenotype is described as a large genomic deletion that spans almost the entire coding sequence of the RALY and EIF2S2 genes upstream of ASIP and that probably causes the lethality observed in homozygous individuals. Thus, this deletion places the expression of ASIP under the control of the promoter of RALY and leads to the expression of a fusion transcript . As a result, ASIP mRNA expression is upregulated in many tissues in heterozygous yellow animals compared to wild-type individuals, although no clearly significant differences were reported in skin samples between these animals [4, 15]. However, the alterations in feather colour are hardly visible and represent only a very small portion of the complex yellow phenotype .
The fawn-2/beige allele is part of a group of alleles responsible for very similar phenotypes that were independently described: fawn, fawn-2 and beige. In Japanese quail, Nichols et al.  reported in 1988 a first fawn dilution phenotype with a light/buff coloration and some darker spots. This fawn allele was identified as incompletely dominant to wild-type and co-dominant to yellow . Later in 1996, Tsudzuki et al.  described a similar fawn-2 phenotype in a population of Japanese quail that had been established at Gifu University from French commercial eggs. In 2003, a beige phenotype similar to fawn-2 was found in a French commercial line of Japanese quail . Both fawn-2 and beige alleles are dominant to wild-type [16, 17] and correspond probably to the same allele. At birth, individuals are characterized by a light yellow plumage with three dark bands on the back (but not on the head) . Adults have a lighter coat color and display a more pronounced sexual dimorphism than wild-type individuals [16, 17] and (see Additional file 1: Figure S1).
Although these studies have contributed to the understanding of the functional activity of ASIP in the yellow and the fawn-2/beige phenotypes, a fine genomic characterization of the causative variants is still lacking. The aims of this study were to characterize the large deletion that causes the yellow phenotype and to describe the fawn-2/beige mutation, which we hypothesized to be due to a structural modification representing a single allele. Thus, we sequenced four individuals i.e. one homozygous beige, one homozygous fawn-2 and two yellow, and compared the data with the recently available Coturnix japonica 2.0 quail annotated reference genome. Then, the molecular consequences of the fawn-2/beige mutation were evaluated.
Whole-genome paired-end sequencing
All animals were produced and maintained at the Inra PEAT experimental unit (Pôle d’Expérimentation Avicole de Tours, authorization # D37–175–1, 2017) in Nouzilly (France) in compliance with the European Union Guidelines for animal care, and with an approval by the local ethical committee in animal experimentation (Val de Loire) and the French Ministry of Higher Education and Scientific Research (authorization # 02411.02). For sequencing, we sampled two heterozygous yellow quails from a line established at Gifu University (Japan)  and maintained in Nouzilly, and one homozygous beige and one homozygous fawn-2 individual. DNA was extracted from blood and fragmented in order to build libraries with fragments of ~ 350 bp. Paired-end sequencing (2 × 150 bp) was done following the Illumina TruSeq DNA PCR-free protocol and using an Illumina Hiseq 3000 instrument.
Alignment of the sequenced fragments to the reference genome
Sequence reads were aligned with BWA-MEM  to the Coturnix japonica 2.0 quail reference genome (NCBI assembly accession GCA_001577835.1; BioProject accession PRJNA314147), which consists of 32 chromosomes or linkage groups and 1095 un-localised scaffolds . We used the Integrative Genome Viewer (IGV) software http://software.broadinstitute.org/software/igv/ to visualize the alignment on the reference genome and to check for compatibility of distance and orientation between read-pairs with the size selection that was set for the construction of the libraries. IGV was also used to evaluate read depth. Close examination of such alignments in IGV can be very informative to characterise deletions or duplications, for details (see Additional file 2).
Analysis of the transcripts
Twenty-five beige and 25 wild-type plumage quail eggs were incubated during 15 days before harvesting the embryos. DNA was extracted from muscle, and after sex determination  five skin samples of each sex were obtained for each phenotype for further RNA analyses.
Quantitative PCR were performed on the ABI 7900HT (Sequence Detection System 7900HT) in one 384-well plate. Since efficiency levels were similar for all the genes measured (including the reference genes RPS13 and GAPDH), results were expressed as 2(Ct_ref − Ct_gene) × 1000 in arbitrary units. For a complete description (see Additional file 3 and Additional file 4: Table S1).
Annotation of the region carrying the ASIP gene
Characterization of the yellow mutation
Validation of both structural modifications as the causal mutation
Characterization of the fawn-2/beige mutation
By examining the alignments of paired-ends obtained from the fawn-2 (SRA biosample SRR8224504) and beige individuals (SRR8224505), we found several paired-ends that had reads in opposite orientations (tail to tail) and that were separated by a large distance from each other in both individuals (see Additional file 5: Figure S3). This suggests a tandem duplication in the ASIP region. The increase in read depth for this region is in favour of a simple tandem duplication [the possibility of three or four copies of this region was excluded, (see Additional file 5: Figure S3)]. The breakpoints that were located upstream and downstream of the duplication were named 5′BKPT-fawn-2 and 3′BKPT-fawn-2, respectively and the junction point was named JUNCT-fawn-2 (Fig. 2b). These three regions were amplified from one homozygous fawn-2 and one homozygous beige individual and sequenced, and a 3-bp (TTG) microhomology was detected in each of these three regions (Fig. 2b). We used the 3′BKPT-fawn-2 and JUNCT-fawn-2 fragments to validate the presence of the duplication in fawn-2 and beige animals (Table 1). Based on our findings, we can conclude that fawn-2 and beige are the same allele, and that it is caused by a 70,895-bp tandem duplication.
Consequences of the 70,895-bp tandem duplication in fawn-2/beige individuals
Transcription of the region carrying the fawn-2/beige mutation
In 2008, Hiragaki et al.  analyzed the expression of ASIP in the skin of neonatal chicks, but in the current study, we sampled embryonic skin to study the phenotypic consequences of the fawn-2/beige mutation at an earlier stage, since they are already visible at hatch. All possible transcripts from this region were sequenced from 15-day embryo skin samples. The fusion gene has three possible TSS, i.e. in the first exon (5′UTR) of ITCH and the TSS-containing ASIP exons e4 (TSS2) and e5 (TSS3) (Figs. 1, 3). Transcripts of the fusion gene from the TSS-containing ASIP exons e4 and e5 (ASIP-tr2 and ASIP-tr3) could not be distinguished from those of the normal ASIP copy because there was no sequence difference. RT-PCR with primers that are located respectively in the 5′UTR of ITCH and in the coding exons of ASIP and sequencing of the products showed that the fusion transcript includes ITCH exon 1 and the three ASIP coding exons but not the ASIP exons e4 and e5. Since the 5′UTR exon 1 of ITCH does not contain a start codon, no fusion protein is expected from this fusion transcript, which supports our hypothesis that the protein produced from the ITCH-ASIP fusion gene is a normal ASIP protein.
We also performed quantitative PCR to evaluate the expression level of each ASIP transcript and the impact of the fusion gene on the amount of transcripts coding for the ASIP protein. We designed primers to selectively amplify three types of ASIP transcripts: the fusion transcript ITCH-ASIP, which can be transcribed only from the ITCH-ASIP fusion gene, and ASIP-tr2 and ASIP-tr3, which can be transcribed from both the ASIP gene and the ITCH-ASIP fusion gene. In addition, we used a primer pair to evaluate the overall amount of all ASIP-coding transcripts (see Additional file 3 and Additional file 4: Table S1).
Relative expression of ASIP-coding transcripts
(Mean ± SD)
(Mean ± SD)
Hiragaki et al. 
1.00 ± 0.35
12.99 ± 9.49
3 animals (triplicate)
3 animals (triplicate) homozygous for fawn-2
1.00 ± 0.83
7.55 ± 3.73
10 animals (duplicate)
9 animals (duplicate) homozygous for beige
The 141,162-bp deletion that we identified for the yellow mutation confirms previous results . It leads to the creation of a RALY-ASIP fusion gene and deletion of the EIF2S2 gene. The strong similarity between the lethal yellow mutation in mouse [5–7] and the yellow mutation in quail, as previously suspected by Nadeau et al. , is confirmed at the genomic level. However, our study reveals that the deletion is also combined with a 241-bp insertion, which is mainly composed of repeated sequences that do not originate from this region. Nadeau et al. (2008) showed that this structural modification preserves the coding structure of the ASIP gene but affects the regulatory 5′ region, and results in the production of transcripts from the RALY-ASIP fusion gene . However the expression of the transcripts coding for ASIP is disorganized with no over-expression in the skin [4, 15]. The transcription of the fusion gene depends on the promoter of the RALY gene but its 5′ region may also contain alternative regulatory sequences. In addition, we cannot exclude the possibility that the loss of the 5′ region of the ASIP gene has additional impacts.
Concerning the fawn-2/beige mutation, a first conclusion is that fawn-2 and beige share the same allele at the ASIP locus, and precedence should be given to the name fawn-2 for these variations. No conclusion can be drawn for the other fawn allele since no molecular data is available for this phenotype. We identified a 71,895-bp duplication (Fig. 3) and although this leads to a full duplication of AHCY, its expression remains unchanged. In contrast, an increased transcription of ASIP is described as responsible for the fawn-2/beige phenotype . We show that the fawn-2/beige animals studied here carry two copies of ASIP, one normal and one truncated (Fig. 3) just upstream of the TSS-2 (ASIP-TSS-2), which generates a fusion gene between the 5′UTR exon 1 of ITCH and the three coding exons of ASIP (Fig. 3). Thus, there is an increased production of ASIP-coding transcripts in the skin, which partly explains the lighter colour of their feathers. However, we also show that the increase in the amount of ASIP transcripts is higher than would be expected from a simple duplication of ASIP (Table 2), and we suggest that the 5′ region of the fusion gene has a higher transcription rate than that of ASIP alone. We found that the ITCH promoter is active for the fusion gene, but we were not able to compare the efficiency of the transcription of ITCH and the transcription of the fusion gene. Nevertheless, we cannot exclude the possibility that the loss of the 5′ region of the ASIP gene itself, putatively containing repressor sequences, could have additional impacts.
The ASIP region is particularly well conserved in birds and in mammals, both in terms of gene order and orientation. In Merino sheep, Norris and Whan  showed that a duplication with breakpoints located downstream from the ITCH-exon-1 and upstream from the ASIP coding exons and resulting in an ITCH-ASIP fusion transcript causes the dominant white|tan phenotype. This rearrangement is very similar to that of the allele we identified in the fawn-2/beige quails, except that, in the dominant white|tan sheep phenotype, the ASIP gene from the original segment appears to be inactivated. We found no evidence of such an effect in the fawn-2/beige quails. In addition, Norris and Whan  detected a SINE-type repeat element at the 5′ and 3′ breakpoints of their duplication and found that the genome of white|tan sheep contained several repetitions of this segment. For the fawn-2/beige quail, we identified only a micro-homology (Fig. 2b) between 5′BKPT-fawn-2 and 3′BKPT-fawn-2. Such events occur very often in duplicated regions and result from the mechanism involved in the duplication process .
The similarities between the yellow mutations in mice and quails and between the fawn-2/beige mutation in quails and the dominant white|tan mutation in sheep, raise the question of whether two independent deletion/duplication events in the same region can occur by chance or whether the presence of some conserved genomic features make this region prone to deletion/duplication events. The early/late feathering phenotype, which is due to a large duplication (K mutation) in chicken [27, 28], and to a 5-bp frameshift deletion in turkey , is another example of such events. Several other mutations involving the ASIP gene have been described in other species. In the white Alpaca, a promoter from another gene (NCOA6) was identified upstream of the ASIP coding exons, which could originate from an inverted duplication such as that characterized in agouti mice , without any other duplication or deletion . In Normande cattle that carry the Abr allele, a full-length L1-B1 element inserted in the 5′ UTR sequence of ASIP was identified as responsible for the over-expression of ASIP . The viable yellow mutation in mouse is due to the insertion of an intracisternal A particle (IAP) type retrotransposon into the promoter region of the ASIP . Although numerous structural genomic modifications in the 5′ UTR region of ASIP are known, we list only those that are not neutral. All of these (agouti/mouse, Abr/Normande cattle, white/alpaca, white|tan/sheep, viable yellow/mouse) are dominant gain-of-function mutations, which support the hypothesis that the 5′ region of ASIP could contain negative regulatory sequences that are active in the skin. This is an additional argument in favour of the loss of repressor sequences in the genome fawn-2/beige quails, which could have an impact on the expression of the fusion gene in addition to the effects brought by the 5′ region of ITCH itself. The loss of a 141-kb region in yellow quails (NC-029535 [1,463,709–1,604,872]) leads to a much diluted feather colour phenotype compared to fawn-2/beige quails. The 5′ region of the ITCH-ASIP fusion gene could also result from a 71-kb deletion (NC-029535 [1,371,395 (or 98)–1,442,295 (or 98)]). Our study suggests that the 21.4-kb region in quail (NC-029535 [1,442,295–1,463,709], indicated by a double pink arrow on Figs. 1b, 3), which contains the ASIP 5′UTR e1, e2, Ne9 and e3 exons (Fig. 1b), could include these repressor sequences. We hope that, in the near future, the improved annotation of livestock genomes resulting from the efforts of the FAANG consortium , will allow us to examine the hypothesis of repressor sequences in the 21.4-kb region.
We have refined the characterization of a 141-kb long deletion in heterozygous quails for the yellow allele and identified a 71-kb long tandem duplication in the ASIP region in fawn-2/beige quails. We conclude that fawn-2 and beige are the same allele, and due to precedence, these should now both be given the name fawn-2. We have shown that the loss (yellow) or the pseudo-loss (fawn-2/beige) of two distinct regions upstream of the coding exons of ASIP leads to the formation of a fusion gene that result in the production of the ASIP transcript, and especially for fawn-2 allele, at a higher rate. Although the fusion gene benefits from a new promoter and a new 5′ region that may contain alternative up-regulatory sequences, we cannot exclude the possibility that the loss of the 5′ region of ASIP itself has additional impacts. Indeed, the hypothesis of the loss of repressor sequences within ASIP should also be considered, since several structural modifications localized upstream of the ASIP coding sequences have been reported in mouse, alpaca, cattle, sheep and now quail, which all cause dominant gain-of-function phenotypes.
This study was initially proposed by MM under the supervision of FP and FM. AR is the major contributor to the analyses included in this paper but all authors made substantial contributions to the conception and design (FP, MM, FM, BB and AV), or acquisition of data (SL, VF, MM, DG and NT), or interpretation of data (AR, MM, FP, BB and TZ). SL performed all the biological experiments under the supervision of AR or MM. NT performed the genome alignments under the supervision of AV. AR wrote the core of manuscript and all the authors provided useful comments and suggestions. All authors read and approved the final manuscript.
The authors greatly appreciate the excellent technical assistance from Florence Plisson-Petit and Damien Trussardi (INRA GenPhySE). We thank the team running the genomic platform of the Génopole Toulouse Midi-Pyrénées (GeT-PlaGe, https://get.genotoul.fr/la-plateforme/get-plage/) and especially Diane Esquerre for her contribution to the results. We are grateful to the genotoul bioinformatics platform Toulouse Midi-Pyrénées (Bioinfo Genotoul, http://bioinfo.genotoul.fr/) for computing and storage resources. Noémie Thebault especially acknowledges AGENAVI, which supported her working position at INRA in 2016–2017.
The authors declare that they have no competing interests.
Availability of data and materials
Sequence data are deposited on SRA, under the Bioproject PRJNA506074.
Ethics approval and consent to participate
Animals were bred at INRA, UE1295 Pôle d’Expérimentation Avicole de Tours (PEAT), F-37380 Nouzilly, in accordance with the European Union Guidelines for animal care, following the Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes, under approval number D-37-175-1. The breeding and study of quail lines for phenotypic diversity were approved by the ethical comity CEEA no. 19, and authorized by the French government as project no. 02411.02.
This work was supported by the French national agency for research (EpiBird ANR-009-GENM-004).
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