Population genomic analysis identifies the complex structural variation at the fibromelanosis (fm) locus in chicken

ABSTRACT Phenotypic diversity and its genetic basis are central questions in biology, with domesticated animals offering valuable insights due to their rapid evolution the last 10,000 years.

In chickens, fibromelanosis (FM) is a striking pigmentation phenotype characterized by hyperpigmentation. A previous study identified a complex structural variant involving both two large

duplications (127.4 and 170.5 kb in size) and inversions associated with upregulated expression of the _Endothelin 3_ (_EDN3_) gene. However, the detailed organization of the structural

arrangements have remained unclear. In this study, we conducted a comprehensive genomic survey of 517 FM chickens representing 44 different populations. Our results elucidate the complex

arrangement of the duplications and inversions at the _FM_ locus based on the large-scale genomic survey, population level genotyping, and linkage disequilibrium analysis, providing

conclusive support for one specific configuration of the two large duplications, resolving a controversy that has been unresolved for more than a decade. Our results show that the birth of

this complex structural variant must have involved an interchromosomal rearrangement creating fixed heterozygosity due to sequence differences between the two copies of the 127.4 kb

duplication. This study shows how population genomics can be used to understand complex structural variations that underlie phenotypic variation. SIMILAR CONTENT BEING VIEWED BY OTHERS THREE

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DOMESTIC YAKS Article Open access 19 September 2023 INTRODUCTION How phenotypic diversity evolves and its genetic basis is a fundamental question in biology. Domesticated animals constitute

a valuable resource to explore this topic due to their rapid phenotypic evolution within the last 10,000 years1. A characteristic feature of domestic animals is altered pigmentation pattern,

a phenotypic change that occurred early during domestications of animals as documented by ancient illustrations and documents. A striking pigmentation phenotype in domestic chicken is

fibromelanosis (FM) that is widespread among Asian chicken breeds, like Chinese Silkie and Ayam Cemani from Indonesia. FM is characterized by a massive expansion of melanocytes resulting in

excessive skin and tissue pigmentation2. The FM trait is highly valued culturally and commercially in Asia. For instance, Silkie chickens are prized in ornamental breeding for their

distinctive appearance, while Ayam Cemani chickens hold cultural significance and command high market prices3. FM chickens are valued for their deep eumelanin deposition, used in food such

as Chinese black-bone soup and in Chinese traditional medicine4,5. A previous study demonstrated that the dominant FM trait in several breeds of chicken is caused by a complex structural

rearrangement involving two duplications, 127.4 and 170.5 kb in size2. One of the duplications encompass the _EDN3_ gene, which has a critical role for melanoblast differentiation and

expansion6. The fact that _EDN3_ shows a highly significant upregulation at the mRNA level in skin from FM chickens strongly suggested that this is the causal mechanism for the massive

expansion of pigment cells in FM chicken2. It was not possible to resolve the organization of the complex rearrangement with the short-read whole genome sequence data available a decade ago,

even with the structural variation detection tools widely used today7, and three possible configurations of the complex structural rearrangement (Fig. 1) were established based on PCR

analysis of breakpoint regions2. A single recombinant found in a pedigree segregating for the _FM_ mutation was only consistent with one of the possible configurations denoted FM-22.

However, more recent data based on long read sequencing assemblies have given conflicting results, in which some studies2,8,9 have supported the FM-2 configuration whereas other studies came

to the conclusion that their assembly of this region from Yeonsan Ogye10 and Silkie11 chicken supported the FM-1 configuration. In order to resolve the structural arrangement of the _FM_

locus, here we have analyzed whole genome sequence data from 517 FM chickens representing 44 chicken breeds worldwide, all with the FM phenotype (Fig. 2A). Our comprehensive population

genomic analysis showed that all these breeds have the same structural variations (SVs) at the _FM_ locus as previously reported2. Our population genomic analysis provides overwhelming

support for the FM-2 model based on the pattern of linkage disequilibrium across the complex rearrangement reflecting where recombination events can occur. This study highlights how

population genomics data can be used to resolve complex structural variations that are challenging to resolve even using long-read sequence data. RESULTS COMPREHENSIVE GENOMIC SURVEY OF THE

_FM_ LOCUS We analysed whole genome sequencing data of 517 FM chickens representing 44 breeds of chicken (Fig. 2A). Our results showed that all these breeds have the same structural variants

(SVs) at the _FM_ locus (Fig. 2B and C). The SVs involve duplications of two genomic regions on chromosome 20:10,766,772–10,894,151 bp (DUP1) and chr20:11,306,686–11,477,501 bp (DUP2) (Fig.

 2C), consistent with previous findings2,12. GENETIC PROFILE OF THE _FM_ ALLELE We performed a comprehensive analysis of genomic data to characterize the arrangement of the complex

chromosomal structural variation of the _FM_ allele (Figs. 3 and 4). Firstly, we analyzed the sequence depth at the two duplications associated with the FM phenotype and confirmed that DUP1

and DUP2 occur as two copies in the _FM_ allele (Fig. 3A and B). This analysis allowed us to identify _FM_ homozygotes that were used in the further analysis. We next determined the genetic

differentiation between wild-type (_FM*N_/_N_) and _FM_ homozygous chickens (_FM*FM_/_FM_) (Fig. 3C). _F_ST results showed that there were significant genetic differences in the two SV

regions, especially in DUP1 and its flanks. Notably, there is an obvious reduction of genetic differentiation at the front segment of the DUP1 region (Fig. 3C). Nucleotide diversity analysis

showed a marked reduction of heterozygosity in _FM_ homozygotes but interestingly only in the DUP1 region and its flanks (Fig. 3D). We next analyzed the frequency of the reference allele

for all SNPs across the region harboring the two duplications. This revealed a remarkable bubble for the DUP1 region in _FM_ homozygotes not present in wild-type chicken (Fig. 4A and B). In

the bubble region, _FM_ homozygotes tended to be fixed for the reference allele or the non-reference allele, or the two alleles occur at exactly the same frequency (50%) (Fig. 4B). A weak

tendency for a similar pattern was noted for the DUP2 region where two regions (marked with red arrows) had an excess of SNPs showing an allele frequency of 50% (Fig. 4B). The non-duplicated

412.54 kb region between DUP1 and DUP2 showed a very similar distribution of allele frequencies as present in wild-type chickens (Fig. 4A and B). These data are consistent with strong

suppression of recombination between FM and wild-type chromosomes but only for the DUP1 region. CHARACTERIZATION OF THE STRUCTURAL ARRANGEMENT OF _FM_ ALLELE The above results refute FM-1 as

a possible organization of the _FM_ allele. This is because the FM-1 configuration (Fig. 1A) implies an inversion involving one copy of DUP1, the connecting region (412.54 kb) and one copy

of DUP2, and the bubble should extend across the entire region which it does not (Fig. 4B). We therefore conclude that the FM-2 configuration must be the correct organization because it is

the only one consistent with a single previously reported recombination event2 and none of the recent reports based on PacBio long reads found support for the FM-3 order10,11. In addition,

if the configuration of FM-3 is correct, we should observe a bubble in the DUP2 region, not in the DUP1 region. Phylogenetic trees constructed for the DUP1 and DUP2 regions revealed

differences in branch lengths between two SV regions which support the hypothesis of an inversion near the DUP1 region (Supplementary Fig. 1), consistent with our proposed structural

configuration of the _FM_ locus. The large number of SNPs showing an allele frequency of 50% in the DUP1 region implies that the complex rearrangement creating the _FM_ allele must have

involved an inter-chromosomal event involving two different haplotypes creating fixed heterozygosity for the positions in regions of severely suppressed recombination (after the

rearrangement) where the two haplotypes differed (Fig. 4C). In conclusion, FM-2 (Fig. 1A) is now the confirmed organization of the _FM_ allele and there is strong suppression of

recombination for most of the DUP1-inverted DUP2-DUP1 region whereas there is no strong suppression of recombination from the end of the second copy of the DUP1 region to the second copy of

DUP2 including the single copy 412.54 kb intervening region (Fig. 4C). This explains the bubble pattern across the DUP1 region (Fig. 4B) in _FM_ homozygotes as well as the corresponding

pattern of genetic differentiation and nucleotide diversity (Fig. 3C and D). TRACING THE FORMATION OF THE _FM_ ALLELE There is a sharp disruption of the region of fixed heterozygosity within

DUP1 in the _FM_ allele, marked by a red arrow in Fig. 4B. This could either reflect a recombination with wild-type chromosomes have occurred or a shift from a large region where the two

donor haplotypes forming the _FM_ duplication showed many sequence differences to a region where they happened to be identical. Similarly, two reference-consistent regions in DUP2 may

reflect regions in which one of the copies of DUP2 not undergoing recombination is identical to the genome reference and thus the frequency of the reference allele among chickens homozygous

for the _FM_ allele will always be 50% or higher (Fig. 4B), because we estimate the allele frequency as the average of sequences from the two copies of DUP2. Linkage disequilibrium (LD)

analysis at the _FM_ locus also showed a clear LD block boundary between two segments within DUP1 which is in perfect agreement with the break of fixed heterozygosity (Figs. 4B and 5). While

LD decay patterns are influenced by recombination rates, population structure, and historical selection events, the significantly elevated LD at the _FM_ locus (Fig. 5) are consistent with

the presence of an inversion. DISCUSSION RESOLVING THE STRUCTURAL CONFIGURATION OF THE _FM_ ALLELE The _FM_ allele constitutes a truly complex structural variant involving two large

duplications (127.4 kb and 170.5 kb), an inversion and translocation of the 170.5 duplication and with a 412.5 kb non-duplicated intervening region (Fig. 1). Previous PCR studies established

three possible configurations of this complex rearrangement (Fig. 1) that could not be resolved using whole-genome sequencing based on short read data2,12. However, one recombinant found in

a pedigree analysis provided strong support for the FM-2 configuration5. Subsequent studies based on long-read sequencing gave conflicting results, some supporting the FM-1

configuration10,11 and others supporting FM-28,9,13. The current study based on extensive whole genome sequence data and the analysis of linkage disequilibrium patterns across more than 40

breeds carrying the _FM_ allele now provides conclusive evidence for the FM-2 configuration and that the _FM_ allele arose only one time. The unique feature with an inverted copy of DUP2

inserted between two tandem copies of DUP1 results in strong suppression of recombination explaining the allele frequency bubble at DUP1 when reads from the _FM_ allele are aligned to the

reference genome (Fig. 4). Furthermore, the data shows that the _FM_ mutation must have involved an inter-chromosomal exchange resulting in fixed heterozygosity in the DUP1 region at the

sites with sequence differences between the two copies (Fig. 4B). In contrast, fixed heterozygosity and an allele frequency bubble is not noted for DUP2 because one of the DUP2 copies is

located outside the region of suppressed recombination (see FM-2 in Fig. 1A). This result is also in complete agreement with the previously reported recombination event between a wild-type

and an _FM_ allele2. Given that the _FM_ allele likely originated thousands of years ago5, sufficient time has passed for sequence variation in the second copy of DUP2 and the corresponding

region of wild-type chromosomes to become randomized due to recombination. This is illustrated by the fact that the nucleotide diversity is clearly reduced only at the DUP1 region and its

flanking region (Fig. 3D) consistent with the observed allele frequency bubble (Fig. 4). EVOLUTION OF THE _FM_ LOCUS Our study did not reveal any genetic heterogeneity at the _FM_ locus

since exactly the same structural rearrangements were detected across all 44 FM chicken breeds analyzed. The _FM_ allele must involve multiple structural rearrangements and it is challenging

to establish the exact mechanism causing these rearrangements with confidence. However, one possible scenario is that the first event involved a duplication of the entire region from DUP1

to DUP2 (710.4 kb) by unequal cross-over, followed by an inversion involving DUP2 and the intervening sequence (412.5 kb) in which the latter was lost. This scenario is consistent with the

fixed heterozygosity for the DUP1 sequence, because the two copies originate from different chromosome homologs. The fixed heterozygosity at DUP1 has since then been maintained due to

suppressed recombination while the fixed heterozygosity for DUP2 has been eroded due to recombination affecting the second copy of DUP2 (Fig. 4). It is still an open question whether the two

events (the duplication and the inversion) occurred in a single meiosis or in a stepwise fashion over multiple generations. LIMITED CAPABILITY OF LONG-READ DATA IN COMPLEX STRUCTURAL

VARIATION For complex structural variations, such as the _FM_ locus, which involve copy number variation, rearrangements, inversion, and translocations, long-read sequencing faces

limitations for instance when the size of a duplication exceeds the read length. In such cases, the inter-sequence assembly strategy plays a critical role and can significantly influence the

accuracy of the assembly14. As more and more research focus on large-scale structural variation15, such as pan-genome16, we suggest that the assembly of regions showing complex structural

variation can be validated using population genomics data. Despite previous conflicting reports2,10,11,12, the population genomic approach employed here successfully clarified the structural

arrangement, demonstrating the value of population-scale sequencing in deciphering complex genomic rearrangements. STRUCTURAL VARIANTS AS DRIVERS OF PHENOTYPIC DIVERSITY The _FM_ locus

exemplifies how structural variants contribute to phenotypic diversity in domesticated animals17,18,19,20,21,22. These are often gain-of-function mutations with a dominant inheritance,

resulting in altered gene expression. Other prominent examples include Dominant white color in pigs21, Greying with age in horses17,18, and all three comb phenotypes in chicken, Pea-comb23,

Rose-comb24, and Duplex comb22. These cases collectively highlight the evolutionary significance of structural variants, particularly in shaping key morphological traits under artificial

selection in domestic animals. MATERIALS AND METHODS SAMPLES COLLECTION In this study, we analyzed 817 chicken samples, comprising 517 FM chicken samples from 44 breeds from various

geographic locations worldwide, and an additional 300 wild-type samples (Supplementary Table 1). All samples included in this analysis were previously reported in

studies9,10,11,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50. DATA PROCESSING Sequencing reads underwent quality control using FastQC (version 0.11.8) to

assess read quality metrics. Index adaptors and raw reads with more than 50% bases in low quality (Q ≤ 5) or 10% “N” content were filtered out using Btrim (version 0.3.0)51 software. Cleaned

reads were then mapped to the reference genome of the red junglefowl (GRCg6a) using the BWA-MEM (version 0.7.17-r1188)52 algorithm with default settings. Bam files were sorted using

SAMtools (version 1.9)53 and PCR duplicates were removed using Picard (version 2.18.6) tools. Sequencing depth relative to the reference genome was calculated using SAMtools (version 1.9)53.

The “RealignerTargetCreator” and “IndelRealigner” tools in Genome Analysis Toolkit (GATK, version 3.7)54 were used to reduce mismatches around INDELs, with base quality score recalibration

(BQSR) performed to reduce mapping errors. Joint calling for SNPs and small INDELs was performed using the “HaplotypeCaller” and “GenotypeGVCFs” tools from GATK (version 3.7)54. Low quality

SNPs were filtered, with parameters following previous research7,55: “QUAL < 30.0 || QD < 2.0 || MQ < 40.0 || FS > 60.0 || MQRankSum < − 12.5 || ReadPosRankSum < − 8.0 ||

SOR > 3.0”. Loci with max-missing rate above 0.10 and minor allele frequency less than 0.05 (–maf 0.05; –max-missing 0.9) were filtered using VCFtools (version 0.1.16)56. Only biallelic

loci were retained. SVS IDENTIFICATION SVs were detected using relative sequence depth analysis. Sequence depth was calculated using SAMtools (version 1.9)53 to identify the genomic regions

of interest, specifically focusing on the DUP1 (chr20:10,766,772–10,894,151) and DUP2 (chr20:11,306,686–11,477,501) regions. The relative sequencing depth is the ratio of the sequencing

depth of the candidate region to the whole genome background. To differentiate between wild type (_FM*N_/_N_), homozygous (_FM*FM_/_FM_), and heterozygous (_FM*FM_/_N_) samples, relative

sequence depth analysis was performed using the average sequence depth of the target region divided by the average sequence depth of whole genome or chromosome 20. NUCLEOTIDE DIVERSITY AND

GENETIC DIFFERENTIATION Nucleotide diversity (π) and genetic differentiation (_F_ST) was calculated using VCFtools (version 0.1.16)56 using a 20 kb window length with 10 kb sliding window,

focusing on the SV regions and their flanking sequences (chr20:10,600,000–11,700,000). To evaluate the statistical significance of _F_ST estimates across different genomic regions, we

performed a _Z_-score transformation relative to the genome-wide distribution. _P_-values were derived from the standard normal distribution, and Benjamini–Hochberg false discovery rate

(FDR) correction was applied to control for multiple testing. _F_ST windows with FDR-adjusted _P_-values < 0.05 were considered significantly different from the genome-wide expectation.

Allele frequency distributions were analyzed using VCFtools (version 0.1.16)56, with particular attention to differences between wild type, heterozygous, and homozygous FM chickens. Regions

showing significant deviations in allele frequency were identified and further examined for recombination breakpoints manually. PHYLOGENETIC ANALYSIS Phylogenetic relationships among FM

chickens were inferred using the identified SV regions. Neighbor-joining (NJ) trees were constructed using and RapidNJ software and visualized using iTOL57. Separate trees were constructed

for the DUP1 and DUP2 regions. LINKAGE DISEQUILIBRIUM ANALYSIS To assess LD decay patterns across the FM locus, we computed pairwise linkage disequilibrium (LD) using r2 between SNPs using

LDBlockShow (version 1.40)58 with default parameters. LD decay patterns were used to infer historical recombination rates, assuming a constant recombination landscape over time. The LD block

structure was analyzed to identify boundaries within the DUP1 and DUP2 regions, indicating possible recombination breakpoints. To determine whether LD decay in the _FM_ locus was

significantly different from the genome-wide background, we performed a genome-wide _Z_-score transformation within 10 kb windows. We then converted _Z_-scores to _P_-values using the

standard normal distribution and applied FDR correction to control for multiple testing. Regions with FDR-adjusted _P_-values < 0.05 were considered significantly different from

genome-wide LD expectations. This analysis assumes that LD patterns reflect both historical recombination events and population demographic history. Specifically, LD tends to persist in

regions under strong selection, low recombination, or with recent population bottlenecks. DATA AVAILABILITY All of the whole genome sequencing (WGS) datasets used in this study have been

previously reported (Supplementary Table 1). The variant call format (VCF) data was deposited in the Genome Variation Map (GVM) under accession project number PRJCA032154. CODE AVAILABILITY

All analyses were performed using previously published or developed tools, as indicated in Methods. No custom code was developed or used. REFERENCES * Andersson, L. & Purugganan, M.

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https://doi.org/10.1093/bib/bbaa227 (2021). Article  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Min-Sheng Peng (Kunming Institute of Zoology,

Chinese Academy of Sciences, Kunming, China) and Xun-He Huang (School of Life Science, Jiaying University, Meizhou, China) for their support and discussion. FUNDING Open access funding

provided by Uppsala University. The project was financially supported by Vetenskapsrådet (2017-02907), Knut and Alice Wallenberg Foundation (KAW 2023.0160), and Erik Philip-Sörensen

Foundation (G2024-033). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden Cheng Ma & Leif Andersson *

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, USA Leif Andersson Authors * Cheng Ma View author publications You can also search for this

author inPubMed Google Scholar * Leif Andersson View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS C.M. and L.A conceived and designed the

project. C.M. performed bioinformatic analyses. C.M. and L.A. wrote the manuscript. CORRESPONDING AUTHORS Correspondence to Cheng Ma or Leif Andersson. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing interests. ETHICAL APPROVAL Data were directly downloaded from published studies and no additional ethics approval was needed. Each study is

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identifies the complex structural variation at the fibromelanosis (_FM_) locus in chicken. _Sci Rep_ 15, 9239 (2025). https://doi.org/10.1038/s41598-025-94250-4 Download citation * Received:

04 December 2024 * Accepted: 12 March 2025 * Published: 18 March 2025 * DOI: https://doi.org/10.1038/s41598-025-94250-4 SHARE THIS ARTICLE Anyone you share the following link with will be

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