Decoding the fibromelanosis locus complex chromosomal rearrangement of black-bone chicken: genetic differentiation, selective sweeps and protein-coding changes in Kadaknath chicken

Abstract

Black-bone chicken (BBC) meat is popular for its distinctive taste and texture. A complex chromosomal rearrangement at the fibromelanosis (Fm) locus on the 20th chromosome results in increased endothelin-3 (EDN3) gene expression and is responsible for melanin hyperpigmentation in BBC. We use public long-read sequencing data of the Silkie breed to resolve high-confidence haplotypes at the Fm locus spanning both Dup1 and Dup2 regions and establish that the Fm_2 scenario is correct of the three possible scenarios of the complex chromosomal rearrangement. The relationship between Chinese and Korean BBC breeds with Kadaknath native to India is underexplored. Our data from whole-genome re-sequencing establish that all BBC breeds, including Kadaknath, share the complex chromosomal rearrangement junctions at the fibromelanosis (Fm) locus. We also identify two Fm locus proximal regions (∼70 Kb and ∼300 Kb) with signatures of selection unique to Kadaknath. These regions harbor several genes with protein-coding changes, with the bactericidal/permeability-increasing-protein-like gene having two Kadaknath-specific changes within protein domains. Our results indicate that protein-coding changes in the bactericidal/permeability-increasing-protein-like gene hitchhiked with the Fm locus in Kadaknath due to close physical linkage. Identifying this Fm locus proximal selective sweep sheds light on the genetic distinctiveness of Kadaknath compared to other BBC.

Keywords: Fm locus; Kadaknath; black-bone chicken; fibromelanosis; genetic linkage.

FIGURE 1

Population structure analysis: (A) Geographical locations of different BBC breeds used in this study are shown on the map using different shapes. The map was generated using rworldmap, map, and mapdata R packages. (B) Genome-wide principal component analysis reveals the genetic relationship of 34 BBC individuals. PC1 and PC2 explained 18.9% and 13% variance, respectively. (C) Population genetic structure and individual ancestry were estimated using NGSadmix for 34 BBCs from different breeds based on best K = 7.

FIGURE 2

Arrangement of the Fm locus region in non-BBC and BBC breeds: two different non-paralogous regions on chromosome 20 are referred to as Duplication 1 (Dup1) and Duplication 2 (Dup2), shown in gold and light green colors, respectively. The length of the Dup1 region is ∼127 Kb, the intermediate (Int) is ∼412 Kb, and Dup2 is ∼170 Kb in size. (A) These regions are neither duplicated nor rearranged in non-BBC (*N). Dup1, Int, and Dup2 regions are collectively referred to as the Fm locus. Dup1 contains five genes, EDN3, ZNF831, SLMO2, ATP5E, and TUBB1, whereas the Dup2 region does not have any protein-coding genes. (B) Three possible scenarios (*Fm_1, *Fm_2, and *Fm_3) for Fm locus have been proposed in the BBC (earlier described in Dorshorst et al., 2011; Dharmayanthi et al., 2017; Sohn et al., 2018). The dark solid purple line represents haplotype-1, which spans across Dup1 from Flank1 to inverted Dup2 (i.e., Flank1 + Dup1 + (inverted Dup2)). The blue line represents haplotype-2, which spans Dup1 from inverted Dup2 to Int (i.e., (inverted Dup2) + Dup1 + Int).

FIGURE 3

Long-read-based haplotypes resolve the sequence spanning the Dup1 region at Fm locus: the two haplotypes spanning the Dup1 region are distinguished by distinct alleles at 24 positions using long sequencing reads. Red dotted vertical boxes highlight the alleles that differ between haplotype-1 and haplotype-2 at the same positions. The alleles at 24 sites that separate these two haplotypes at various positions along Dup1 are presented sequentially between the red dotted boxes. (A) Haplotype-1 of Dup1 spans from Flank1 to inverted Dup2 (i.e., Flank1 + Dup1 + (inverted Dup2)). The purple lines represent overlapping reads of Nanopore containing Dup1 haplotype-1 alleles. The light blue dotted line represents the span of the same read from the end of haplotype-1 of Dup1 to the end of haplotype-2 of Dup2. (B) Haplotype-2 spans Dup1 from haplotype-2 of Dup2 to the Int region (i.e., (inverted Dup2) + Dup1 + Int). The blue lines represent the overlapping reads of Nanopore containing haplotype-2 alleles. The light blue dotted line represents the span of the same read from the start of haplotype-2 of Dup1 to the start of haplotype-2 of Dup2. Both haplotypes of Dup1 are connected to the single haplotype of Dup2 (i.e., haplotype-2 of Dup2) but at different ends.

FIGURE 4

Long-read-based haplotypes resolve the sequence spanning the Dup2 region at Fm locus: The two haplotypes spanning the Dup2 region are distinguished by distinct alleles at 25 positions using long sequencing reads. Red dotted vertical boxes highlight the alleles that differ between haplotype-1 and haplotype-2 at the same positions. The alleles at 25 sites that separate these two haplotypes at various positions along Dup2 are presented sequentially between the red dotted boxes. (A) Haplotype-1 of Dup2 spans from Int to Flank2 (i.e., Int + Dup2 + Flank2). The gray lines represent overlapping reads of Nanopore containing Dup2 haplotype-1 alleles. (B) Haplotype-2 of Dup2 spans from the start of haplotype-2 of Dup1 to the end of haplotype-1 of the Dup1 region (i.e., Dup1 +(inverted Dup2)+ Dup1). The red lines represent the overlapping reads of Nanopore containing Dup2 haplotype-2 alleles. The light blue dotted line represents the span of the same reads from the start of haplotype-2 of Dup1 to the start of haplotype-2 of Dup2. Similarly, the same reads span the end of haplotype-1 of Dup1 to the end of haplotype-2 of Dup2.

FIGURE 5

Screenshot of the UCSC genome browser of two haplotypes at Dup1 and Dup2 regions highlighted in cyan color identified using Nanopore long reads. Haplotype-1 of Dup1, shown in purple, spans from Flank1 to the end of inverted Dup2 (i.e., Flank1 + Dup1 + (inverted Dup2)). Haplotype-2 of Dup1, shown in blue, spans from the start of inverted Dup2 to the Int region (i.e, (inverted Dup2) + Dup1 + Int). Haplotype-1 of Dup2, shown in gray, spans from Int to Flank2 (i.e., Int + Dup2 + Flank2). Haplotype-2 of Dup2, shown in red, spans from the start of haplotype-2 of Dup1 to the end of haplotype-1 of the Dup1 region (i.e., Dup1 +(inverted Dup2)+ Dup1).

FIGURE 6

Comparison of Fm locus read coverage in black-bone vs. non-black-bone chicken breeds: read coverage along chromosome 20 at the Fm locus is shown in 1 Kb sliding windows. Two duplicated genomic loci, Dup1 and Dup2, are denoted by vertical dotted red lines and have a higher coverage in BBC breeds: (A) Kadaknath, (B) Xichuan, (C) Yeonsan Ogye, (D) Tianfu, (E) Ayam Cemani than non-BBC, and (F) red jungle fowl (RJF).

FIGURE 7

Nucleotide sequence of rearranged junctions at the Fm locus in black-bone vs. non-black-bone chicken: (A) Dup1 + (inverted Dup2) (B) (inverted Dup1) + Dup2. Brown arrows represent the direction of rearranged junction sequence, which is present as a continuous sequence (verified by their presence in a single read) in BBC breeds. In contrast, non-BBC breeds did not contain these junctions. Sequences highlighted in red and blue represent the Dup1 and Dup2 sequences, respectively. Sequences in black color represent the subsequent nucleotides.

FIGURE 8

Within and between population comparison of KADK with CHIN: (A) genome-wide landscape of pairwise genetic differentiation (F_ST) between KADK and CHIN in 50 Kb non-overlapping windows. The dark slate gray and deep sky blue colors represent the alternative chromosomes. The dotted horizontal black line marks the 99th percentile outlier of estimated F_ST for autosome and the Z chromosome, respectively. (B) Highest F_ST region with a prominent pattern occurs on chromosome 20. The panels from top to bottom show estimates of F_ST in 10 Kb non-overlapping windows within population pairwise nucleotide diversity (π) and integrated haplotype score (iHS) with the major allele as ancestral and the minor allele as derived. The genes are represented as black boxes with names. The horizontal red dotted line marks the 99th percentile 10 Kb F_ST outlier. The transparent gray color highlights the Dup1 region in all panes in the B panel, while R1 and R2 represent the selective sweep regions. (C) Non-synonymous changes (shown above the exon) within genes in the regions R1 and R2 are denoted by vertical orange lines. Blue boxes are the exons connected by black lines representing introns.