Physical Linkage Research Articles

Opto-chemogenetic inhibition of L-type CaV1 channels in neurons through a membrane-assisted molecular linkage.

Genetically encoded inhibitors of CaV1 channels that operate via C-terminus-mediated inhibition (CMI) have been actively pursued. Here, we advance the design of CMI peptides by proposing a membrane-anchoring tag that is sufficient to link the inhibitory modules to the target channel as well as chemical and optogenetic modes of system control. We designed and implemented the constitutive and inducible CMI modules with appropriate dynamic ranges for the short and long variants of CaV1.3, both naturally occurring in neurons. Upon optical (near-infrared-responsive nanoparticles) and/or chemical (rapamycin) induction of FRB/FKBP binding, the designed peptides translocated onto the membrane via FRB-Ras, where the physical linkage requirement for CMI could be satisfied. The peptides robustly produced acute, potent, and specific inhibitions on both recombinant and neuronal CaV1 activities, including Ca2+ influx-neuritogenesis coupling. Validated through opto-chemogenetic induction, this prototype demonstrates Ca2+ channel modulation via membrane-assisted molecular linkage, promising broad applicability to diverse membrane proteins.

Historic breeding practices contribute to germplasm divergence in leaf specialized metabolism and ecophysiology in cultivated sunflower (Helianthus annuus).

The use of hybrid breeding systems to increase crop yields has been the cornerstone of modern agriculture and is exemplified in the breeding and improvement of cultivated sunflower (Helianthus annuus). However, it is poorly understood what effect supporting separate breeding pools in such systems, combined with continued selection for yield, may have on leaf ecophysiology and specialized metabolite variation. We analyzed 288 lines of cultivated H. annuus to examine the genomic basis of several specialized metabolites and agronomically important traits across major heterotic groups. Heterotic group identity supports phenotypic divergences between fertility restoring and cytoplasmic male-sterility maintainer lines in leaf ecophysiology and specialized metabolism. However, the divergence is not associated with physical linkage to nuclear genes that support current hybrid breeding practices in cultivated H. annuus. Additionally, we identified four genomic regions associated with leaf ecophysiology and specialized metabolism that colocalize with previously identified QTLs for quantitative self-compatibility traits and with S-protein homolog (SPH) proteins, a recently discovered family of proteins associated with self-incompatibility and self/nonself recognition in Papaver rhoeas (common poppy) with suggested conserved downstream mechanisms among eudicots. Further work is necessary to confirm the self-incompatibility mechanisms in cultivated H. annuus and their relationship to the integrative and polygenic architecture of leaf ecophysiology and specialized metabolism in cultivated sunflower. However, because self-compatibility is a derived quantitative trait in cultivated H. annuus, trait linkage to divergent phenotypic traits may have partially arisen as a potential unintended consequence of historical breeding practices and selection for yield.

Generation of Antibody Libraries for Phage Display: Library Reamplification.

Phage display of Fab libraries enables the de novo discovery and in vitro evolution of monoclonal antibodies. Fab libraries are collections of millions to billions of different antibodies that collectively cover a large antigen or epitope binding space. To preserve the diversity of the Fab library for repeated selection campaigns, it is recommended to use the original phage from the Fab library generation rather than reamplified phage, if practically possible. This is because reamplification will bias the Fab library for clones that are expressed at higher rates. Fab-phage, however, should only be used if they have been prepared on the same day, to avoid proteolytic cleavage of the physical linkage of phenotype (phage-displayed Fab protein) and genotype (phage-encapsulated Fab DNA). Thus, in practice, reamplification of a Fab-phage library cannot usually be avoided. Here, we describe the steps for the reamplification of an original Fab-phage library prior to its selection. The protocol can also be used to reamplify Fab-phage from the third or later panning rounds when enriched clones are unlikely to be lost by reamplification biases.

Peptide Bundlemer Networks or Lattices: Controlling Cross-Linking and Self-Assembly Using Protein-like Display of Chemistry.

Coiled-coil 'bundlemer' peptides were selectively modified with allyloxycarbonyl (alloc)-protected lysine, a non-natural amino acid containing an alkene on its side chain. The specific display of this alkene from the coiled-coil surface with protein-like specificity enabled this residue to be used as a covalent linkage for creating peptide networks with controllable properties or as a physical linkage for the self-assembly of bundlemers into unexpected, intricate lattices driven by the hydrophobic nature of the side chain. For network formation, peptides were modified with both alloc-protected lysine and cysteine amino acids for solution assembly into solvent-swollen films and subsequent covalent cross-linking via thiol-ene photo click reactions. The degree of network cross-linking, as determined by rheometry, was finely tuned by varying the specific spatial display of reactive groups on the bundlemer building block particles, transitioning between intrabundle and interbundle cross-linking. The designed display of alloc groups from the center of the bundlemer building block also prompted particle self-assembly into an unexpected intricate lattice with a porous morphology. The lattices were studied in a variety of solution conditions using transmission electron microscopy, cryotransmission electron microscopy, and small-angle X-ray scattering. The approximate particle arrangement in the lattice was determined by using coarse-grained modeling and machine learning optimization techniques along with experimental methods. The proposed truss-like face-centered cubic packing of the alloc-functionalized bundlemers agrees well with the experimental results.

Estimation of effective number of breeders and effective population size in an abundant and heavily exploited marine teleost.

Obtaining reliable estimates of the effective number of breeders (N b) and generational effective population size (N e) for fishery-important species is challenging because they are often iteroparous and highly abundant, which can lead to bias and imprecision. However, recent advances in understanding of these parameters, as well as the development of bias correction methods, have improved the capacity to generate reliable estimates. We utilized samples of both single-cohort young of the year and mixed-age adults from two geographically and genetically isolated stocks of the Australasian snapper (Chrysophrys auratus) to investigate the feasibility of generating reliable N b and N e estimates for a fishery species. Snapper is an abundant, iteroparous broadcast spawning teleost that is heavily exploited by recreational and commercial fisheries. Employing neutral genome-wide SNPs and the linkage-disequilibrium method, we determined that the most reliable N b and N e estimates could be derived by genotyping at least 200 individuals from a single cohort. Although our estimates made from the mixed-age adult samples were generally lower and less precise than those based on a single cohort, they still proved useful for understanding relative differences in genetic effective size between stocks. The correction formulas applied to adjust for biases due to physical linkage of loci and age structure resulted in substantial upward modifications of our estimates, demonstrating the importance of applying these bias corrections. Our findings provide important guidelines for estimating N b and N e for iteroparous species with large populations. This work also highlights the utility of samples originally collected for stock structure and stock assessment work for investigating genetic effective size in fishery-important species.

A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment

In this report, we present OLAF-Seq, a novel strategy to construct a long-read sequencing library such that adjacent fragments are linked with end-terminal duplications. We use the CRISPR-Cas9 nickase enzyme and a pool of multiple sgRNAs to perform non-random fragmentation of targeted long DNA molecules (> 300kb) into smaller library-sized fragments (about 20 kbp) in a manner so as to retain physical linkage information (up to 1000 bp) between adjacent fragments. DNA molecules targeted for fragmentation are preferentially ligated with adaptors for sequencing, so this method can enrich targeted regions while taking advantage of the long-read sequencing platforms. This enables the sequencing of target regions with significantly lower total coverage, and the genome sequence within linker regions provides information for assembly and phasing. We demonstrated the validity and efficacy of the method first using phage and then by sequencing a panel of 100 full-length cancer-related genes (including both exons and introns) in the human genome. When the designed linkers contained heterozygous genetic variants, long haplotypes could be established. This sequencing strategy can be readily applied in both PacBio and Oxford Nanopore platforms for both long and short genes with an easy protocol. This economically viable approach is useful for targeted enrichment of hundreds of target genomic regions and where long no-gap contigs need deep sequencing.

Northward Extension of East Asian Summer Monsoon Since the Miocene Set by the Uplift of Tibetan Plateau

AbstractThe modern East Asian summer monsoon (EASM) features an extension from tropical to subtropical areas. However, the fundamental process that determines the northward extension of EASM in the geological history remains unclear. Here, we showed evidence from proxy data, climate modeling, and theoretical solutions that the northward extension of EASM to today's boundary emerged no later than the Miocene. The extension was driven by the monsoon seasonal march which features stepwise northward rainfall stages. The seasonal progression of monsoon was determined by Rossby wave responses from early summer to late summer and caused by the weakening of westerly jet colliding with the Tibetan Plateau (TP). The Rossby wave responses further led to a northward migration of the western Pacific subtropical high and thereby monsoon precipitation. Our findings propose a novel physical linkage between the geological evolution of EASM and the TP uplift in the context of monsoon seasonal march.

Metazoa-level USCOs as markers in species delimitation and classification.

Metazoa-level universal single-copy orthologs (mzl-USCOs) are universally applicable markers for DNA taxonomy in animals that can replace or supplement single-gene barcodes. Previously, mzl-USCOs from target enrichment data were shown to reliably distinguish species. Here, we tested whether USCOs are an evenly distributed, representative sample of a given metazoan genome and therefore able to cope with past hybridization events and incomplete lineage sorting. This is relevant for coalescent-based species delimitation approaches, which critically depend on the assumption that the investigated loci do not exhibit autocorrelation due to physical linkage. Based on 239 chromosome-level assembled genomes, we confirmed that mzl-USCOs are genetically unlinked for practical purposes and a representative sample of a genome in terms of reciprocal distances between USCOs on a chromosome and of distribution across chromosomes. We tested the suitability of mzl-USCOs extracted from genomes for species delimitation and phylogeny in four case studies: Anopheles mosquitos, Drosophila fruit flies, Heliconius butterflies and Darwin's finches. In almost all instances, USCOs allowed delineating species and yielded phylogenies that corresponded to those generated from whole genome data. Our phylogenetic analyses demonstrate that USCOs may complement single-gene DNA barcodes and provide more accurate taxonomic inferences. Combining USCOs from sources that used different versions of ortholog reference libraries to infer marker orthology may be challenging and, at times, impact taxonomic conclusions. However, we expect this problem to become less severe as the rapidly growing number of reference genomes provides a better representation of the number and diversity of organismal lineages.

Practical application of the linkage disequilibrium method for estimating contemporary effective population size: A review.

The method to estimate contemporary effective population size (Ne ) based on patterns of linkage disequilibrium (LD) at unlinked loci has been widely applied to natural and managed populations. The underlying model makes many simplifying assumptions, most of which have been evaluated in numerous studies published over the last two decades. Here, these performance evaluations are reviewed and summarized, with a focus on information that facilitates practical application to real populations in nature. Potential sources of bias that are discussed include calculation of r2 (a measure of LD), adjustments for sampling error, physical linkage, age structure, migration and spatial structure, mutation and selection, mating systems, changes in abundance, rare alleles, missing data, genotyping errors, data filtering choices and methods for combining multiple Ne estimates. Factors that affect precision are reviewed, including pseudoreplication that limits the information gained from large genomics datasets, constraints imposed by small samples of individuals, and the challenges in obtaining robust estimates for large populations. Topics that merit further research include the potential to weight r2 values by allele frequency, lump samples of individuals, use genotypic likelihoods rather than called genotypes, prune large LD values and apply the method to species practising partial monogamy.

Semmelweis Caring University Model Program: A Holistic Approach to Promoting Healthy Ageing through a Longitudinal Cohort Study, Preventive Services and Workplace Health Promotion

Abstract Hungary is facing an alarming public health situation due to the unhealthy ageing of its population, calling for innovative and effective approaches to promote healthy ageing. This presentation showcases the Semmelweis Caring University Model Program, a unique and holistic program developed by the leading health sciences university in Hungary to address this societal challenge. The program serves as a model for the ongoing primary care reform, which incorporates preventive services, the first study to address determinants of unhealthy ageing in Hungary, and includes a workplace-based health promotion program that could be implemented nationally. The Semmelweis Caring University Model Program aims to promote the health and wellbeing of its 8500 employees and provides potential answers to the unfavorable public health situation in Hungary. The Semmelweis Study, a longitudinal occupational cohort of university employees, investigates the complex relationships between lifestyle, environmental, and occupational risk factors and the development and progression of chronic age-associated diseases. Data is collected through computer-assisted questionnaires, physical health examination, physiological measurements, biomarker assessment and data linkage to national registers. The results of the Semmelweis Study inform the development of the Centre of Preventive Services (CPS), which provides health risk assessment, screening, and counseling services to employees, with medical specialists in preventive medicine and primary care, public health professionals, dieticians, physiotherapists, and psychologists. The CPS units also serve as practical training sites for medical and health sciences students. The Semmelweis Workplace Health Promotion Program is a personalized public health model program that utilizes infrastructure and human resources of Semmelweis University to encourage healthy behaviors and environments in the workplace, with a potential for nationwide implementation.

Convergences between India-ASEAN Relations in the Realm of Indo-Pacific

The "Indo-Pacific" as a strategic concept has gained fresh impetus due to the rising significance of the Indian and Pacific oceans. The Association of Southeast Asian Nations states provide a physical and strategic linkage between both the regions. In India's view of the Indo-Pacific, ASEAN prominence and cohesion" are at the core. This research focuses on identifying developments in India-ASEAN relations in the Indo-Pacific region and expounding on their connection. As a result of the study, the researchers have outlined and analysed different "Convergences" between India and ASEAN that may safeguard their vision of a peaceful, dynamic, and progressive Indo-Pacific.

Smartphone application with 3D-printed needle guide for faster and more accurate CT-guided interventions in a phantom.

This study is to determine whether a needle guidance device combining a 3D-printed component with a smartphone would decrease the number of passes and time required to perform a standard CT-guided needle procedure in a phantom study. A 3D-printed mechanical guide with built-in apertures for various needle sizes was designed and printed. It was mounted on a smartphone and used to direct commercially available spring-loaded biopsy devices. A smartphone software application was developed to use the phone's sensors to provide the real-time location of a lesion in space, based on parameters derived from preprocedural CT images. The physical linkage of the guide, smartphone, and needle allowed the operator to manipulate the assembly as a single unit, with real-time graphical representation of the lesion shown on the smartphone display. Two radiology trainees and 3 staff radiologists targeted 5 lesions with and without the device (50 total procedures). The number of passes and time taken to reach each lesion were determined. Use of the smartphone needle guide decreased the mean number of passes (with guide, 1.8; without guide, 3.4; P < 0.001) and mean time taken (with guide, 1.6 min; without guide, 2.7 min; P = 0.005) to perform a standard CT-guided procedure. On average, the decreases in number of passes and procedure time were more pronounced among trainees (P < 0.001). The combination of a mechanical guide and smartphone can reduce the number of needle passes and the amount of time needed to reach a lesion in a phantom for both trainees and experienced radiologists.

The Rossby Normal Mode as a Physical Linkage in a Machine Learning Forecast Model for the SST and SSH of South China Sea Deep Basin

AbstractMachine learning (ML) has been widely applied in Earth sciences, but its black‐box nature still remains. Existing techniques for interpretable ML developed by deep learning researchers are not satisfactory in understanding physical problems. In this study, our objective is to establish the physical linkage between features extracted from ML model and results derived from physical equations that describe the problem. This approach aims to provide a more comprehensible way of understanding an ML model. We select a less complex ML forecasting model, consisting of traditional statistical algorithms, which effectively forecast sea surface temperature anomaly (SSTA) and sea level anomaly (SLA) of the South China Sea (SCS) in 30 days ahead. Here, we focus on the SLA prediction and detect the physical mechanism of model capability to predict SCS SLA. Previous study has identified Rossby normal modes as the physical mechanism for long‐lived eddies in SCS in previous study. We here demonstrate the relationship between the ML model and Rossby normal modes in SCS in terms of temporal variations and spatial distributions of water mass. The model's skill in predicting Rossby normal modes partially explains its skill in predicting SLA, thereby providing a more transparent and interpretable basis for forecasts in SCS.

Genome structure and population genomics of the canine heartworm Dirofilaria immitis

The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one sex-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between-population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped single nucleotide polymorphisms previously associated with macrocyclic lactone resistance in the new genome assembly, revealing the physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment.

CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-β mimic

Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through release of an immunosuppressive TGF-β mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-β receptors, acting on T cells to induce Foxp3+ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type-specific potency of TGF-β signaling in mammalian cells.

Emergence of linkage between cooperative RNA replicators encoding replication and metabolic enzymes through experimental evolution.

The integration of individually replicating genes into a primitive chromosome is a key evolutionary transition in the development of life, allowing the simultaneous inheritance of genes. However, how this transition occurred is unclear because the extended size of primitive chromosomes replicate slower than unlinked genes. Theoretical studies have suggested that a primitive chromosome can evolve in the presence of cell-like compartments, as the physical linkage prevents the stochastic loss of essential genes upon division, but experimental support for this is lacking. Here, we demonstrate the evolution of a chromosome-like RNA from two cooperative RNA replicators encoding replication and metabolic enzymes. Through their long-term replication in cell-like compartments, linked RNAs emerged with the two cooperative RNAs connected end-to-end. The linked RNAs had different mutation patterns than the two unlinked RNAs, suggesting that they were maintained as partially distinct lineages in the population. Our results provide experimental evidence supporting the plausibility of the evolution of a primitive chromosome from unlinked gene fragments, an important step in the emergence of complex biological systems.

Variability of volume transport in the Taiwan Strait and its response to tropical MJO convection: A numerical approach

Taiwan Strait (TWS) plays a crucial role in material exchange and nutrient budget between the South China Sea and the East China Sea. In this study, we investigate the variability of volume transport in the TWS and its response to tropical Madden–Julian Oscillation (MJO) convection based on the simulated results from a three-dimensional operational numerical model. Validated by the observational data, the model generally reproduced the physical field well. The volume transport in the TWS has strong seasonal cycles as well as higher-frequency variations. Intra-seasonal fluctuations dominate the along-strait currents variability, while secondary and the last signal are seasonal one and inter-annual variability, respectively. Overall, the along-strait wind stress plays a more important role in controlling the variability of volume transport in the TWS than the pressure gradient induced by north-to-south sea level slope. At intra-seasonal time scales, the volume transport in the TWS varies with the movement of the tropical MJO convection from Indian Ocean eastward to the western Pacific Ocean. These oceanic anomalies are related to atmospheric anomalies, with a distinct physical linkage from the tropical atmosphere to the mid-latitude ocean. The tropical MJO deep convection can modify the upper tropospheric heights and generate a wave train pattern that propagates to mid-latitudes. In addition, these anomalous upper tropospheric heights modulate the surface pressure, resulting in a cyclonic anomaly. The upper tropospheric heights anomaly and its corresponding mean sea level pressure anomaly move eastward in the MJO cycle following the migrating MJO heat source in the tropics from phase 2 to 5. Consequently, surface winds change as the cyclonic anomaly moves from central China to the east of Japan, resulting in a northeast volume transport anomaly during MJO phase 2 and 3 and a southwest volume transport anomaly during MJO phase 4 and 5.

Nuclear elongation during spermiogenesis depends on physical linkage of nuclear pore complexes to bundled microtubules by Drosophila Mst27D.

Spermatozoa in animal species are usually highly elongated cells with a long motile tail attached to a head that contains the haploid genome in a compact and often elongated nucleus. In Drosophila melanogaster, the nucleus is compacted two hundred-fold in volume during spermiogenesis and re-modeled into a needle that is thirty-fold longer than its diameter. Nuclear elongation is preceded by a striking relocalization of nuclear pore complexes (NPCs). While NPCs are initially located throughout the nuclear envelope (NE) around the spherical nucleus of early round spermatids, they are later confined to one hemisphere. In the cytoplasm adjacent to this NPC-containing NE, the so-called dense complex with a strong bundle of microtubules is assembled. While this conspicuous proximity argued for functional significance of NPC-NE and microtubule bundle, experimental confirmation of their contributions to nuclear elongation has not yet been reported. Our functional characterization of the spermatid specific Mst27D protein now resolves this deficit. We demonstrate that Mst27D establishes physical linkage between NPC-NE and dense complex. The C-terminal region of Mst27D binds to the nuclear pore protein Nup358. The N-terminal CH domain of Mst27D, which is similar to that of EB1 family proteins, binds to microtubules. At high expression levels, Mst27D promotes bundling of microtubules in cultured cells. Microscopic analyses indicated co-localization of Mst27D with Nup358 and with the microtubule bundles of the dense complex. Time-lapse imaging revealed that nuclear elongation is accompanied by a progressive bundling of microtubules into a single elongated bundle. In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal. Thus, we propose that Mst27D permits normal nuclear elongation by promoting the attachment of the NPC-NE to the microtubules of the dense complex, as well as the progressive bundling of these microtubules.

Wintertime sea surface temperature variability modulated by Arctic Oscillation in the northwestern part of the East/Japan Sea and its relationship with marine heatwaves

The northwestern part of the East/Japan Sea (EJS) is a region with large sea surface temperature (SST) variability and is known as a hotspot of marine heatwaves (MHW) stress for marine environments that peaked in boreal winter (January-February-March). This could have profound impacts on the marine ecosystems over the EJS. Here, we used a set of high-resolution satellite and reanalysis products to systematically analyze the spatiotemporal SST variations and examine their linkage to a large-scale mode of climate variability, such as the Arctic Oscillation (AO). The results show that AO-related wind forcing modulates the SST variability over the EJS via the oceanic dynamic adjustment processes. In particular, the abnormally warm SSTs in the northwestern part of the EJS are driven by the anomalous anticyclonic eddy-like circulation and Ekman downwelling during a positive AO phase. This physical linkage between a positive AO and the abnormally warm SST could be conducive to MHW occurrences in the EJS as in the extremely positive AO event during the winter of 2020. These results have implications that the MHW occurrences in the EJS could be amplified by natural climate variability along with long-term SST warming.

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

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 ∼300Kb) 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.