Despite the significant phenotypic divergence between domestic ducks and their wild progenitors, the structural variants (SVs) underlying this differentiation remain largely unexplored due to the limitations of linear references in capturing genetic diversity across the genus Anas. Current pangenome efforts either lack base-level resolution or are constrained by genomic anchoring, hindering the exploration of genetic signatures of domestication. Here, we assembled chromosome-level genomes for a mallard (Anas platyrhynchos) and an eastern spot-billed duck (Anas zonorhyncha) that retained substantial domestic components. By integrating these with 14 high-quality Anas assemblies, we constructed a base-level resolution pangenome graph encompassing 243.4 Mb of non-reference sequences. Our graph-based pipeline demonstrated superior mapping performance and identified 67,165 population-level SVs, representing a five-fold increase in detection sensitivity compared to linear-based methods. Notably, SVs explained more genetic variance (PC1: 8.75% vs. 3.25%) and provided finer resolution of local ancestry than single nucleotide polymorphisms (SNPs), highlighting the unique advantage of SVs in resolving duck population structure. Leveraging the graph, we recovered 163 ancestral SVs that are prevalent in distant species but persist at low frequencies in domestic and wild populations. The loss of these ancestral sequences in gravity sensing and cilium assembly likely provided the physiological plasticity necessary for domestication. Moreover, highly divergent exonic SVs exhibited a non-random distribution, preferentially accumulating in UTRs over CDS regions. Specifically, highly divergent SVs located in UTRs of TMEM123 and FAM13A may be associated with viral resistance and the regulation of adipogenesis. Similarly, we identified a highly divergent 75 bp insertion located 3.66 kb upstream of the FAM184B gene, which may be associated with changes in muscle growth during duck domestication. In conclusion, the study establishes a comprehensive base-resolution pangenome resource for the Anas. Our findings reveal that SVs are essential for resolving complex evolutionary histories and suggest that SV-mediated regulatory evolution is an important driver of rapid phenotypic change during duck domestication.

Phenotypic plasticity enables organisms to modify their phenotypes in response to environmental fluctuations. Interspecific hybrids can inherit parental phenotypic plasticity and even exhibit enhanced physiological performance under analogous environmental shifts, a phenomenon termed as plastic heterosis. However, the genetic mechanisms underlying plastic heterosis remain poorly understood. Here, we explore the transcriptional profiling of overfeeding-induced fatty liver in Peking ducks (Anas platyrhynchos), Muscovy ducks (Cairina moschata), and their reciprocal hybrids. We find that genes associated with fatty acid catabolism retained their ancestral plasticity in Muscovy ducks but lost it in Peking ducks. The parental transcriptional plasticity is reshuffled in hybrid ducks during hybridization. Specifically, the expression pattern of 129 Peking-derived alleles is converted to those of Muscovy-derived alleles in both reciprocal hybrid ducks in response to overfeeding. Among these, eight fatty acid catabolism-related alleles that had lost plasticity in Peking ducks restore ancestral plasticity in hybrids. Our findings systematically characterize transcriptional changes linked to parental phenotypic plasticity and illuminate its evolutionary trajectory. In particular, the restoration of ancestral plasticity in interspecific hybrids may represent a mechanism contributing to plastic heterosis.

Chickens are one of the most important domesticated animals, serving as an important protein source. Studying genetic variations in chickens to enhance their production performance is of great potential value. The emergence of next-generation sequencing has enabled precise analysis of single nucleotide polymorphisms and insertions/deletions in chicken, while third-generation sequencing achieves the accurate structural variant identification. However, the high cost of third-generation sequencing technology limits its application in population studies. The graph-based pan-genome strategy can overcome this challenge by enabling the detection of structural variations using cost-effective next-generation sequencing data. This study constructed a graph-based pan-genome for chickens using 12 high-quality genomes. This pan-genome used linear genome GRCg6a as the reference genome, containing variant information from two commercial and nine native chicken breeds. Compared to the linear genome, the pan-genome provided significant improvements in the efficiency of structural variation identification. On the basis of the graph-based pan-genome, high-frequency structural variations related to high egg production in Leghorn chicken were predicted. Additionally, it was discovered that potential structural variations was associated with highland adaptation in Tibetan chickens according to next-generation sequencing and transcriptomics data. Using the pan-genome graph, a new strategy to identify structural variations related to traits of interest in chickens is presented.

The influenza A viruses (IAV) are the principal pathogens for annual (seasonal) influenza, which cause world-wide outbreaks in poultry and pose a persistent threat to public health. The Hemagglutinin protein (HA) of IAV promotes virus infection by binding the host membrane receptor and mediating virus-host membrane fusion. Immunoprecipitation-mass spectrometry (IP-MS) provides global insights into IAV HA-host protein interactions. However, various experimental conditions might affect the identification of interactions. Here, we performed a serial IP-MS to compare interactors of IAV HA in accidental host human, chicken and reservoir host duck cells. We found that the positive ratio of interactors identified by the IP-MS was improved when the transfected HA plasmid had a similar expression level to HA proteins found in IAV virus infection. Comparing interactors in human, chicken and duck cells, we found that HA-interacting host factors might play a role in the susceptibility of accidental hosts (human and chicken) to IAV infection compared to reservoir hosts (duck). We then focused on the function of two heat shock proteins (HSPA5 and HSPA8), which interacted with IAV HA proteins in all three species (human, chicken and duck). We found that both HSPA5 and HSPA8 promoted the IAV replication by enhancing the viral attachment and internalization. These findings extend our knowledge about the mechanisms of IAV entry to host cells and provide target genes to create chickens resistant to avian influenza.

Evolutionary studies of wild and domestic organisms have yielded fascinating discoveries, while the species diversity and the domestication of ducks remain unclear. Here, we assembled eight chromosome-level Anas genomes, combined with the Pekin duck genome, to investigate Anas evolution and domestication. We found that, compared to autosomes, the Z chromosome was less affected by introgression and exhibited relatively stable local phylogenies. From the Z chromosome perspective, we proposed that the speciation of Anas platyrhynchos and Anas zonorhyncha was accompanied by continuous female-biased gene flow and remodeled duck domestication history. Moreover, we constructed an Anas pan-genome and identified several differentiated structural variations (SVs) between domestic and wild ducks. These SVs likely regulate their neighboring genes (i.e., GHR and FER), which represented the promising "domestication genes." Furthermore, a long terminal repeat (LTR) retrotransposon burst was found to have accelerated duck domestication, specifically contributing to functional shifts of the notable MITF and IGF2BP1 genes. These findings presented a live example for understanding animal evolutionary processes.

The Muscovy duck (Cairina moschata) is a waterfowl indigenous to the neotropical regions of Central and South America. It has low demand for concentrated feed and strong adaptability to different rearing conditions. After introduced to China through Eurasian commercial trade, Muscovy ducks have a domestication history of around 300 years in the Fujian Province of China. In the 1990s, the commodity Muscovy duck breed "Crimo," cultivated in Europe, entered the Chinese market for consumption and breeding purposes. Due to the different selective breeding processes, Muscovy ducks have various populational traits and lack transparency of their genetic background. To remove this burden in the Muscovy duck breeding process, we analyzed genomic data from 8 populations totaling 83 individuals. We identify 11.24 million single nucleotide polymorphisms (SNPs) and categorized these individuals into the Fujian-bred and the Crimo populations according to phylogenetic analyses. We then delved deeper into their evolutionary relationships through assessing population structure, calculating fixation index (FST) values, and measuring genetic distances. Our exploration of runs of homozygosity (ROHs) and homozygous-by-descent (HBD) uncovered genomic regions enriched for genes implicated in fatty acid metabolism, development, and immunity pathways. Selective sweep analyses further indicated strong selective pressures exerted on genes including TECR, STAT2, and TRAF5. These findings provide insights into genetic variations of Muscovy ducks, thus offering valuable information regarding genetic diversity, population conservation, and genome associated with the breeding of Muscovy ducks.

The interaction between migratory birds and domestic waterfowl facilitates viral co-infections, leading to viral reassortment and the emergence of novel viruses. In 2022, samples were collected from duck farms around Poyang Lake in Jiangxi Province, China, which is located within the East Asia–Australasia flyway. Three strains of H4N6 avian influenza virus (AIV) were isolated. Genetic and phylogenetic analyses showed that the isolated H4N6 avian influenza viruses (AIVs) belonged to new genotypes, G23 and G24. All isolated strains demonstrated dual receptor binding properties. Additionally, the isolated strains were able to replicate efficiently not only in avian cells but also in mammalian cells. Furthermore, the H4N6 AIV isolates could infect chickens, with viral replication detected in the lungs and extrapulmonary organs, and could transmit within chicken flocks through contact, with viral shedding detected only in oropharyngeal swabs from chickens in the contact group. Notably, the H4N6 AIV could infect mice without prior adaptation and replicate in the lungs with high viral titers, suggesting that it is a potential threat to humans. In conclusion, this study provides valuable insight into the characteristics of H4N6 strains currently circulating in China.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease. Little is known about how gene expression and chromatin structure are regulated in NAFLD due to lack of suitable model. Ducks naturally develop fatty liver similar to serious human non-alcoholic fatty liver (NAFL) without adipose inflammation and liver fibrosis, thus serves as a good model for investigating molecular mechanisms of adipose metabolism and anti-inflammation. Here, we constructed a NAFLD model without adipose inflammation and liver fibrosis in ducks. By performing dynamic pathological and transcriptomic analyses, we identified critical genes involving in regulation of the NF-κB and MHCII signaling, which usually lead to adipose inflammation and liver fibrosis. We further generated dynamic three-dimensional chromatin maps during liver fatty formation and recovery. This showed that ducks enlarged hepatocyte cell nuclei to reduce inter-chromosomal interaction, decompress chromatin structure, and alter strength of intra-TAD and loop interactions during fatty liver formation. These changes partially contributed to the tight control the NF-κB and the MHCII signaling. Our analysis uncovers duck chromatin reorganization might be advantageous to maintain liver regenerative capacity and reduce adipose inflammation. These findings shed light on new strategies for NAFLD control.

Background The duck (Anas platyrhynchos) is one of the principal natural hosts of influenza A virus (IAV), harbors almost all subtypes of IAVs and resists to many IAVs which cause extreme virulence in chicken and human. However, the response of duck’s adaptive immune system to IAV infection is poorly characterized due to lack of a detailed gene map of the major histocompatibility complex (MHC). Results We herein reported a chromosome-scale Beijing duck assembly by integrating Nanopore, Bionano, and Hi-C data. This new reference genome SKLA1.0 covers 40 chromosomes, improves the contig N50 of the previous duck assembly with highest contiguity (ZJU1.0) of more than a 5.79-fold, surpasses the chicken and zebra finch references in sequence contiguity and contains a complete genomic map of the MHC. Our 3D MHC genomic map demonstrated that gene family arrangement in this region was primordial; however, families such as AnplMHCI, AnplMHCIIβ, AnplDMB, NKRL (NK cell receptor-like genes) and BTN underwent gene expansion events making this area complex. These gene families are distributed in two TADs and genes sharing the same TAD may work in a co-regulated model. Conclusions These observations supported the hypothesis that duck’s adaptive immunity had been optimized with expanded and diversified key immune genes which might help duck to combat influenza virus. This work provided a high-quality Beijing duck genome for biological research and shed light on new strategies for AIV control.

BACKGROUND:Non-alcoholic fatty liver disease (NAFLD) is one of most common diseases in the world. Recently, alternative splicing (AS) has been reported to play a key role in NAFLD processes in mammals. Ducks can quickly form fatty liver similar to human NAFLD after overfeeding and restore to normal liver in a short time, suggesting that ducks are an excellent model to unravel molecular mechanisms of lipid metabolism for NAFLD. However, how alternative splicing events (ASEs) affect the fatty liver process in ducks is still unclear. RESULTS:Here we identify 126,277 unique transcripts in liver tissue from an overfed duck (77,237 total transcripts) and its sibling control (69,618 total transcripts). We combined these full-length transcripts with Illumina RNA-seq data from five pairs of overfed ducks and control individuals. Full-length transcript sequencing provided us with structural information of transcripts and Illumina RNA-seq data reveals the expressional profile of each transcript. We found, among these unique transcripts, 30,618 were lncRNAs and 1,744 transcripts including 155 lncRNAs and 1,589 coding transcripts showed significantly differential expression in liver tissues between overfed ducks and control individuals. We also detected 27,317 ASEs and 142 of them showed significant relative abundance changes in ducks under different feeding conditions. Full-length transcript profiles together with Illumina RNA-seq data demonstrated that 10 genes involving in lipid metabolism had ASEs with significantly differential abundance in normally fed (control) and overfed ducks. Among these genes, protein products of five genes (CYP4F22, BTN, GSTA2, ADH5, and DHRS2 genes) were changed by ASEs. CONCLUSIONS:This study presents an example of how to identify ASEs related to important biological processes, such as fatty liver formation, using full-length transcripts alongside Illumina RNA-seq data. Based on these data, we screened out ASEs of lipid-metabolism related genes which might respond to overfeeding. Our future ability to explore the function of genes showing AS differences between overfed ducks and their sibling controls, using genetic manipulations and co-evolutionary studies, will certainly extend our knowledge of genes related to the non-pathogenic fatty liver process.

PURPOSE. Diabetic retinopathy (DR) is a common complication of diabetes and has a high prevalence. Dysregulation of circadian rhythmicity is associated with the development of DR. This research aimed to investigate rhythmical transcriptome alterations in the retina of diabetic mice. METHODS. C57BL/6J mice were used to establish a diabetes model by intraperitoneal injection of streptozotocin (STZ). After 12 weeks, retinas were collected continuously at 4-hour intervals over 1 day. Total RNA was extracted from normal and STZ-treated retinas and RNA sequencing was performed. Meta2d algorithm, Kyoto Encyclopedia of Genes, Phase Set Enrichment Analysis, and time-series cluster analysis were used to identify, analyze and annotate the composition, phase, and molecular functions of rhythmic transcripts in retinas. RESULTS. The retina exhibited powerful transcriptome rhythmicity. STZ-induced diabetes markedly modified the transcriptome characteristics of the circadian transcriptome in the retina, including composition, phase, and amplitude. Moreover, the diabetic mice led to re-organized temporal and clustering enrichment pathways in space and time and affected core clock machinery. CONCLUSIONS. Diabetes impairs the circadian rhythm of the transcriptomic profile of retinas. This study offers new perspectives on the negative effects of diabetes on the retina, which may provide important information for the development of new treatments for DR.

OASs play critical roles in immune response against virus infection by polymerizing ATP into 2-5As,which initiate the classical OAS/RNase L pathway and induce degradation of viral RNA.OAS members are functionally diverged in four known innate immune pathways(OAS/RNase L,OASL/IRF7,OASL/RIG-I,and OASL/cGAS),but how they functionally diverged is unclear.Here,we focus on evolutionary patterns and explore the link between evolutionary processes and functional divergence of Tetrapod OAS1.We show that Palaeognathae and Primate OAS1 genes are conserved in genomic and protein structures but differ in function.The former(i.e.,ostrich)efficiently synthesized long 2-5A and activated RNase L,while the latter(i.e.,human)synthesized short 2-5A and did not activate RNase L.We predicted and verified that two in-frame indels and one positively selected site in the active site pocket contributed to the functional divergence of Palaeognathae and Primate OAS 1.Moreover,we discovered and validated that an in-frame indel in the C-terminus of Palaeognathae OAS 1 affected the binding affinity of dsRNA and enzymatic activity,and contributed to the functional divergence of Palaeognathae OAS1 proteins.Our findings unravel the molecular mechanism for functional divergence and give insights into the emergence of novel functions in Tetrapod OAS1.

Avian H9N2 influenza virus, although primarily restricted to chicken populations, is a major threat to human public health by acting as a donor of variant viral genes through reassortment to cocirculating influenza viruses. We established that the high prevalence of evolving H9N2 virus in vaccinated flocks played a key role as a donor of new subclade PB2 and PA genes in the generation of a dominant H7N9 virus genotype (genotype 72 [G72]) with enhanced infectivity in humans during the 2016–2017 N7N9 virus epidemic.

This datasheet on Anas platyrhynchos covers Identity, Distribution.

Table S2. DElncRNAs detected between duck infected with DK/49 or GS/65 H5N1 virus and control individuals. (XLSX 176 kb)

2´-5´-Oligoadenylate synthetase like protein (OASL) plays a key role in response to viral infections through selectively activating the OAS/RNase L or OASL/RIG-I signaling pathway. Although classic pathway of OASL is well-known, its regulated genes or co-actors are largely unknown. To study the possible molecular mechanism of duck OASL (dOASL), we performed RNA-sequencing (RNA-seq) and immunoprecipitation and mass spectrometry (IP-MS) at the level of mRNA and protein, respectively. For RNA-seq, we used DF1 cell lines (DF1dOASL+/+, DF1cOASL–/–, and DF1) with or without the CK/0513 H5N1 virus (A/chicken/huabei/0513/2007) infection. 1 737 differentially expressed genes (DEGs) were identified as candidate target genes regulated by dOASL. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Weighted Correlation Network Analysis (WGCNA) were performed. We identified one important yellow co-expression module correlated with antiviral immune response. In this module, Ankyrin repeat and FYVE domain containing 1 (ANKFY1), harboring a BTB domain similar to the methyl CpG-binding protein 1 (MBD1) which bound to OASL in human, was regulated by dOASL. At protein level, 133 host proteins were detected. Interestingly, ANKFY1 was one of them binding to dOASL protein. Further phylogenomic and chromosomal syntenic analysis demonstrated MBD1 was absent in birds, while mammals retained. It is suggested that OASL-ANKFY1 interaction might act as a compensatory mechanism to regulate gene expression in birds. Our findings will provide a useful resource for the molecular mechanism research of dOASL.

BackgroundLong non-coding RNAs (lncRNAs) are important component of mammalian genomes, where their numbers are even larger than that of protein-coding genes. For example, human (Homo sapiens) (96,308 vs. 20,376) and mouse (Mus musculus) (87,774 vs. 22,630) have more lncRNA genes than protein-coding genes in the NONCODEv5 database. Recently, mammalian lncRNAs were reported to play critical roles in immune response to influenza A virus infections. Such observation inspired us to identify lncRNAs related to immune response to influenza A virus in duck, which is the most important natural host of influenza A viruses.ResultsWe explored features of 62,447 lncRNAs from human, mouse, chicken, zebrafish and elegans, and developed a pipeline to identify lncRNAs using the identified features with transcriptomic data. We then collected 151,970 assembled transcripts from RNA-Seq data of 21 individuals from three tissues and annotated 4094 duck lncRNAs. Comparing to duck protein-coding transcripts, we found that 4094 lncRNAs had smaller number of exons (2.4 vs. 10.2) and longer length of transcripts (1903.0bp vs. 1686.9bp) on average. Among them, 3586 (87.6%) lncRNAs located in intergenic regions and 619 lncRNAs showed differential expression in ducks infected by H5N1 virus when compared to control individuals. 58 lncRNAs were involved into two co-expressional modules related to anti-influenza A virus immune response. Moreover, we confirmed that eight lncRNAs showed remarkably differential expression both in vivo (duck individuals) and in vitro (duck embryo fibroblast cells, DEF cells) after infected with H5N1 viruses, implying they might play important roles in response to influenza A virus infection.ConclusionsThis study presented an example to annotate lncRNA in new species based on model species using transcriptome data. These data and analysis provide information for duck lncRNAs' function in immune response to influenza A virus.

H5N1 influenza represents one of the great challenges to public health. Some H5N1 viruses (i.e., A/goose/Hubei/65/05, GS/65) are weakly pathogenic, while the others (i.e., A/duck/Hubei/49/05, DK/49) are highly pathogenic to their natural hosts. Here, we performed brain and spleen transcriptomic analyses of control ducks and ones infected by the DK/49 or the GS/65 H5N1 virus. We demonstrated that, compared to the GS/65 virus, the DK/49 virus infection changed more numerous immune genes’ expression and caused continuous increasing of immune pathways (i.e., RIG-I and MDA5) in ducks. We found that both H5N1 virus strains might escape or subvert host immune response through affecting alternative translation of immune genes, while the DK/49 virus seemed to induce alternative translation of more immune genes than the GS/65 virus. We also identified five co-expressional modules associated with H5N1 virus replication through the weight correlation network analysis (WGCNA). Moreover, we first demonstrated that the duck BCL2L15 and DCSTAMP in one of these five modules inhibited both the highly pathogenic and weakly pathogenic H5N1 virus replication efficiently. These analyses, in combination with our comprehensive transcriptomic data, provided global view of the molecular architecture for the interaction between host and H5N1 viruses.

Host cells develop the OAS/RNase L [2'-5'-oligoadenylate synthetase (OAS)/ribonuclease L] system to degrade cellular and viral RNA, and/or the OASL/RIG-I (2'-5'-OAS like/retinoic acid inducible protein I) system to enhance RIG-I-mediated IFN induction, thus providing the first line of defense against viral infection. The 2'-5'-OAS-like (OASL) protein may activate the OAS/RNase L system using its typical OAS-like domain (OLD) or mimic the K63-linked pUb to enhance antiviral activity of the OASL/RIG-I system using its two tandem ubiquitin-like domains (UBLs). We first describe that divergent avian (duck and ostrich) OASL inhibit the replication of a broad range of RNA viruses by activating and magnifying the OAS/RNase L pathway in a UBL-dependent manner. This is in sharp contrast to mammalian enzymatic OASL, which activates and magnifies the OAS/RNase L pathway in a UBL-independent manner, similar to 2'-5'-oligoadenylate synthetase 1 (OAS1). We further show that both avian and mammalian OASL can reversibly exchange to activate and magnify the OAS/RNase L and OASL/RIG-I system by introducing only three key residues, suggesting that ancient OASL possess 2-5A [px5' A(2'p5'A)(n); x = 1-3; n >= 2] activity and has functionally switched to the OASL/RIG-I pathway recently. Our findings indicate the molecular mechanisms involved in the switching of avian and mammalian OASL molecules to activate and enhance the OAS/RNase L and OASL/RIG-I pathways in response to infection by RNA viruses.