The duck (Anas platyrhynchos) is one of the principal natural hosts of influenza A viruses. We present the duck genome sequence and perform deep transcriptome analyses to investigate immune-related genes. Our data indicate that the duck possesses a contractive immune gene repertoire, as in chicken and zebra finch, and this repertoire has been shaped through lineage-specific duplications. We identify genes that are responsive to influenza A viruses using the lung transcriptomes of control ducks and ones that were infected with either a highly pathogenic (A/duck/Hubei/49/05) or a weakly pathogenic (A/goose/Hubei/65/05) H5N1 virus. Further, we show how the duck’s defense mechanisms against influenza infection have been optimized through the diversification of its β-defensin and butyrophilin-like repertoires. These analyses, in combination with the genomic and transcriptomic data, provide a resource for characterizing the interaction between host and influenza viruses.

The Y chromosome plays key roles in male fertility and reflects the evolutionary history of paternal lineages. Here, we present a de novo genome assembly of the Hu sheep with the first draft assembly of ovine Y chromosome (oMSY), using nanopore sequencing and Hi-C technologies. The oMSY that we generated spans 10.6 Mb from which 775 Y-SNPs were identified by applying a large panel of whole genome sequences from worldwide sheep and wild Iranian mouflons. Three major paternal lineages (HY1a, HY1b and HY2) were defined across domestic sheep, of which HY2 was newly detected. Surprisingly, HY2 forms a monophyletic clade with the Iranian mouflons and is highly divergent from both HY1a and HY1b. Demographic analysis of Y chromosomes, mitochondrial and nuclear genomes confirmed that HY2 and the maternal counterpart of lineage C represented a distinct wild mouflon population in Iran that diverge from the direct ancestor of domestic sheep, the wild mouflons in Southeastern Anatolia. Our results suggest that wild Iranian mouflons had introgressed into domestic sheep and thereby introduced this Iranian mouflon specific lineage carrying HY2 to both East Asian and Africa sheep populations.

Rumen microbiota are essential for maintaining digestive and metabolic functions, producing methane as a byproduct. Dairy heifers produce large amounts of methane based on fermentation of digested organic matter, with adverse consequences for feed efficiency and the environment. It is therefore important to understand the influence of host age on the relationship between microbiota and methane production. This study explored the age effect on the relationship between microbial communities and enteric methane production in dairy cows and heifers using high-throughput sequencing. Methane production and volatile fatty acid concentrations were age-related. Heifers (9–10 months) had lower methane production but higher methane production per dry matter intake (DMI). The acetate:propionate ratio decreased significantly with increasing age. Age-related microbiota changes in the rumen were reflected by a significant shift in bacterial taxa, but relatively stable archaeal taxa. Prevotella, Ruminococcus, Flavonifractor, Succinivibrio, and Methanobrevibacter were affected by age. This study revealed different associations between predominant bacterial phylotypes and Methanobrevibacter with increasing age. Prevotella was strongly correlated with Methanobrevibacter in heifers; howerver, in older cows (96–120 months) this association was replaced by a correlation between Succinivibrio and Methanobrevibacter. This shift may account for the age-related difference in rumen fermentation and methane production per DMI.

Structural variations (SVs) are a major contributor of genetic diversity and phenotypic variations, however their prevalence and functions in domestic animals are largely unexplored. Here, we assembled 26 haplotype-resolved genome assemblies from 13 genetically diverse sheep breeds using PacBio HiFi sequencing. We then constructed an ovine graph pan-genome and demonstrated its advantage in discovering 142,593 biallelic SVs (Insertions and deletions), 7,028 divergent alleles and 13,419 multiallelic variations with high accuracy and sensitivity. To link the SVs to genotypes, we genotyped the SVs in 687 resequenced individuals of domestic and wild sheep using a graph-based approach and identified numerous population-stratified variants, of which expression-associated SVs were detected by integrating RNA-seq data. Taking the varying sheep tail morphology as example, we located a putative causative insertion in HOXB13 gene responsible for the long tail and reported multiple large SVs associated with the fat tail. Beyond generating a benchmark resource for ovine structural variants, our study also highlighted that the population genetics analysis based on graph pan-genome rather than reference genome will greatly benefit the animal genetic research.

The diverse functions of long noncoding RNAs (lncRNAs), which execute their functions mainly through modulating the activities of their target genes, have been have been widely studied for many years (including a number of studies involving lncRNAs in the ovary and uterus). Herein, for the first time, we detect lncRNAs in sheep hypothalami with FecB++ through RNA Sequencing (RNA-Seq) and identify a number of known and novel lncRNAs, with 622 and 809 found to be differentially expressed in polytocous sheep in the follicular phase (PF) vs. monotocous sheep in the follicular phase (MF) and polytocous sheep in the luteal phase (PL) vs. monotocous sheep in the luteal phase (ML), respectively. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed based on the predicted target genes. The most highly enriched GO terms (at the molecular function level) included carbonyl reductase (NADPH), 15-hydroxyprostaglandin dehydrogenase (NADP+), and prostaglandin-E2 9-reductase activity in PF vs. MF, and phosphatidylinositol-3,5-bisphosphate binding in PL vs. ML was associated with sheep fecundity. Interestingly, the phenomena of valine, leucine, and isoleucine degradation in PL vs. ML, and valine, leucine, and isoleucine biosynthesis in PF vs. MF, were present. In addition, the interactome of lncRNA and its targets showed that MSTRG.26777 and its cis-targets ENSOARG00000013744, ENSOARG00000013700, and ENSOARG00000013777, and MSTRG.105228 and its target WNT7A may participate in the sheep reproductive process at the hypothalamus level. Significantly, MSTRG.95128 and its cis-target Forkhead box L1 (FOXG1) were shown to be upregulated in PF vs. MF but downregulated in PL vs. ML. All of these results may be attributed to discoveries of new candidate genes and pathways related to sheep reproduction, and they may provide new views for understanding sheep reproduction without the effects of the FecB mutation.

The myostatin gene of seven important meat (Beltex (Australia), Beltex×Huyang (F1), Meat and Multi-Prolific Chinese Merino Fine Wool, Meat Chinese Merino Fine Wool and Dorper (South Africa)) and non-meat (Huyang and Kazak) sheep breeds was analyzed to study the genetic basis of muscular hypertrophy (double muscling) phenotype in sheep. SNPs, four in regulatory regions and several in the introns in the myostatin gene, were identified, and the former four SNPs were used for further studies. Twelve haplotypes were predicted by PHASE program, of which four main haplotypes (1, 3, 7, 9) were present in 90% of the 364 sheep in the study. Haplotypes 1-4 were mainly present in meat breeds while haplotypes 7 and 9 dominated the non-meat breeds. The association between haplotypes and average daily gain (ADG) was analyzed among 116 sheep with production data, Haplo2 (CGAA) and Haplo8 (TGAA) were identified to have significant (p<0.05) effect on ADG by the model (JMP5.1 software) taking into account the effects of breed, family background, haplotype, birth weight and sex. ADG of these haplotype groups also correlated well (r = 0.82) with hypertrophic phenotype scores. In conclusion, the mutations -956 (T→C), -41 (C→A) and 6223 (G→A) involved in Haplo2 and 8 may be associated with the double-muscling trait by influencing myostatin function and be suitable markers in selecting meat sheep.

Early studies have provided a wealth of information on the functions of microRNAs (miRNAs). However, less is known regarding their functions in the hypothalamus involved in sheep reproduction. To explore the potential roles of hypothalamic messenger RNAs (mRNAs) and miRNAs in sheep without FecB mutation, in total, 172 and 235 differentially expressed genes (DEGs) and 42 and 79 differentially expressed miRNAs (DE miRNAs) were identified in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase versus monotocous sheep in the luteal phase (PL vs. ML), respectively, using RNA sequencing. We also identified several key mRNAs (e.g., POMC, GNRH1, PRL, GH, TRH, and TTR) and mRNA-miRNAs pairs (e.g., TRH co-regulated by oar-miR-379-5p, oar-miR-30b, oar-miR-152, oar-miR-495-3p, oar-miR-143, oar-miR-106b, oar-miR-218a, oar-miR-148a, and PRL regulated by oar-miR-432) through functional enrichment analysis, and the identified mRNAs and miRNAs may function, conceivably, by influencing gonadotropinreleasing hormone (GnRH) activities and nerve cell survival associated with reproductive hormone release via direct and indirect ways. This study represents an integral analysis between mRNAs and miRNAs in sheep hypothalamus and provides a valuable resource for elucidating sheep prolificacy.

Structural variations (SVs) are a major contributor to genetic diversity and phenotypic variations, but their prevalence and functions in domestic animals are largely unexplored. Here we generated high-quality genome assemblies for 15 individuals from genetically diverse sheep breeds using Pacific Biosciences (PacBio) high-fidelity sequencing, discovering 130.3 Mb nonreference sequences, from which 588 genes were annotated. A total of 149,158 biallelic insertions/deletions, 6531 divergent alleles, and 14,707 multiallelic variations with precise breakpoints were discovered. The SV spectrum is characterized by an excess of derived insertions compared to deletions (94,422 vs. 33,571), suggesting recent active LINE expansions in sheep. Nearly half of the SVs display low to moderate linkage disequilibrium with surrounding single-nucleotide polymorphisms (SNPs) and most SVs cannot be tagged by SNP probes from the widely used ovine 50K SNP chip. We identified 865 population-stratified SVs including 122 SVs possibly derived in the domestication process among 690 individuals from sheep breeds worldwide. A novel 168-bp insertion in the 5′ untranslated region (5′ UTR) ofHOXB13is found at high frequency in long-tailed sheep. Further genome-wide association study and gene expression analyses suggest that this mutation is causative for the long-tail trait. In summary, we have developed a panel of high-quality de novo assemblies and present a catalog of structural variations in sheep. Our data capture abundant candidate functional variations that were previously unexplored and provide a fundamental resource for understanding trait biology in sheep.