Search

The annotation of animal genomes plays an important role in elucidating molecular mechanisms behind the genetic control of economically important traits. Here, we employed long-read sequencing technology, Oxford Nanopore Technology, to annotate the pig transcriptome across 17 tissues from two Yorkshire littermate pigs. More than 9.8 million reads were obtained from a single flow cell, and 69 781 unique transcripts at 50 108 loci were identified. Of these transcripts, 16 255 were found to be novel isoforms, and 22 344 were found at loci that were novel and unannotated in the Ensembl (release 102) and NCBI (release 106) annotations. Novel transcripts were mostly expressed in cerebellum, followed by lung, liver, spleen, and hypothalamus. By comparing the unannotated transcripts to existing databases, there were 21 285 (95.3%) transcripts matched to the NT database (v5) and 13 676 (61.2%) matched to the NR database (v5). Moreover, there were 4324 (19.4%) transcripts matched to the SwissProt database (v5), corresponding to 11 356 proteins. Tissue-specific gene expression analyses showed that 9749 transcripts were highly tissue-specific, and cerebellum contained the most tissue-specific transcripts. As the same samples were used for the annotation of cis-regulatory elements in the pig genome, the transcriptome annotation generated by this study provides an additional and complementary annotation resource for the Functional Annotation of Animal Genomes effort to comprehensively annotate the pig genome.

A comprehensive characterization of regulatory elements in the chicken genome across tissues will have substantial impacts on both fundamental and applied research. Here, we systematically identified and characterized regulatory elements in the chicken genome by integrating 377 genome-wide sequencing datasets from 23 adult tissues. In total, we annotated 1.57 million regulatory elements, representing 15 distinct chromatin states, and predicted about 1.2 million enhancer-gene pairs and 7662 super-enhancers. This functional annotation of the chicken genome should have wide utility on identifying regulatory elements accounting for gene regulation underlying domestication, selection, and complex trait regulation, which we explored. In short, this comprehensive atlas of regulatory elements provides the scientific community with a valuable resource for chicken genetics and genomics.

To comprehensively identify and annotate transcript isoforms in the chicken genome, we generated Nanopore long-read sequencing data from a diverse set of 19 chicken tissues comprising 68 samples collected from experimental line 6 × line 7 F1 adult males and females. More than 23.8 million reads with mean read length of 790 bases and average quality of 18.2 were generated. The annotation and subsequent filtering resulted in identification of 55,382 transcripts with mean length of 1,700 bases at 40,547 loci, representing ∼1.4 transcripts per locus. Among them, we predicted 30,967 potential coding transcripts at 19,461 loci and 16,495 potential lncRNA transcripts at 15,512 loci. Compared to reference annotations, we found 52% of annotated transcripts could partially to fully match while 47% were novel and potentially transcribed from lncRNA loci. Based on our annotation, we quantified transcript expression across tissues and found brain tissues (i.e. cerebellum, cortex) expressed highest number of transcripts and loci. The further tissue specificity revealed that ∼22% of the transcripts displaying tissue specificity. Of them, the reproductive tissues (i.e. testis, ovary) contained the most tissue-specific transcripts. Despite sequencing 68 transcriptomes derived from 19 tissues, still ∼20% of Ensembl reference loci were not detected. This suggests that including additional samples from different cell types, developmental and physiological conditions, is needed to fully annotate the chicken genome. The application of Nanopore sequencing transcriptomes in this study demonstrated the usefulness of long-read data in discovering additional novel loci (e.g., lncRNA loci) and resolving complex transcripts (e.g., the longest transcript for the TTN locus).

It remains poorly understood how histone modifications regulate changes in gene expression during preimplantation development. Using a bovine model, we profiled changes in two activating (H3K4me3 and H3K27ac) and two repressive (H3K9me3 and H3K27me3) marks in oocytes, 2-, 4- and 8-cell embryos (that developed in the presence or absence of the transcription inhibitor a-amanitin), morula, blastocysts, inner cell mass cells and trophectoderm. In oocytes, we find that broad bivalent domains of H3K4me3 and H3K27me3 mark developmental genes, and that prior to genome activation, H3K9me3 and H3K27me3 co-occupy gene bodies. During genome activation, chromatin accessibility is established before canonical H3K4me3 and H3K27ac, and although embryonic transcription is required for this active remodeling, it is dispensable for maintenance of pre-established histone marks. Finally, blastocyst lineages are defined by differential Polycomb repression and transcription factor activity. Overall, these results further support the use of bovine as a more appropriate model system than the mouse to study genome activation and cell lineage specification during human preimplantation development.

There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programs. To address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We used the “Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq)” to identify putative regulatory regions in flash-frozen semitendinosus muscle from 24 male piglets. We collected samples from the smallest-, average-, and largest-sized male piglets from each litter through five developmental time points. Of the 4661 ATAC-Seq peaks identified that represent regions of open chromatin, >50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with downregulation of genes involved in muscle development that were present in small-sized fetal piglets but absent in large-sized fetal piglets at day 90 of gestation. The dataset that we have generated provides a resource for studies of genome regulation in pigs and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programs.

Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs was only recently reported. Although successful, bESC culture relies on complex culture conditions that require a custom-made base medium and mouse embryonic fibroblasts (MEF) feeders, limiting the widespread use of bESCs. We report here simplified bESC culture conditions based on replacing custom base medium with a commercially available alternative and eliminating the need for MEF feeders by using a chemically-defined substrate. bESC lines were cultured and derived using a base medium consisting of N2B27 supplements and 1% BSA (NBFR-bESCs). Newly derived bESC lines were easy to establish, simple to propagate and stable after long-term culture. These cells expressed pluripotency markers and actively proliferated for more than 35 passages while maintaining normal karyotype and the ability to differentiate into derivatives of all three germ lineages in embryoid bodies and teratomas. In addition, NBFR-bESCs grew for multiple passages in a feeder-free culture system based on vitronectin and Activin A medium supplementation while maintaining pluripotency. Simplified conditions will facilitate the use of bESCs for gene editing applications and pluripotency and lineage commitment studies.

Gene regulatory elements are central drivers of phenotypic variation and thus of critical importance towards understanding the genetics of complex traits. The Functional Annotation of Animal Genomes consortium was formed to collaboratively annotate the functional elements in animal genomes, starting with domesticated animals. Here we present an expansive collection of datasets from eight diverse tissues in three important agricultural species: chicken (Gallus gallus), pig (Sus scrofa), and cattle (Bos taurus). Comparative analysis of these datasets and those from the human and mouse Encyclopedia of DNA Elements projects reveal that a core set of regulatory elements are functionally conserved independent of divergence between species, and that tissue-specific transcription factor occupancy at regulatory elements and their predicted target genes are also conserved. These datasets represent a unique opportunity for the emerging field of comparative epigenomics, as well as the agricultural research community, including species that are globally important food resources., In order to interpret non-coding variants, information about regulatory elements in the genome is essential. Here, the authors annotate regulatory elements in chicken, pig and cattle, and characterize conservation of these elements between species.

The functional annotation of livestock genomes is crucial for understanding the molecular mechanisms that underpin complex traits of economic importance, adaptive evolution and comparative genomics. Here, we provide the most comprehensive catalogue to date of regulatory elements in the pig (Sus scrofa) by integrating 223 epigenomic and transcriptomic data sets, representing 14 biologically important tissues. We systematically describe the dynamic epigenetic landscape across tissues by functionally annotating 15 different chromatin states and defining their tissue-specific regulatory activities. We demonstrate that genomic variants associated with complex traits and adaptive evolution in pig are significantly enriched in active promoters and enhancers. Furthermore, we reveal distinct tissue-specific regulatory selection between Asian and European pig domestication processes. Compared with human and mouse epigenomes, we show that porcine regulatory elements are more conserved in DNA sequence, under both rapid and slow evolution, than those under neutral evolution across pig, mouse, and human. Finally, we provide novel biological insights on tissue-specific regulatory conservation and demonstrate that, depending on the traits, mouse or pig might be more appropriate biomedical models for different complex traits and diseases in humans through integrating comparative epigenomes with 47 human genome-wide association studies.

There is very little species-specific information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programmes. In order to address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We have optimised the ′Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-seq)′, to profile regions of open chromatin, in flash frozen pig muscle tissue samples. This protocol has allowed us to identify putative regulatory regions in semitendinosus muscle from 24 piglets. We collected samples from the smallest, average, and largest sized piglets from each litter through five developmental time points. The ATAC-Seq data was mapped to Sscrofa11.1 with Bowtie2 and Genrich used for post-alignment peak-calling. Of the 4661 putative regions of accessible chromatin identified, >50% were within 1 kb of known transcription start sites. The size of each open chromatin region varied according to the developmental time point. At day 90 of gestation, we investigated chromatin openness relative to foetal piglet size. In parallel we measured genome-wide gene expression and allele-specific expression using RNA-Seq analysis of the same muscle samples. We found regions of open chromatin associated with down regulation of genes involved in muscle development in small sized foetal piglets. The dataset we have generated here provides a resource for studies of genome regulation in pigs and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programmes. Future work could leverage the ATAC-Seq data with very large datasets of genetic variants from phenotyped pigs. This approach could improve chromatin-informed genomic prediction models and determine whether regions of open chromatin are enriched for trait-linked variants, and especially for muscle and meat traits.

Background Although considerable progress has been made towards annotating the noncoding portion of the human and mouse genomes, regulatory elements in other species, such as livestock, remain poorly characterized. This lack of functional annotation poses a substantial roadblock to agricultural research and diminishes the value of these species as model organisms. As active regulatory elements are typically characterized by chromatin accessibility, we implemented the Assay for Transposase Accessible Chromatin (ATAC-seq) to annotate and characterize regulatory elements in pigs and cattle, given a set of eight adult tissues. Results Overall, 306,304 and 273,594 active regulatory elements were identified in pig and cattle, respectively. 71,478 porcine and 47,454 bovine regulatory elements were highly tissue-specific and were correspondingly enriched for binding motifs of known tissue-specific transcription factors. However, in every tissue the most prevalent accessible motif corresponded to the insulator CTCF, suggesting pervasive involvement in 3-D chromatin organization. Taking advantage of a similar dataset in mouse, open chromatin in pig, cattle, and mice were compared, revealing that the conservation of regulatory elements, in terms of sequence identity and accessibility, was consistent with evolutionary distance; whereas pig and cattle shared about 20% of accessible sites, mice and ungulates only had about 10% of accessible sites in common. Furthermore, conservation of accessibility was more prevalent at promoters than at intergenic regions. Conclusions The lack of conserved accessibility at distal elements is consistent with rapid evolution of enhancers, and further emphasizes the need to annotate regulatory elements in individual species, rather than inferring elements based on homology. This atlas of chromatin accessibility in cattle and pig constitutes a substantial step towards annotating livestock genomes and dissecting the regulatory link between genome and phenome.

The shift from maternal to embryonic control is a critical developmental milestone in preimplantation development. Widespread transcriptomic and epigenetic remodeling facilitate this transition from terminally differentiated gametes to totipotent blastomeres, but the identity of transcription factors (TF) and genomic elements regulating embryonic genome activation (EGA) are poorly defined. The timing of EGA is species-specific, e.g., the timing of murine and human EGA differ significantly. To deepen our understanding of mammalian EGA, here we profile changes in open chromatin during bovine preimplantation development. Before EGA, open chromatin is enriched for maternal TF binding, similar to that observed in humans and mice. During EGA, homeobox factor binding becomes more prevalent and requires embryonic transcription. A cross-species comparison of open chromatin during preimplantation development reveals strong similarity in the regulatory circuitry underlying bovine and human EGA compared to mouse. Moreover, TFs associated with murine EGA are not enriched in cattle or humans, indicating that cattle may be a more informative model for human preimplantation development than mice., Preimplantation embryos undergo extensive transcriptomic and epigenomic remodeling. Here the authors assay open chromatin in bovine oocytes, embryos, and embryonic stem cells, and compare the transcriptomes and epigenomes of cattle, human and mouse embryos, revealing species-specific regulation of genome activation.

Characterizing transcription start sites is essential for understanding the regulatory mechanisms that control gene expression. Recently, a new bovine genome assembly (ARS-UCD1.2) with high continuity, accuracy, and completeness was released; however, the functional annotation of the bovine genome lacks precise transcription start sites and contains a low number of transcripts in comparison to human and mouse. Using the RAMPAGE approach, this study identified transcription start sites at high resolution in a large collection of bovine tissues. We found several known and novel transcription start sites attributed to promoters of protein coding and lncRNA genes that were validated through experimental and in silico evidence. With these findings, the annotation of transcription start sites in cattle reached a level comparable to the mouse and human genome annotations. In addition, we identified and characterized transcription start sites for antisense transcripts derived from bidirectional promoters, potential lncRNAs, mRNAs, and pre-miRNAs. We also analyzed the quantitative aspects of RAMPAGE to produce a promoter activity atlas, reaching highly reproducible results comparable to traditional RNA-seq. Co-expression networks revealed considerable use of tissue specific promoters, especially between brain and testicle, which expressed several genes in common from alternate loci. Furthermore, regions surrounding co-expressed modules were enriched in binding factor motifs representative of each tissue. The comprehensive annotation of promoters in such a large collection of tissues will substantially contribute to our understanding of gene expression in cattle and other mammalian species, shortening the gap between genotypes and phenotypes.

Early mammalian embryonic transcriptomes are dynamic throughout the process of preimplantation development. Cataloging of primate transcriptomics during early development has been accomplished in humans, but global characterization of transcripts is lacking in the rhesus macaque: a key model for human reproductive processes. We report here the systematic classification of individual macaque transcriptomes using RNA-Seq technology from the germinal vesicle stage oocyte through the blastocyst stage embryo. Major differences in gene expression were found between sequential stages, with the 4- to 8-cell stages showing the highest level of differential gene expression. Analysis of putative transcription factor binding sites also revealed a striking increase in key regulatory factors in 8-cell embryos, indicating a strong likelihood of embryonic genome activation occurring at this stage. Furthermore, clustering analyses of gene co-expression throughout this period resulted in distinct groups of transcripts significantly associated to the different embryo stages assayed. The sequence data provided here along with characterizations of major regulatory transcript groups present a comprehensive atlas of polyadenylated transcripts that serves as a useful resource for comparative studies of preimplantation development in humans and other species.

Full-grown oocytes are transcriptionally quiescent. Following maturation and fertilization, the early stages of embryonic development occur in the absence (or low levels) of transcription that results in a period of development relying on maternally derived products (e.g., mRNAs and proteins). Two critical steps occur during the transition from maternal to embryo control of development: maternal mRNA clearance and embryonic genome activation with an associated dramatic reprogramming of gene expression required for further development. By combining an RNA polymerase II inhibitor with RNA sequencing, we were able not only to distinguish maternally derived from embryonic transcripts in bovine preimplantation embryos but also to establish that embryonic gene activation is required for clearance of maternal mRNAs as well as to identify putative transcription factors that are likely critical for early bovine development.

Avian gastrointestinal tracts are highly populated with a diverse array of microorganisms that share a symbiotic relationship with their hosts and contribute to the overall health and disease state of the intestinal tract. The microbiome of the young chick is easily prone to alteration in its composition by both exogenous and endogenous factors especially during the early post-hatch period. The genetic background of the host and exposure to pathogens can impact the diversity of the microbial profile that consequently contributes to the disease progression in the host. The objective of this study was to profile the composition and structure of the gut microbiota in young chickens from two genetically distinct highly inbred lines. Furthermore, the effect of the Salmonella Enteritidis infection on altering the composition makeup of the chicken microbiome was evaluated through the 16S rRNA gene sequencing analysis. One-day-old layer chicks were challenged with S. Enteritidis and the host cecal microbiota profile as well as the degree of susceptibility to Salmonella infection was examined at 2 and 7 days post-infection. Our result indicated that host genotype had a limited effect on resistance to S. Enteritidis infection. Alpha diversity, beta diversity, and overall microbiota composition were analyzed for four factors: host genotype, age, treatment, and post-infection time-points. S. Enteritidis infection in young chicks was found to significantly reduce the overall diversity of the microbiota population with expansion of Enterobacteriaceae family. These changes indicated that Salmonella colonization in the gastrointestinal tract of the chickens has a direct effect on altering the natural development of the gastrointestinal microbiota. The impact of S. Enteritidis infection on microbial communities was also more substantial in late stage of infection. Significant inverse correlation between Enterobacteriaceae and Lachnospiraceae family in both non-infected and infected groups, suggested possible antagonistic interaction between members of these two taxa, which could potentially influences the overall microbial population in the gut. Our results also revealed that genetic difference between two lines had minimal effect on the establishment of microbiota population. Overall, this study provided preliminary insights into the contributing role of S. Enteritidis in influencing the overall makeup of chicken's gut microbiota.