Long read isoform sequencing reveals hidden transcriptional complexity between cattle subspecies.

The Iso-Seq method of full-length cDNA sequencing is suitable to quantify differentially expressed genes (DEGs), transcripts (DETs) and transcript usage (DTU). However, the higher cost of Iso-Seq relative to RNA-seq has limited the comparison of both methods. Transcript abundance estimated by RNA-seq and deep Iso-Seq data for fetal liver from two cattle subspecies were compared to evaluate concordance. Inter-sample correlation of gene- and transcript-level abundance was higher within technology than between technologies. Identification of DEGs between the cattle subspecies depended on sequencing method with only 44 genes identified by both that included 6 novel genes annotated by Iso-Seq. There was a pronounced difference between Iso-Seq and RNA-seq results at transcript-level wherein Iso-Seq revealed several magnitudes more transcript abundance and usage differences between subspecies. Factors influencing DEG identification included size selection during Iso-Seq library preparation, average transcript abundance, multi-mapping of RNA-seq reads to the reference genome, and overlapping coordinates of genes. Some DEGs called by RNA-seq alone appear to be sequence duplication artifacts. Among the 44 DEGs identified by both technologies some play a role in immune system, thyroid function and cell growth. Iso-Seq revealed hidden transcriptional complexity in DEGs, DETs and DTU genes between cattle subspecies previously missed by RNA-seq.

MicroRNA breed and parent-of-origin effects provide insights into biological pathways differentiating cattle subspecies in fetal liver.

Introduction: MicroRNAs (miRNAs) play a crucial role in regulating gene expression during key developmental processes, including fetal development. Brahman (Bos taurus indicus) and Angus (Bos taurus taurus) cattle breeds represent two major cattle subspecies with strikingly different phenotypes. Methods: We analyzed miRNA expression in liver samples of purebred and reciprocal crosses of Angus and Brahman to investigate breed and parent-of-origin effects at the onset of accelerated fetal growth. Results: We identified eight novel miRNAs in fetal liver samples and 14 differentially expressed miRNAs (DEMs) between purebred samples. Correlation of gene expression modules and miRNAs by breed and parent-of-origin effects revealed an enrichment of genes associated with breed-specific differences in traits such as heat tolerance (Brahman) and fat deposition (Angus). We demonstrate that genes predicted to be targets of DEMs were more likely to be differentially expressed than non-targets (p-value < 0.05). We identified several miRNAs (bta-miR-187, bta-miR-216b, bta-miR-2284c, bta-miR-2285c, bta-miR-2285cp, bta-miR-2419-3p, bta-miR-2419-5p, and bta-miR-11984) that showed similar correlation patterns as bta-miR-2355-3p, which has been associated with the glutamatergic synapse pathway, a key facilitator of heat tolerance. Furthermore, we report Angus-breed-specific miRNAs (bta-miR-2313-5p, btamiR-490, bta-miR-2316, and bta-miR-11990) that may be involved in fat deposition. Finally, we showed that the DEMs identified in fetal liver are involved in Rap1, MAPK, and Ras signalling pathways, which are important for fetal development, muscle development and metabolic traits such as fat metabolism. Conclusion: Our work sheds light on the miRNA expression patterns that contribute to gene expression differences driving phenotypic differences in indicine and taurine cattle.

Cross-species enhancer prediction using machine learning.

Cis-regulatory elements (CREs) are non-coding parts of the genome that play a critical role in gene expression regulation. Enhancers, as an important example of CREs, interact with genes to influence complex traits like disease, heat tolerance and growth rate. Much of what is known about enhancers come from studies of humans and a few model organisms like mouse, with little known about other mammalian species. Previous studies have attempted to identify enhancers in less studied mammals using comparative genomics but with limited success. Recently, Machine Learning (ML) techniques have shown promising results to predict enhancer regions. Here, we investigated the ability of ML methods to identify enhancers in three non-model mammalian species (cattle, pig and dog) using human and mouse enhancer data from VISTA and publicly available ChIP-seq. We tested nine models, using four different representations of the DNA sequences in cross-species prediction using both the VISTA dataset and species-specific ChIP-seq data. We identified between 809,399 and 877,278 enhancer-like regions (ELRs) in the study species (11.6-13.7% of each genome). These predictions were close to the ~8% proportion of ELRs that covered the human genome. We propose that our ML methods have predictive ability for identifying enhancers in non-model mammalian species. We have provided a list of high confidence enhancers at https://github.com/DaviesCentreInformatics/Cross-species-enhancer-prediction and believe these enhancers will be of great use to the community.

Gaur genome reveals expansion of sperm odorant receptors in domesticated cattle.

BACKGROUND: The gaur (Bos gaurus) is the largest extant wild bovine species, native to South and Southeast Asia, with unique traits, and is listed as vulnerable by the International Union for Conservation of Nature (IUCN). RESULTS: We report the first gaur reference genome and identify three biological pathways including lysozyme activity, proton transmembrane transporter activity, and oxygen transport with significant changes in gene copy number in gaur compared to other mammals. These may reflect adaptation to challenges related to climate and nutrition. Comparative analyses with domesticated indicine (Bos indicus) and taurine (Bos taurus) cattle revealed genomic signatures of artificial selection, including the expansion of sperm odorant receptor genes in domesticated cattle, which may have important implications for understanding selection for male fertility. CONCLUSIONS: Apart from aiding dissection of economically important traits, the gaur genome will also provide the foundation to conserve the species.

Antimicrobial susceptibility and genomic analysis of Histophilus somni isolated from cases of bovine respiratory disease in Autralian feedlot cattle.

Histophilus somni is a prevalent commensal organism of the upper respiratory tract of cattle and a major causative agent of bovine respiratory disease (BRD) and other syndromes including myocarditis and infectious thromboembolic meningoencephalitis. This study investigated the antimicrobial susceptibility and phylogenetic relationships of H. somni isolates obtained from lung, heart, and other tissues at post-mortem as well as nasal mucosa swabs from cases of BRD in Australian feedlots (2004-2019). Broth microdilution Minimal Inhibitory Concentration (MIC) assays were determined for 19 antimicrobials using three different media (CLSI approved Veterinary Fastidious Medium [VFM], Mueller-Hinton fastidious broth medium supplemented with yeast extract [MHF-Y] and Columbia Broth [CB] supplemented with 5% lysed horse blood). For all antimicrobials, MICs obtained using CB medium were identical or within 1 dilution step of the MICs obtained for VFM and MHF-Y media. Therefore, CB may be a suitable medium for H. somni antimicrobial susceptibility testing similar to MHF-Y medium. None of the 70 Australian H. somni isolates exhibited resistance to antimicrobials with CLSI breakpoints including those commonly used in the treatment of BRD in Australia (first-line tetracyclines [chlortetracycline and oxytetracycline], second-line macrolides [tulathromycin], and third-line extended-spectrum cephalosporin [ceftiofur]). Whole-genome sequence analysis of 65 H. somni isolates for genomic single nucleotide polymorphism differences identified four phylogenetic clusters, each containing isolates from different Australian states, feedlots and tissue sources that clustered together. These findings demonstrate limited genetic diversity and the absence of significant antimicrobial resistance among Australian isolates of H. somni isolated from feedlot cattle.

Newcastle disease virus genotype VII gene expression in experimentally infected birds.

Newcastle disease virus genotype VII (NDV-GVII) is a highly contagious pathogen responsible for pandemics that have caused devastating economic losses in the poultry industry. Several features in the transcription of NDV mRNA, including differentially expressed genes across the viral genome, are shared with that for other single, non-segmented, negative-strand viruses. Previous studies measuring viral gene expression using northern blotting indicated that the NDV transcription produced non-equimolar levels of viral mRNAs. However, deep high-throughput sequencing of virus-infected tissues can provide a better insight into the patterns of viral transcription. In this report, the transcription pattern of virulent NDV-GVII was analysed using RNA-seq and qRT-PCR. This study revealed the transcriptional profiling of these highly pathogenic NDV-GVII genes: NP:P:M:F:HN:L, in which there was a slight attenuation at the NP:P and HN:L gene boundaries. Our result also provides a fully comprehensive qPCR protocol for measuring viral transcript abundance that may be more convenient for laboratories where accessing RNA-seq is not feasible.

Indicators of the molecular pathogenesis of virulent Newcastle disease virus in chickens revealed by transcriptomic profiling of spleen.

Newcastle disease virus (NDV) has caused significant outbreaks in South-East Asia, particularly in Indonesia in recent years. Recently emerged genotype VII NDVs (NDV-GVII) have shifted their tropism from gastrointestinal/respiratory tropism to a lymphotropic virus, invading lymphoid organs including spleen and bursa of Fabricius to cause profound lymphoid depletion. In this study, we aimed to identify candidate genes and biological pathways that contribute to the disease caused by this velogenic NDV-GVII. A transcriptomic analysis based on RNA-Seq of spleen was performed in chickens challenged with NDV-GVII and a control group. In total, 6361 genes were differentially expressed that included 3506 up-regulated genes and 2855 down-regulated genes. Real-Time PCR of ten selected genes validated the RNA-Seq results as the correlation between them is 0.98. Functional and network analysis of Differentially Expressed Genes (DEGs) showed altered regulation of ElF2 signalling, mTOR signalling, proliferation of cells of the lymphoid system, signalling by Rho family GTPases and synaptogenesis signalling in spleen. We have also identified modified expression of IFIT5, PI3K, AGT and PLP1 genes in NDV-GVII infected chickens. Our findings in activation of autophagy-mediated cell death, lymphotropic and synaptogenesis signalling pathways provide new insights into the molecular pathogenesis of this newly emerged NDV-GVII.

Seeing the forest through the trees: prioritising potentially functional interactions from Hi-C.

Eukaryotic genomes are highly organised within the nucleus of a cell, allowing widely dispersed regulatory elements such as enhancers to interact with gene promoters through physical contacts in three-dimensional space. Recent chromosome conformation capture methodologies such as Hi-C have enabled the analysis of interacting regions of the genome providing a valuable insight into the three-dimensional organisation of the chromatin in the nucleus, including chromosome compartmentalisation and gene expression. Complicating the analysis of Hi-C data, however, is the massive amount of identified interactions, many of which do not directly drive gene function, thus hindering the identification of potentially biologically functional 3D interactions. In this review, we collate and examine the downstream analysis of Hi-C data with particular focus on methods that prioritise potentially functional interactions. We classify three groups of approaches: structural-based discovery methods, e.g. A/B compartments and topologically associated domains, detection of statistically significant chromatin interactions, and the use of epigenomic data integration to narrow down useful interaction information. Careful use of these three approaches is crucial to successfully identifying potentially functional interactions within the genome.

Adaptive selection signatures in river buffalo with emphasis on immune and major histocompatibility complex genes.

River buffalo is an agriculturally important species with many traits, such as disease tolerance, which promote its use worldwide. Highly contiguous genome assemblies of the river buffalo, goat, pig, human and two cattle subspecies were aligned to study gene gains and losses and signs of positive selection. The gene families that have changed significantly in river buffalo since divergence from cattle play important roles in protein degradation, the olfactory receptor system, detoxification and the immune system. We used the branch site model in PAML to analyse single-copy orthologs to identify positively selected genes that may be involved in skin differentiation, mammary development and bone formation in the river buffalo branch. The high contiguity of the genomes enabled evaluation of differences among species in the major histocompatibility complex. We identified a Babesia-like L1 LINE insertion in the DRB1-like gene in the river buffalo and discuss the implication of this finding.

Distinctive gene expression patterns and imprinting signatures revealed in reciprocal crosses between cattle sub-species.

BACKGROUND: There are two genetically distinct subspecies of cattle, Bos taurus taurus and Bos taurus indicus, which arose from independent domestication events. The two types of cattle show substantial phenotypic differences, some of which emerge during fetal development and are reflected in birth outcomes, including birth weight. We explored gene expression profiles in the placenta and four fetal tissues at mid-gestation from one taurine (Bos taurus taurus; Angus) and one indicine (Bos taurus indicus; Brahman) breed and their reciprocal crosses. RESULTS: In total 120 samples were analysed from a pure taurine breed, an indicine breed and their reciprocal cross fetuses, which identified 6456 differentially expressed genes (DEGs) between the two pure breeds in at least one fetal tissue of which 110 genes were differentially expressed in all five tissues examined. DEGs shared across tissues were enriched for pathways related to immune and stress response functions. Only the liver had a substantial number of DEGs when reciprocal crossed were compared among which 310 DEGs were found to be in common with DEGs identified between purebred livers; these DEGs were significantly enriched for metabolic process GO terms. Analysis of DEGs across purebred and crossbred tissues suggested an additive expression pattern for most genes, where both paternal and maternal alleles contributed to variation in gene expression levels. However, expression of 5% of DEGs in each tissue was consistent with parent of origin effects, with both paternal and maternal dominance effects identified. CONCLUSIONS: These data identify candidate genes potentially driving the tissue-specific differences between these taurine and indicine breeds and provide a biological insight into parental genome effects underlying phenotypic differences in bovine fetal development.

Molecular Diagnosis of Koala Retrovirus (KoRV) in South Australian Koalas (Phascolarctos cinereus).

Koala retrovirus, a recent discovery in Australian koalas, is endogenised in 100% of northern koalas but has lower prevalence in southern populations, with lower proviral and viral loads, and an undetermined level of endogenisation. KoRV has been associated with lymphoid neoplasia, e.g., lymphoma. Recent studies have revealed high complexity in southern koala retroviral infections, with a need to clarify what constitutes positive and negative cases. This study aimed to define KoRV infection status in Mount Lofty Ranges koalas in South Australia using RNA-seq and proviral analysis (n = 216). The basis for positivity of KoRV was deemed the presence of central regions of the KoRV genome (gag 2, pol, env 1, and env 2) and based on this, 41% (89/216) koalas were positive, 57% (124/216) negative, and 2% inconclusive. These genes showed higher expression in lymph node tissue from KoRV positive koalas with lymphoma compared with other KoRV positive koalas, which showed lower, fragmented expression. Terminal regions (LTRs, partial gag, and partial env) were present in SA koalas regardless of KoRV status, with almost all (99.5%, 215/216) koalas positive for gag 1 by proviral PCR. Further investigation is needed to understand the differences in KoRV infection in southern koala populations.

Extended haplotype-phasing of long-read de novo genome assemblies using Hi-C.

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. To date, these assemblies have been best created with complex protocols, such as cultured cells that contain a single-haplotype (haploid) genome, single cells where haplotypes are separated, or co-sequencing of parental genomes in a trio-based approach. These approaches are impractical in most situations. To address this issue, we present FALCON-Phase, a phasing tool that uses ultra-long-range Hi-C chromatin interaction data to extend phase blocks of partially-phased diploid assembles to chromosome or scaffold scale. FALCON-Phase uses the inherent phasing information in Hi-C reads, skipping variant calling, and reduces the computational complexity of phasing. Our method is validated on three benchmark datasets generated as part of the Vertebrate Genomes Project (VGP), including human, cow, and zebra finch, for which high-quality, fully haplotype-resolved assemblies are available using the trio-based approach. FALCON-Phase is accurate without having parental data and performance is better in samples with higher heterozygosity. For cow and zebra finch the accuracy is 97% compared to 80-91% for human. FALCON-Phase is applicable to any draft assembly that contains long primary contigs and phased associate contigs.

Opportunities and Challenges for Improving the Productivity of Swamp Buffaloes in Southeastern Asia.

The swamp buffalo is a domesticated animal commonly found in Southeast Asia. It is a highly valued agricultural animal for smallholders, but the production of this species has unfortunately declined in recent decades due to rising farm mechanization. While swamp buffalo still plays a role in farmland cultivation, this species' purposes has shifted from draft power to meat, milk, and hide production. The current status of swamp buffaloes in Southeast Asia is still understudied compared to its counterparts such as the riverine buffaloes and cattle. This review discusses the background of swamp buffalo, with an emphasis on recent work on this species in Southeast Asia, and associated genetics and genomics work such as cytogenetic studies, phylogeny, domestication and migration, genetic sequences and resources. Recent challenges to realize the potential of this species in the agriculture industry are also discussed. Limited genetic resource for swamp buffalo has called for more genomics work to be done on this species including decoding its genome. As the economy progresses and farm mechanization increases, research and development for swamp buffaloes are focused on enhancing its productivity through understanding the genetics of agriculturally important traits. The use of genomic markers is a powerful tool to efficiently utilize the potential of this animal for food security and animal conservation. Understanding its genetics and retaining and maximizing its adaptability to harsher environments are a strategic move for food security in poorer nations in Southeast Asia in the face of climate change.

Nucleotide substitution rates of diatom plastid encoded protein genes are positively correlated with genome architecture.

Diatoms are the largest group of heterokont algae with more than 100,000 species. As one of the single-celled photosynthetic organisms that inhabit marine, aquatic and terrestrial ecosystems, diatoms contribute ~ 45% of global primary production. Despite their ubiquity and environmental significance, very few diatom plastid genomes (plastomes) have been sequenced and studied. This study explored patterns of nucleotide substitution rates of diatom plastids across the entire suite of plastome protein-coding genes for 40 taxa representing the major clades. The highest substitution rate was lineage-specific within the araphid 2 taxon Astrosyne radiata and radial 2 taxon Proboscia sp. Rate heterogeneity was also evident in different functional classes and individual genes. Similar to land plants, proteins genes involved in photosynthetic metabolism have lower synonymous and nonsynonymous substitutions rates than those involved in transcription and translation. Significant positive correlations were identified between substitution rates and measures of genomic rearrangements, including indels and inversions, which is a similar result to what was found in legume plants. This work advances the understanding of the molecular evolution of diatom plastomes and provides a foundation for future studies.

Molecular evolutionary and 3D protein structural analyses of Lactobacillus fermentum elongation factor Tu, a novel brain health promoting factor.

The role of microbiota in gut-brain communication has led to the development of probiotics promoting brain health. Here we report a genomic study of a Lactobacillus fermentum PS150 and its patented bioactive protein, elongation factor Tu (EF-Tu), which is associated with cognitive improvement in rats. The L. fermentum PS150 circular chromosome is 2,238,401 bp and it consists of 2281 genes. Chromosome comparisons with other L. fermentum strains highlighted a cluster of glycosyltransferases as potential candidate probiotic factors besides EF-Tu. Molecular evolutionary analyses on EF-Tu genes (tuf) in 235 bacteria species revealed one to three copies of the gene per genome. Seven tuf pseudogenes were found and three species only possessed pseudogenes, which is an unprecedented finding. Protein variability analysis of EF-Tu showed five highly variable residues (40 K, 41G, 42 L, 44 K, and 46E) on the protein surface, which warrant further investigation regarding their potential roles as binding sites.

Transcriptomic analysis of multi-drug resistant Escherichia coli K-12 strain in response to Lavandula angustifolia essential oil.

To better understand the synergistic antibacterial activity between piperacillin and Lavandula angustifolia essential oil (LEO) against multidrug-resistant Escherichia coli, we performed microarray transcriptomic analysis of LEO when used alone and in combination with piperacillin against the non-treated control. In total, 90 genes were differentially expressed after the combination of LEO and piperacillin treatment. Among the up-regulated genes, nfsB, nemA, fruA, nfsB, nemA are known to control microbial metabolism and nitrotoluene degradation, which were observed only in the LEO-piperacillin combinatory treatment. Four candidate genes from the microarray result, srIA, srID, waaR and nfsB, were validated by qRT-PCR as these genes showed differential expression consistently in the two methods. Biochemical pathway analysis showed that there was upregulation of genes involved in several biological processes including fructose and mannose metabolism, phosphotransferase system (PTS), lipopolysaccharide biosynthesis and nitrotoluene degradation. Genes involved in microbial metabolism in diverse environments were found both up- and down-regulated in LEO-piperacillin combinatory treatment. Our study provides new information concerning the transcriptional changes that occur during the LEO and piperacillin interaction against the multidrug-resistant bacteria and contributes to unravel the mechanisms underlying this synergism.

Genome Sequences of Newly Emerged Newcastle Disease Virus Strains Isolated from Disease Outbreaks in Indonesia.

Here, we report two genomes of newly emerged strains of Newcastle disease virus (NDV), Chicken/Indonesia/Tangerang/004WJ/14 and Chicken/Indonesia/VD/003WJ/11, from disease outbreaks in chickens in Indonesia. Phylogenetic study results of the fusion (F) protein's gene-coding sequences of different genotypes of NDV revealed that these two strains belong to genotype VII.1 in the class II cluster of avian paramyxoviruses.

Genome Sequences of Newcastle Disease Virus Strains from Two Outbreaks in Indonesia.

The genomes of two newly emerged Newcastle disease virus strains, chicken/Indonesia/Mega/001WJ/2013 and chicken/Indonesia/Cimanglid/002WJ/2015, from disease outbreaks in chickens in Indonesia are reported. Phylogenetic analysis of different genotypes of Newcastle disease virus using the F gene coding sequences suggests that these two strains belong to genotype VII.2, in class II of avian paramyxoviruses.