BACKGROUND: Structural variants (SV) are causative for some prominent phenotypic traits of livestock as different comb types in chickens or color patterns in pigs. Their effects on production traits are also increasingly studied. Nevertheless, accurately calling SV remains challenging. It is therefore of interest, whether close-by single nucleotide polymorphisms (SNPs) are in strong linkage disequilibrium (LD) with SVs and can serve as markers. Literature comes to different conclusions on whether SVs are in LD to SNPs on the same level as SNPs to other SNPs. The present study aimed to generate a precise SV callset from whole-genome short-read sequencing (WGS) data for three commercial chicken populations and to evaluate LD patterns between the called SVs and surrounding SNPs. It is thereby the first study that assessed LD between SVs and SNPs in chickens. RESULTS: The final callset consisted of 12,294,329 bivariate SNPs, 4,301 deletions (DEL), 224 duplications (DUP), 218 inversions (INV) and 117 translocation breakpoints (BND). While average LD between DELs and SNPs was at the same level as between SNPs and SNPs, LD between other SVs and SNPs was strongly reduced (DUP: 40%, INV: 27%, BND: 19% of between-SNP LD). A main factor for the reduced LD was the presence of local minor allele frequency differences, which accounted for 50% of the difference between SNP - SNP and DUP - SNP LD. This was potentially accompanied by lower genotyping accuracies for DUP, INV and BND compared with SNPs and DELs. An evaluation of the presence of tag SNPs (SNP in highest LD to the variant of interest) further revealed DELs to be slightly less tagged by WGS SNPs than WGS SNPs by other SNPs. This difference, however, was no longer present when reducing the pool of potential tag SNPs to SNPs located on four different chicken genotyping arrays. CONCLUSIONS: The results implied that genomic variance due to DELs in the chicken populations studied can be captured by different SNP marker sets as good as variance from WGS SNPs, whereas separate SV calling might be advisable for DUP, INV, and BND effects.

BACKGROUND: The hard clam Mercenaria mercenaria is a major marine resource along the Atlantic coasts of North America and has been introduced to other continents for resource restoration or aquaculture activities. Significant mortality events have been reported in the species throughout its native range as a result of diseases (microbial infections, leukemia) and acute environmental stress. In this context, the characterization of the hard clam genome can provide highly needed resources to enable basic (e.g., oncogenesis and cancer transmission, adaptation biology) and applied (clam stock enhancement, genomic selection) sciences. RESULTS: Using a combination of long and short-read sequencing technologies, a 1.86 Gb chromosome-level assembly of the clam genome was generated. The assembly was scaffolded into 19 chromosomes, with an N50 of 83 Mb. Genome annotation yielded 34,728 predicted protein-coding genes, markedly more than the few other members of the Venerida sequenced so far, with coding regions representing only 2% of the assembly. Indeed, more than half of the genome is composed of repeated elements, including transposable elements. Major chromosome rearrangements were detected between this assembly and another recent assembly derived from a genetically segregated clam stock. Comparative analysis of the clam genome allowed the identification of a marked diversification in immune-related proteins, particularly extensive tandem duplications and expansions in tumor necrosis factors (TNFs) and C1q domain-containing proteins, some of which were previously shown to play a role in clam interactions with infectious microbes. The study also generated a comparative repertoire highlighting the diversity and, in some instances, the specificity of LTR-retrotransposons elements, particularly Steamer elements in bivalves. CONCLUSIONS: The diversity of immune molecules in M. mercenaria may allow this species to cope with varying and complex microbial and environmental landscapes. The repertoire of transposable elements identified in this study, particularly Steamer elements, should be a prime target for the investigation of cancer cell development and transmission among bivalve mollusks.

BACKGROUND: MicroRNAs (miRNAs) are a family of short non-coding RNA molecules and play important roles in various biological processes. However, knowledge of the expression profiles and function of miRNAs on the regulation of brown adipose tissue (BAT) thermogenesis remains largely unknown. RESULTS: In this study, we found that brown adipose tissue (BAT) existed within the perirenal fat at 1 day after birth (D1) and transferred into white adipose tissue (WAT) at 30 days after birth (D30) by UCP1 protein expression and immunohistochemistry analysis. After that, we performed RNA sequencing on six libraries of goat BAT and WAT. A total of 238 known miRNAs and 1834 goat novel miRNAs were identified. Moreover, 395 differentially expressed miRNAs including 167 up-regulated and 228 down-regulated miRNAs were obtained in BAT. For the known BAT enriched miRNA, 30 miRNAs were enriched in goat BAT but not in mouse BAT. In addition, miR-433 was enriched in goat BAT but not in mouse BAT. Gain- and loss-of-function experiments reveal that miR-433 reduced the lipid accumulation of brown adipocytes and decreased the expression of BAT marker and mitochondrial related genes. However, miR-433 had no effect on lipid accumulation and thermogenesis in white adipocytes. In addition, miR-433 inhibited the expression of MAPK8 by targeting to the 3'UTR of MAPK8 gene. These data demonstrate that miR-433 acts as a negative regulator in controlling brown adipocytes differentiation and thermogenesis. CONCLUSION: The present study provides a detailed miRNAs expression landscape in BAT and WAT. Furthermore, we found that miR-433, which was highly expressed on BAT had a negative regulatory function on the thermogenesis and adipogenesis in goat brown adipocytes. This study provides evidence for understanding the role of miRNAs in regulating BAT thermogenesis and energy expenditure in goats.

BACKGROUND: Walnuts (Juglans regia L.) are known for their nutrient-rich nuts and are one of the important economic tree species in the world. However, due to global warming and soil salinization, walnuts suffer from various abiotic stresses. TIFY (TIF[F/Y]XG) proteins play an essential role in the growth and development of plants, signal transduction, and stress response in plants. At present, although the TIFY gene family of a number of plants has been identified and studied, how TIFY takes part in stress tolerance remains obscure and many functions of TIFY require further investigation. RESULT: In this study, twenty-one TIFY transcription factors were identified in the walnut genome database, and they were divided into four subfamilies (TIFY, JAZ, ZML, and PPD) by bioinformatics analysis. Chromosome location revealed tandem duplication of some genes. Phylogenetic tree analysis showed JrTIFYs were closely related to the TIFY gene family of Arabidopsis thaliana (A. thaliana). qRT-PCR (quantitative real-time PCR) analysis revealed the TIFY genes have different expression patterns in 'Qingxiang' and 'Xiangling' walnut varieties under drought, heat, and salt stress. JAZ subfamily was more expressed in different abiotic stress than other subfamilies. The expressions of JrTIFY14 under heat and salt stress were significantly higher than those under drought stress. However, the expression of JrTIFYs was not significant in 'Xiangling'. CONCLUSION: This study reveals the TIFY gene family plays an important role in walnuts facing abiotic stresses and provides a theoretical basis for walnut breeding.

BACKGROUND: Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively. RESULTS: We found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals. CONCLUSIONS: Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

BACKGROUND: A variety of protocols exist for producing whole genome run-on transcription datasets. However, little is known about how differences between these protocols affect the signal within the resulting libraries. RESULTS: Using run-on transcription datasets generated from the same biological system, we show that a variety of GRO- and PRO-seq preparation methods leave identifiable signatures within each library. Specifically we show that the library preparation method results in differences in quality control metrics, as well as differences in the signal distribution at the 5 ' end of transcribed regions. These shifts lead to disparities in eRNA identification, but do not impact analyses aimed at inferring the key regulators involved in changes to transcription. CONCLUSIONS: Run-on sequencing protocol variations result in technical signatures that can be used to identify both the enrichment and library preparation method of a particular data set. These technical signatures are batch effects that limit detailed comparisons of pausing ratios and eRNAs identified across protocols. However, these batch effects have only limited impact on our ability to infer which regulators underlie the observed transcriptional changes.

BACKGROUND: Citrus is one of the most important fresh fruit crops worldwide. Juice sac granulation is a physiological disorder, which leads to a reduction in soluble solid concentration, total sugar, and titratable acidity of citrus fruits. Pectin methylesterase (PME) catalyzes the de-methylesterification of homogalacturonans and plays crucial roles in cell wall modification during plant development and fruit ripening. Although PME family has been well investigated in various model plants, little is known regarding the evolutionary property and biological function of PME family genes in citrus. RESULTS: In this study, 53 non-redundant PME genes were identified from Citrus sinensis genome, and these PME genes were divided into four clades based on the phylogenetic relationship. Subsequently, bioinformatics analyses of gene structure, conserved domain, chromosome localization, gene duplication, and collinearity were performed on CsPME genes, providing important clues for further research on the functions of CsPME genes. The expression profiles of CsPME genes in response to juice sac granulation and low-temperature stress revealed that CsPME genes were involved in the low temperature-induced juice sac granulation in navel orange fruits. Subcellular localization analysis suggested that CsPME genes were localized on the apoplast, endoplasmic reticulum, plasma membrane, and vacuole membrane. Moreover, yeast one-hybrid screening and dual luciferase activity assay revealed that the transcription factor CsRVE1 directly bound to the promoter of CsPME3 and activated its activity. CONCLUSION: In summary, this study conducts a comprehensive analysis of the PME gene family in citrus, and provides a novel insight into the biological functions and regulation patterns of CsPME genes during juice sac granulation of citrus.

BACKGROUND: Tibetan cashmere goats are served as a valuable model for high altitude adaptation and hypoxia complications related studies, while the cashmere produced by these goats is an important source of income for the herders. The aim of this study was to investigate the differences in protein abundance underlying the fine (average 12.20 ± 0.03 μm of mean fiber diameter) and coarse cashmere (average 14.67 ± 0.05 μm of mean fiber diameter) producing by Tibetan cashmere goats. We systematically investigated the genetic determinants of fiber diameter by integrated analysis with proteomic and transcriptomic datasets from skin tissues of Tibetan cashmere goats. RESULTS: We identified 1980 proteins using a label-free proteomics approach. They were annotated to three different databases, while 1730 proteins were mapped to the original protein coding genes (PCGs) of the transcriptomic study. Comparative analyses of cashmere with extremely fine vs. coarse phenotypes yielded 29 differentially expressed proteins (DEPs), for instance, APOH, GANAB, AEBP1, CP, CPB2, GPR142, VTN, IMPA1, CTSZ, GLB1, and HMCN1. Functional enrichment analysis of these DEPs revealed their involvement in oxidation-reduction process, cell redox homeostasis, metabolic, PI3K-Akt, MAPK, and Wnt signaling pathways. Transcription factors enrichment analysis revealed the proteins mainly belong to NF-YB family, HMG family, CSD family. We further validated the protein abundance of four DEPs (GC, VTN, AEBP1, and GPR142) through western blot, and considered they were the most potential candidate genes for cashmere traits in Tibetan cashmere goats. CONCLUSIONS: These analyses indicated that the major biological variations underlying the difference of cashmere fiber diameter in Tibetan cashmere goats were attributed to the inherent adaptations related to metabolic, hypoxic, and stress response differences. This study provided novel insights into the breeding strategies for cashmere traits and enhance the understanding of the biological and genetic mechanisms of cashmere traits in Tibetan cashmere goats.

BACKGROUND: The repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported. RESULTS: RGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to 'adherens junsctions' and 'extracellular-cell adhesion', while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation. CONCLUSIONS: These results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.

BACKGROUND: Maize is one of the most important food crops worldwide. Roots play important role in maize productivity through water and nutrient uptake from the soil. Improving maize root traits for efficient water uptake will help to optimize irrigation and contribute to sustainable maize production. Therefore, we investigated the protein profiles of maize cv. Anyu308 root system divided into Upper root zone (UR), Middle root (MR), and Lower root (LR), by label free quantitative shotgun proteomic approach (LFQ). The aim of our study was to identify proteins and mechanisms associated with enhanced water uptake in different maize root zones under automatic irrigation system. RESULTS: At field capacity, MR had the highest water uptake than the UR and LR. We identified a total of 489 differentially abundant proteins (DAPs) by pairwise comparison of MR, LR, and UR. Cluster analysis of DAPs revealed MR and UR had similar protein abundance patterns different from LR. More proteins were differentially abundant in MR/UR compared to LR/MR and LR/UR. Comparisons of protein profiles indicate that the DAPs in MR increased in abundance, compared to UR and LR which had more downregulated DAPs. The abundance patterns, functional category, and pathway enrichment analyses highlight chromatin structure and dynamics, ribosomal structures, polysaccharide metabolism, energy metabolism and transport, induction of water channels, inorganic ion transport, intracellular trafficking, and vesicular transport, and posttranslational modification as primary biological processes related to enhanced root water uptake in maize. Specifically, the abundance of histones, ribosomal proteins, and aquaporins, including mitochondrion electron transport proteins and the TCA cycle, underpinned MR's enhanced water uptake. Furthermore, proteins involved in folding and vascular transport supported the radial transport of solute across cell membranes in UR and MR. Parallel reaction monitoring analysis was used to confirmed profile of the DAPs obtained by LFQ-based proteomics. CONCLUSION: The list of differentially abundant proteins identified in MR are interesting candidates for further elucidation of their role in enhanced water uptake in maize root. Overall, the current results provided an insight into the mechanisms of maize root water uptake.

BACKGROUND: The cyclical nature of gene expression in the intraerythrocytic development cycle (IDC) of the malaria parasite, Plasmodium falciparum, confounds the accurate detection of specific transcriptional differences, e.g. as provoked by the development of drug resistance. In lab-based studies, P. falciparum cultures are synchronized to remove this confounding factor, but the rapid detection of emerging resistance to artemisinin therapies requires rapid analysis of transcriptomes extracted directly from clinical samples. Here we propose the use of cyclical regression covariates (CRC) to eliminate the major confounding effect of developmentally driven transcriptional changes in clinical samples. We show that elimination of this confounding factor reduces both Type I and Type II errors and demonstrate the effectiveness of this approach using a published dataset of 1043 transcriptomes extracted directly from patient blood samples with different patient clearance times after treatment with artemisinin. RESULTS: We apply this method to two publicly available datasets and demonstrate its ability to reduce the confounding of differences in transcript levels due to misaligned intraerythrocytic development time. Adjusting the clinical 1043 transcriptomes dataset with CRC results in detection of fewer functional categories than previously reported from the same data set adjusted using other methods. We also detect mostly the same functional categories, but observe fewer genes within these categories. Finally, the CRC method identifies genes in a functional category that was absent from the results when the dataset was adjusted using other methods. Analysis of differential gene expression in the clinical data samples that vary broadly for developmental stage resulted in the detection of far fewer transcripts in fewer functional categories while, at the same time, identifying genes in two functional categories not present in the unadjusted data analysis. These differences are consistent with the expectation that CRC reduces both false positives and false negatives with the largest effect on datasets from samples with greater variance in developmental stage. CONCLUSIONS: Cyclical regression covariates have immediate application to parasite transcriptome sequencing directly from clinical blood samples and to cost-constrained in vitro experiments.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder and is the most common cause of late-onset dementia. Microglia, the primary innate immune cells of the central nervous system (CNS), have a complex role in AD neuropathology. In the initial stages of AD, microglia play a role in limiting pathology by removing amyloid-β (Aβ) by phagocytosis. In contrast, microglia also release pro-inflammatory cytokines and chemokines to promote neuroinflammation and exacerbate AD neuropathology. Therefore, investigating microglial gene networks could identify new targets for therapeutic strategies for AD. RESULTS: We identified 465 differentially expressed genes (DEG) in 5XFAD versus wild-type mice by microarray, 354 DEG in lipopolysaccharide (LPS)-stimulated N9 microglia versus unstimulated control cells using RNA-sequencing (RNA-seq), with 32 DEG common between both datasets. Analyses of the 32 common DEG uncovered numerous molecular functions and pathways involved in Aβ phagocytosis and neuroinflammation associated with AD. Furthermore, multiplex ELISA confirmed the induction of several cytokines and chemokines in LPS-stimulated microglia. CONCLUSIONS: In summary, AD triggered multiple signaling pathways that regulate numerous genes in microglia, contributing to Aβ phagocytosis and neuroinflammation. Overall, these data identified several regulatory factors and biomarkers in microglia that could be useful in further understanding AD neuropathology.

BACKGROUND: Meiotic recombination is one of the important phenomena contributing to gamete genome diversity. However, except for human and a few model organisms, it is not well studied in livestock, including cattle. RESULTS: To investigate their distributions in the cattle sperm genome, we sequenced 143 single sperms from two Holstein bulls. We mapped meiotic recombination events at high resolution based on phased heterozygous single nucleotide polymorphism (SNP). In the absence of evolutionary selection pressure in fertilization and survival, recombination events in sperm are enriched near distal chromosomal ends, revealing that such a pattern is intrinsic to the molecular mechanism of meiosis. Furthermore, we further validated these findings in single sperms with results derived from sequencing its family trio of diploid genomes and our previous studies of recombination in cattle. CONCLUSIONS: To our knowledge, this is the first large-scale single sperm whole-genome sequencing effort in livestock, which provided useful information for future studies of recombination, genome instability, and male infertility.

BACKGROUND: The TIFY gene family is a group of plant-specific transcription factors involved in regulation of plant growth and development and a variety of stress responses. However, the TIFY family has not yet been well characterized in kiwifruit, a popular fruit with important nutritional and economic value. RESULTS: A total of 27 and 21 TIFY genes were identified in the genomes of Actinidia eriantha and A. chinensis, respectively. Phylogenetic analyses showed that kiwifruit TIFY genes could be classified into four major groups, JAZ, ZML, TIFY and PPD, and the JAZ group could be further clustered into six subgroups (JAZ I to JAZ VI). Members within the same group or subgroup have similar exon-intron structures and conserved motif compositions. The kiwifruit TIFY genes are unevenly distributed on the chromosomes, and the segmental duplication events played a vital role in the expansion of the TIFY genes in kiwifruit. Syntenic analyses of TIFY genes between kiwifruit and other five plant species (including Arabidopsis thaliana, Camellia sinensis, Oryza sativa, Solanum lycopersicum and Vitis vinifera) and between the two kiwifruit species provided valuable clues for understanding the potential evolution of the kiwifruit TIFY family. Molecular evolutionary analysis showed that the evolution of kiwifruit TIFY genes was primarily constrained by intense purifying selection. Promoter cis-element analysis showed that most kiwifruit TIFY genes possess multiple cis-elements related to stress-response, phytohormone signal transduction and plant growth and development. The expression pattern analyses indicated that TIFY genes might play a role in different kiwifruit tissues, including fruit at specific development stages. In addition, several TIFY genes with high expression levels during Psa (Pseudomonas syringae pv. actinidiae) infection were identified, suggesting a role in the process of Pas infection. CONCLUSIONS: In this study, the kiwifruit TIFY genes were identified from two assembled kiwifruit genomes. In addition, their basic physiochemical properties, chromosomal localization, phylogeny, gene structures and conserved motifs, synteny analyses, promoter cis-elements and expression patters were systematically examined. The results laid a foundation for further understanding the function of TIFY genes in kiwifruit, and provided a new potential approach for the prevention and treatment of Psa infection.

BACKGROUND: Lipid droplets (LDs) present in land plants serve as an essential energy and carbon reserve for seed germination and seedling development. Oleosins, the most abundant structural proteins of LDs, comprise a small family involved in LD formation, stabilization and degradation. Despite their importance, our knowledge on oleosins is still poor in Euphorbiaceae, a large plant family that contains several important oil-bearing species. RESULTS: To uncover lineage-specific evolution of oleosin genes in Euphorbiaceae, in this study, we performed a genome-wide identification and comprehensive comparison of the oleosin family in Euphorbiaceae species with available genome sequences, i.e. castor bean (Ricinus communis), physic nut (Jatropha curcas), tung tree (Vernicia fordii), Mercurialis annua, cassava (Manihot esculenta) and rubber tree (Hevea brasiliensis), and a number of five, five, five, five, eight and eight members were found, respectively. Synteny analysis revealed one-to-one collinear relationship of oleosin genes between the former four (i.e. castor bean, physic nut, tung tree and M. annua) as well as latter two species (i.e. cassava and rubber tree), whereas one-to-one and one-to-two collinear relationships were observed between physic nut and cassava, reflecting the occurrence of one recent whole-genome duplication (WGD) in the last common ancestor of cassava and rubber tree. The presence of five ortholog groups representing three previously defined clades (i.e. U, SL and SH) dates back at least to the Malpighiales ancestor, because they are also conserved in poplar (Populus trichocarpa), a tree having experienced one Salicaceae-specific recent WGD. As observed in poplar, WGD was shown to be the main driver for the family expansion in both cassava and rubber tree. Nevertheless, same retention patterns of WGD-derived duplicates observed in cassava and rubber tree are somewhat different from that of poplar, though certain homologous fragments are still present in rubber tree. Further transcriptional profiling revealed an apparent seed-predominant expression pattern of oleosin genes in physic nut, castor bean and rubber tree. Moreover, structure and expression divergence of paralogous pairs were also observed in both cassava and rubber tree. CONCLUSION: Comparative genomics analysis of oleosin genes reported in this study improved our knowledge on lineage-specific family evolution in Euphorbiaceae, which also provides valuable information for further functional analysis and utilization of key members and their promoters.

BACKGROUND: Equol, an isoflavonoid metabolite with possible health benefits in humans, is known to be produced by some human gut bacteria. While the genes encoding the equol production pathway have been characterized in a few bacterial strains, a systematic analysis of the equol production pathway is currently lacking. RESULTS: This study presents an analysis of the taxonomic distribution and evolutionary history of the gene cluster encoding the equol production pathway. A survey for equol gene clusters within the Genome Taxonomy Database bacterial genomes and human gut metagenomes resulted in the identification of a highly conserved gene cluster found in nine bacterial species from the Eggerthellaceae family. The identified gene clusters from human gut metagenomes revealed potential variations in the equol gene cluster organization and gene content within the equol-producing Eggerthellaceae clades. Subsequent analysis showed that in addition to the four genes directly involved in equol production, multiple other genes were consistently found in the equol gene clusters. These genes were predicted to encode a putative electron transport complex and hydrogenase maturase system, suggesting potential roles for them in the equol production pathway. Analysis of the gene clusters and a phylogenetic reconstruction of a putative NAD kinase gene provided evidence of the recent transfer of the equol gene cluster from a basal Eggerthellaceae species to Slackia_A equolifaciens, Enteroscipio sp000270285, and Lactococcus garvieae 20-92. CONCLUSIONS: This analysis demonstrates that the highly conserved equol gene cluster is taxonomically restricted to the Eggerthellaceae family of bacteria and provides evidence of the role of horizontal gene transfer in the evolutionary history of these genes. These results provide a foundation for future studies of equol production in the human gut and future efforts related to bioengineering and the use of equol-producing bacteria as probiotics.

BACKGROUND: Long noncoding RNAs (lncRNAs) are now proven as essential regulatory elements, playing diverse roles in many biological processes including mammary gland development. However, little is known about their roles in the bovine lactation process. RESULTS: To identify and characterize the roles of lncRNAs in bovine lactation, high throughput RNA sequencing data from Jersey (high milk yield producer), and Kashmiri cattle (low milk yield producer) were utilized. Transcriptome data from three Kashmiri and three Jersey cattle throughout their lactation stages were utilized for differential expression analysis. At each stage (early, mid and late) three samples were taken from each breed. A total of 45 differentially expressed lncRNAs were identified between the three stages of lactation. The differentially expressed lncRNAs were found co-expressed with genes involved in the milk synthesis processes such as GPAM, LPL, and ABCG2 indicating their potential regulatory effects on milk quality genes. KEGG pathways analysis of potential cis and trans target genes of differentially expressed lncRNAs indicated that 27 and 48 pathways were significantly enriched between the three stages of lactation in Kashmiri and Jersey respectively, including mTOR signaling, PI3K-Akt signaling, and RAP1 signaling pathways. These pathways are known to play key roles in lactation biology and mammary gland development. CONCLUSIONS: Expression profiles of lncRNAs across different lactation stages in Jersey and Kashmiri cattle provide a valuable resource for the study of the regulatory mechanisms involved in the lactation process as well as facilitate understanding of the role of lncRNAs in bovine lactation biology.

BACKGROUND: High temperature shock is becoming increasingly common in our climate, affecting plant growth and productivity. The ability of a plant to survive stress is a complex phenomenon. One of the essential tissues for plant performance under various environmental stimuli is the crown. However, the molecular characterization of this region remains poorly investigated. Gibberellins play a fundamental role in whole-plant stature formation. This study identified plant stature modifications and crown-specific transcriptome re-modeling in gibberellin-deficient barley sdw1.a (BW827) and sdw1.d (BW828) mutants exposed to increased temperature. RESULTS: The deletion around the sdw1 gene in BW827 was found to encompass at least 13 genes with primarily regulatory functions. A bigger genetic polymorphism of BW828 than of BW827 in relation to wild type was revealed. Transcriptome-wide sequencing (RNA-seq) revealed several differentially expressed genes involved in gibberellin metabolism and heat response located outside of introgression regions. It was found that HvGA20ox4, a paralogue of the HvGA20ox2 gene, was upregulated in BW828 relative to other genotypes, which manifested as basal internode elongation. The transcriptome response to elevated temperature differed in the crown of sdw1.a and sdw1.d mutants; it was most contrasting for HvHsf genes upregulated under elevated temperature in BW828, whereas those specific to BW827 were downregulated. In-depth examination of sdw1 mutants revealed also some differences in their phenotypes and physiology. CONCLUSIONS: We concluded that despite the studied sdw1 mutants being genetically related, their heat response seemed to be genotype-specific and observed differences resulted from genetic background diversity rather than single gene mutation, multiple gene deletion, or allele-specific expression of the HvGA20ox2 gene. Differences in the expressional reaction of genes to heat in different sdw1 mutants, found to be independent of the polymorphism, could be further explained by in-depth studies of the regulatory factors acting in the studied system. Our findings are particularly important in genetic research area since molecular response of crown tissue has been marginally investigated, and can be useful for wide genetic research of crops since barley has become a model plant for them.

BACKGROUND: Single molecule measurements of DNA polymerization kinetics provide a sensitive means to detect both secondary structures in DNA and deviations from primary chemical structure as a result of modified bases. In one approach to such analysis, deviations can be inferred by monitoring the behavior of DNA polymerase using single-molecule, real-time sequencing with zero-mode waveguide. This approach uses a Single Molecule Real Time (SMRT)-sequencing measurement of time between fluorescence pulse signals from consecutive nucleosides incorporated during DNA replication, called the interpulse duration (IPD). RESULTS: In this paper we present an analysis of loci with high IPDs in two genomes, a bacterial genome (E. coli) and a eukaryotic genome (C. elegans). To distinguish the potential effects of DNA modification on DNA polymerization speed, we paired an analysis of native genomic DNA with whole-genome amplified (WGA) material in which DNA modifications were effectively removed. Adenine modification sites for E. coli are known and we observed the expected IPD shifts at these sites in the native but not WGA samples. For C. elegans, such differences were not observed. Instead, we found a number of novel sequence contexts where IPDs were raised relative to the average IPDs for each of the four nucleotides, but for which the raised IPD was present in both native and WGA samples. CONCLUSION: The latter results argue strongly against DNA modification as the underlying driver for high IPD segments for C. elegans, and provide a framework for separating effects of DNA modification from context-dependent DNA polymerase kinetic patterns inherent in underlying DNA sequence for a complex eukaryotic genome.

BACKGROUND: The recent big data revolution in Genomics, coupled with the emergence of Deep Learning as a set of powerful machine learning methods, has shifted the standard practices of machine learning for Genomics. Even though Deep Learning methods such as Convolutional Neural Networks (CNNs) and Recurrent Neural Networks (RNNs) are becoming widespread in Genomics, developing and training such models is outside the ability of most researchers in the field. RESULTS: Here we present ENNGene-Easy Neural Network model building tool for Genomics. This tool simplifies training of custom CNN or hybrid CNN-RNN models on genomic data via an easy-to-use Graphical User Interface. ENNGene allows multiple input branches, including sequence, evolutionary conservation, and secondary structure, and performs all the necessary preprocessing steps, allowing simple input such as genomic coordinates. The network architecture is selected and fully customized by the user, from the number and types of the layers to each layer's precise set-up. ENNGene then deals with all steps of training and evaluation of the model, exporting valuable metrics such as multi-class ROC and precision-recall curve plots or TensorBoard log files. To facilitate interpretation of the predicted results, we deploy Integrated Gradients, providing the user with a graphical representation of an attribution level of each input position. To showcase the usage of ENNGene, we train multiple models on the RBP24 dataset, quickly reaching the state of the art while improving the performance on more than half of the proteins by including the evolutionary conservation score and tuning the network per protein. CONCLUSIONS: As the role of DL in big data analysis in the near future is indisputable, it is important to make it available for a broader range of researchers. We believe that an easy-to-use tool such as ENNGene can allow Genomics researchers without a background in Computational Sciences to harness the power of DL to gain better insights into and extract important information from the large amounts of data available in the field.

BACKGROUND: With advances in next-generation sequencing technologies, an unprecedented amount of soybean accessions has been sequenced by many individual studies and made available as raw sequencing reads for post-genomic research. RESULTS: To develop a consolidated and user-friendly genomic resource for post-genomic research, we consolidated the raw resequencing data of 1465 soybean genomes available in the public and 91 highly diverse wild soybean genomes newly sequenced. These altogether provided a collection of 1556 sequenced genomes of 1501 diverse accessions (1.5 K). The collection comprises of wild, landraces and elite cultivars of soybean that were grown in East Asia or major soybean cultivating areas around the world. Our extensive sequence analysis discovered 32 million single nucleotide polymorphisms (32mSNPs) and revealed a SNP density of 30 SNPs/kb and 12 non-synonymous SNPs/gene reflecting a high structural and functional genomic diversity of the new collection. Each SNP was annotated with 30 categories of structural and/or functional information. We further identified paired accessions between the 1.5 K and 20,087 (20 K) accessions in US collection as genomic "equivalent" accessions sharing the highest genomic identity for minimizing the barriers in soybean germplasm exchange between countries. We also exemplified the utility of 32mSNPs in enhancing post-genomics research through in-silico genotyping, high-resolution GWAS, discovering and/or characterizing genes and alleles/mutations, identifying germplasms containing beneficial alleles that are potentially experiencing artificial selection. CONCLUSION: The comprehensive analysis of publicly available large-scale genome sequencing data of diverse cultivated accessions and the newly in-house sequenced wild accessions greatly increased the soybean genome-wide variation resolution. This could facilitate a variety of genetic and molecular-level analyses in soybean. The 32mSNPs and 1.5 K accessions with their comprehensive annotation have been made available at the SoyBase and Ag Data Commons. The dataset could further serve as a versatile and expandable core resource for exploring the exponentially increasing genome sequencing data for a variety of post-genomic research.

BACKGROUND: Apple Glomerella leaf spot (GLS) and apple bitter rot (ABR) are two devastating foliar and fruit diseases on apples. The different symptoms of GLS and ABR could be related to different transcriptome patterns. Thus, the objectives of this study were to compare the transcriptome profiles of Colletotrichum gloeosporioides species complex isolates GC20190701, FL180903, and FL180906, the pathogen of GLS and ABR, and to evaluate the involvement of the genes on pathogenicity. RESULTS: A relatively large difference was discovered between the GLS-isolate GC20190701 and ABR-isolates FL180903, FL180906, and quite many differential expression genes associated with pathogenicity were revealed. The DEGs between the GLS- and ABR-isolate were significantly enriched in GO terms of secondary metabolites, however, the categories of degradation of various cell wall components did not. Many genes associated with secondary metabolism were revealed. A total of 17 Cytochrome P450s (CYP), 11 of which were up-regulated while six were down-regulated, and five up-regulated methyltransferase genes were discovered. The genes associated with the secretion of extracellular enzymes and melanin accumulation were up-regulated. Four genes associated with the degradation of the host cell wall, three genes involved in the degradation of cellulose, and one gene involved in the degradation of xylan were revealed and all up-regulated. In addition, genes involved in melanin syntheses, such as tyrosinase and glucosyltransferase, were highly up-regulated. CONCLUSIONS: The penetration ability, pathogenicity of GLS-isolate was greater than that of ABR-isolate, which might indicate that GLS-isolate originated from ABR-isolates by mutation. These results contributed to highlighting the importance to investigate such DEGs between GLS- and ABR-isolate in depth.

BACKGROUND: Chromatin architecture is critical for gene expression during development. Matrix attachment regions (MARs) control and regulate chromatin dynamics. The position of MARs in the genome determines the expression of genes in the organism. In this study, we set out to elucidate how MARs temporally regulate the expression of the fibroin heavy chain (FIBH) gene during development. We addressed this by identifying MARs and studying their distribution and differentiation, in the posterior silk glands of Bombyx mori during 5th instar development. RESULTS: Of the MARs identified on three different days, 7.15% MARs were common to all 3 days, whereas, 1.41, 19.27 and 52.47% MARs were unique to day 1, day 5, and day 7, respectively highlighting the dynamic nature of the matrix associated DNA. The average chromatin loop length based on the chromosome wise distribution of MARs and the distances between these MAR regions decreased from day 1 (253.91 kb) to day 5 (73.54 kb) to day 7 (39.19 kb). Further significant changes in the MARs in the vicinity of the FIBH gene were found during different days of 5th instar development which implied their role in the regulation and expression of the FIBH gene. CONCLUSIONS: The presence of MARs in the flanking regions of genes found to exhibit differential expression during 5th instar development indicates their possible role in the regulation of their expression. This reiterates the importance of MARs in the genomic functioning as regulators of the molecular mechanisms in the nucleus. This is the first study that takes into account the tissue specific genome-wide MAR association and the potential role of these MARs in developmentally regulated gene expression. The current study lays a foundation to understand the genome wide regulation of chromatin during development.

BACKGROUND: Melanoma is a highly heterogeneous and aggressive cutaneous malignancy. Ferroptosis, a new pathway of cell death depending on the intracellar iron, has been shown to be significantly associated with apoptosis of a number of tumors, including melanoma. Nevertheless, the relationship between ferroptosis-related genes (FRGs) and the melanoma patients' prognosis needs to be explored. METHODS: Download expression profiles of FRGs and clinical data from The Cancer Genome Atlas (TCGA) database. 70% data were randomly selected from the TCGA database and utilized the univariate Cox analysis and the least absolute shrinkage and selection operator (LASSO) regression model to create a prognostic model, and the remaining 30% was used to validate the predictive power of the model. In addition, GSE65904 and GSE22153 date sets as the verification cohort to testify the predictive ability of the signature. RESULTS: We identified nine FRGs relating with melanoma patients' overall survival (OS) and established a prognostic model based on their expression. During the research, patients were divided into group of high-risk and low-risk according to the results of LASSO regression analysis. Survival time was significantly longer in the low-risk group than that of in the high-risk group (P < 0.001). Enrichment analysis of different risk groups demonstrated that the reasons for the difference were related to immune-related pathways, and the degree of immune cell infiltration in the low-risk group was significantly higher than that in the high-risk group. CONCLUSIONS: The FRG prognostic model we established can predict the prognosis of melanoma patients and may further guide subsequent treatment.

BACKGROUND: Diaporthe caulivora is a fungal pathogen causing stem canker in soybean worldwide. The generation of genomic and transcriptomic information of this ascomycete, together with a comparative genomic approach with other pathogens of this genus, will contribute to get insights into the molecular basis of pathogenicity strategies used by D. caulivora and other Diaporthe species. RESULTS: In the present work, the nuclear genome of D. caulivora isolate (D57) was resolved, and a comprehensive annotation based on gene expression and genomic analysis is provided. Diaporthe caulivora D57 has an estimated size of 57,86 Mb and contains 18,385 predicted protein-coding genes, from which 1501 encode predicted secreted proteins. A large array of D. caulivora genes encoding secreted pathogenicity-related proteins was identified, including carbohydrate-active enzymes (CAZymes), necrosis-inducing proteins, oxidoreductases, proteases and effector candidates. Comparative genomics with other plant pathogenic Diaporthe species revealed a core secretome present in all Diaporthe species as well as Diaporthe-specific and D. caulivora-specific secreted proteins. Transcriptional profiling during early soybean infection stages showed differential expression of 2659 D. caulivora genes. Expression patterns of upregulated genes and gene ontology enrichment analysis revealed that host infection strategies depends on plant cell wall degradation and modification, detoxification of compounds, transporter activities and toxin production. Increased expression of effectors candidates suggests that D. caulivora pathogenicity also rely on plant defense evasion. A high proportion of the upregulated genes correspond to the core secretome and are represented in the pathogen-host interaction (PHI) database, which is consistent with their potential roles in pathogenic strategies of the genus Diaporthe. CONCLUSIONS: Our findings give novel and relevant insights into the molecular traits involved in pathogenicity of D. caulivora towards soybean plants. Some of these traits are in common with other Diaporthe pathogens with different host specificity, while others are species-specific. Our analyses also highlight the importance to have a deeper understanding of pathogenicity functions among Diaporthe pathogens and their interference with plant defense activation.

Sex has proven to be one of the most intriguing areas of research across evolution, development, and ecology. Intersex or sex change occurs frequently in molluscs. The deep-sea mussel Gigantidas haimaensis often dominates within Haima cold seep ecosystems, but details of their reproduction remain unknown. Herein, we conducted a combined proteomic and transcriptomic analysis of G. haimaensis gonads to provide a systematic understanding of sexual development in deep-sea bivalves. A total of 2,452 out of 42,238 genes (5.81%) and 288 out of 7,089 proteins (4.06%) were significantly differentially expressed between ovaries and testes with a false discovery rate (FDR) <0.05. Candidate genes involved in sexual development were identified; among 12 differentially expressed genes between sexes, four ovary-biased genes (β-catenin, fem-1, forkhead box L2 and membrane progestin receptor α) were expressed significantly higher in males than females. Combining histological characteristics, we speculate that the males maybe intersex undergoing sex change, and implied that these genes may be involved in the process of male testis converting into female gonads in G. haimaensis. The results suggest that this adaptation may be based on local environmental factors, sedentary lifestyles, and patchy distribution, and sex change may facilitate adaptation to a changing environment and expansion of the population. The findings provide a valuable genetic resource to better understand the mechanisms of sex change and survival strategies in deep-sea bivalves.

BACKGROUND: The emerging epitranscriptome plays an essential role in female fertility. As the most prevalent internal mRNA modification, N6-methyladenine (m6A) methylation regulate mRNA fate and translational efficiency. However, whether m6A methylation was involved in the aging-related ovarian reserve decline has not been investigated. Herein, we performed m6A transcriptome-wide profiling in the ovarian granulosa cells of younger women (younger group) and older women (older group). RESULTS: m6A methylation distribution was highly conserved and enriched in the CDS and 3'UTR region. Besides, an increased number of m6A methylated genes were identified in the older group. Bioinformatics analysis indicated that m6A methylated genes were enriched in the FoxO signaling pathway, adherens junction, and regulation of actin cytoskeleton. A total of 435 genes were differently expressed in the older group, moreover, 58 of them were modified by m6A. Several specific genes, including BUB1B, PHC2, TOP2A, DDR2, KLF13, and RYR2 which were differently expressed and modified by m6A, were validated using qRT-PCR and might be involved in the decreased ovarian functions in the aging ovary. CONCLUSIONS: Hence, our finding revealed the transcriptional significance of m6A modifications and provide potential therapeutic targets to promote fertility reservation for aging women.

BACKGROUND: An important aspect of studying evolution is to understand how new species are formed and their uniqueness is maintained. Hybridization can lead to the formation of new species through reorganization of the adaptive system and significant changes in phenotype. Interestingly, eight stable strains of 2nNCRC derived from interspecies hybridization have been established in our laboratory. To examine the phylogeographical pattern of the widely distributed genus Carassius across Eurasia and investigate the possible homoploid hybrid origin of the Carassius auratus complex lineage in light of past climatic events, the mitochondrial genome (mtDNA) and one nuclear DNA were used to reconstruct the phylogenetic relationship between the C. auratus complex and 2nNCRC and to assess how demographic history, dispersal and barriers to gene flow have led to the current distribution of the C. auratus complex. RESULTS: As expected, 2nNCRC had a very close relationship with the C. auratus complex and similar morphological characteristics to those of the C. auratus complex, which is genetically distinct from the other three species of Carassius. The estimation of divergence time and ancestral state demonstrated that the C. auratus complex possibly originated from the Yangtze River basin in China. There were seven sublineages of the C. auratus complex across Eurasia and at least four mtDNA lineages endemic to particular geographical regions in China. The primary colonization route from China to Mongolia and the Far East (Russia) occurred during the Late Pliocene, and the diversification of other sublineages of the C. auratus complex specifically coincided with the interglacial stage during the Early and Mid-Pleistocene in China. CONCLUSION: Our results support the origin of the C. auratus complex in China, and its wide distribution across Eurasia was mainly due to natural Pleistocene dispersal and recent anthropogenic translocation. The sympatric distribution of the ancestral area for both parents of 2nNCRC and the C. auratus complex, as well as the significant changes in the structure of pharyngeal teeth and morphological characteristics between 2nNCRC and its parents, imply that homoploid hybrid speciation (HHS) for C. auratus could likely have occurred in nature. The diversification pattern indicated an independent evolutionary history of the C. auratus complex, which was not separated from the most recent common ancestor of C. carassius or C. cuvieri. Considering that the paleoclimate oscillation and the development of an eastward-flowing drainage system during the Pliocene and Pleistocene in China provided an opportunity for hybridization between divergent lineages, the formation of 2nNCRC in our laboratory could be a good candidate for explaining the HHS of C. auratus in nature.

BACKGROUND: External environmental factors, such as salt, alkali and drought, severely limit the acreage and yield of alfalfa. The mining of tolerance-related genes in alfalfa and improving the stress resistance of this plant are essential for increasing alfalfa yield. PLD is the main phospholipid hydrolase in plants and plays an important role in plant growth, development, signaling, and resistance to adverse stress. With the availability of whole genome sequences, the annotation and expression of PLDs in alfalfa can now be achieved. At present, few studies have investigated PLDs in alfalfa. Here, we conducted a study of PLDs in alfalfa and identified and analyzed the expression pattern of PLDs under different treatments. RESULTS: Fifty-nine MsPLDs were identified in alfalfa and classified into six subtypes: MsPLDα, β, γ, δ and ε belong to the C2-PLD subfamily, and MsPLDζ belongs to the PXPH-PLD subfamily. Members of the same PLD subtype have similar physicochemical properties, sequence structure and domains, but their cis-acting elements are different. A qRT-PCR analysis revealed that MsPLDs are expressed in multiple tissues. MsPLDs can respond to alkali, drought, ABA, IAA, and GA3 treatments and particularly to salt stress. Different expression patterns were found for the same gene under different treatments and different genes under the same treatment. Expression of MsPLD05 improved salt tolerance in yeast. CONCLUSION: This study represents the first genome-wide characterization of MsPLDs in alfalfa. Most MsPLDs are expressed mainly in mature leaves and respond positively to abiotic stresses and hormonal treatments. This study further expands the resistance gene pool in legume forage grasses and provides a reference for further in-depth study of MsPLDs in alfalfa.

BACKGROUND: Peanut is the most essential oil and food crop globally due to its high oil and protein content. Root-knot nematode infects peanut roots, causing poor development and severely limiting peanut yields worldwide. The discovery of peanut genome identified a considerable number of genetic loci controlling the peanut root-knot nematode; however, the molecular mechanism of root-knot nematode remains unknown. RESULTS: The heterogeneous response to root-knot nematode stress in peanut roots was identified using whole-transcriptome RNA-seq. A total of 430 mRNAs, 111 miRNAs, 4453 lncRNAs, and 123 circRNAs were found to have differential expression between infected and non-infected peanuts. The expression profiles of the lncRNA/circRNA-miRNA-mRNA network were developed to understand the potential pathways that lead to root-knot nematodes in peanut roots. During root-knot nematodes stress, a total of 10 lncRNAs, 4 circRNAs, 5 miRNAs, and 13 mRNAs can create competing endogenous RNA and participate in the oxidation-reduction process as well as other biological metabolism processes in peanuts. The findings will highlight the role of peanut ceRNAs in response to root-knot nematodes. CONCLUSION: The GO classification and KEGG pathway enrichment study of core regulatory networks revealed that ceRNAs are involved in oxidation-reduction, peroxidase activity, lignin synthesis in the xylem, and flavonoid synthesis. Overall, these findings may help researchers better understand the role of non-coding RNAs in response to root-knot nematodes.

Basic helix-loop-helix (bHLH) proteins are transcription factors (TFs) that have been shown to regulate anthocyanin biosynthesis in many plant species. However, the bHLH gene family in Populus deltoids has not yet been reported. In this study, 185 PdbHLH genes were identified in the Populus deltoids genome and were classified into 15 groups based on their sequence similarity and phylogenetic relationships. Analysis of the gene structure, chromosome location and conserved motif of the PdbHLH genes were performed by bioinformatic methods. Gene duplication analyses revealed that 114 PdbHLH were expanded and retained after WGD/segmental and proximal duplication. Investigation of cis-regulatory elements of PdbHLH genes indicated that many PdbHLH genes are involved in the regulation of anthocyanin biosynthesis. The expression patterns of PdbHLHs were obtained from previous data in two colored-leaf poplar (QHP and JHP) and green leaf poplar (L2025). Further analysis revealed that 12 candidate genes, including 3 genes (PdbHLH57, PdbHLH143, and PdbHLH173) from the subgroup III(f) and 9 gene from other groups, were positively associated with anthocyanin biosynthesis. In addition, 4 genes (PdbHLH4, PdbHLH1, PdbHLH18, and PdbHLH164) may be involved in negatively regulating the anthocyanin biosynthesis. These results provide a basis for the functional characterization of bHLH genes and investigations on the molecular mechanisms of anthocyanin biosynthesis in colored-leaf poplar.

BACKGROUND: Ribosomal DNAs (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosomes during generation of genome assembly. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution. RESULTS: Here we generate ultra-long Oxford Nanopore Technologies (ONT) and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA's repeating units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform assembly of the cluster by taking advantage of the long reads that carry these units, which led to an assembly of 5S rDNA cluster consisting of up to 167 consecutive 5S rDNA units in the N2 strain. The ordering and copy number of various rDNA units are consistent with the separation time between strains. Surprisingly, we observed a drastically reduced level of variation in the unit composition in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than in the C. elegans N2 strain, suggesting that N2, a widely used reference strain, is likely to be defective in maintaining the 5S rDNA cluster stability compared with other wild isolates of C. elegans or C. briggsae. CONCLUSIONS: The results demonstrate that Nanopore DNA sequencing reads are capable of generating assembly of highly repetitive sequences, and rDNA units are highly dynamic both within and between population(s) of the same species in terms of sequence and copy number. The detailed structure and variation of the 5S rDNA units within the rDNA cluster pave the way for functional and evolutionary studies.

BACKGROUND: Skin pigmentation is a broadly appearing phenomenon of most animals and humans in nature. Here we used a bird model to investigate why melanin spot deposits on the skin. RESULTS: Our result showed that growth age and the sunlight might induce melanin deposition in bird beak skin which was determined by genetic factors. GWAS helped us to identify two major loci affecting melanin deposition, located on chromosomes 13 and 25, respectively. The fine mapping works narrowed the candidate regions to 0.98 Mb and 1.0 Mb on chromosomes 13 and 25. The MITF and POU2F3 may be the causative genes and synergistically affect melanin deposition during duck beak skin. Furthermore, our data strongly demonstrated that the pathway of melanin metabolism contributes to melanin deposition on the skin. CONCLUSIONS: We demonstrated that age and sunlight induce melanin deposition in bird beak skin, while heredity is fundamental. The MITF and POU2F3 likely played a synergistic effect on the regulation of melanin synthesis, and their mutations contribute to phenotypic differences in beak melanin deposition among individuals. It is pointed out that melanin deposition in the skin is related to the pathway of melanin metabolism, which provided insights into the molecular regulatory mechanisms and the genetic improvement of the melanin deposition in duck beak.

BACKGROUND: Novel antimalarials should be effective across all species of malaria parasites that infect humans, especially the two species that bear the most impact, Plasmodium falciparum and Plasmodium vivax. Protein kinases encoded by pathogens, as well as host kinases required for survival of intracellular pathogens, carry considerable potential as targets for antimalarial intervention (Adderley et al. Trends Parasitol 37:508-524, 2021;  Wei et al. Cell Rep Med 2:100423, 2021). To date, no comprehensive P. vivax kinome assembly has been conducted; and the P. falciparum kinome, first assembled in 2004, requires an update. The present study, aimed to fill these gaps, utilises a recently published structurally-validated multiple sequence alignment (MSA) of the human kinome (Modi et al. Sci Rep 9:19790, 2019). This MSA is used as a scaffold to assist the alignment of all protein kinase sequences from P. falciparum and P. vivax, and (where possible) their assignment to specific kinase groups/families. RESULTS: We were able to assign six P. falciparum previously classified as OPK or 'orphans' (i.e. with no clear phylogenetic relation to any of the established ePK groups) to one of the aforementioned ePK groups. Direct phylogenetic comparison established that despite an overall high level of similarity between the P. falciparum and P. vivax kinomes, which will help in selecting targets for intervention, there are differences that may underlie the biological specificities of these species. Furthermore, we highlight a number of Plasmodium kinases that have a surprisingly high level of similarity with their human counterparts and therefore not well suited as targets for drug discovery. CONCLUSIONS: Direct comparison of the kinomes of Homo sapiens, P. falciparum and P. vivax sheds additional light on the previously documented divergence of many P. falciparum and P. vivax kinases from those of their human host. We provide the first direct kinome comparison between the phylogenetically distinct species of P. falciparum and P. vivax, illustrating the key similarities and differences which must be considered in the context of kinase-directed antimalarial drug discovery, and discuss the divergences and similarities between the human and Plasmodium kinomes to inform future searches for selective antimalarial intervention.

BACKGROUND: Whole genome sequencing analyzed by core genome multi-locus sequence typing (cgMLST) is widely used in surveillance of the pathogenic bacteria Listeria monocytogenes. Given the heterogeneity of available bioinformatics tools to define cgMLST alleles, our aim was to identify parameters influencing the precision of cgMLST profiles. METHODS: We used three L. monocytogenes reference genomes from different phylogenetic lineages and assessed the impact of in vitro (i.e. tested genomes, successive platings, replicates of DNA extraction and sequencing) and in silico parameters (i.e. targeted depth of coverage, depth of coverage, breadth of coverage, assembly metrics, cgMLST workflows, cgMLST completeness) on cgMLST precision made of 1748 core loci. Six cgMLST workflows were tested, comprising assembly-based (BIGSdb, INNUENDO, GENPAT, SeqSphere and BioNumerics) and assembly-free (i.e. kmer-based MentaLiST) allele callers. Principal component analyses and generalized linear models were used to identify the most impactful parameters on cgMLST precision. RESULTS: The isolate's genetic background, cgMLST workflows, cgMLST completeness, as well as depth and breadth of coverage were the parameters that impacted most on cgMLST precision (i.e. identical alleles against reference circular genomes). All workflows performed well at ≥40X of depth of coverage, with high loci detection (> 99.54% for all, except for BioNumerics with 97.78%) and showed consistent cluster definitions using the reference cut-off of ≤7 allele differences. CONCLUSIONS: This highlights that bioinformatics workflows dedicated to cgMLST allele calling are largely robust when paired-end reads are of high quality and when the sequencing depth is ≥40X.

BACKGROUND: The application of RNA-seq technology has become more extensive and the number of analysis procedures available has increased over the past years. Selecting an appropriate workflow has become an important issue for researchers in the field. METHODS: In our study, six popular analytical procedures/pipeline were compared using four RNA-seq datasets from mouse, human, rat, and macaque, respectively. The gene expression value, fold change of gene expression, and statistical significance were evaluated to compare the similarities and differences among the six procedures. qRT-PCR was performed to validate the differentially expressed genes (DEGs) from all six procedures. RESULTS: Cufflinks-Cuffdiff demands the highest computing resources and Kallisto-Sleuth demands the least. Gene expression values, fold change, p and q values of differential expression (DE) analysis are highly correlated among procedures using HTseq for quantification. For genes with medium expression abundance, the expression values determined using the different procedures were similar. Major differences in expression values come from genes with particularly high or low expression levels. HISAT2-StringTie-Ballgown is more sensitive to genes with low expression levels, while Kallisto-Sleuth may only be useful to evaluate genes with medium to high abundance. When the same thresholds for fold change and p value are chosen in DE analysis, StringTie-Ballgown produce the least number of DEGs, while HTseq-DESeq2, -edgeR or -limma generally produces more DEGs. The performance of Cufflinks-Cuffdiff and Kallisto-Sleuth varies in different datasets. For DEGs with medium expression levels, the biological verification rates were similar among all procedures. CONCLUSION: Results are highly correlated among RNA-seq analysis procedures using HTseq for quantification. Difference in gene expression values mainly come from genes with particularly high or low expression levels. Moreover, biological validation rates of DEGs from all six procedures were similar for genes with medium expression levels. Investigators can choose analytical procedures according to their available computer resources, or whether genes of high or low expression levels are of interest. If computer resources are abundant, one can utilize multiple procedures to obtain the intersection of results to get the most reliable DEGs, or to obtain a combination of results to get a more comprehensive DE profile for transcriptomes.

BACKGROUND: As human activity alters the planet, there is a pressing need to understand how organisms adapt to environmental change. Of growing interest in this area is the role of epigenetic modifications, such as DNA methylation, in tailoring gene expression to fit novel conditions. Here, we reanalyzed nine invertebrate (Anthozoa and Hexapoda) datasets to validate a key prediction of this hypothesis: changes in DNA methylation in response to some condition correlate with changes in gene expression. RESULTS: In accord with previous observations, baseline levels of gene body methylation (GBM) positively correlated with transcription, and negatively correlated with transcriptional variation between conditions. Correlations between changes in GBM and transcription, however, were negligible. There was also no consistent negative correlation between methylation and transcription at the level of gene body methylation class (either highly- or lowly-methylated), anticipated under the previously described "seesaw hypothesis". CONCLUSION: Our results do not support the direct involvement of GBM in regulating dynamic transcriptional responses in invertebrates. If changes in DNA methylation regulate invertebrate transcription, the mechanism must involve additional factors or regulatory influences.

Abscisic acid (ABA) is a phytohormone that plays important roles in the regulation of plant growth, seed germination, and stress responses. The pyrabactin resistance 1-like (PYR/PYL) protein, an ABA receptor, was involved in the initial step in ABA signal transduction. However, the evolutionary history and characteristics of PYL genes expression remain unclear in Chinese white pear (Pyrus bretschneideri) or other Rosaceae species. In this study, 67 PYL genes were identified in eight Rosaceae species, and have been classified into three subgroups based on specific motifs and phylogenetic analysis. Intriguingly, we observed that whole-genome duplication (WGD) and dispersed duplication (DSD) have a major contribution to PYL family expansion. Purifying selection was the major force in PYL genes evolution. Expression analysis finds that PYL genes may function in multiple pear tissues. qRT-PCR validation of 11 PbrPYL genes indicates their roles in seed germination and abiotic stress responses. Our study provides a basis for further elucidation of the function of PYL genes and analysis of their expansion, evolution and expression patterns, which helps to understand the molecular mechanism of pear response to seed germination and seedling abiotic stress.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) is an emerging health challenge worldwide and outbreaks caused by this pathogen poses a serious public health concern. Shiga toxin (Stx) is the major virulence factor of EHEC, and the stx genes are carried by temperate bacteriophages (Stx phages). The switch between lysogenic and lytic life cycle of the phage, which is crucial for Stx production and for severity of the disease, is regulated by the CI repressor which maintain latency by preventing transcription of the replication proteins. Three EHEC phage replication units (Eru1-3) in addition to the classical lambdoid replication region have been described previously, and Stx phages carrying the Eru1 replication region were associated with highly virulent EHEC strains. RESULTS: In this study, we have classified the Eru replication region of 419 Stx phages. In addition to the lambdoid replication region and three already described Erus, ten novel Erus (Eru4 to Eru13) were detected. The lambdoid type, Eru1, Eru4 and Eru7 are widely distributed in Western Europe. Notably, EHEC strains involved in severe outbreaks in England and Norway carry Stx phages with Eru1, Eru2, Eru5 and Eru7 replication regions. Phylogenetic analysis of CI repressors from Stx phages revealed eight major clades that largely separate according to Eru type. CONCLUSION: The classification of replication regions and CI proteins of Stx phages provides an important platform for further studies aimed to assess how characteristics of the replication region influence the regulation of phage life cycle and, consequently, the virulence potential of the host EHEC strain.

BACKGROUND: The cryptochromes (CRY) are specific blue light receptors of plants and animals, which play crucial roles in physiological processes of plant growth, development, and stress tolerance. RESULTS: In the present work, a systematic analysis of the CRY gene family was performed on twelve cotton species, resulting in 18, 17, 17, 17, and 17 CRYs identified in five alloteraploid cottons (Gossypium hirsutum, G. barbadense, G. tomentosum, G. mustelinum and G. darwinii), respectively, and five to nine CRY genes in the seven diploid species. Phylogenetic analysis of protein-coding sequences revealed that CRY genes from cottons and Arabidopsis thaliana could be classified into seven clades. Synteny analysis suggested that the homoeolog of G. hirsutum Gh_A02G0384 has undergone an evolutionary loss event in the other four allotetraploid cotton species. Cis-element analysis predicated the possible functions of CRY genes in G. hirsutum. RNA-seq data revealed that Gh_D09G2225, Gh_A09G2012 and Gh_A11G1040 had high expressions in fiber cells of different developmental states. In addition, the expression levels of one (Gh_A03G0120), 15 and nine GhCRY genes were down-regulated following the PEG, NaCl and high-temperature treatments, respectively. For the low-temperature treatment, five GhCRY genes were induced, and five were repressed. These results indicated that most GhCRY genes negatively regulate the abiotic stress treatments. CONCLUSION: We report the structures, domains, divergence, synteny, and cis-elements analyses systematically of G. hirsutum CRY genes. Possible biological functions of GhCRY genes in differential tissues as well as in response to abiotic stress during the cotton plant life cycle were predicted.

BACKGROUND: BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains. RESULTS: In this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1-3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their 'accessory regions' can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains. CONCLUSION: This study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains.

BACKGROUND: Genetic variants have been found to influence red blood cell (RBC) susceptibility to hemolytic stress and affect transfusion outcomes and the severity of blood diseases. Males have a higher susceptibility to hemolysis than females, but little is known about the genetic mechanism contributing to the difference. RESULTS: To investigate the sex differences in RBC susceptibility to hemolysis, we conducted a sex-stratified genome-wide association study and a genome-wide gene-by-sex interaction scan in a multi-ethnic dataset with 12,231 blood donors who have in vitro osmotic hemolysis measurements during routine blood storage. The estimated SNP-based heritability for osmotic hemolysis was found to be significantly higher in males than in females (0.46 vs. 0.41). We identified SNPs associated with sex-specific susceptibility to osmotic hemolysis in five loci (SPTA1, KCNA6, SLC4A1, SUMO1P1, and PAX8) that impact RBC function and hemolysis. CONCLUSION: Our study established a best practice to identify sex-specific genetic modifiers for sexually dimorphic traits in datasets with mixed ancestries, providing evidence of different genetic regulations of RBC susceptibility to hemolysis between sexes. These and other variants may help explain observed sex differences in the severity of hemolytic diseases, such as sickle cell and malaria, as well as the viability of red cell storage and recovery.

BACKGROUND: The tall wheatgrass species Thinopyrum elongatum carries a strong fusarium head blight (FHB) resistance locus located on the long arm of chromosome 7 (7EL) as well as resistance to leaf and stem rusts, all diseases with a significant impact on wheat production. Towards understanding the contribution of Th. elongatum 7EL to improvement of disease resistance in wheat, the genomic sequence of the 7EL fragment present in the wheat Chinese Spring (CS) telosomic addition line CS-7EL was determined and the contribution and impact of 7EL on the rachis transcriptome during FHB infection was compared between CS and CS-7EL. RESULTS: We assembled the Th. elongatum 7EL chromosome arm using a reference-guided approach. Combining this assembly with the available reference sequence for CS hexaploid wheat provided a reliable reference for interrogating the transcriptomic differences in response to infection conferred by the 7EL fragment. Comparison of the transcriptomes of rachis tissues from CS and CS-7EL showed expression of Th. elongatum transcripts as well as modulation of wheat transcript expression profiles in the CS-7EL line. Expression profiles at 4 days after infection with Fusarium graminearum, the causal agent of FHB, showed an increased in expression of genes associated with an effective defense response, in particular glucan endo-1,3-beta-glucosidases and chitinases, in the FHB-resistant line CS-7EL while there was a larger increase in differential expression for genes associated with the level of fungal infection in the FHB-susceptible line CS. One hundred and seven 7EL transcripts were expressed in the smallest 7EL region defined to carry FHB resistance. CONCLUSION: 7EL contributed to CS-7EL transcriptome by direct expression and through alteration of wheat transcript profiles. FHB resistance in CS-7EL was associated with transcriptome changes suggesting a more effective defense response. A list of candidate genes for the FHB resistance locus on 7EL has been established.

The soil-dwelling nematode Caenorhabditis elegans serves as a model system to study innate immunity against microbial pathogens. C. elegans have been collected from around the world, where they, presumably, adapted to regional microbial ecologies. Here we use survival assays and RNA-sequencing to better understand how two isolates from disparate climates respond to pathogenic bacteria. We found that, relative to N2 (originally isolated in Bristol, UK), CB4856 (isolated in Hawaii), was more susceptible to the Gram-positive microbe, Staphylococcus epidermidis, but equally susceptible to Staphylococcus aureus as well as two Gram-negative microbes, Providencia rettgeri and Pseudomonas aeruginosa. We performed transcriptome analysis of infected worms and found gene-expression profiles were considerably different in an isolate-specific and microbe-specific manner. We performed GO term analysis to categorize differential gene expression in response to S. epidermidis. In N2, genes that encoded detoxification enzymes and extracellular matrix proteins were significantly enriched, while in CB4856, genes that encoded detoxification enzymes, C-type lectins, and lipid metabolism proteins were enriched, suggesting they have different responses to S. epidermidis, despite being the same species. Overall, discerning gene expression signatures in an isolate by pathogen manner can help us to understand the different possibilities for the evolution of immune responses within organisms.

BACKGROUND: The ability of animals and their microbiomes to adapt to starvation and then restore homeostasis after refeeding is fundamental to their continued survival and symbiosis. The intestine is the primary site of nutrient absorption and microbiome interaction, however our understanding of intestinal adaptations to starvation and refeeding remains limited. Here we used RNA sequencing and 16S rRNA gene sequencing to uncover changes in the intestinal transcriptome and microbiome of zebrafish subjected to long-term starvation and refeeding compared to continuously fed controls. RESULTS: Starvation over 21 days led to increased diversity and altered composition in the intestinal microbiome compared to fed controls, including relative increases in Vibrio and reductions in Plesiomonas bacteria. Starvation also led to significant alterations in host gene expression in the intestine, with distinct pathways affected at early and late stages of starvation. This included increases in the expression of ribosome biogenesis genes early in starvation, followed by decreased expression of genes involved in antiviral immunity and lipid transport at later stages. These effects of starvation on the host transcriptome and microbiome were almost completely restored within 3 days after refeeding. Comparison with published datasets identified host genes responsive to starvation as well as high-fat feeding or microbiome colonization, and predicted host transcription factors that may be involved in starvation response. CONCLUSIONS: Long-term starvation induces progressive changes in microbiome composition and host gene expression in the zebrafish intestine, and these changes are rapidly reversed after refeeding. Our identification of bacterial taxa, host genes and host pathways involved in this response provides a framework for future investigation of the physiological and ecological mechanisms underlying intestinal adaptations to food restriction.

Erratum in BMC Genomics. 2022 May 24;23(1):395.

BACKGROUND: Musaceae is an economically important family consisting of 70-80 species. Elucidation of the interspecific relationships of this family is essential for a more efficient conservation and utilization of genetic resources for banana improvement. However, the scarcity of herbarium specimens and quality molecular markers have limited our understanding of the phylogenetic relationships in wild species of Musaceae. Aiming at improving the phylogenetic resolution of Musaceae, we analyzed a comprehensive set of 49 plastomes for 48 species/subspecies representing all three genera of this family. RESULTS: Musaceae plastomes have a relatively well-conserved genomic size and gene content, with a full length ranging from 166,782 bp to 172,514 bp. Variations in the IR borders were found to show phylogenetic signals to a certain extent in Musa. Codon usage bias analysis showed different preferences for the same codon between species and three genera and a common preference for A/T-ending codons. Among the two genes detected under positive selection (dN/dS > 1), ycf2 was indicated under an intensive positive selection. The divergent hotspot analysis allowed the identification of four regions (ndhF-trnL, ndhF, matK-rps16, and accD) as specific DNA barcodes for Musaceae species. Bayesian and maximum likelihood phylogenetic analyses using full plastome resulted in nearly identical tree topologies with highly supported relationships between species. The monospecies genus Musella is sister to Ensete, and the genus Musa was divided into two large clades, which corresponded well to the basic number of n = x = 11 and n = x =10/9/7, respectively. Four subclades were divided within the genus Musa. A dating analysis covering the whole Zingiberales indicated that the divergence of Musaceae family originated in the Palaeocene (59.19 Ma), and the genus Musa diverged into two clades in the Eocene (50.70 Ma) and then started to diversify from the late Oligocene (29.92 Ma) to the late Miocene. Two lineages (Rhodochlamys and Australimusa) radiated recently in the Pliocene /Pleistocene periods. CONCLUSIONS: The plastome sequences performed well in resolving the phylogenetic relationships of Musaceae and generated new insights into its evolution. Plastome sequences provided valuable resources for population genetics and phylogenetics at lower taxon.

BACKGROUND: Soybean mosaic virus (SMV) is one of the most devastating pathogens of soybean. MicroRNAs (miRNAs) are a class of non-coding RNAs (21-24 nucleotides) which are endogenously produced by the plant host as part of a general gene expression regulatory mechanisms, but also play roles in regulating plant defense against pathogens. However, miRNA-mediated plant response to SMV in soybean is not as well documented. RESULT: In this study, we analyzed 18 miRNA libraries, including three biological replicates from two soybean lines (Resistant and susceptible lines to SMV strain SC3 selected from the near-isogenic lines of Qihuang No. 1 × Nannong1138-2) after virus infection at three different time intervals (0 dpi, 7 dpi and 14 dpi). A total of 1,092 miRNAs, including 608 known miRNAs and 484 novel miRNAs were detected. Differential expression analyses identified the miRNAs profile changes during soybean-SMV interaction. Then, miRNAs potential target genes were predicted via data mining, and functional annotation was done by Gene Ontology (GO) analysis. The expression patterns of several miRNAs were validated by quantitative real-time PCR. We also validated the miRNA-target gene interaction by agrobacterium-mediated transient expression in Nicotiana benthamiana. CONCLUSION: We have identified a large number of miRNAs and their target genes and also functional annotations. We found that multiple miRNAs were differentially expressed in the two lines and targeted a series of NBS-LRR resistance genes. It is worth mentioning that many of these genes exist in the previous fine-mapping interval of the resistance gene locus. Our study provides additional information on soybean miRNAs and an insight into the role of miRNAs during SMV-infection in soybean.

BACKGROUND: Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited. RESULTS: Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. CONCLUSIONS: Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.

BACKGROUND: Melanin is an important antioxidant in food and has been used in medicine and cosmetology. Chicken meat with high melanin content from black-boned chickens have been considered a high nutritious food with potential medicinal properties. The molecular mechanism of melanogenesis of skeletal muscle in black-boned chickens remain poorly understood. This study investigated the biological gene-metabolite associations regulating the muscle melanogenesis pathways in Wuliangshan black-boned chickens with two normal boned chicken breeds as control. RESULTS: We identified 25 differentially expressed genes and 11 transcription factors in the melanogenesis pathways. High levels of the meat flavor compounds inosine monophosphate, hypoxanthine, lysophospholipid, hydroxyoctadecadienoic acid, and nicotinamide mononucleotide were found in Wuliangshan black-boned chickens. CONCLUSION: Integrative analysis of transcriptomics and metabolomics revealed the dual physiological functions of the PDZK1 gene, involved in pigmentation and/or melanogenesis and regulating the phospholipid signaling processes in muscle of black boned chickens.

BACKGROUND: Jasmonate-ZIM domain (JAZ) repressors negatively regulate signal transduction of jasmonates, which regulate plant development and immunity. However, no comprehensive analysis of the JAZ gene family members has been done in the common fig (Ficus carica L.) during fruit development and hormonal treatment. RESULTS: In this study, 10 non-redundant fig JAZ family genes (FcJAZs) distributed on 7 chromosomes were identified in the fig genome. Phylogenetic and structural analysis showed that FcJAZ genes can be grouped into 5 classes. All the classes contained relatively complete TIFY and Jas domains. Yeast two hybrid (Y2H) results showed that all FcJAZs proteins may interact with the identified transcription factor, FcMYC2. Tissue-specific expression analysis showed that FcJAZs were highly expressed in the female flowers and roots. Expression patterns of FcJAZs during the fruit development were analyzed by RNA-Seq and qRT-PCR. The findings showed that, most FcJAZs were significantly downregulated from stage 3 to 5 in the female flower, whereas downregulation of these genes was observed in the fruit peel from stage 4 to 5. Weighted-gene co-expression network analysis (WGCNA) showed the expression pattern of FcJAZs was correlated with hormone signal transduction and plant-pathogen interaction. Putative cis-elements analysis of FcJAZs and expression patterns of FcJAZs which respond to hormone treatments revealed that FcJAZs may regulate fig fruit development by modulating the effect of ethylene or gibberellin. CONCLUSIONS: This study provides a comprehensive analysis of the FcJAZ family members and provides information on FcJAZs contributions and their role in regulating the common fig fruit development.

BACKGROUND: Flavonoid glycosides extracted from roots of Scutellaria baicalensis exhibit strong pharmaceutical antitumor, antioxidative, anti-inflammatory, and antiviral activities. UDP glycosyltransferase (UGT) family members are responsible for the transfer of a glycosyl moiety from UDP sugars to a wide range of acceptor flavonoids. Baicalin is the major flavonoid glycoside found in S. baicalensis roots, and its aglycone baicalein is synthesized from a specially evolved pathway that has been elucidated. However, it is necessary to carry out a genome-wide study of genes involved in 7-O-glucuronidation, the final biosynthesis step of baicalin, which might elucidate the relationship between the enzymes and the metabolic accumulation patterns in this medicinal plant. RESULTS: We reported the phylogenetic analysis, tissue-specific expression, biochemical characterization and evolutionary analysis of glucosyltransferases (SbUGTs) and glucuronosyltransferases (SbUGATs) genes based on the recently released genome of S. baicalensis. A total of 124 UGTs were identified, and over one third of them were highly expressed in roots. In vitro enzyme assays showed that 6 SbUGTs could use UDP-glucose as a sugar donor and convert baicalein to oroxin A (baicalein 7-O-glucoside), while 4 SbUGATs used only UDP-glucuronic acid as the sugar donor and catalyzed baicalein to baicalin. SbUGAT4 and SbUGT2 are the most highly expressed SbUGAT and SbUGT genes in root tissues, respectively. Kinetic measurements revealed that SbUGAT4 had a lower Km value and higher Vmax/Km ratio to baicalein than those of SbUGT2. Furthermore, tandem duplication events were detected in SbUGTs and SbUGATs. CONCLUSIONS: This study demonstrated that glucosylation and glucuronidation are two major glycosylated decorations in the roots of S. baicalensis. Higher expression level and affinity to substrate of SbUGAT4, and expansion of this gene family contribute high accumulation of baicalin in the root of S. baicalensis.

BACKGROUND: Mastitis is a very common disease in the dairy industry that producers encounter daily. Transcriptomics, using RNA-Sequencing (RNA - Seq) technology, can be used to study the functional aspect of mastitis resistance to identify animals that have a better immune response to mastitis. When the cow has mastitis, not only genes but also specific mRNA isoforms generated via alternative splicing (AS) could be differentially expressed (DE), leading to the phenotypic variation observed. Therefore, the objective of this study was to use large gap read mapping to identify mRNA isoforms DE between healthy and mastitic milk somatic cell samples (N = 12). These mRNA isoforms were then categorized based on being 1) annotated mRNA isoforms for gene name and length, 2) annotated mRNA isoforms with different transcript length and 3) novel mRNA isoforms of non - annotated genes. RESULTS: Analysis identified 333 DE transcripts (with at least 2 mRNA isoforms annotated, with at least one being DE) between healthy and mastitic samples corresponding to 303 unique genes. Of these 333 DE transcripts between healthy and mastitic samples, 68 mRNA isoforms are annotated in the bovine genome reference (ARS.UCD.1.2), 249 mRNA isoforms had novel transcript lengths of known genes and 16 were novel transcript lengths of non - annotated genes in the bovine genome reference (ARS.UCD.1.2). Functional analysis including gene ontology, gene network and metabolic pathway analysis was performed on the list of 288 annotated and unique DE mRNA isoforms. In total, 67 significant metabolic pathways were identified including positive regulation of cytokine secretion and immune response. Additionally, numerous DE novel mRNA isoforms showed potential involvement with the immune system or mastitis. Lastly, QTL annotation analysis was performed on coding regions of the DE mRNA isoforms, identifying overlapping QTLs associated with clinical mastitis and somatic cell score. CONCLUSION: This study identified novel mRNA isoforms generated via AS that could lead to differences in the immune response of Holstein dairy cows and be potentially implemented in future breeding programs.

BACKGROUND: Salmonella spp. is a major foodborne pathogen with a wide variety of serovars associated with human cases and food sources. Nevertheless, in Europe a panel of ten serovars is responsible for up to 80% of confirmed human cases. Clustering studies by single nucleotide polymorphism (SNP) core-genome phylogenetic analysis of outbreaks due to these major serovars are simplified by the availability of many complete genomes in the free access databases. This is not the case for outbreaks due to less common serovars, such as Welikade, for which no reference genomes are available. In this study, we propose a method to solve this problem. We propose to perform a core genome MLST (cgMLST) analysis based on hierarchical clustering using the free-access EnteroBase to select the most suitable genome to use as a reference for SNP phylogenetic analysis. In this study, we applied this protocol to a retrospective analysis of a Salmonella enterica serovar Welikade (S. Welikade) foodborne outbreak that occurred in France in 2016. Finally, we compared the cgMLST and SNP analyses. SNP phylogenetic reconstruction was carried out considering the effect of recombination events identified by the ClonalFrameML tool. The accessory genome was also explored by phage content and virulome analyses. RESULTS: Our findings revealed high clustering concordance using cgMLST and SNP analyses. Nevertheless, SNP analysis allowed for better assessment of the genetic distance among strains. The results revealed epidemic clones of S. Welikade circulating within the poultry and dairy sectors in France, responsible for sporadic and non-sporadic human cases between 2012 and 2019. CONCLUSIONS: This study increases knowledge on this poorly described serovar and enriches public genome databases with 42 genomes from human and non-human S. Welikade strains, including the isolate collected in 1956 in Sri Lanka, which gave the name to this serovar. This is the first genomic analysis of an outbreak due to S. Welikade described to date.

BACKGROUND: Colocasia gigantea, Caladium bicolor and Xanthosoma sagittifolium are three worldwide famous ornamental and/or vegetable plants in the Araceae family, these species in the subfamily Aroideae are phylogenetically perplexing due to shared interspecific morphological traits and variation. RESULT: This study, for the first time ever, assembled and analyzed complete chloroplast genomes of C. gigantea, C. bicolor and X. sagittifolium with genome sizes of 165,906 bp, 153,149 bp and 165,169 bp in length, respectively. The genomes were composed of conserved quadripartite circular structures with a total of 131 annotated genes, including 8 rRNA, 37 tRNA and 86 protein-coding genes. A comparison within Aroideae showed seven protein-coding genes (accD, ndhF, ndhK, rbcL, rpoC1, rpoC2 and matK) linked to environmental adaptation. Phylogenetic analysis confirmed a close relationship of C. gigantea with C. esculenta and S. colocasiifolia, and the C. bicolor with X. sagittifolium. Furthermore, three DNA barcodes (atpH-atpI + psaC-ndhE, atpH-atpI + trnS-trnG, atpH-atpI + psaC-ndhE + trnS-trnG) harbored highly variable regions to distinguish species in Aroideae subfamily. CONCLUSION: These results would be beneficial for species identification, phylogenetic relationship, genetic diversity, and potential of germplasm resources in Aroideae.

BACKGROUND: Adipose tissue is an important endocrine and energy-storage organ in organisms, and it plays a crucial role in the energy-metabolism balance. Previous studies have found that sex-linked dwarf (SLD) chickens generally have excessively high abdominal fat deposition during the growing period, which increases feeding costs. However, the underlying mechanism of this fat deposition during the growth of SLD chickens remains unknown. RESULTS: The Oil Red O staining showed that the lipid-droplet area of SLD chickens was larger than that of normal chickens in E15 and 14d. Consistently, TG content in the livers of SLD chickens was higher than that of normal chickens in E15 and 14d. Further, lower ΔΨm and lower ATP levels and higher MDA levels were observed in SLD chickens than normal chickens in both E15 and 14d. We also found that overexpression of GHR reduced the expression of genes related to lipid metabolism (AMPK, PGC1α, PPARγ, FAS, C/EBP) and oxidative phosphorylation (CYTB, CYTC, COX1, ATP), as well as reducing ΔΨm and ATP levels and increasing MDA levels. In addition, overexpression of GHR inhibited fat deposition in CPPAs, as measured by Oil Red O staining. On the contrary, knockdown of GHR had the opposite effects in vitro. CONCLUSIONS: In summary, we demonstrate that GHR promotes mitochondrial function and inhibits lipid peroxidation as well as fat deposition in vivo and in vitro. Therefore, GHR is essential for maintaining the stability of lipid metabolism and regulating mitochondrial function in chicken.

Author information: (1)Population Health Research Institute, David Braley Cardiac, Vascular and Stroke Research Institute, 237 Barton Street East, Hamilton, ON, Canada. (2)Department of Medicine, McMaster University, 1280 Main Street West, L8S 4K1, Hamilton, ON, Canada. (3)Department of Pediatrics, University of Alberta, Edmonton, AB, Canada. (4)Department of Pediatrics and Child Health, Children's Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, MB, Canada. (5)Department of Pediatrics, Hospital for Sick Children, ON, Toronto, Canada. (6)Department of Pediatrics, BC Children's Hospital, University of British Columbia, Vancouver, BC, Canada. (7)Thrombosis and Atherosclerosis Research Institute, David Braley Cardiac, Vascular and Stroke Research Institute, 237 Barton Street East, Hamilton, ON, Canada. (8)Department of Pathology and Molecular Medicine, Michael G. DeGroote School of Medicine, McMaster University, 1280 Main Street West, Hamilton, ON, Canada. (9)Department of Clinical Epidemiology & Biostatistics, McMaster University, 1280 Main Street West, Hamilton, ON, Canada. (10)Department of Health Research Methods, Evidence, and Impact, McMaster University, Hamilton, Ontario, Canada. (11)Population Health Research Institute, David Braley Cardiac, Vascular and Stroke Research Institute, 237 Barton Street East, Hamilton, ON, Canada. anands@mcmaster.ca. (12)Department of Medicine, McMaster University, 1280 Main Street West, L8S 4K1, Hamilton, ON, Canada. anands@mcmaster.ca. (13)Department of Health Research Methods, Evidence, and Impact, McMaster University, Hamilton, Ontario, Canada. anands@mcmaster.ca. (#)Contributed equally

BACKGROUND: MYBs are among the largest transcription factor families in plants. Consequently, members of this family are involved in a plethora of processes including development and specialized metabolism. The MYB families of many plant species were investigated in the last two decades since the first investigation looked at Arabidopsis thaliana. This body of knowledge and characterized sequences provide the basis for the identification, classification, and functional annotation of candidate sequences in new genome and transcriptome assemblies. RESULTS: A pipeline for the automatic identification and functional annotation of MYBs in a given sequence data set was implemented in Python. MYB candidates are identified, screened for the presence of a MYB domain and other motifs, and finally placed in a phylogenetic context with well characterized sequences. In addition to technical benchmarking based on existing annotation, the transcriptome assembly of Croton tiglium and the annotated genome sequence of Castanea crenata were screened for MYBs. Results of both analyses are presented in this study to illustrate the potential of this application. The analysis of one species takes only a few minutes depending on the number of predicted sequences and the size of the MYB gene family. This pipeline, the required bait sequences, and reference sequences for a classification are freely available on github: https://github.com/bpucker/MYB_annotator . CONCLUSIONS: This automatic annotation of the MYB gene family in novel assemblies makes genome-wide investigations consistent and paves the way for comparative studies in the future. Candidate genes for in-depth analyses are presented based on their orthology to previously characterized sequences which allows the functional annotation of the newly identified MYBs with high confidence. The identification of orthologs can also be harnessed to detect duplication and deletion events.

BACKGROUND: In eukaryote transcriptomes, a significant amount of transcript diversity comes from genes' capacity to generate different transcripts through alternative splicing. Identifying orthologous alternative transcripts across multiple species is of particular interest for genome annotators. However, there is no formal definition of transcript orthology based on the splicing structure conservation. Likewise there is no public dataset benchmark providing groups of orthologous transcripts sharing a conserved splicing structure. RESULTS: We introduced a formal definition of splicing structure orthology and we predicted transcript orthologs in human, mouse and dog. Applying a selective strategy, we analyzed 2,167 genes and their 18,109 known transcripts and identified a set of 253 gene orthologs that shared a conserved splicing structure in all three species. We predicted 6,861 transcript CDSs (coding sequence), mainly for dog, an emergent model species. Each predicted transcript was an ortholog of a known transcript: both share the same CDS splicing structure. Evidence for the existence of the predicted CDSs was found in external data. CONCLUSIONS: We generated a dataset of 253 gene triplets, structurally conserved and sharing all their CDSs in human, mouse and dog, which correspond to 879 triplets of spliced CDS orthologs. We have released the dataset both as an SQL database and as tabulated files. The data consists of the 879 CDS orthology groups with their detailed splicing structures, and the predicted CDSs, associated with their experimental evidence. The 6,861 predicted CDSs are provided in GTF files. Our data may contribute to compare highly conserved genes across three species, for comparative transcriptomics at the isoform level, or for benchmarking splice aligners and methods focusing on the identification of splicing orthologs. The data is available at https://data-access.cesgo.org/index.php/s/V97GXxOS66NqTkZ .

BACKGROUND: Copy number variation (CNV) has been routinely studied using bulk-cell sequencing. However, CNV is not well studied on the single-cell level except for humans and a few model organisms. RESULTS: We sequenced 143 single sperms of two Holstein bulls, from which we predicted CNV events using 14 single sperms with deep sequencing. We then compared the CNV results derived from single sperms with the bulk-cell sequencing of one bull's family trio of diploid genomes. As a known CNV hotspot, segmental duplications were also predicted using the bovine ARS-UCD1.2 genome. Although the trio CNVs validated only some single sperm CNVs, they still showed a distal chromosomal distribution pattern and significant associations with segmental duplications and satellite repeats. CONCLUSION: Our preliminary results pointed out future research directions and highlighted the importance of uniform whole genome amplification, deep sequence coverage, and dedicated software pipelines for CNV detection using single cell sequencing data.

BACKGROUND: The "Assay for Transposase Accessible Chromatin sequencing" (ATAC-seq) is an efficient and easy to implement protocol to measure chromatin accessibility that has been widely used in multiple applications studying gene regulation. While several modifications or variants of the protocol have been published since it was first described, there has not yet been an extensive evaluation of the effects of specific protocol choices head-to-head in a consistent experimental setting. In this study, we tested multiple protocol options for major ATAC-seq components (including three reaction buffers, two reaction temperatures, two enzyme sources, and the use of either native or fixed nuclei) in a well-characterized cell line. With all possible combinations of components, we created 24 experimental conditions with four replicates for each (a total of 96 samples). In addition, we tested the 12 native conditions in a primary sample type (mouse lung tissue) with two different input amounts. Through these extensive comparisons, we were able to observe the effect of different ATAC-seq conditions on data quality and to examine the utility and potential redundancy of various quality metrics. RESULTS: In general, native samples yielded more peaks (particularly at loci not overlapping transcription start sites) than fixed samples, and the temperature at which the enzymatic reaction was carried out had a major impact on data quality metrics for both fixed and native nuclei. However, the effect of various conditions tested was not always consistent between the native and fixed samples. For example, the Nextera and Omni buffers were largely interchangeable across all other conditions, while the THS buffer resulted in markedly different profiles in native samples. In-house and commercial enzymes performed similarly. CONCLUSIONS: We found that the relationship between commonly used measures of library quality differed across temperature and fixation, and so evaluating multiple metrics in assessing the quality of a sample is recommended. Notably, we also found that these choices can bias the functional class of elements profiled and so we recommend evaluating several formulations in any new experiments. Finally, we hope the ATAC-seq workflow formulated in this study on crosslinked samples will help to profile archival clinical specimens.

BACKGROUND: Streptococcus thermophilus is a Gram-positive bacterium widely used as starter in the dairy industry as well as in many traditional fermented products. In addition to its technological importance, it has also gained interest in recent years as beneficial bacterium due to human health-promoting functionalities. The objective of this study was to inventory the main health-promoting properties of S. thermophilus and to study their intra-species diversity at the genomic and genetic level within a collection of representative strains. RESULTS: In this study various health-related functions were analyzed at the genome level from 79 genome sequences of strains isolated over a long time period from diverse products and different geographic locations. While some functions are widely conserved among isolates (e.g., degradation of lactose, folate production) suggesting their central physiological and ecological role for the species, others including the tagatose-6-phosphate pathway involved in the catabolism of galactose, and the production of bioactive peptides and gamma-aminobutyric acid are strain-specific. Most of these strain-specific health-promoting properties seems to have been acquired via horizontal gene transfer events. The genetic basis for the phenotypic diversity between strains for some health related traits have also been investigated. For instance, substitutions in the galK promoter region correlate with the ability of some strains to catabolize galactose via the Leloir pathway. Finally, the low occurrence in S. thermophilus genomes of genes coding for biogenic amine production and antibiotic resistance is also a contributing factor to its safety status. CONCLUSIONS: The natural intra-species diversity of S. thermophilus, therefore, represents an interesting source for innovation in the field of fermented products enriched for healthy components that can be exploited to improve human health. A better knowledge of the health-promoting properties and their genomic and genetic diversity within the species may facilitate the selection and application of strains for specific biotechnological and human health-promoting purpose. Moreover, by pointing out that a substantial part of its functional potential still defies us, our work opens the way to uncover additional health-related functions through the intra-species diversity exploration of S. thermophilus by comparative genomics approaches.

BACKGROUND: Recent evidences have suggested that human microorganisms participate in important biological activities in the human body. The dysfunction of host-microbiota interactions could lead to complex human disorders. The knowledge on host-microbiota interactions can provide valuable insights into understanding the pathological mechanism of diseases. However, it is time-consuming and costly to identify the disorder-specific microbes from the biological "haystack" merely by routine wet-lab experiments. With the developments in next-generation sequencing and omics-based trials, it is imperative to develop computational prediction models for predicting microbe-disease associations on a large scale. RESULTS: Based on the known microbe-disease associations derived from the Human Microbe-Disease Association Database (HMDAD), the proposed model shows reliable performance with high values of the area under ROC curve (AUC) of 0.9456 and 0.8866 in leave-one-out cross validations and five-fold cross validations, respectively. In case studies of colorectal carcinoma, 80% out of the top-20 predicted microbes have been experimentally confirmed via published literatures. CONCLUSION: Based on the assumption that functionally similar microbes tend to share the similar interaction patterns with human diseases, we here propose a group based computational model of Bayesian disease-oriented ranking to prioritize the most potential microbes associating with various human diseases. Based on the sequence information of genes, two computational approaches (BLAST+ and MEGA 7) are leveraged to measure the microbe-microbe similarity from different perspectives. The disease-disease similarity is calculated by capturing the hierarchy information from the Medical Subject Headings (MeSH) data. The experimental results illustrate the accuracy and effectiveness of the proposed model. This work is expected to facilitate the characterization and identification of promising microbial biomarkers.

BACKGROUND: Respectively, prostate cancer (PCa) and breast cancer (BC) are the second most and most commonly diagnosed cancer in men and women, and they account for a majority of cancer-related deaths world-wide. Cancer cells typically exhibit much-facilitated growth that necessitates upregulated glycolysis and augmented amino acid metabolism, that of glutamine and aspartate in particular, which is tightly coupled with an increased flux of the tricarboxylic acid (TCA) cycle. Epidemiological studies have exploited metabolomics to explore the etiology and found potentially effective biomarkers for early detection or progression of prostate and breast cancers. However, large randomized controlled trials (RCTs) to establish causal associations between amino acid metabolism and prostate and breast cancers have not been reported. OBJECTIVE: Utilizing two-sample Mendelian randomization (MR), we aimed to estimate how genetically predicted glutamate and aspartate levels could impact upon prostate and breast cancers development. METHODS: Single nucleotide polymorphisms (SNPs) as instrumental variables (IVs), associated with the serum levels of glutamate and aspartate were extracted from the publicly available genome-wide association studies (GWASs), which were conducted to associate genetic variations with blood metabolite levels using comprehensive metabolite profiling in 1,960 adults; and the glutamate and aspartate we have chosen were two of 644 metabolites. The summary statistics for the largest and latest GWAS datasets for prostate cancer (61,106 controls and 79,148 cases) were from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) consortium, and datasets for breast cancer (113,789 controls and 133,384 cases) were from Breast Cancer Association Consortium (BCAC). The study was performed through two-sample MR method. RESULTS: Causal estimates were expressed as odds ratios (OR) and 95% confidence interval (CI) per standard deviation increment in serum level of aspartate or glutamate. Aspartate was positively associated with prostate cancer (Effect = 1.043; 95% confidence interval, 1.003 to 1.084; P = 0.034) and breast cancer (Effect = 1.033; 95% confidence interval, 1.004 to 1.063; P = 0.028); however, glutamate was neither associated with prostate cancer nor with breast cancer. The potential causal associations were robust to the sensitivity analysis. CONCLUSIONS: Our study found that the level of serum aspartate could serve as a risk factor that contributed to the development of prostate and breast cancers. Efforts on a detailed description of the underlying biochemical mechanisms would be extremely valuable in early assessment and/or diagnosis, and strategizing clinical intervention, of both cancers.

BACKGROUND: The diversified aquaporin (AQP) family that was derived from gene duplication and subsequent functional differentiation play critical roles in multiple physiological processes and in adaptation to the dynamic environments during the evolutionary process. Oysters are a group of bivalve fauna in Mollusca that were widely distributed around the world and show extraordinary adaptation to harsh environments. However, knowledge is lacking with the diversity and evolution of the AQP family in oysters, even in molluscs. RESULTS: Here, we performed a comprehensive analysis of the AQP family in three geographical isolated oyster species that are native to different environments. Genome distribution and phylogenetic analysis revealed that the expansion of the AQP family in oysters were attributed to tandem duplication. Synteny analysis indicated that large-scale inversions lead to the independent duplication or deletion of the AQPs after speciation. As a consequence, these independent duplication events contributed to the diversification of the AQP family in different oysters. Pore pattern analysis suggested that the duplicated AQPs in oysters were highly diversified in inner surface profiles, implying the subsequent functional differentiation. The comparison conducted based on the transcriptome data demonstrated that the functional differentiated AQP family members in oysters may play critical roles in maintaining the balance between the stationary homeostasis and dynamic environments. CONCLUSIONS: Our observation provides evidence for the correlation between the duplicated and functional differentiated AQP family and the adaptation to stationary life under dynamic environments in oysters. Additionally, it also broadens our knowledge of the evolution of AQP family in molluscs.

BACKGROUND: PRDM9 is a key regulator of meiotic recombination in most metazoans, responsible for reshuffling parental genomes. During meiosis, the PRDM9 protein recognizes and binds specific target motifs via its array of C2H2 zinc-fingers encoded by a rapidly evolving minisatellite. The gene coding for PRDM9 is the only speciation gene identified in vertebrates to date and shows high variation, particularly in the DNA-recognizing positions of the zinc-finger array, within and between species. Across all vertebrate genomes studied for PRDM9 evolution, only one genome lacks variability between repeat types - that of the North Pacific minke whale. This study aims to understand the evolution and diversity of Prdm9 in minke whales, which display the most unusual genome reference allele of Prdm9 so far discovered in mammals. RESULTS: Minke whales possess all the features characteristic of PRDM9-directed recombination, including complete KRAB, SSXRD and SET domains and a rapidly evolving array of C2H2-type-Zincfingers (ZnF) with evidence of rapid evolution, particularly at DNA-recognizing positions that evolve under positive diversifying selection. Seventeen novel PRDM9 variants were identified within the Antarctic minke whale species, plus a single distinct PRDM9 variant in Common minke whales - shared across North Atlantic and North Pacific minke whale subspecies boundaries. CONCLUSION: The PRDM9 ZnF array evolves rapidly, in minke whales, with at least one DNA-recognizing position under positive selection. Extensive PRDM9 diversity is observed, particularly in the Antarctic in minke whales. Common minke whales shared a specific Prdm9 allele across subspecies boundaries, suggesting incomplete speciation by the mechanisms associated with PRDM9 hybrid sterility.

BACKGROUND: A decline in the level of genetic diversity in livestock can result in reduced response to selection, greater incidence of genetic defects, and inbreeding depression. In this context, various metrics have been proposed to assess the level of genetic diversity in selected populations. Therefore, the main goals of this study were to: 1) investigate the population structure of 16 cattle populations from 15 different pure breeds or composite populations, which have been selected for different breeds goals; and, 2) identify and compare runs of homozygosity (ROH) and heterozygosity-enriched regions (HER) based on different single nucleotide polymorphism (SNP) panels and whole-genome sequence data (WGS), followed by functional genomic analyses. RESULTS: A total of 24,187 ROH were found across all cattle populations, with 55% classified in the 2-4 Mb size group. Fourteen homozygosity islands were found in five populations, where four ROH islands located on BTA1, BTA5, BTA16, and BTA19 overlapped between the Brahman (BRM) and Gyr (GIR) breeds. A functional analysis of the genes found in these islands revealed candidate genes known to play a role in the melanogenesis, prolactin signaling, and calcium signaling pathways. The correlations between inbreeding metrics ranged from 0.02 to 0.95, where the methods based on homozygous genotypes (FHOM), uniting of gametes (FUNI), and genotype additive variance (FGRM) showed strong correlations among them. All methods yielded low to moderate correlations with the inbreeding coefficients based on runs of homozygosity (FROH). For the HER, 3576 runs and 26 islands, distributed across all autosomal chromosomes, were found in regions containing genes mainly related to the immune system, indicating potential balancing selection. Although the analyses with WGS did not enable detection of the same island patterns, it unraveled novel regions not captured when using SNP panel data. CONCLUSIONS: The cattle populations that showed the largest amount of ROH and HER were Senepol (SEN) and Montana (MON), respectively. Overlapping ROH islands were identified between GIR and BRM breeds, indicating a possible historical connection between the populations. The distribution and pattern of ROH and HER are population specific, indicating that different breeds have experienced divergent selection processes or different genetic processes.

BACKGROUND: Frequent occurrence of extreme high temperature is a major threat to crop production. Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) have important biological functions in the regulation of the response to heat stress. However, the regulatory mechanism of lncRNAs involved in heat response requires further exploration and the regulatory network remains poorly understood in maize. RESULTS: In this research, high-throughput sequencing was adopted to systematically identify lncRNAs in maize inbred line CM1. In total, 53,249 lncRNAs (259 known lncRNAs and 52,990 novel lncRNAs) were detected, of which 993 lncRNAs showed significantly differential expression (DElncRNAs) under heat stress. By predicting the target genes, 953 common targets shared by cis- and trans-regulation of the DElncRNAs were identified, which exhibited differential expression between the control and the heat stress treatments. Functional annotation indicated that a number of important biological processes and pathways, including photosynthesis, metabolism, translation, stress response, hormone signal transduction, and spliceosome, were enriched for the common targets, suggesting that they play important roles in heat response. A lncRNA-mediated regulatory network was constructed to visualize the molecular response mechanism in response to heat stress, which represented the direct regulatory relationships of DElncRNAs, differentially expressed miRNAs, target genes, and functional annotations. CONCLUSIONS: This study lays a foundation for further elucidation of the regulatory mechanism for the response to heat stress in the maize inbred line CM1. The findings provide important information for identification of heat-responsive genes, which will be beneficial for the molecular breeding in the cultivation of heat-tolerant maize germplasm.

Erratum in BMC Genomics. 2022 May 16;23(1):370.

BACKGROUND: Ilex (Aquifoliaceae) are of great horticultural importance throughout the world for their foliage and decorative berries, yet a dearth of genetic information has hampered our understanding of phylogenetic relationships and evolutionary history. Here, we compare chloroplast genomes from across Ilex and estimate phylogenetic relationships. RESULTS: We sequenced the chloroplast genomes of seven Ilex species and compared them with 34 previously published Ilex plastomes. The length of the seven newly sequenced Ilex chloroplast genomes ranged from 157,182 bp to 158,009 bp, and contained a total of 118 genes, including 83 protein-coding, 31 rRNA, and four tRNA genes. GC content ranged from 37.6 to 37.69%. Comparative analysis showed shared genomic structures and gene rearrangements. Expansion and contraction of the inverted repeat regions at the LSC/IRa and IRa/SSC junctions were observed in 22 and 26 taxa, respectively; in contrast, the IRb boundary was largely invariant. A total of 2146 simple sequence repeats and 2843 large repeats were detected in the 41 Ilex plastomes. Additionally, six genes (psaC, rbcL, trnQ, trnR, trnT, and ycf1) and two intergenic spacer regions (ndhC-trnV and petN-psbM) were identified as hypervariable, and thus potentially useful for future phylogenetic studies and DNA barcoding. We recovered consistent phylogenetic relationships regardless of inference methodology or choice of loci. We recovered five distinct, major clades, which were inconsistent with traditional taxonomic systems. CONCLUSION: Our findings challenge traditional circumscriptions of the genus Ilex and provide new insights into the evolutionary history of this important clade. Furthermore, we detail hypervariable and repetitive regions that will be useful for future phylogenetic and population genetic studies.

BACKGROUND: Chilling injury of mung bean (Vigna radiata (L.)) during the blooming and podding stages is a major agricultural threat in Northeast China. Uniconazole (UNZ) can alleviate water deficit stress in soybean and waterlogging stress in mung bean. However, there has been no report on the effect of UNZ application on the growth and transcriptomic profile of mung bean under chilling stress. RESULTS: UNZ application before chilling stress at the R1 stage alleviated the decline in mung bean yield. UNZ delayed the decrease in leaf chlorophyll content under chilling stress at the R1 stage and accelerated the increase in leaf chlorophyll content during the recovery period. Eighteen separate RNA-Seq libraries were generated from RNA samples collected from leaves exposed to six different treatment schemes. The numbers of DEGs specific for UNZ treatment between D1 + S vs. D1 and D4 + S vs. D4 were 708 and 810, respectively. GO annotations showed that photosynthesis genes were obviously enriched among the genes affected by chilling stress and UNZ application. KEGG pathway enrichment analysis indicated that 4 pathways (cutin, suberin and wax biosynthesis; photosynthesis; porphyrin and chlorophyll metabolism; and ribosome) were downregulated, while plant-pathogen interaction was upregulated, by chilling stress. UNZ application effectively prevented the further downregulation of the gene expression of members of these 4 KEGG pathways under chilling stress. CONCLUSIONS: UNZ application effectively delayed the decrease in photosynthetic pigment content under chilling stress and accelerated the increase in photosynthetic pigment content during the recovery period, thus effectively limiting the decline in mung bean yield. UNZ application effectively prevented the further downregulation of the gene expression of members of 4 KEGG pathways under chilling stress and increased mung bean tolerance to chilling stress.

BACKGROUND: Rapid development of high-throughput omics technologies generates an increasing interest in algorithms for cutoff point identification. Existing cutoff methods and tools identify cutoff points based on an association of continuous variables with another variable, such as phenotype, disease state, or treatment group. These approaches are not applicable for descriptive studies in which continuous variables are reported without known association with any biologically meaningful variables. RESULTS: The most common shape of the ranked distribution of continuous variables in high-throughput descriptive studies corresponds to a biphasic curve, where the first phase includes a big number of variables with values slowly growing with rank and the second phase includes a smaller number of variables rapidly growing with rank. This study describes an easy algorithm to identify the boundary between these phases to be used as a cutoff point. DISCUSSION: The major assumption of that approach is that a small number of variables with high values dominate the biological system and determine its major processes and functions. This approach was tested on three different datasets: human genes and their expression values in the human cerebral cortex, mammalian genes and their values of sensitivity to chemical exposures, and human proteins and their expression values in the human heart. In every case, the described cutoff identification method produced shortlists of variables (genes, proteins) highly relevant for dominant functions/pathways of the analyzed biological systems. CONCLUSIONS: The described method for cutoff identification may be used to prioritize variables in descriptive omics studies for a focused functional analysis, in situations where other methods of dichotomization of data are inaccessible.

BACKGROUND: Apoptosis plays important roles in a variety of functions, including immunity and response to environmental stress. The Inhibitor of Apoptosis (IAP) gene family of apoptosis regulators is expanded in molluscs, including eastern, Crassostrea virginica, and Pacific, Crassostrea gigas, oysters. The functional importance of IAP expansion in apoptosis and immunity in oysters remains unknown. RESULTS: Phylogenetic analysis of IAP genes in 10 molluscs identified lineage specific gene expansion in bivalve species. Greater IAP gene family expansion was observed in C. virginica than C. gigas (69 vs. 40), resulting mainly from tandem duplications. Functional domain analysis of oyster IAP proteins revealed 3 novel Baculoviral IAP Repeat (BIR) domain types and 14 domain architecture types across gene clusters, 4 of which are not present in model organisms. Phylogenetic analysis of bivalve IAPs suggests a complex history of domain loss and gain. Most IAP genes in oysters (76% of C. virginica and 82% of C. gigas), representing all domain architecture types, were expressed in response to immune challenge (Ostreid Herpesvirus OsHV-1, bacterial probionts Phaeobacter inhibens and Bacillus pumilus, several Vibrio spp., pathogenic Aliiroseovarius crassostreae, and protozoan parasite Perkinsus marinus). Patterns of IAP and apoptosis-related differential gene expression differed between the two oyster species, where C. virginica, in general, differentially expressed a unique set of IAP genes in each challenge, while C. gigas differentially expressed an overlapping set of IAP genes across challenges. Apoptosis gene expression patterns clustered mainly by resistance/susceptibility of the oyster host to immune challenge. Weighted Gene Correlation Network Analysis (WGCNA) revealed unique combinations of transcripts for 1 to 12 IAP domain architecture types, including novel types, were significantly co-expressed in response to immune challenge with transcripts in apoptosis-related pathways. CONCLUSIONS: Unprecedented diversity characterized by novel BIR domains and protein domain architectures was observed in oyster IAPs. Complex patterns of gene expression of novel and conserved IAPs in response to a variety of ecologically-relevant immune challenges, combined with evidence of direct co-expression of IAP genes with apoptosis-related transcripts, suggests IAP expansion facilitates complex and nuanced regulation of apoptosis and other immune responses in oysters.

BACKGROUND: Sphaerophoria rueppellii, a European species of hoverfly, is a highly effective beneficial predator of hemipteran crop pests including aphids, thrips and coleopteran/lepidopteran larvae in integrated pest management (IPM) programmes. It is also a key pollinator of a wide variety of important agricultural crops. No genomic information is currently available for S. rueppellii. Without genomic information for such beneficial predator species, we are unable to perform comparative analyses of insecticide target-sites and genes encoding metabolic enzymes potentially responsible for insecticide resistance, between crop pests and their predators. These metabolic mechanisms include several gene families - cytochrome P450 monooxygenases (P450s), ATP binding cassette transporters (ABCs), glutathione-S-transferases (GSTs), UDP-glycosyltransferases (UGTs) and carboxyl/choline esterases (CCEs). METHODS AND FINDINGS: In this study, a high-quality near-chromosome level de novo genome assembly (as well as a mitochondrial genome assembly) for S. rueppellii has been generated using a hybrid approach with PacBio long-read and Illumina short-read data, followed by super scaffolding using Hi-C data. The final assembly achieved a scaffold N50 of 87Mb, a total genome size of 537.6Mb and a level of completeness of 96% using a set of 1,658 core insect genes present as full-length genes. The assembly was annotated with 14,249 protein-coding genes. Comparative analysis revealed gene expansions of CYP6Zx P450s, epsilon-class GSTs, dietary CCEs and multiple UGT families (UGT37/302/308/430/431). Conversely, ABCs, delta-class GSTs and non-CYP6Zx P450s showed limited expansion. Differences were seen in the distributions of resistance-associated gene families across subfamilies between S. rueppellii and some hemipteran crop pests. Additionally, S. rueppellii had larger numbers of detoxification genes than other pollinator species. CONCLUSION AND SIGNIFICANCE: This assembly is the first published genome for a predatory member of the Syrphidae family and will serve as a useful resource for further research into selectivity and potential tolerance of insecticides by beneficial predators. Furthermore, the expansion of some gene families often linked to insecticide resistance and selectivity may be an indicator of the capacity of this predator to detoxify IPM selective insecticides. These findings could be exploited by targeted insecticide screens and functional studies to increase effectiveness of IPM strategies, which aim to increase crop yields by sustainably and effectively controlling pests without impacting beneficial predator populations.

BACKGROUND: RAD51 proteins, which are conserved in all eukaryotes, repair DNA double-strand breaks. This is critical to homologous chromosome pairing and recombination enabling successful reproduction. Work in Arabidopsis suggests that RAD51 also plays a role in plant defense; the Arabidopsis rad51 mutant is more susceptible to Pseudomonas syringae. However, the defense functions of RAD51 and the proteins interacting with RAD51 have not been thoroughly investigated in maize. Uncovering ligands of RAD51 would help to understand meiotic recombination and possibly the role of RAD51 in defense. This study used phage display, a tool for discovery of protein-protein interactions, to search for proteins interacting with maize RAD51A1. RESULTS: Maize RAD51A1 was screened against a random phage library. Eleven short peptide sequences were recovered from 15 phages which bound ZmRAD51A1 in vitro; three sequences were found in multiple successfully binding phages. Nine of these phage interactions were verified in vitro through ELISA and/or dot blotting. BLAST searches did not reveal any maize proteins which contained the exact sequence of any of the selected phage peptides, although one of the selected phages had a strong alignment (E-value = 0.079) to a binding domain of maize BRCA2. Therefore, we designed 32 additional short peptides using amino acid sequences found in the predicted maize proteome. These peptides were not contained within phages. Of these synthesized peptides, 14 bound to ZmRAD51A1 in a dot blot experiment. These 14 sequences are found in known maize proteins including transcription factors putatively involved in defense. CONCLUSIONS: These results reveal several peptides which bind ZmRAD51A1 and support a potential role for ZmRAD51A1 in transcriptional regulation and plant defense. This study also demonstrates the applicability of phage display to basic science questions, such as the search for binding partners of a known protein, and raises the possibility of an iterated approach to test peptide sequences that closely but imperfectly align with the selected phages.

BACKGROUND: Shotgun sequencing of cultured microbial isolates/individual eukaryotes (whole-genome sequencing) and microbial communities (metagenomics) has become commonplace in biology. Very often, sequenced samples encompass organisms spanning multiple domains of life, necessitating increasingly elaborate software for accurate taxonomic classification of assembled sequences. RESULTS: While many software tools for taxonomic classification exist, SprayNPray offers a quick and user-friendly, semi-automated approach, allowing users to separate contigs by taxonomy (and other metrics) of interest. Easy installation, usage, and intuitive output, which is amenable to visual inspection and/or further computational parsing, will reduce barriers for biologists beginning to analyze genomes and metagenomes. This approach can be used for broad-level overviews, preliminary analyses, or as a supplement to other taxonomic classification or binning software. SprayNPray profiles contigs using multiple metrics, including closest homologs from a user-specified reference database, gene density, read coverage, GC content, tetranucleotide frequency, and codon-usage bias. CONCLUSIONS: The output from this software is designed to allow users to spot-check metagenome-assembled genomes, identify, and remove contigs from putative contaminants in isolate assemblies, identify bacteria in eukaryotic assemblies (and vice-versa), and identify possible horizontal gene transfer events.

BACKGROUND: Genomic prediction (GP) based on single nucleotide polymorphisms (SNP) has become a broadly used tool to increase the gain of selection in plant breeding. However, using predictors that are biologically closer to the phenotypes such as transcriptome and metabolome may increase the prediction ability in GP. The objectives of this study were to (i) assess the prediction ability for three yield-related phenotypic traits using different omic datasets as single predictors compared to a SNP array, where these omic datasets included different types of sequence variants (full-SV, deleterious-dSV, and tolerant-tSV), different types of transcriptome (expression presence/absence variation-ePAV, gene expression-GE, and transcript expression-TE) sampled from two tissues, leaf and seedling, and metabolites (M); (ii) investigate the improvement in prediction ability when combining multiple omic datasets information to predict phenotypic variation in barley breeding programs; (iii) explore the predictive performance when using SV, GE, and ePAV from simulated 3'end mRNA sequencing of different lengths as predictors. RESULTS: The prediction ability from genomic best linear unbiased prediction (GBLUP) for the three traits using dSV information was higher than when using tSV, all SV information, or the SNP array. Any predictors from the transcriptome (GE, TE, as well as ePAV) and metabolome provided higher prediction abilities compared to the SNP array and SV on average across the three traits. In addition, some (di)-similarity existed between different omic datasets, and therefore provided complementary biological perspectives to phenotypic variation. Optimal combining the information of dSV, TE, ePAV, as well as metabolites into GP models could improve the prediction ability over that of the single predictors alone. CONCLUSIONS: The use of integrated omic datasets in GP model is highly recommended. Furthermore, we evaluated a cost-effective approach generating 3'end mRNA sequencing with transcriptome data extracted from seedling without losing prediction ability in comparison to the full-length mRNA sequencing, paving the path for the use of such prediction methods in commercial breeding programs.

BACKGROUND: Expression quantitative trait loci (eQTLs) analyses have been widely used to identify genetic variants associated with gene expression levels to understand what molecular mechanisms underlie genetic traits. The resultant eQTLs might affect the expression of associated genes through transcriptional or post-transcriptional regulation. In this study, we attempt to distinguish these two types of regulation by identifying genetic variants associated with mRNA stability of genes (stQTLs). RESULTS: Here, we presented a computational framework that takes advantage of recently developed methods to infer the mRNA stability of genes based on RNA-seq data and performed association analysis to identify stQTLs. Using the Genotype-Tissue Expression (GTEx) lung RNA-Seq data, we identified a total of 142,801 stQTLs for 3942 genes and 186,132 eQTLs for 4751 genes from 15,122,700 genetic variants for 13,476 genes on the autosomes, respectively. Interestingly, our results indicated that stQTLs were enriched in the CDS and 3'UTR regions, while eQTLs are enriched in the CDS, 3'UTR, 5'UTR, and upstream regions. We also found that stQTLs are more likely than eQTLs to overlap with RNA binding protein (RBP) and microRNA (miRNA) binding sites. Our analyses demonstrate that simultaneous identification of stQTLs and eQTLs can provide more mechanistic insight on the association between genetic variants and gene expression levels.

BACKGROUND: Chemogenomic profiling is a powerful approach for understanding the genome-wide cellular response to small molecules. First developed in Saccharomyces cerevisiae, chemogenomic screens provide direct, unbiased identification of drug target candidates as well as genes required for drug resistance. While many laboratories have performed chemogenomic fitness assays, few have been assessed for reproducibility and accuracy. Here we analyze the two largest independent yeast chemogenomic datasets comprising over 35 million gene-drug interactions and more than 6000 unique chemogenomic profiles; the first from our own academic laboratory (HIPLAB) and the second from the Novartis Institute of Biomedical Research (NIBR). RESULTS: Despite substantial differences in experimental and analytical pipelines, the combined datasets revealed robust chemogenomic response signatures, characterized by gene signatures, enrichment for biological processes and mechanisms of drug action. We previously reported that the cellular response to small molecules is limited and can be described by a network of 45 chemogenomic signatures. In the present study, we show that the majority of these signatures (66%) are also found in the companion dataset, providing further support for their biological relevance as conserved systems-level, small molecule response systems. CONCLUSIONS: Our results demonstrate the robustness of chemogenomic fitness profiling in yeast, while offering guidelines for performing other high-dimensional comparisons including parallel CRISPR screens in mammalian cells.

BACKGROUND: One of the most promising strategies of Pine Pitch Canker (PPC) management is the use of reproductive plant material resistant to the disease. Understanding the complexity of plant transcriptome that underlies the defence to the causal agent Fusarium circinatum, would greatly facilitate the development of an accurate breeding program. Long non-coding RNAs (lncRNAs) are emerging as important transcriptional regulators under biotic stresses in plants. However, to date, characterization of lncRNAs in conifer trees has not been reported. In this study, transcriptomic identification of lncRNAs was carried out using strand-specific paired-end RNA sequencing, from Pinus radiata samples inoculated with F. circinatum at an early stage of infection. RESULTS: Overall, 13,312 lncRNAs were predicted through a bioinformatics approach, including long intergenic non-coding RNAs (92.3%), antisense lncRNAs (3.3%) and intronic lncRNAs (2.9%). Compared with protein-coding RNAs, pine lncRNAs are shorter, have lower expression, lower GC content and harbour fewer and shorter exons. A total of 164 differentially expressed (DE) lncRNAs were identified in response to F. circinatum infection in the inoculated versus mock-inoculated P. radiata seedlings. The predicted cis-regulated target genes of these pathogen-responsive lncRNAs were related to defence mechanisms such as kinase activity, phytohormone regulation, and cell wall reinforcement. Co-expression network analysis of DE lncRNAs, DE protein-coding RNAs and lncRNA target genes also indicated a potential network regulating pectinesterase activity and cell wall remodelling. CONCLUSIONS: This study presents the first comprehensive genome-wide analysis of P. radiata lncRNAs and provides the basis for future functional characterizations of lncRNAs in relation to pine defence responses against F. circinatum.

BACKGROUND: The investigation of molecular mechanisms involved in polysaccharides and saponin metabolism is critical for genetic engineering of Polygonatum cyrtonema Hua to raise major active ingredient content. Up to now, the transcript sequences are available for different tissues of P. cyrtonema, a wide range scanning about temporal transcript at different ages' rhizomes was still absent in P. cyrtonema. RESULTS: Transcriptome sequencing for rhizomes at different ages was performed. Sixty-two thousand six hundred thirty-five unigenes were generated by assembling transcripts from all samples. A total of 89 unigenes encoding key enzymes involved in polysaccharide biosynthesis and 56 unigenes encoding key enzymes involved in saponin biosynthesis. The content of total polysaccharide and total saponin was positively correlated with the expression patterns of mannose-6-phosphate isomerase (MPI), GDP-L-fucose synthase (TSTA3), UDP-apiose/xylose synthase (AXS), UDP-glucose 6-dehydrogenase (UGDH), Hydroxymethylglutaryl CoA synthase (HMGS), Mevalonate kinase (MVK), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF), (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (ispG), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH), Farnesyl diphosphate synthase (FPPS). Finally, a number of key genes were selected and quantitative real-time PCR were performed to validate the transcriptome analysis results. CONCLUSIONS: These results create the link between polysaccharides and saponin biosynthesis and gene expression, provide insight for underlying key active substances, and reveal novel candidate genes including TFs that are worth further exploration for their functions and values.

BACKGROUND: Head of fish species, an exquisitely complex anatomical system, is important not only for studying fish evolution and development, but also for economic values. Currently, although some studies have been made on fish growth and body shapes, very limited information is available on the molecular mechanism of head development. RESULTS: In this study, RNA sequencing (RNA-Seq) and small RNA sequencing (sRNA-Seq) technologies were used to conduct integrated analysis for the head of bighead carp at different development stages, including 1, 3, 5, 15 and 30 Dph (days post hatch). By RNA-Seq data, 26 pathways related to growth and bone formation were identified as the main physiological processes during early development. Coupling this to sRNA-Seq data, we picked out six key pathways that may be responsible for head development, namely ECM receptor interaction, TNF signaling pathway, osteoclast differentiation, PI3K-Akt signaling pathway, Neuroactive ligand-receptor interaction and Jak-STAT signaling pathway. Totally, 114 important candidate genes from the six pathways were obtained. Then we found the top 20 key genes according to the degree value by cytohubba, which regulated cell growth, skeletal formation and blood homeostasis, such as pik3ca, pik3r1, egfr, vegfa, igf1 and itga2b. Finally, we also acquired 19 key miRNAs playing multiple roles in the perfection of various tissues in the head (such as brain, eye and mouth) and mineralization of head bone system, such as let-7e, miR-142a-5p, miR-144-3p, miR-23a-3p and miR-223. CONCLUSIONS: Results of this study will be informative for genetic mechanisms of head development and also provide potential candidate targets for the interaction regulation during early growth in bighead carp.