BACKGROUND: GC pairs are generally more stable than AT pairs; GC-rich genomes were proposed to be more adapted to high temperatures than AT-rich genomes. Previous studies consistently showed positive correlations between growth temperature and the GC contents of structural RNA genes. However, for the whole genome sequences and the silent sites of the codons in protein-coding genes, the relationship between GC content and growth temperature is in a long-lasting debate. RESULTS: With a dataset much larger than previous studies (681 bacteria and 155 archaea with completely assembled genomes), our phylogenetic comparative analyses showed positive correlations between optimal growth temperature (Topt) and GC content both in bacterial and archaeal structural RNA genes and in bacterial whole genome sequences, chromosomal sequences, plasmid sequences, core genes, and accessory genes. However, in the 155 archaea, we did not observe a significant positive correlation of Topt with whole-genome GC content (GCw) or GC content at four-fold degenerate sites. We randomly drew 155 samples from the 681 bacteria for 1000 rounds. In most cases (> 95%), the positive correlations between Topt and genomic GC contents became statistically nonsignificant (P > 0.05). This result suggested that the small sample sizes might account for the lack of positive correlations between growth temperature and genomic GC content in the 155 archaea and the bacterial samples of previous studies. Comparing the GC content among four categories (psychrophiles/psychrotrophiles, mesophiles, thermophiles, and hyperthermophiles) also revealed a positive correlation between GCw and growth temperature in bacteria. By including the GCw of incompletely assembled genomes, we expanded the sample size of archaea to 303. Positive correlations between GCw and Topt appear especially after excluding the halophilic archaea whose GC contents might be strongly shaped by intense UV radiation. CONCLUSIONS: This study explains the previous contradictory observations and ends a long debate. Prokaryotes growing in high temperatures have higher GC contents. Thermal adaptation is one possible explanation for the positive association. Meanwhile, we propose that the elevated efficiency of DNA repair in response to heat mutagenesis might have the by-product of increasing GC content like that happens in intracellular symbionts and marine bacterioplankton.

BACKGROUND: Gamma-irradiated mutants of Triticum aestivum L., hexaploid wheat, provide novel and agriculturally important traits and are used as breeding materials. However, the identification of causative genomic regions of mutant phenotypes is challenging because of the large and complicated genome of hexaploid wheat. Recently, the combined use of high-quality reference genome sequences of common wheat and cost-effective resequencing technologies has made it possible to evaluate genome-wide polymorphisms, even in complex genomes. RESULTS: To investigate whether the genome sequencing approach can effectively detect structural variations, such as deletions, frequently caused by gamma irradiation, we selected a grain-hardness mutant from the gamma-irradiated population of Japanese elite wheat cultivar "Kitahonami." The Hardness (Ha) locus, including the puroindoline protein-encoding genes Pina-D1 and Pinb-D1 on the short arm of chromosome 5D, primarily regulates the grain hardness variation in common wheat. We performed short-read genome sequencing of wild-type and grain-hardness mutant plants, and subsequently aligned their short reads to the reference genome of the wheat cultivar "Chinese Spring." Genome-wide comparisons of depth-of-coverage between wild-type and mutant strains detected ~ 130 Mbp deletion on the short arm of chromosome 5D in the mutant genome. Molecular markers for this deletion were applied to the progeny populations generated by a cross between the wild-type and the mutant. A large deletion in the region including the Ha locus was associated with the mutant phenotype, indicating that the genome sequencing is a powerful and efficient approach for detecting a deletion marker of a gamma-irradiated mutant phenotype. In addition, we investigated a pre-harvest sprouting tolerance mutant and identified a 67.8 Mbp deletion on chromosome 3B where Viviparous-B1 and GRAS family transcription factors are located. Co-dominant markers designed to detect the deletion-polymorphism confirmed the association with low germination rate, leading to pre-harvest sprouting tolerance. CONCLUSIONS: Short read-based genome sequencing of gamma-irradiated mutants facilitates the identification of large deletions linked to mutant phenotypes when combined with segregation analyses in progeny populations. This method allows effective application of mutants with agriculturally important traits in breeding using marker-assisted selection.

BACKGROUND: Obesity has been recognized as a worldwide growing problem, producing many pathologies including the promotion of "proinflammatory state." The etiology of human obesity is still only partially understood; however, the genetic background has been proved. Its nature is complex, and currently, it appears that the combined effects of the interactions among multiple genes should receive more attention. Due to the fact that obesity promotes proinflammatory conditions, in this study, we investigated the genetic polymorphism of IL-1 family genes in healthy people with normal and elevated body mass index (BMI) and fat %. RESULTS: The single-nucleotide polymorphisms (SNPs) within the IL1A -889C > T (rs1800587), IL1B + 3954 T > C (rs1143634), and IL1RN -87G > A (rs2234677) genes alone were associated neither with BMI nor fat % values in tested group. The associations between SNP-SNP interaction and BMI for the IL1B × IL1RN interactions were significant for dominant model (p = 0.02) and codominant model (p = 0.03). The same SNP-SNP interaction (IL1B × IL1RN) was associated also with fat % for codominant (p = 0.01) and recessive (p = 0.002) models. CONCLUSIONS: This study further confirmed that IL-1 family genes are involved in genetic background of obesity. It has been shown that interaction IL1B × IL1RN was associated with both BMI and fat % with rare T allele protecting form higher values. Thus, even if certain polymorphisms in single genes of IL-1 family cannot be defined as related to obesity in examined population, the genetic interrelationships should be analyzed.

BACKGROUND: Chemical signaling between a mammalian host and intestinal microbes is health and maintenance of 'healthy' intestinal microbiota. Escherichia coli O157:H7 can hijack host- and microbiota-produced chemical signals for survival in a harsh and nutritionally competitive gastrointestinal environment and for intestinal colonization. Norepinephrine (NE) produced by sympathetic neurons of the enteric nervous system has been shown in vitro to induce expression of genes controlling E. coli O157:H7 swimming motility, acid resistance, and adherence to epithelial cells. A previous study used a microarray approach to identify differentially expressed genes in E. coli O157:H7 strain EDL933 in response to NE. To elucidate a comprehensive transcriptional response to NE, we performed RNA-Seq on rRNA-depleted RNA of E. coli O157:H7 strain NADC 6564, an isolate of a foodborne E. coli O157:H7 strain 86-24. The reads generated by RNA-Seq were mapped to NADC 6564 genome using HiSat2. The mapped reads were quantified by htseq-count against the genome of strain NADC 6564. The differentially expressed genes were identified by analyzing quantified reads by DESeq2. RESULTS: Of the 585 differentially expressed genes (≥ 2.0-fold; p < 0.05), many encoded pathways promoting ability of E. coli O157:H7 strain NADC 6564 to colonize intestines of carrier animals and to produce disease in an incidental human host through increased adherence to epithelial cells and production of Shiga toxins. In addition, NE exposure also induced the expression of genes encoding pathways conferring prolonged survival at extreme acidity, controlling influx/efflux of specific nutrients/metabolites, and modulating tolerance to various stressors. A correlation was also observed between the EvgS/EvgA signal transduction system and the ability of bacterial cells to survive exposure to high acidity for several hours. Many genes involved in nitrogen, sulfur, and amino acid uptake were upregulated while genes linked to iron (Fe3+) acquisition and transport were downregulated. CONCLUSION: The availability of physiological levels of NE in gastrointestinal tract could serve as an important cue for E. coli O157:H7 to engineer its virulence, stress, and metabolic pathways for colonization in reservoir animals, such as cattle, causing illness in humans, and surviving outside of a host.

BACKGROUND: Circular RNA (circRNA), a class of RNA molecule with a loop structure, has recently attracted researchers due to its diverse biological functions and potential biomarkers of human diseases. Most of the current circRNA detection methods from RNA-sequencing (RNA-Seq) data utilize the mapping information of paired-end (PE) reads to eliminate false positives. However, much of the practical RNA-Seq data such as cross-linking immunoprecipitation sequencing (CLIP-Seq) data usually contain single-end (SE) reads. It is not clear how well these tools perform on SE RNA-Seq data. RESULTS: In this study, we present a systematic evaluation of six advanced RNA-based methods and two CLIP-Seq based methods for detecting circRNAs from SE RNA-Seq data. The performances of the methods are rigorously assessed based on precision, sensitivity, F1 score, and true discovery rate. We investigate the impacts of read length, false positive ratio, sequencing depth and PE mapping information on the performances of the methods using simulated SE RNA-Seq simulated datasets. The real datasets used in this study consist of four experimental RNA-Seq datasets with ≥100bp read length and 124 CLIP-Seq samples from 45 studies that contain mostly short-read (≤50bp) RNA-Seq data. The simulation study shows that the sensitivities of most of the methods can be improved by increasing either read length or sequencing depth, and that the levels of false positive rates significantly affect the precision of all methods. Furthermore, the PE mapping information can improve the method's precision but can not always guarantee the increase of F1 score. Overall, no method is dominant for all SE RNA-Seq data. The RNA-based methods perform better for the long-read datasets but are worse for the short-read datasets. In contrast, the CLIP-Seq based methods outperform the RNA-Seq based methods for all the short-read samples. Combining the results of these methods can significantly improve precision in the CLIP-Seq data. CONCLUSIONS: The results provide a systematic evaluation of circRNA detection methods on SE RNA-Seq data that would facilitate researchers' strategies in circRNA analysis.

BACKGROUND: The epigenetic modifications play important regulatory roles in tissue development, maintenance of physiological functions and pathological process. RNA methylations, including newly identified m1A, m5C, m6A and m7G, are important epigenetic modifications. However, how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study, we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. RESULTS: By performing unbiased analyses of the m1A, m5C, m6A and m7G methylation of mRNA, we found that within the whole brain transcriptome, with the increase of the gene expression levels, the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia-treated zebrafish brains, the proportion of m1A is decreased, affecting the RNA splicing and zebrafish endogenous retroviruses. CONCLUSIONS: Our study presents the first comprehensive atlas of m1A, m5C, m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A, m5C, m6A and m7G in the regulation of miRNA and repeat elements in vertebrates, and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome.

BACKGROUND: Seed germination is a series of ordered physiological and morphogenetic processes and a critical stage in plant life cycle. Tamarix hispida is one of the most salt-tolerant plant species; however, its seed germination has not been analysed using combined transcriptomics and metabolomics. RESULTS: Transcriptomic sequencing and widely targeted metabolomics were used to detect the transcriptional metabolic profiles of T. hispida at different stages of seed germination and young seedling growth. Transcriptomics showed that 46,538 genes were significantly altered throughout the studied development period. Enrichment study revealed that plant hormones, such as auxin, ABA, JA and SA played differential roles at varying stages of seed germination and post-germination. Metabolomics detected 1022 metabolites, with flavonoids accounting for the highest proportion of differential metabolites. Combined analysis indicated that flavonoid biosynthesis in young seedling growth, such as rhoifolin and quercetin, may improve the plant's adaptative ability to extreme desert environments. CONCLUSIONS: The differential regulation of plant hormones and the accumulation of flavonoids may be important for the seed germination survival of T. hispida in response to salt or arid deserts. This study enhanced the understanding of the overall mechanism in seed germination and post-germination. The results provide guidance for the ecological value and young seedling growth of T. hispida.

BACKGROUND: Genetic markers like the nuclear ribosomal RNA (rRNA) genes, internal transcribed spacer regions, mitochondrial protein-coding genes, and genomes have been utilized for molecular identification of parasitic trematodes. However, challenges such as the design of broadly applicable primers for the vast number of species within Digenea and the genetic markers' ability to provide sufficient species-level resolution limited their utility. This study presented novel and broadly applicable primers using the mitochondrial 12S and 16S rRNA genes for Digenea and aimed to show their suitability as alternative genetic markers for molecular identification of orders Plagiorchiida, Echinostomida, and Strigeida. RESULTS: Our results revealed that the mitochondrial 12S and 16S rRNA genes are suitable for trematode molecular identification, with sufficient resolution to discriminate closely related species and achieve accurate species identification through phylogenetic placements. Moreover, the robustness of our newly designed primers to amplify medically important parasitic trematodes encompassing three orders was demonstrated through successful amplification. The convenience and applicability of the newly designed primers and adequate genetic variation of the mitochondrial rRNA genes can be useful as complementary markers for trematode molecular-based studies. CONCLUSIONS: We demonstrated that the mitochondrial rRNA genes could be alternative genetic markers robust for trematode molecular identification and potentially helpful for DNA barcoding where our primers can be widely applied across the major Digenea orders. Furthermore, the potential of the mitochondrial rRNA genes for molecular systematics can be explored, enhancing their appeal for trematode molecular-based studies. The novelty of utilizing the mitochondrial rRNA genes and the designed primers in this study can potentially open avenues for species identification, discovery, and systematics in the future.

BACKGROUND: Very little is known about the etiology of personality and psychiatric disorders. Because the core neurobiology of many such traits is evolutionarily conserved, dogs present a powerful model. We previously reported genome scans of breed averages of ten traits related to fear, anxiety, aggression and social behavior in multiple cohorts of pedigree dogs. As a second phase of that discovery, here we tested the ability of markers at 13 of those loci to predict canine behavior in a community sample of 397 pedigree and mixed-breed dogs with individual-level genotype and phenotype data. RESULTS: We found support for all markers and loci. By including 122 dogs with veterinary behavioral diagnoses in our cohort, we were able to identify eight loci associated with those diagnoses. Logistic regression models showed subsets of those loci could predict behavioral diagnoses. We corroborated our previous findings that small body size is associated with many problem behaviors and large body size is associated with increased trainability. Children in the home were associated with anxiety traits; illness and other animals in the home with coprophagia; working-dog status with increased energy and separation-related problems; and competitive dogs with increased aggression directed at familiar dogs, but reduced fear directed at humans and unfamiliar dogs. Compared to other dogs, Pit Bull-type dogs were not defined by a set of our markers and were not more aggressive; but they were strongly associated with pulling on the leash. Using severity-threshold models, Pit Bull-type dogs showed reduced risk of owner-directed aggression (75th quantile) and increased risk of dog-directed fear (95th quantile). CONCLUSIONS: Our association analysis in a community sample of pedigree and mixed-breed dogs supports the interbreed mapping. The modeling shows some markers are predictive of behavioral diagnoses. Our findings have broad utility, including for clinical and breeding purposes, but we caution that thorough understanding is necessary for their interpretation and use.

BACKGROUND: Pochonia chlamydosporia is an endophytic fungus used for nematode biocontrol that employs its cellular and molecular machinery to degrade the nematode egg-shell. Chitosanases, among other enzymes, are involved in this process. In this study, we improve the genome sequence assembly of P. chlamydosporia 123, by utilizing long Pacific Biosciences (PacBio) sequence reads. Combining this improved genome assembly with previous RNA-seq data revealed alternative isoforms of a chitosanase in the presence of chitosan. This study could open new insights into understanding fungal resistance to chitosan and root-knot nematode (RKN) egg infection processes. RESULTS: The P. chlamydosporia 123 genome sequence assembly has been updated using long-read PacBio sequencing and now includes 12,810 predicted protein-coding genes. Compared with the previous assembly based on short reads, there are 701 newly annotated genes, and 69 previous genes are now split. Eight of the new genes were differentially expressed in fungus interactions with Meloidogyne javanica eggs or chitosan. A survey of the RNA-seq data revealed alternative splicing in the csn3 gene that encodes a chitosanase, with four putative splicing variants: csn3_v1, csn3_v2, csn3_v3 and csn3_v4. When P. chlamydosporia is treated with 0.1 mg·mL- 1 chitosan for 4 days, csn3 is expressed 10-fold compared with untreated controls. Furthermore, the relative abundances of each of the four transcripts are different in chitosan treatment compared with controls. In controls, the abundances of each transcript are nil, 32, 55, and 12% for isoforms csn3_v1, csn3_v2, csn3_v3 and csn3_v4 respectively. Conversely, in chitosan-treated P. chlamydosporia, the abundances are respectively 80, 15%, 2-3%, 2-3%. Since isoform csn3_v1 is expressed with chitosan only, the putatively encoded enzyme is probably induced and likely important for chitosan degradation. CONCLUSIONS: Alternative splicing events have been discovered and described in the chitosanase 3 encoding gene from P. chlamydosporia 123. Gene csn3 takes part in RKN parasitism process and chitosan enhances its expression. The isoform csn3_v1 would be related to the degradation of this polymer in bulk form, while other isoforms may be related to the degradation of chitosan in the nematode egg-shell.

BACKGROUND: Previous reports have shown that soil salinity is a growing threat to cowpea production, and thus the need for breeding salt-tolerant cowpea cultivars. A total of 234 Multi-Parent Advanced Generation Inter-Cross (MAGIC) lines along with their 8 founders were evaluated for salt tolerance under greenhouse conditions. The objectives of this study were to evaluate salt tolerance in a multi-parent advanced generation inter-cross (MAGIC) cowpea population, to identify single nucleotide polymorphism (SNP) markers associated with salt tolerance, and to assess the accuracy of genomic selection (GS) in predicting salt tolerance, and to explore possible epistatic interactions affecting salt tolerance in cowpea. Phenotyping was validated through the use of salt-tolerant and salt-susceptible controls that were previously reported. Genome-wide association study (GWAS) was conducted using a total of 32,047 filtered SNPs. The epistatic interaction analysis was conducted using the PLINK platform. RESULTS: Results indicated that: (1) large variation in traits evaluated for salt tolerance was identified among the MAGIC lines, (2) a total of 7, 2, 18, 18, 3, 2, 5, 1, and 23 were associated with number of dead plants, salt injury score, leaf SPAD chlorophyll under salt treatment, relative tolerance index for leaf SPAD chlorophyll, fresh leaf biomass under salt treatment, relative tolerance index for fresh leaf biomass, relative tolerance index for fresh stem biomass, relative tolerance index for the total above-ground fresh biomass, and relative tolerance index for plant height, respectively, with overlapping SNP markers between traits, (3) candidate genes encoding for proteins involved in ion transport such as Na+/Ca2+ K+ independent exchanger and H+/oligopeptide symporter were identified, and (4) epistatic interactions were identified. CONCLUSIONS: These results will have direct applications in breeding programs aiming at improving salt tolerance in cowpea through marker-assisted selection. To the best of our knowledge, this study was one of the earliest reports using a MAGIC population to investigate the genetic architecture of salt tolerance in cowpea.

BACKGROUND: Mixed models are used to correct for confounding due to population stratification and hidden relatedness in genome-wide association studies. This class of models includes linear mixed models and generalized linear mixed models. Existing mixed model approaches to correct for population substructure have been previously investigated with both continuous and case-control response variables. However, they have not been investigated in the context of extreme phenotype sampling (EPS), where genetic covariates are only collected on samples having extreme response variable values. In this work, we compare the performance of existing binary trait mixed model approaches (GMMAT, LEAP and CARAT) on EPS data. Since linear mixed models are commonly used even with binary traits, we also evaluate the performance of a popular linear mixed model implementation (GEMMA). RESULTS: We used simulation studies to estimate the type I error rate and power of all approaches assuming a population with substructure. Our simulation results show that for a common candidate variant, both LEAP and GMMAT control the type I error rate while CARAT's rate remains inflated. We applied all methods to a real dataset from a Québec, Canada, case-control study that is known to have population substructure. We observe similar type I error control with the analysis on the Québec dataset. For rare variants, the false positive rate remains inflated even after correction with mixed model approaches. For methods that control the type I error rate, the estimated power is comparable. CONCLUSIONS: The methods compared in this study differ in their type I error control. Therefore, when data are from an EPS study, care should be taken to ensure that the models underlying the methodology are suitable to the sampling strategy and to the minor allele frequency of the candidate SNPs.

BACKGROUND: Most dementia disorders have a clear genetic background and a number of disease genes have been identified. Mutations in the tau gene (MAPT) lead to frontotemporal dementia (FTD), whereas mutations in the genes for the amyloid-β precursor protein (APP) and the presenilins (PSEN1, PSEN2) cause early-onset, dominantly inherited forms of Alzheimer's disease (AD). Even if mutations causing Mendelian forms of these diseases are uncommon, elucidation of the pathogenic effects of such mutations have proven important for understanding the pathogenic processes. Here, we performed a screen to identify novel pathogenic mutations in known disease genes among patients undergoing dementia investigation. RESULTS: Using targeted exome sequencing we have screened all coding exons in eleven known dementia genes (PSEN1, PSEN2, APP, MAPT, APOE, GRN, TARDBP, CHMP2B, TREM2, VCP and FUS) in 102 patients with AD, FTD, other dementia diagnoses or mild cognitive impairment. We found three AD patients with two previously identified pathogenic mutations in PSEN1 (Pro264Leu and Met146Val). In this screen, we also identified the recently reported APP mutation in two siblings with AD. This mutation, named the Uppsala mutation, consists of a six amino acid intra-amyloid β deletion. In addition, we found several potentially pathogenic mutations in PSEN2, FUS, MAPT, GRN and APOE. Finally, APOE ε4 was prevalent in this patient group with an allele frequency of 54%. CONCLUSIONS: Among the 102 screened patients, we found two disease causing mutations in PSEN1 and one in APP, as well as several potentially pathogenic mutations in other genes related to neurodegenerative disorders. Apart from giving important information to the clinical investigation, the identification of disease mutations can contribute to an increased understanding of disease mechanisms.

BACKGROUND: RNA-seq is being increasingly adopted for gene expression studies in a panoply of non-model organisms, with applications spanning the fields of agriculture, aquaculture, ecology, and environment. For organisms that lack a well-annotated reference genome or transcriptome, a conventional RNA-seq data analysis workflow requires constructing a de-novo transcriptome assembly and annotating it against a high-confidence protein database. The assembly serves as a reference for read mapping, and the annotation is necessary for functional analysis of genes found to be differentially expressed. However, assembly is computationally expensive. It is also prone to errors that impact expression analysis, especially since sequencing depth is typically much lower for expression studies than for transcript discovery. RESULTS: We propose a shortcut, in which we obtain counts for differential expression analysis by directly aligning RNA-seq reads to the high-confidence proteome that would have been otherwise used for annotation. By avoiding assembly, we drastically cut down computational costs - the running time on a typical dataset improves from the order of tens of hours to under half an hour, and the memory requirement is reduced from the order of tens of Gbytes to tens of Mbytes. We show through experiments on simulated and real data that our pipeline not only reduces computational costs, but has higher sensitivity and precision than a typical assembly-based pipeline. A Snakemake implementation of our workflow is available at: https://bitbucket.org/project_samar/samar . CONCLUSIONS: The flip side of RNA-seq becoming accessible to even modestly resourced labs has been that the time, labor, and infrastructure cost of bioinformatics analysis has become a bottleneck. Assembly is one such resource-hungry process, and we show here that it can be avoided for quick and easy, yet more sensitive and precise, differential gene expression analysis in non-model organisms.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) plays essential roles in the development, hormone regulation and abiotic stress response of plants. Nevertheless, a comprehensive study on MAPK family members has thus far not been performed in Tartary buckwheat. RESULTS: Here, we identified 16 FtMAPKs in the Fagopyrum tataricum genome. Phylogenetic analysis showed that the FtMAPK family members could be classified into Groups A, B, C and D, in which A, B and C members contain a Thr-Glu-Tyr (TEY) signature motif and Group D members contain a Thr-Asp-Tyr (TDY) signature motif. Promoter cis-acting elements showed that most ProFtMAPks contain light response elements, hormone response elements and abiotic stress response elements, and several ProFtMAPks have MYB-binding sites, which may be involved in the regulation of flavonoid biosynthesis-related enzyme gene expression. Synteny analysis indicated that FtMAPKs have a variety of biological functions. Protein interaction prediction suggested that MAPKs can interact with proteins involved in development and stress resistance. Correlation analysis further confirmed that most of the FtMAPK genes and transcription factors involved in the stress response have the same expression pattern. The transient transformation of FtMAPK1 significantly increased the antioxidant enzymes activity in Tartary buckwheat leaves. In addition, we also found that FtMAPK1 can respond to salt stress by up-regulating the transcription abundance of downstream genes. CONCLUSIONS: A total of 16 MAPKs were identified in Tartary buckwheat, and the members of the MAPK family containing the TDY motif were found to have expanded. The same subfamily members have relatively conserved gene structures and similar protein motifs. Tissue-specific expression indicated that the expression of all FtMAPK genes varied widely in the roots, stems, leaves and flowers. Most FtMAPKs can regulate the expression of other transcription factors and participate in the abiotic stress response. Our findings comprehensively revealed the FtMAPK gene family and laid a theoretical foundation for the functional characterization of FtMAPKs.

BACKGROUND: Leaf angle is an important plant architecture trait, affecting plant density, light interception efficiency, photosynthetic rate, and yield. The "smart canopy" model proposes more vertical leaves in the top plant layers and more horizontal leaves in the lower canopy, maximizing conversion efficiency and photosynthesis. Sorghum leaf arrangement is opposite to that proposed in the "smart canopy" model, indicating the need for improvement. Although leaf angle quantitative trait loci (QTL) have been previously reported, only the Dwarf3 (Dw3) auxin transporter gene, colocalizing with a major-effect QTL on chromosome 7, has been validated. Additionally, the genetic architecture of leaf angle across canopy layers remains to be elucidated. RESULTS: This study characterized the canopy-layer specific transcriptome of five sorghum genotypes using RNA sequencing. A set of 284 differentially expressed genes for at least one layer comparison (FDR < 0.05) co-localized with 69 leaf angle QTL and were consistently identified across genotypes. These genes are involved in transmembrane transport, hormone regulation, oxidation-reduction process, response to stimuli, lipid metabolism, and photosynthesis. The most relevant eleven candidate genes for layer-specific angle modification include those homologous to genes controlling leaf angle in rice and maize or genes associated with cell size/expansion, shape, and cell number. CONCLUSIONS: Considering the predicted functions of candidate genes, their potential undesirable pleiotropic effects should be further investigated across tissues and developmental stages. Future validation of proposed candidates and exploitation through genetic engineering or gene editing strategies targeted to collar cells will bring researchers closer to the realization of a "smart canopy" sorghum.

BACKGROUND: Ticks are obligate haematophagous ectoparasites considered second to mosquitos as vectors and reservoirs of multiple pathogens of global concern. Individual variation in tick infestation has been reported in indigenous sheep, but its genetic control remains unknown. RESULTS: Here, we report 397 genome-wide signatures of selection overlapping 991 genes from the analysis, using ROH, LR-GWAS, XP-EHH, and FST, of 600 K SNP genotype data from 165 Tunisian sheep showing high and low levels of tick infestations and piroplasm infections. We consider 45 signatures that are detected by consensus results of at least two methods as high-confidence selection regions. These spanned 104 genes which included immune system function genes, solute carriers and chemokine receptor. One region spanned STX5, that has been associated with tick resistance in cattle, implicating it as a prime candidate in sheep. We also observed RAB6B and TF in a high confidence candidate region that has been associated with growth traits suggesting natural selection is enhancing growth and developmental stability under tick challenge. The analysis also revealed fine-scale genome structure indicative of cryptic divergence in Tunisian sheep. CONCLUSIONS: Our findings provide a genomic reference that can enhance the understanding of the genetic architecture of tick resistance and cryptic divergence in indigenous African sheep.

BACKGROUND: Aeromonas veronii is a Gram-negative rod-shaped motile bacterium that inhabits mainly freshwater environments. A. veronii is a pathogen of aquatic animals, causing diseases in fish. A. veronii is also an emerging human enteric pathogen, causing mainly gastroenteritis with various severities and also often being detected in patients with inflammatory bowel disease. Currently, limited information is available on the genomic information of A. veronii strains that cause human gastrointestinal diseases. Here we sequenced, assembled and analysed 25 genomes (one complete genome and 24 draft genomes) of A. veronii strains isolated from patients with gastrointestinal diseases using combine sequencing technologies from Illumina and Oxford Nanopore. We also conducted comparative analysis of genomes of 168 global A. veronii strains isolated from different sources. RESULTS: We found that most of the A. veronii strains isolated from patients with gastrointestinal diseases were closely related to each other, and the remaining were closely related to strains from other sources. Nearly 300 putative virulence factors were identified. Aerolysin, microbial collagenase and multiple hemolysins were present in all strains isolated from patients with gastrointestinal diseases. Type III Secretory System (T3SS) in A. veronii was in AVI-1 genomic island identified in this study, most likely acquired via horizontal transfer from other Aeromonas species. T3SS was significantly less present in A. veronii strains isolated from patients with gastrointestinal diseases as compared to strains isolated from fish and domestic animals. CONCLUSIONS: This study provides novel information on source of infection and virulence of A. veronii in human gastrointestinal diseases.

BACKGROUND: Several studies have been performed to study transcriptome profiles after dengue virus infections with partly different results. Due to slightly different settings of the individual studies, different genes and enriched gene sets are reported in these studies. The main aim of this network meta-analysis was to aggregate a selection of these studies to identify genes and gene sets that are more generally associated with dengue virus infection, i.e. with less dependence on the individual study settings. METHODS: We performed network meta-analysis by different approaches using publicly available gene expression data of five selected studies from the Gene Expression Omnibus database. The study network includes dengue fever (DF), hemorrhagic fever (DHF), shock syndrome (DSS) patients as well as convalescent and healthy control individuals. After data merging and missing value imputation, study-specific batch effects were removed. Pairwise differential expression analysis and subsequent gene-set enrichment analysis were performed between the five study groups. Furthermore, mutual information networks were derived from the top genes of each group comparison, and the separability between the three patient groups was studied by machine learning models. RESULTS: From the 10 possible pairwise group comparisons in the study network, six genes (IFI27, TPX2, CDT1, DTL, KCTD14 and CDCA3) occur with a noticeable frequency among the top listed genes of each comparison. Thus, there is an increased evidence that these genes play a general role in dengue virus infections. IFI27 and TPX2 have also been highlighted in the context of dengue virus infection by other studies. A few of the identified gene sets from the network meta-analysis overlap with findings from the original studies. Mutual information networks yield additional genes for which the observed pairwise correlation is different between the patient groups. Machine learning analysis shows a moderate separability of samples from the DF, DHF and DSS groups (accuracy about 80%). CONCLUSIONS: Due to an increased sample size, the network meta-analysis could reveal additional genes which are called differentially expressed between the studied groups and that may help to better understand the molecular basis of this disease.

BACKGROUND: Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. However, 5' ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. The implementation of the template-switching mechanism at the reverse transcription stage along with 5' rapid amplification of cDNA ends (RACE) constitutes the most prominent and efficient strategy to specify the actual 5' ends of cDNAs. In the current study, we developed a 5' RACE-seq method by coupling a custom template-switching and 5' RACE assay with targeted nanopore sequencing, to accurately unveil 5' termini of mRNA targets. RESULTS: The optimization of the described 5' RACE-seq method was accomplished using the human BCL2L12 as control gene. We unveiled that the selection of hybrid DNA/RNA template-switching oligonucleotides as well as the complete separation of the cDNA extension incubation from the template-switching process, significantly increase the overall efficiency of the downstream 5' RACE. Collectively, our results support the existence of two distinct 5' termini for BCL2L12, being in complete accordance with the results derived from both direct RNA and PCR-cDNA sequencing approaches from Oxford Nanopore Technologies. As proof of concept, we implemented the described 5' RACE-seq methodology to investigate the 5' UTRs of several kallikrein-related peptidases (KLKs) gene family members. Our results confirmed the existence of multiple annotated 5' UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones. CONCLUSIONS: In this work we present an in-house developed 5' RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. This approach enables the broad and in-depth study of 5' UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits.

BACKGROUND: The ability of plants to withstand and thrive in an adverse environment is crucial to ensure their survivability and yield performance. The WRKY transcription factors (TFs) have crucial roles in plant growth, development and stress response, particularly drought stress. In oil palm, drought is recognized as one of the major yield limiting factors. However, the roles of WRKY TFs in the drought response of oil palm is unclear. RESULTS: Herein, we studied the transcriptome of drought treated oil palm leaf and identified 40 differentially expressed genes (DEGs) of WRKY TFs, of which 32 DEGs were upregulated and 8 DEGs were downregulated in response to drought stress in oil palm. They were categorized into Groups I to IV based on the numbers of WRKY domain and the structural difference in the zinc finger domain. Multiple stress- and hormone-responsive cis-regulatory elements were detected in the drought responsive oil palm EgWRKY (Dro-EgWRKY) genes. Fourteen of the 15 selected oil palm WRKY (EgWRKY) genes demonstrated a tissue-specific expression profile except for EgWRKY28 (Group I), which was expressed in all tissues tested. The expression levels of 15 candidate EgWRKYs were upregulated upon salinity and heat treatments, while several genes were also inducible by abscisic acid, methyl jasmonate, salicylic acid and hydrogen peroxide treatments. Members of the Group III WRKY TFs including EgWRKY07, 26, 40, 52, 59, 73 and 81 displayed multiple roles in drought- and salinity-response under the modulation of phytohormones. CONCLUSIONS: EgWRKY TFs of oil palm are involved in phytohormones and abiotic stress responses including drought, salinity and heat. EgWRKY07, 26, 59 and 81 from Group III maybe important regulators in modulating responses of different abiotic stresses. Further functional analysis is required to understand the underlying mechanism of WRKY TFs in the regulatory network of drought stress.

BACKGROUND: Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia. A new blunt snout bream strain, "Pujiang No.2", was developed to overcome this shortcoming. As a proteasome inhibitor, bortezomib (PS-341) has been shown to affect the adaptation of cells to a hypoxic environment. In the present study, bortezomib was used to explore the hypoxia adaptation mechanism of "Pujiang No.2". We examined how acute hypoxia alone (hypoxia-treated, HN: 1.0 mg·L- 1), and in combination with bortezomib (hypoxia-bortezomib-treated, HB: Use 1 mg bortezomib for 1 kg fish), impacted the hepatic ultrastructure and transcriptome expression compared to control fish (normoxia-treated, NN). RESULTS: Hypoxia tolerance was significantly decreased in the bortezomib-treated group (LOEcrit, loss of equilibrium, 1.11 mg·L- 1 and 1.32 mg·L- 1) compared to the control group (LOEcrit, 0.73 mg·L- 1 and 0.85 mg·L- 1). The HB group had more severe liver injury than the HN group. Specifically, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the HB group (52.16 U/gprot, 32 U/gprot) were significantly (p < 0.01) higher than those in the HN group (32.85 U/gprot, 21. 68 U/gprot). In addition, more severe liver damage such as vacuoles, nuclear atrophy, and nuclear lysis were observed in the HB group. RNA-seq was performed on livers from the HN, HB and NN groups. KEGG pathway analysis disclosed that many DEGs (differently expressed genes) were enriched in the HIF-1, FOXO, MAPK, PI3K-Akt and AMPK signaling pathway and their downstream. CONCLUSION: We explored the adaptation mechanism of "Pujiang No.2" to hypoxia stress by using bortezomib, and combined with transcriptome analysis, accurately captured the genes related to hypoxia tolerance advantage.

BACKGROUND: Paris polyphylla is a herb widely used in traditional Chinese medicine to treat various diseases. Stem rot diseases seriously affected the yield of P. polyphylla in subtropical areas of China. Therefore, cost-effective, chemical-free, eco-friendly strategies to control stem rot on P. polyphylla are valuable and urgently needed. RESULTS: In this paper, we reported the biocontrol efficiency of Paenibacillus peoriae HJ-2 and its complete genome sequence. Strain HJ-2 could serve as a potential biocontrol agent against stem rot on P. polyphylla in the greenhouse and field. The genome of HJ-2 consists of a single 6,001,192 bp chromosome with an average GC content of 45% and 5,237 predicted protein coding genes, 39 rRNAs and 108 tRNAs. The phylogenetic tree indicated that HJ-2 is most closely related to P. peoriae IBSD35. Functional analysis of genome revealed numerous genes/gene clusters involved in plant colonization, biofilm formation, plant growth promotion, antibiotic and resistance inducers synthesis. Moreover, metabolic pathways that potentially contribute to biocontrol mechanisms were identified. CONCLUSIONS: This study revealed that P. peoriae HJ-2 could serve as a potential BCA against stem rot on P. polyphylla. Based on genome analysis, the genome of HJ-2 contains more than 70 genes and 12 putative gene clusters related to secondary metabolites, which have previously been described as being involved in chemotaxis motility, biofilm formation, growth promotion, antifungal activity and resistance inducers biosynthesis. Compared with other strains, variation in the genes/gene clusters may lead to different antimicrobial spectra and biocontrol efficacies.

BACKGROUND: Whole genomes are commonly assembled into a collection of scaffolds and often lack annotations of autosomes, sex chromosomes, and organelle genomes (i.e., mitochondrial and chloroplast). As these chromosome types differ in effective population size and can have highly disparate evolutionary histories, it is imperative to take this information into account when analysing genomic variation. Here we assessed the accuracy of four methods for identifying the homogametic sex chromosome in a small population using two whole genome sequences (WGS) and 133 RAD sequences of white-tailed eagles (Haliaeetus albicilla): i) difference in read depth per scaffold in a male and a female, ii) heterozygosity per scaffold in a male and a female, iii) mapping to the reference genome of a related species (chicken) with annotated sex chromosomes, and iv) analysis of SNP-loadings from a principal components analysis (PCA), based on the low-depth RADseq data. RESULTS: The best performing approach was the reference mapping (method iii), which identified 98.12% of the expected homogametic sex chromosome (Z). Read depth per scaffold (method i) identified 86.41% of the homogametic sex chromosome with few false positives. SNP-loading scores (method iv) identified 78.6% of the Z-chromosome and had a false positive discovery rate of more than 10%. Heterozygosity per scaffold (method ii) did not provide clear results due to a lack of diversity in both the Z and autosomal chromosomes, and potential interference from the heterogametic sex chromosome (W). The evaluation of these methods also revealed 10 Mb of putative PAR and gametologous regions. CONCLUSION: Identification of the homogametic sex chromosome in a small population is best accomplished by reference mapping or examining differences in read depth between sexes.

BACKGROUND: 'Nebbiolo' is a grapevine cultivar typical of north-western Italy, appreciated for producing high-quality red wines. Grapevine cultivars are characterized by possessing highly heterozygous genomes, including a great incidence of genomic rearrangements larger than 50 bp, so called structural variations (SVs). Even though abundant, SVs are an under-explored source of genetic variation mainly due to methodological limitations at their detection. RESULTS: We employed a multiple platform approach to produce long-range genomic data for two different 'Nebbiolo' clones, namely: optical mapping, long-reads and linked-reads. We performed a haplotype-resolved de novo assembly for cultivar 'Nebbiolo' (clone CVT 71) and used an ab-initio strategy to annotate it. The annotated assembly enhanced our ability to detect SVs, enabling the study of genomic regions not present in the grapevines' reference genome and accounting for their functional implications. We performed variant calling analyses at three different organizational levels: i) between haplotypes of clone CVT 71 (primary assembly vs haplotigs), ii) between 'Nebbiolo' and 'Cabernet Sauvignon' assemblies and iii) between clones CVT 71 and CVT 185, representing different 'Nebbiolo' biotypes. The cumulative size of non-redundant merged SVs indicated a total of 79.6 Mbp for the first comparison and 136.1 Mbp for the second one, while no SVs were detected for the third comparison. Interestingly, SVs differentiating cultivars and haplotypes affected similar numbers of coding genes. CONCLUSIONS: Our results suggest that SVs accumulation rate and their functional implications in 'Nebbiolo' genome are highly-dependent on the organizational level under study. SVs are abundant when comparing 'Nebbiolo' to a different cultivar or the two haplotypes of the same individual, while they turned absent between the two analysed clones.

BACKGROUND: Accurate variant detection in the coding regions of the human genome is a key requirement for molecular diagnostics of Mendelian disorders. Efficiency of variant discovery from next-generation sequencing (NGS) data depends on multiple factors, including reproducible coverage biases of NGS methods and the performance of read alignment and variant calling software. Although variant caller benchmarks are published constantly, no previous publications have leveraged the full extent of available gold standard whole-genome (WGS) and whole-exome (WES) sequencing datasets. RESULTS: In this work, we systematically evaluated the performance of 4 popular short read aligners (Bowtie2, BWA, Isaac, and Novoalign) and 9 novel and well-established variant calling and filtering methods (Clair3, DeepVariant, Octopus, GATK, FreeBayes, and Strelka2) using a set of 14 "gold standard" WES and WGS datasets available from Genome In A Bottle (GIAB) consortium. Additionally, we have indirectly evaluated each pipeline's performance using a set of 6 non-GIAB samples of African and Russian ethnicity. In our benchmark, Bowtie2 performed significantly worse than other aligners, suggesting it should not be used for medical variant calling. When other aligners were considered, the accuracy of variant discovery mostly depended on the variant caller and not the read aligner. Among the tested variant callers, DeepVariant consistently showed the best performance and the highest robustness. Other actively developed tools, such as Clair3, Octopus, and Strelka2, also performed well, although their efficiency had greater dependence on the quality and type of the input data. We have also compared the consistency of variant calls in GIAB and non-GIAB samples. With few important caveats, best-performing tools have shown little evidence of overfitting. CONCLUSIONS: The results show surprisingly large differences in the performance of cutting-edge tools even in high confidence regions of the coding genome. This highlights the importance of regular benchmarking of quickly evolving tools and pipelines. We also discuss the need for a more diverse set of gold standard genomes that would include samples of African, Hispanic, or mixed ancestry. Additionally, there is also a need for better variant caller assessment in the repetitive regions of the coding genome.

BACKGROUND: Fusarium langsethiae is a T-2 and HT-2 mycotoxins producing species firstly characterised in 2004. It is commonly isolated from oats in Northern Europe. T-2 and HT-2 mycotoxins exhibit immunological and haemotological effects in animal health mainly through inhibition of protein, RNA and DNA synthesis. The development of a high-quality and comprehensively annotated assembly for this species is therefore essential in providing the molecular understanding and the mechanism of T-2 and HT-2 biosynthesis in F. langsethiae to help develop effective control strategies. RESULTS: The F. langsethiae assembly was produced using PacBio long reads, which were then assembled independently using Canu, SMARTdenovo and Flye. A total of 19,336 coding genes were identified using RNA-Seq informed ab-initio gene prediction. Finally, predicting genes were annotated using the basic local alignment search tool (BLAST) against the NCBI non-redundant (NR) genome database and protein hits were annotated using InterProScan. Genes with blast hits were functionally annotated with Gene Ontology. CONCLUSIONS: We developed a high-quality genome assembly of a total length of 59 Mb and N50 of 3.51 Mb. Raw sequence reads and assembled genome is publicly available and can be downloaded from: GenBank under the accession JAFFKB000000000. All commands used to generate this assembly are accessible via GitHub: https://github.com/FadyMohareb/fusarium_langsethiae .

BACKGROUND: Patient-derived xenografts (PDX) mice models play an important role in preclinical trials and personalized medicine. Sharing data on the models is highly valuable for numerous reasons - ethical, economical, research cross validation etc. The EurOPDX Consortium was established 8 years ago to share such information and avoid duplicating efforts in developing new PDX mice models and unify approaches to support preclinical research. EurOPDX Data Portal is the unified data sharing platform adopted by the Consortium. MAIN BODY: In this paper we describe the main features of the EurOPDX Data Portal ( https://dataportal.europdx.eu/ ), its architecture and possible utilization by researchers who look for PDX mice models for their research. The Portal offers a catalogue of European models accessible on a cooperative basis. The models are searchable by metadata, and a detailed view provides molecular profiles (gene expression, mutation, copy number alteration) and treatment studies. The Portal displays the data in multiple tools (PDX Finder, cBioPortal, and GenomeCruzer in future), which are populated from a common database displaying strictly mutually consistent views. (SHORT) CONCLUSION: EurOPDX Data Portal is an entry point to the EurOPDX Research Infrastructure offering PDX mice models for collaborative research, (meta)data describing their features and deep molecular data analysis according to users' interests.

BACKGROUND: Pacific Biosciences HiFi read technology is currently the industry standard for high accuracy long-read sequencing that has been widely adopted by large sequencing and assembly initiatives for generation of de novo assemblies in non-model organisms. Though adapter contamination filtering is routine in traditional short-read analysis pipelines, it has not been widely adopted for HiFi workflows. RESULTS: Analysis of 55 publicly available HiFi datasets revealed that a read-sanitation step to remove sequence artifacts derived from PacBio library preparation from read pools is necessary as adapter sequences can be erroneously integrated into assemblies. CONCLUSIONS: Here we describe the nature of adapter contaminated reads, their consequences in assembly, and present HiFiAdapterFilt, a simple and memory efficient solution for removing adapter contaminated reads prior to assembly.

BACKGROUND: global trade in living plants and plant material has significantly increased the geographic distribution of many plant pathogens. As a consequence, several pathogens have been first found and described in their introduced range where they may cause severe damage on naïve host species. Knowing the center of origin and the pathways of spread of a pathogen is of importance for several reasons, including identifying natural enemies and reducing further spread. Several Phytophthora species are well-known invasive pathogens of natural ecosystems, including Phytophthora multivora. Following the description of P. multivora from dying native vegetation in Australia in 2009, the species was subsequently found to be common in South Africa where it does not cause any remarkable disease. There are now reports of P. multivora from many other countries worldwide, but not as a commonly encountered species in natural environments. RESULTS: a global collection of 335 isolates from North America, Europe, Africa, Australia, the Canary Islands, and New Zealand was used to unravel the worldwide invasion history of P. multivora, using 10 microsatellite markers for all isolates and sequence data from five loci from 94 representative isolates. Our population genetic analysis revealed an extremely low heterozygosity, significant non-random association of loci and substantial genotypic diversity suggesting the spread of P. multivora readily by both asexual and sexual propagules. The P. multivora populations in South Africa, Australia, and New Zealand show the most complex genetic structure, are well established and evolutionary older than those in Europe, North America and the Canary Islands. CONCLUSIONS: according to the conducted analyses, the world invasion of P. multivora most likely commenced from South Africa, which can be considered the center of origin of the species. The pathogen was then introduced to Australia, which acted as bridgehead population for Europe and North America. Our study highlights a complex global invasion pattern of P. multivora, including both direct introductions from the native population and secondary spread/introductions from bridgehead populations.

BACKGROUND: Plant miRNAs are a class of small non-coding RNAs that can repress gene expression at the post-transcriptional level by targeting RNA degradation or promoting translational repression. There is increasing evidence that some miRNAs can derive from a group of non-autonomous class II transposable elements called Miniature Inverted-repeat Transposable Elements (MITEs). RESULTS: We used public small RNA and degradome libraries from Triticum aestivum to screen for microRNAs production and predict their cleavage target sites. In parallel, we also created a comprehensive wheat MITE database by identifying novel elements and compiling known ones. When comparing both data sets, we found high homology between MITEs and 14% of all the miRNAs production sites detected. Furthermore, we show that MITE-derived miRNAs have preference for targeting degradation sites with MITE insertions in the 3' UTR regions of the transcripts. CONCLUSIONS: Our results revealed that MITE-derived miRNAs can underlay the origin of some miRNAs and potentially shape a regulatory gene network. Since MITEs are found in millions of insertions in the wheat genome and are closely linked to genic regions, this kind of regulatory network could have a significant impact on the post-transcriptional control of gene expression.

BACKGROUND: Zinc finger homeodomain (ZHD) protein is a plant-specific transcription factor and a potential regulator of phosphoenolpyruvate carboxylase (PEPCase)-coding genes, and it also participates in plant growth regulation and abiotic stress responses. To study the function of MsZF-HD genes in the alkaline stress response, this paper assessed biological information and performed transcriptome analysis of the MsZF-HD gene family by using the genomes of two different varieties of alfalfa (XinJiangDa Ye and Zhongmu No. 1). RESULTS: In total, 49 and 11 MsZF-HD genes were identified in the two different varieties respectively, including the alleles of XinJiangDa Ye. According to their phylogenetic relationships, the 60 MsZF-HD genes were divided into 5 ZHD subfamilies and 1 MIF subfamily. A total of 88.3% of MsZF-HD genes do not contain introns and are unevenly distributed among the 6 chromosomes of alfalfa. A collinearity analysis indicated that 26 genes of XinJiangDa Ye have no orthologous genes in Zhongmu No. 1, although these genes (such as ZHD-X1-2, ZHD-X3-2 and ZHD-X4-2) have homologous genes in Arabidopsis thaliana, Medicago truncatula and Glycine max. Through RNA-seq and qRT-PCR verification, it was found that MsZF-HD genes are downregulated to participate in the alkaline stress response. CONCLUSION: The results of this study may lay the foundation for the cloning and functional study of MsZF-HD genes and provide a theoretical basis for revealing the difference between XinJiangDa Ye and Zhongmu No. 1 at the genome level.

BACKGROUND: Biocontrol agents are sustainable eco-friendly alternatives for chemical pesticides that cause adverse effects in the environment and toxicity in animals including humans. An improved understanding of the phyllosphere microbiology is of vital importance for biocontrol development. Most studies have been directed towards beneficial plant-microbe interactions and ignore the pathogens that might affect humans when consuming vegetables. In this study we extended this perspective and investigated potential biocontrol strains isolated from tomato and lettuce phyllosphere that can promote plant growth and potentially antagonize human pathogens as well as plant pathogens. Subsequently, we mined into their genomes for discovery of antimicrobial biosynthetic gene clusters (BGCs), that will be further characterized. RESULTS: The antimicrobial activity of 69 newly isolated strains from a healthy tomato and lettuce phyllosphere against several plant and human pathogens was screened. Three strains with the highest antimicrobial activity were selected and characterized (Bacillus subtilis STRP31, Bacillus velezensis SPL51, and Paenibacillus sp. PL91). All three strains showed a plant growth promotion effect on tomato and lettuce. In addition, genome mining of the selected isolates showed the presence of a large variety of biosynthetic gene clusters. A total of 35 BGCs were identified, of which several are already known, but also some putative novel ones were identified. Further analysis revealed that among the novel BGCs, one previously unidentified NRPS and two bacteriocins are encoded, the gene clusters of which were analyzed in more depth. CONCLUSIONS: Three recently isolated strains of the Bacillus genus were identified that have high antagonistic activity against lettuce and tomato plant pathogens. Known and unknown antimicrobial BGCs were identified in these antagonistic bacterial isolates, indicating their potential to be used as biocontrol agents. Our study serves as a strong incentive for subsequent purification and characterization of novel antimicrobial compounds that are important for biocontrol.

BACKGROUND: The oocyte development ability of prepubertal animals is significantly lower than that of adult animals. Granulosa cells (GCs) have an important function on regulation of follicular and oocyte development. Therefore, analysis of GC characteristics can be used to explore the developmental mechanism of follicles and oocytes. RESULTS: In order to understand the possible reasons for the differences in follicle and oocyte development between lambs and adult sheep, we utilized high-throughput sequencing technique to analyze the transcriptome of GCs from follicle-stimulating hormone (FSH) superstimulated adult ewes and prepubertal lambs. Adult ewes were treated with FSH for 3 days (group A) and lambs were FSH-treated for 2 days (group B) or 3 days (group C). Transcriptome analysis of GCs showed that there were 405 and 159 differentially expressed genes from A vs. B and A vs. C, respectively. The results indicated that prolonging the FSH-treatment of lambs made the GC state of lambs more similar to the adult ewes, but there were still a large number of differentially expressed genes between adult ewes and lambs. Further analysis showed that many differently expressed genes were implicated in cell proliferation and apoptosis, oocyte development and follicular ovulation. Cellular examination demonstrated that fatty acid binding protein 4 (FABP4), which was highly expressed in lamb GCs, had a potential of promoting cell apoptosis. Cytoplasmic phospholipase A2 (PLA2G4A), which was expressed lowly in lamb GCs, may be responsible for reduced synthesis of prostaglandins in cells and impaired follicle/oocyte development. In contrast, glutathione S-transferase β-1 (GSTT2B) and forkhead boxO6 (FOXO6) had no apparent effect on the proliferation and apoptosis of GCs. CONCLUSIONS: Our study found dramatic transcriptomic differences in GCs between lambs and adult sheep, which may explain the possible reasons for the defects of follicle and oocyte development in lambs compared to adult sheep. Our data provides important information for further understanding the mechanism of follicular development in prepubertal animals and improving their oocyte developmental competence.

BACKGROUND: Transgenic animal models are crucial for the study of gene function and disease, and are widely utilized in basic biological research, agriculture and pharma industries. Since the current methods for generating transgenic animals result in the random integration of the transgene under study, the phenotype may be compromised due to disruption of known genes or regulatory regions. Unfortunately, most of the tools that predict transgene insertion sites from high-throughput data are not publicly available or not properly maintained. RESULTS: We implemented TC-hunter, Transgene-Construct hunter, an open tool that identifies transgene insertion sites and provides simple reports and visualization aids. It relies on common tools used in the analysis of high-throughput data and makes use of chimeric reads and discordant read pairs to identify and support the transgenic insertion site. To demonstrate its applicability, we applied TC-hunter to four transgenic mice samples harboring the human PPM1D gene, a model used in the study of malignant tumor development. We identified the transgenic insertion site in each sample and experimentally validated them with Touchdown-polymerase chain reaction followed by Sanger sequencing. CONCLUSIONS: TC-hunter is an accessible bioinformatics tool that can automatically identify transgene insertion sites from DNA sequencing data with high sensitivity (98%) and precision (92.45%). TC-hunter is a valuable tool that can aid in evaluating any potential phenotypic complications due to the random integration of the transgene and can be accessed at https://github.com/bcfgothenburg/SSF .

BACKGROUND: The broad application of high-resolution chromosome detection technology in prenatal diagnosis has identified copy number loss (CNL) involving autosomal dominant (AD) genes in certain fetuses. Exon sequencing of fetuses exhibiting structural anomalies yields diagnostic information in up to 20% of cases. However, there is currently no relevant literature about the genetic origin and pregnancy outcome of CNL involving AD genes in fetuses without structural abnormalities. RESULTS: This was a prospective study involving pregnant women who underwent amniocentesis for fetal copy number variation sequencing (CNVseq). Detection of parent-of-origin was suggested in cases of samples with CNL involving AD genes and the pregnancy outcome was monitored. Amniotic fluid samples from 24,844 fetuses without structural abnormalities were successfully tested via CNVseq. The results showed that 134 fetuses (0.5%) had small CNL (< 10 Mb) containing AD genes, after excluding microdeletion and microduplication syndrome and polymorphisms. By monitoring the pregnancy outcomes of the 134 fetuses, we found that 104 (77.6%) were good, 13 (9.7%) were adverse, and 17 (12.7%) pregnant women voluntarily chose to terminate pregnancy. Of the 13 fetuses with adverse pregnancy outcomes, only 2 fetuses had phenotypes consistent with those of diseases caused by AD genes involved in CNL. CONCLUSIONS: The overall prognosis for fetuses without family history or structural abnormalities but with small CNL containing AD genes detected during pregnancy is good. The genetic origin, overlap status of established haploinsufficient gene and/or region, size of the CNL, and genetic mode may affect the pathogenicity of the CNL.

BACKGROUND: Deciphering the hereditary mechanism of seed iron (Fe) and zinc (Zn) content in soybean is important and sustainable to address the "hidden hunger" that presently affects approximately 2 billion people worldwide. Therefore, in order to detect genomic regions related to soybean seed Fe and Zn content, a recombinant inbred line (RIL) population with 248 lines was assessed in four environments to detect Quantitative Trait Loci (QTLs) related to soybean seed Fe and Zn content. RESULT: Wide variation was found in seed Fe and Zn content in four environments, and genotype, environment, and genotype × environment interactions had significant influences on both the seed Fe and Zn content. A positive correlation was observed between seed Fe content and seed Zn content, and broad-sense heritability (H2) of seed Fe and Zn content were 0.73 and 0.75, respectively. In this study, five QTLs for seed Fe content were detected with 4.57 - 32.71% of phenotypic variation explained (PVE) and logarithm of odds (LOD) scores ranging from 3.60 to 33.79. Five QTLs controlling the seed Zn content were detected, and they individually explained 3.35 to 26.48% of the phenotypic variation, with LOD scores ranging from 3.64 to 20.4. Meanwhile, 409,541 high-quality single-nucleotide variants (SNVs) and 85,102 InDels (except intergenic regions) between two bi-parental lines were identified by whole genome resequencing. A total of 12 candidate genes were reported in one major QTL for seed Fe content and two major QTLs for seed Zn content, with the help of RNA-Seq analysis, gene ontology (GO) enrichment, gene annotation, and bi-parental whole genome sequencing (WGS) data. CONCLUSIONS: Limited studies were performed about microelement of soybean, so these results may play an important role in the biofortification of Fe and Zn and accelerate the development of marker-assisted selection (MAS) for breeding soybeans fortified with iron and zinc.

BACKGROUND: While large genome-wide association studies have identified nearly one thousand loci associated with variation in blood pressure, rare variant identification is still a challenge. In family-based cohorts, genome-wide linkage scans have been successful in identifying rare genetic variants for blood pressure. This study aims to identify low frequency and rare genetic variants within previously reported linkage regions on chromosomes 1 and 19 in African American families from the Trans-Omics for Precision Medicine (TOPMed) program. Genetic association analyses weighted by linkage evidence were completed with whole genome sequencing data within and across TOPMed ancestral groups consisting of 60,388 individuals of European, African, East Asian, Hispanic, and Samoan ancestries. RESULTS: Associations of low frequency and rare variants in RCN3 and multiple other genes were observed for blood pressure traits in TOPMed samples. The association of low frequency and rare coding variants in RCN3 was further replicated in UK Biobank samples (N = 403,522), and reached genome-wide significance for diastolic blood pressure (p = 2.01 × 10- 7). CONCLUSIONS: Low frequency and rare variants in RCN3 contributes blood pressure variation. This study demonstrates that focusing association analyses in linkage regions greatly reduces multiple-testing burden and improves power to identify novel rare variants associated with blood pressure traits.

BACKGROUND: Among the Mangifera species, mango (Mangifera indica) is an important commercial fruit crop. However, very few studies have been conducted on the Mangifera mitochondrial genome. This study reports and compares the newly sequenced mitochondrial genomes of three Mangifera species. RESULTS: Mangifera mitochondrial genomes showed partial similarities in the overall size, genomic structure, and gene content. Specifically, the genomes are circular and contain about 63-69 predicted functional genes, including five ribosomal RNA (rRNA) genes and 24-27 transfer RNA (tRNA) genes. The GC contents of the Mangifera mitochondrial genomes are similar, ranging from 44.42-44.66%. Leucine (Leu) and serine (Ser) are the most frequently used, while tryptophan (Trp) and cysteine (Cys) are the least used amino acids among the protein-coding genes in Mangifera mitochondrial genomes. We also identified 7-10 large chloroplast genomic fragments in the mitochondrial genome, ranging from 1407 to 6142 bp. Additionally, four intact mitochondrial tRNAs genes (tRNA-Cys, tRNA-Trp, tRNA-Pro, and tRNA-Met) and intergenic spacer regions were identified. Phylogenetic analysis based on the common protein-coding genes of most branches provided a high support value. CONCLUSIONS: We sequenced and compared the mitochondrial genomes of three Mangifera species. The results showed that the gene content and the codon usage pattern of Mangifera mitochondrial genomes is similar across various species. Gene transfer from the chloroplast genome to the mitochondrial genome were identified. This study provides valuable information for evolutionary and molecular studies of Mangifera and a basis for further studies on genomic breeding of mango.

BACKGROUNDS: Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered posttranslational modification (PTM) and has been identified in several prokaryotic and eukaryotic organisms. Fusarium graminearum, a major pathogen of Fusarium head blight (FHB) in cereal crops, can cause considerable yield loss and produce various mycotoxins that threaten human health. The application of chemical fungicides such as tebuconazole (TEC) remains the major method to control this pathogen. However, the distribution of Khib in F. graminearum and whether Khib is remodified in response to fungicide stress remain unknown. RESULTS: Here, we carried out a proteome-wide analysis of Khib in F. graminearum, identifying the reshaping of the lysine 2-hydroxyisobutyrylome by tebuconazole, using the most recently developed high-resolution LC-MS/MS technique in combination with high-specific affinity enrichment. Specifically, 3501 Khib sites on 1049 proteins were identified, and 1083 Khib sites on 556 modified proteins normalized to the total protein content were changed significantly after TEC treatment. Bioinformatics analysis showed that Khib proteins are involved in a wide range of biological processes and may be involved in virulence and deoxynivalenol (DON) production, as well as sterol biosynthesis, in F. graminearum. CONCLUSIONS: Here, we provided a wealth of resources for further study of the roles of Khib in the fungicide resistance of F. graminearum. The results enhanced our understanding of this PTM in filamentous ascomycete fungi and provided insight into the remodification of Khib sites during azole fungicide challenge in F. graminearum.

BACKGROUND: DNA methylation is thought to influence the expression of genes, especially in response to changing environmental conditions and developmental changes. Sugar beet (Beta vulgaris ssp. vulgaris), and other biennial or perennial plants are inevitably exposed to fluctuating temperatures throughout their lifecycle and might even require such stimulus to acquire floral competence. Therefore, plants such as beets, need to fine-tune their epigenetic makeup to ensure phenotypic plasticity towards changing environmental conditions while at the same time steering essential developmental processes. Different crop species may show opposing reactions towards the same abiotic stress, or, vice versa, identical species may respond differently depending on the specific kind of stress. RESULTS: In this study, we investigated common effects of cold treatment on genome-wide DNA methylation and gene expression of two Beta vulgaris accessions via multi-omics data analysis. Cold exposure resulted in a pronounced reduction of DNA methylation levels, which particularly affected methylation in CHH context (and to a lesser extent CHG) and was accompanied by transcriptional downregulation of the chromomethyltransferase CMT2 and strong upregulation of several genes mediating active DNA demethylation. CONCLUSION: Integration of methylomic and transcriptomic data revealed that, rather than methylation having directly influenced expression, epigenetic modifications correlated with changes in expression of known players involved in DNA (de)methylation. In particular, cold triggered upregulation of genes putatively contributing to DNA demethylation via the ROS1 pathway. Our observations suggest that these transcriptional responses precede the cold-induced global DNA-hypomethylation in non-CpG, preparing beets for additional transcriptional alterations necessary for adapting to upcoming environmental changes.

BACKGROUND: Animal genomes are strikingly conserved in terms of local gene order (microsynteny). While some of these microsyntenies have been shown to be coregulated or to form gene regulatory blocks, the diversity of their genomic and regulatory properties across the metazoan tree of life remains largely unknown. RESULTS: Our comparative analyses of 49 animal genomes reveal that the largest gains of synteny occurred in the last common ancestor of bilaterians and cnidarians and in that of bilaterians. Depending on their node of emergence, we further show that novel syntenic blocks are characterized by distinct functional compositions (Gene Ontology terms enrichment) and gene density properties, such as high, average and low gene density regimes. This is particularly pronounced among bilaterian novel microsyntenies, most of which fall into high gene density regime associated with higher gene coexpression levels. Conversely, a majority of vertebrate novel microsyntenies display a low gene density regime associated with lower gene coexpression levels. CONCLUSIONS: Our study provides first evidence for evolutionary transitions between different modes of microsyntenic block regulation that coincide with key events of metazoan evolution. Moreover, the microsyntenic profiling strategy and interactive online application (Syntenic Density Browser, available at: http://synteny.csb.univie.ac.at/ ) we present here can be used to explore regulatory properties of microsyntenic blocks and predict their coexpression in a wide-range of animal genomes.

BACKGROUND: Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. RESULTS: To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2'-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. CONCLUSIONS: miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.

BACKGROUND: Cashmere goat is famous for its high-quality fibers. The growth of cashmere in secondary hair follicles exhibits a seasonal pattern arising from circannual changes in the natural photoperiod. Although several studies have compared and analyzed the differences in gene expression between different hair follicle growth stages, the selection of samples in these studies relies on research experience or morphological evidence. Distinguishing hair follicle growth cycle according to gene expression patterns may help to explore the regulation mechanisms related to cashmere growth and the effect of melatonin from a molecular level more accurately. RESULTS: In this study, we applied RNA-sequencing to the hair follicles of three normal and three melatonin-treated Inner Mongolian cashmere goats sampled every month during a whole hair follicle growth cycle. A total of 3559 and 988 genes were subjected as seasonal changing genes (SCGs) in the control and treated groups, respectively. The SCGs in the normal group were divided into three clusters, and their specific expression patterns help to group the hair follicle growth cycle into anagen, catagen and telogen stages. Some canonical pathways such as Wnt, TGF-beta and Hippo signaling pathways were detected as promoting the hair follicle growth, while Cell adhesion molecules (CAMs), Cytokine-cytokine receptor interaction, Jak-STAT, Fc epsilon RI, NOD-like receptor, Rap1, PI3K-Akt, cAMP, NF-kappa B and many immune-related pathways were detected in the catagen and telogen stages. The PI3K-Akt signaling, ECM-receptor interaction and Focal adhesion were found in the transition stage between telogen to anagen, which may serve as candidate biomarkers for telogen-anagen regeneration. A total of 16 signaling pathways, 145 pathway mRNAs, and 93 lncRNAs were enrolled to construct the pathway-mRNA-lncRNA network, which indicated the function of lncRNAs through interacting with their co-expressed mRNAs. Pairwise comparisons between the control and melatonin-treated groups also indicated 941 monthly differentially expressed genes (monthly DEGs). These monthly DEGs were mainly distributed from April and September, which revealed a potential signal pathway map regulating the anagen stage triggered by melatonin. Enrichment analysis showed that Wnt, Hedgehog, ECM, Chemokines and NF-kappa B signaling pathways may be involved in the regulation of non-quiescence and secondary shedding under the influence of melatonin. CONCLUSIONS: Our study decoded the key regulators of the whole hair follicle growth cycle, laying the foundation for the control of hair follicle growth and improvement of cashmere yield.

BACKGROUND: Molecular breeding accelerates the speed of animal breeding. Screening molecular markers that can affect economic traits through genome-wide association studies (GWAS) can provide a theoretical basis for molecular breeding. At present, a large number of molecular markers have been screened in poultry research, but few reports on how molecular markers affect economic traits exist. It is particularly important to reveal the action mechanisms of molecular markers, which can provide more accurate information for molecular breeding. RESULTS: The aim of this study was to investigate the relationships between two indels (NUDT15-indel-2777 and NUDT15-indel-1673) in the promoter region of NUDT15 and growth and carcass traits in chickens and to explore the regulatory mechanism of NUDT15. Significant differences were found in genotype and allele frequencies among commercial broilers, commercial laying hens and dual-purpose chickens. The results of association analyses showed that these two indel loci could significantly affect growth traits, such as body weight, and carcass traits. Tissue expression profiling at E12 showed that the expression of NUDT15 was significantly higher in skeletal muscle, and time-expression profiling of leg muscle showed that the expression of NUDT15 in myoblasts was significantly higher in the E10 and E12 proliferation stages than in other stages. Promoter activity analysis showed that pro-1673-I and pro-1673-D significantly inhibited promoter activity, and the promoter activity of pro-1673-D was significantly lower than that of pro-1673-I. In addition, when NUDT15 was overexpressed or underwent interference in chicken primary myoblasts (CPMs), NUDT15 could inhibit the proliferation of CPMs. CONCLUSION: The results suggest that the studied indels in the promoter region of NUDT15 may regulate the proliferation of CPMs by affecting NUDT15 expression, ultimately affecting the growth and carcass traits of chickens. These indel polymorphisms may be used together as molecular markers for improving economic traits in chickens.

BACKGROUND: The collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data. RESULTS: Here, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights. CONCLUSIONS: This novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns .

BACKGROUND: Wucai suffers from low temperature during the growth period, resulting in a decline in yield and poor quality. But the molecular mechanisms of cold tolerance in wucai are still unclear. RESULTS: According to the phenotypes and physiological indexes, we screened out the cold-tolerant genotype "W18" (named CT) and cold-sensitive genotype "Sw-1" (named CS) in six wucai genotypes. We performed transcriptomic analysis using seedling leaves after 24 h of cold treatment. A total of 3536 and 3887 differentially expressed genes (DEGs) were identified between the low temperature (LT) and control (NT) comparative transcriptome in CT and CS, respectively, with 1690 DEGs specific to CT. The gene ontology (GO) analysis showed that the response to cadmium ion (GO:0,046,686), response to jasmonic acid (GO:0,009,753), and response to wounding (GO:0,009,611) were enriched in CT (LT vs NT). The DEGs were enriched in starch and sucrose metabolism and glutathione metabolism in both groups, and α-linolenic acid metabolism was enriched only in CT (LT vs NT). DEGs in these processes, including glutathione S-transferases (GSTs), 13S lipoxygenase (LOX), and jasmonate ZIM-domain (JAZ), as well as transcription factors (TFs), such as the ethylene-responsive transcription factor 53 (ERF53), basic helix-loop-helix 92 (bHLH92), WRKY53, and WRKY54.We hypothesize that these genes play important roles in the response to cold stress in this species. CONCLUSIONS: Our data for wucai is consistent with previous studies that suggest starch and sucrose metabolism increased the content of osmotic substances, and the glutathione metabolism pathway enhance the active oxygen scavenging. These two pathways may participated in response to cold stress. In addition, the activation of α-linolenic acid metabolism may promote the synthesis of methyl jasmonate (MeJA), which might also play a role in the cold tolerance of wucai.

BACKGROUND: Brachyspira (B.) hyodysenteriae is a fastidious anaerobe spirochete that can cause swine dysentery, a severe mucohaemorragic colitis that affects pig production and animal welfare worldwide. In Switzerland, the population of B. hyodysenteriae is characterized by the predominance of macrolide-lincosamide-resistant B. hyodysenteriae isolates of sequence type (ST) ST196, prompting us to obtain deeper insights into the genomic structure and variability of ST196 using pangenome and whole genome variant analyses. RESULTS: The draft genome of 14 B. hyodysenteriae isolates of ST196, sampled during a 7-year period from geographically distant pig herds, was obtained by whole-genome sequencing (WGS) and compared to the complete genome of the B. hyodysenteriae isolate Bh743-7 of ST196 used as reference. Variability results revealed the existence of 30 to 52 single nucleotide polymorphisms (SNPs), resulting in eight sublineages of ST196. The pangenome analysis led to the identification of a novel prophage, pphBhCH20, of the Siphoviridae family in a single isolate of ST196, which suggests that horizontal gene transfer events may drive changes in genomic structure. CONCLUSIONS: This study contributes to the catalogue of publicly available genomes and provides relevant bioinformatic tools and information for further comparative genomic analyses for B. hyodysenteriae. It reveals that Swiss B. hyodysenteriae isolates of the same ST may have evolved independently over time by point mutations and acquisition of larger genetic elements. In line with this, the third type of mobile genetic element described so far in B. hyodysenteriae, the novel prophage pphBhCH20, has been identified in a single isolate of B. hyodysenteriae of ST196.

BACKGROUND: Imputation from genotyping array to whole-genome sequence variants using resequencing of representative reference populations enhances our ability to map genetic factors affecting complex phenotypes in livestock species. The accumulation of knowledge about gene function in human and laboratory animals can provide substantial advantage for genomic research in livestock species. RESULTS: In this study, 201,388 pigs from three commercial Danish breeds genotyped with low to medium (8.5k to 70k) SNP arrays were imputed to whole genome sequence variants using a two-step approach. Both imputation steps achieved high accuracies, and in total this yielded 26,447,434 markers on 18 autosomes. The average estimated imputation accuracy of markers with minor allele frequency ≥ 0.05 was 0.94. To overcome the memory consumption of running genome-wide association study (GWAS) for each breed, we performed within-breed subpopulation GWAS then within-breed meta-analysis for average daily weight gain (ADG), followed by a multi-breed meta-analysis of GWAS summary statistics. We identified 15 quantitative trait loci (QTL). Our post-GWAS analysis strategy to prioritize of candidate genes including information like gene ontology, mammalian phenotype database, differential expression gene analysis of high and low feed efficiency pig and human GWAS catalog for height, obesity, and body mass index, we proposed MRAP2, LEPROT, PMAIP1, ENSSSCG00000036234, BMP2, ELFN1, LIG4 and FAM155A as the candidate genes with biological support for ADG in pigs. CONCLUSION: Our post-GWAS analysis strategy helped to identify candidate genes not just by distance to the lead SNP but also by multiple sources of biological evidence. Besides, the identified QTL overlap with genes which are known for their association with human growth-related traits. The GWAS with this large data set showed the power to map the genetic factors associated with ADG in pigs and have added to our understanding of the genetics of growth across mammalian species.

BACKGROUND: Long non-coding RNAs (lncRNAs) play essential roles in developmental processes and disease development at the transcriptional and post-transcriptional levels across diverse taxa. However, only few studies have profiled fungal lncRNAs in a genome-wide manner during host infection. RESULTS: Infection-associated lncRNAs were identified using lncRNA profiling over six stages of host infection (e.g., vegetative growth, pre-penetration, biotrophic, and necrotrophic stages) in the model pathogenic fungus, Magnaporthe oryzae. We identified 2,601 novel lncRNAs, including 1,286 antisense lncRNAs and 980 intergenic lncRNAs. Among the identified lncRNAs, 755 were expressed in a stage-specific manner and 560 were infection-specifically expressed lncRNAs (ISELs). To decipher the potential roles of lncRNAs during infection, we identified 365 protein-coding genes that were associated with 214 ISELs. Analysis of the predicted functions of these associated genes suggested that lncRNAs regulate pathogenesis-related genes, including xylanases and effectors. CONCLUSIONS: The ISELs and their associated genes provide a comprehensive view of lncRNAs during fungal pathogen-plant interactions. This study expands new insights into the role of lncRNAs in the rice blast fungus, as well as other plant pathogenic fungi.

BACKGROUND: Accurate haplotype reconstruction is required in many applications in quantitative and population genomics. Different phasing methods are available but their accuracy must be evaluated for samples with different properties (population structure, marker density, etc.). We herein took advantage of whole-genome sequence data available for a Holstein cattle pedigree containing 264 individuals, including 98 trios, to evaluate several population-based phasing methods. This data represents a typical example of a livestock population, with low effective population size, high levels of relatedness and long-range linkage disequilibrium. RESULTS: After stringent filtering of our sequence data, we evaluated several population-based phasing programs including one or more versions of AlphaPhase, ShapeIT, Beagle, Eagle and FImpute. To that end we used 98 individuals having both parents sequenced for validation. Their haplotypes reconstructed based on Mendelian segregation rules were considered the gold standard to assess the performance of population-based methods in two scenarios. In the first one, only these 98 individuals were phased, while in the second one, all the 264 sequenced individuals were phased simultaneously, ignoring the pedigree relationships. We assessed phasing accuracy based on switch error counts (SEC) and rates (SER), lengths of correctly phased haplotypes and the probability that there is no phasing error between a pair of SNPs as a function of their distance. For most evaluated metrics or scenarios, the best software was either ShapeIT4.1 or Beagle5.2, both methods resulting in particularly high phasing accuracies. For instance, ShapeIT4.1 achieved a median SEC of 50 per individual and a mean haplotype block length of 24.1 Mb (scenario 2). These statistics are remarkable since the methods were evaluated with a map of 8,400,000 SNPs, and this corresponds to only one switch error every 40,000 phased informative markers. When more relatives were included in the data (scenario 2), FImpute3.0 reconstructed extremely long segments without errors. CONCLUSIONS: We report extremely high phasing accuracies in a typical livestock sample. ShapeIT4.1 and Beagle5.2 proved to be the most accurate, particularly for phasing long segments and in the first scenario. Nevertheless, most tools achieved high accuracy at short distances and would be suitable for applications requiring only local haplotypes.

BACKGROUND: Single-cell CRISPR screens are powerful tools to understand genome function by linking genetic perturbations to transcriptome-wide phenotypes. However, since few cells can be affordably sequenced in these screens, biased sampling of cells could affect data interpretation. One potential source of biased sampling is clonal cell expansion. RESULTS: Here, we identify clonal cells in single cell screens using multiplexed sgRNAs as barcodes. We find that the cells in each clone share transcriptional similarities and bear segmental copy number changes. These analyses suggest that clones are genetically distinct. Finally, we show that the transcriptional similarities of clonally expanded cells contribute to false positives in single-cell CRISPR screens. CONCLUSIONS: Experimental conditions that reduce clonal expansion or computational filtering of clonal cells will improve the reliability of single-cell CRISPR screens.

BACKGROUND: All birds reproduce via internal fertilization, but only ~3% of male birds possess the external genitalia that allows for intromission. Waterfowl (e.g., duck and goose) are representatives of them, and the external genitalia development of male geese is directly related to mating ability. Notably, some male geese show abnormal external genitalia development during ontogenesis. However, until now little is known about the molecular mechanisms of the external genitalia development in goose. In the present study, comparative transcriptomic analyses were performed on the hypothalamus, pituitary gland, testis, and external genitalia isolated from the 245-day-old male Tianfu meat geese showing normal (NEGG, n = 3) and abnormal (AEGG, n = 3) external genitals in order to provide a better understanding of the mechanisms controlling the development of the external genitalia in aquatic bird species. RESULTS: There were 107, 284, 2192, and 1005 differentially expressed genes (DEGs) identified in the hypothalamus, pituitary gland, testis and external genitalia between NEGG and AEGG. Functional enrichment analysis indicated that the DEGs identified in the hypothalamus were mainly enriched in the ECM-receptor interaction pathway. The ECM-receptor interaction, focal adhesion, and neuroactive ligand-receptor interaction pathways were significantly enriched by the DEGs in the pituitary gland. In the testis, the DEGs were enriched in the neuroactive ligand-receptor interaction, cell cycle, oocyte meiosis, and purine metabolism. In the external genitalia, the DEGs were enriched in the metabolic, neuroactive ligand-receptor interaction, and WNT signaling pathways. Furthermore, through integrated analysis of protein-protein interaction (PPI) network and co-expression network, fifteen genes involved in the neuroactive ligand-receptor interaction and WNT signaling pathways were identified, including KNG1, LPAR2, LPAR3, NPY, PLCB1, AVPR1B, GHSR, GRM3, HTR5A, FSHB, FSHR, WNT11, WNT5A, WIF1, and WNT7B, which could play crucial roles in the development of goose external genitalia. CONCLUSIONS: This study is the first systematically comparing the hypothalamus, pituitary gland, testis, and external genitalia transcriptomes of male geese exhibiting normal and abnormal external genitals. Both bioinformatic analysis and validation experiments indicated that the neuroactive ligand-receptor interaction pathway could regulate the WNT signaling pathway through PLCB1 to control male goose external genitalia development.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that differentiates from acute tachyzoite stages to latent bradyzoite forms in response to environmental cues that modify the epigenome. We studied the distribution of the histone variants CenH3, H3.3, H2A.X, H2A.Z and H2B.Z, by genome-wide chromatin immunoprecipitation to understand the role of variant histones in developmental transitions of T. gondii parasites. RESULTS: H3.3 and H2A.X were detected in telomere and telomere associated sequences, whereas H3.3, H2A.X and CenH3 were enriched in centromeres. Histones H2A.Z and H2B.Z colocalize with the transcriptional activation mark H3K4me3 in promoter regions surrounding the nucleosome-free region upstream of the transcription start site. The H2B.Z/H2A.Z histone pair also localizes to the gene bodies of genes that are silent but poised for activation, including bradyzoite stage-specific genes. The majority of H2A.X and H2A.Z/H2B.Z loci do not overlap, consistent with variant histones demarcating specific functional regions of chromatin. The extent of enrichment of H2A.Z/H2B.Z (and H3.3 and H2A.X) within the entire gene (5'UTR and gene body) reflects the timing of gene expression during the cell cycle, suggesting that dynamic turnover of H2B.Z/H2A.Z occurs during the tachyzoite cell cycle. Thus, the distribution of the variant histone H2A.Z/H2B.Z dimer defines active and developmentally silenced regions of the T. gondii epigenome including genes that are poised for expression. CONCLUSIONS: Histone variants mark functional regions of parasite genomes with the dynamic placement of the H2A.Z/H2B.Z dimer implicated as an evolutionarily conserved regulator of parasite and eukaryotic differentiation.

BACKGROUND: The advent of next-generation sequencing technologies empowered a wide variety of transcriptomics studies. A widely studied topic is gene fusion which is observed in many cancer types and suspected of having oncogenic properties. Gene fusions are the result of structural genomic events that bring two genes closely located and result in a fused transcript. This is different from fusion transcripts created during or after the transcription process. These chimeric transcripts are also known as read-through and trans-splicing transcripts. Gene fusion discovery with short reads is a well-studied problem, and many methods have been developed. But the sensitivity of these methods is limited by the technology, especially the short read length. Advances in long-read sequencing technologies allow the generation of long transcriptomics reads at a low cost. Transcriptomic long-read sequencing presents unique opportunities to overcome the shortcomings of short-read technologies for gene fusion detection while introducing new challenges. RESULTS: We present Genion, a sensitive and fast gene fusion detection method that can also detect read-through events. We compare Genion against a recently introduced long-read gene fusion discovery method, LongGF, both on simulated and real datasets. On simulated data, Genion accurately identifies the gene fusions and its clustering accuracy for detecting fusion reads is better than LongGF. Furthermore, our results on the breast cancer cell line MCF-7 show that Genion correctly identifies all the experimentally validated gene fusions. CONCLUSIONS: Genion is an accurate gene fusion caller. Genion is implemented in C++ and is available at https://github.com/vpc-ccg/genion .

BACKGROUND: The Natterin protein family was first discovered in the venom of the medically significant fish Thalassophryne nattereri, and over the last decade natterin-like genes have been identified in various organisms, notably performing immune-related functions. Previous findings support natterin-like genes as effector defense molecules able to activate multiprotein complexes driving the host innate immune response, notably due to the pore-forming function of the aerolysin superfamily members. Herein, employing a combination of the CRISPR/Cas9 depletion system, phenotype-based screening, and morphometric methods, we evaluated the role of one family member, LOC795232, in the embryonic development of zebrafish since it might be implicated in multiple roles and characterization of the null mutant is central for analysis of gene activity. RESULTS: Multiple sequence alignment revealed that the candidate natterin-like has the highest similarity to zebrafish aep1, a putative and better characterized fish-specific defense molecule from the same family. Compared to other species, zebrafish have many natterin-like copies. Whole-mount in situ hybridization confirmed the knockout and mutant embryos exhibited epiboly delay, growth retardation, yolk sac and heart edema, absent or diminished swim bladder, spinal defects, small eyes and head, heart dysfunction, and behavioral impairment. As previously demonstrated, ribonucleoproteins composed of Cas9 and duplex guide RNAs are effective at inducing mutations in the F0 zebrafish. CONCLUSIONS: The considerably high natterin-like copies in zebrafish compared to other species might be due to the teleost-specific whole genome duplication and followed by subfunctionalization or neofunctionalization. In the present work, we described some of the natterin-like features in the zebrafish development and infer that natterin-like proteins potentially contribute to the embryonary development and immune response.

BACKGROUND: Plant volatiles play an important role in both plant-pollinator and plant-herbivore interactions. Intraspecific polymorphisms in volatile production are ubiquitous, but studies that explore underlying differential gene expression are rare. Oenothera harringtonii populations are polymorphic in floral emission of the monoterpene (R)-(-)-linalool; some plants emit (R)-(-)-linalool (linalool+ plants) while others do not (linalool- plants). However, the genes associated with differential production of this floral volatile in Oenothera are unknown. We used RNA-Seq to broadly characterize differential gene expression involved in (R)-(-)-linalool biosynthesis. To identify genes that may be associated with the polymorphism for this trait, we used RNA-Seq to compare gene expression in six different Oenothera harringtonii tissues from each of three linalool+ and linalool- plants. RESULTS: Three clusters of differentially expressed genes were enriched for terpene synthase activity: two were characterized by tissue-specific upregulation and one by upregulation only in plants with flowers that produce (R)-(-)-linalool. A molecular phylogeny of all terpene synthases identified two putative (R)-(-)-linalool synthase transcripts in Oenothera harringtonii, a single allele of which is found exclusively in linalool+ plants. CONCLUSIONS: By using a naturally occurring polymorphism and comparing different tissues, we were able to identify candidate genes putatively involved in the biosynthesis of (R)-(-)-linalool. Expression of these genes in linalool- plants, while low, suggests a regulatory polymorphism, rather than a population-specific loss-of-function allele. Additional terpene biosynthesis-related genes that are up-regulated in plants that emit (R)-(-)-linalool may be associated with herbivore defense, suggesting a potential economy of scale between plant reproduction and defense.

Identifying the genes underlying fitness-related traits such as body size and male ornamentation can provide tools for conservation and management and are often subject to various selective pressures. Here we performed high-depth whole genome re-sequencing of pools of individuals representing the phenotypic extremes for antler and body size in white-tailed deer (Odocoileus virginianus). Samples were selected from a tissue repository containing phenotypic data for 4,466 male white-tailed deer from Anticosti Island, Quebec, with four pools representing the extreme phenotypes for antler and body size after controlling for age. Our results revealed a largely homogenous population but detected highly divergent windows between pools for both traits, with the mean allele frequency difference of 14% for and 13% for antler and body SNPs in outlier windows, respectively. Genes in outlier antler windows were enriched for pathways associated with cell death and protein metabolism and some of the most differentiated windows included genes associated with oncogenic pathways and reproduction, processes consistent with antler evolution and growth. Genes associated with body size were more nuanced, suggestive of a highly complex trait. Overall, this study revealed the complex genomic make-up of both antler morphology and body size in free-ranging white-tailed deer and identified target loci for additional analyses.

BACKGROUND: As one of the largest transcription factor families in plants, the APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily is involved in various biological processes and plays significant roles in plant growth, development and responses to various stresses. Although identification and characterization of AP2/ERF superfamily genes have been accomplished in many plant species, very little is known regarding the structure and function of AP2/ERF genes in maize. RESULTS: In this study, a total of 214 genes encoding ZmAP2/ERF proteins with complete AP2/ERF domain were eventually identified according to the AGPv4 version of the maize B73 genome. Based on the number of AP2/ERF domain and similarities of amino acid sequences among AP2/ERF proteins from Arabidopsis, rice and maize, all 214 putative ZmAP2/ERF proteins were categorized into three distinct families, including the AP2 family (44), the ERF family (166) and the RAV family (4), respectively. Among them, the ERF family was further subdivided into two diverse subfamilies, including the DREB and ERF subfamilies with 61 and 105 members, respectively. Further, based on phylogenetic analysis, the members of DREB and ERF subfamilies were subdivided into four (Group I-IV) and eight (Group V-XII) groups, respectively. The characteristics of exon-intron structure of these putative ZmAP2/ERF genes and conserved protein motifs of their encoded ZmAP2/ERF proteins were also presented respectively, which was in accordance with the results of group classification. Promoter analysis suggested that ZmAP2/ERF genes shared many stress- and hormone-related cis-regulatory elements. Gene duplication and synteny analysis revealed that tandem or segmental duplication and purifying selection might play significant roles in evolution and functional differentiation of AP2/ERF superfamily genes among three various gramineous species (maize, rice and sorghum). Using RNA-seq data, transcriptome analysis indicated that the majority of ZmAP2/ERF genes displayed differential expression patterns at different developmental stages of maize. In addition, the following analyses of co-expression network among ZmAP2/ERF genes and protein protein interaction between ZmAP2 and ZmERF proteins further enabled us to understand the regulatory relationship among members of the AP2/ERF superfamily in maize. Furthermore, by quantitative real-time PCR analysis, twenty-seven selected ZmAP2/ERF genes were further confirmed to respond to three different abiotic stresses, suggesting their potential roles in various abiotic stress responses. Collectively, these results revealed that these ZmAP2/ERF genes play essential roles in abiotic stress tolerance. CONCLUSIONS: Taken together, the present study will serve to present an important theoretical basis for further exploring the function and regulatory mechanism of ZmAP2/ERF genes in the growth, development, and adaptation to abiotic stresses in maize.

BACKGROUND: More than 2 million SARS-CoV-2 genome sequences have been generated and shared since the start of the COVID-19 pandemic and constitute a vital information source that informs outbreak control, disease surveillance, and public health policy. The Pango dynamic nomenclature is a popular system for classifying and naming genetically-distinct lineages of SARS-CoV-2, including variants of concern, and is based on the analysis of complete or near-complete virus genomes. However, for several reasons, nucleotide sequences may be generated that cover only the spike gene of SARS-CoV-2. It is therefore important to understand how much information about Pango lineage status is contained in spike-only nucleotide sequences. Here we explore how Pango lineages might be reliably designated and assigned to spike-only nucleotide sequences. We survey the genetic diversity of such sequences, and investigate the information they contain about Pango lineage status. RESULTS: Although many lineages, including the main variants of concern, can be identified clearly using spike-only sequences, some spike-only sequences are shared among tens or hundreds of Pango lineages. To facilitate the classification of SARS-CoV-2 lineages using subgenomic sequences we introduce the notion of designating such sequences to a "lineage set", which represents the range of Pango lineages that are consistent with the observed mutations in a given spike sequence. CONCLUSIONS: We find that many lineages, including the main variants-of-concern, can be reliably identified by spike alone and we define lineage-sets to represent the lineage precision that can be achieved using spike-only nucleotide sequences. These data provide a foundation for the development of software tools that can assign newly-generated spike nucleotide sequences to Pango lineage sets.

BACKGROUND: Mammalian sex chromosomes provide dosage compensation, but avian lack a global mechanism of dose compensation. Herein, we employed nanopore sequencing to investigate the genetic basis of gene expression and gene dosage effects in avian Z chromosomes at the posttranscriptional level. RESULTS: In this study, the gonad and head skin of female and male duck samples (n = 4) were collected at 16 weeks of age for Oxford nanopore sequencing. Our results revealed a dosage effect and local regulation of duck Z chromosome gene expression. Additionally, AS and APA achieve tissue-specific gene expression, and male-biased lncRNA regulates its Z-linked target genes, with a positive regulatory role for gene dosage effects on the duck Z chromosome. In addition, GO enrichment and KEGG pathway analysis showed that the dosage effects of Z-linked genes were mainly associated with the cellular response to hormone stimulus, melanin biosynthetic, metabolic pathways, and melanogenesis, resulting in sex differences. CONCLUSIONS: Our data suggested that post transcriptional regulation (AS, APA and lncRNA) has a potential impact on the gene expression effects of avian Z chromosomes. Our study provides a new view of gene regulation underlying the dose effects in avian Z chromosomes at the RNA post transcriptional level.

BACKGROUND: Aedes aegypti is a medically-important mosquito vector that transmits arboviruses including yellow fever, dengue, chikungunya, and Zika viruses to humans. The mosquito exhibits typical sexually dimorphic behaviors such as courtship, mating, host seeking, bloodfeeding, and oviposition. All these behaviors are mainly regulated by the brain; however, little is known about the function and neuron composition of the mosquito brain. In this study, we generated an initial atlas of the adult male and female brain of Ae. aegypti using 10xGenomics based single-nucleus RNA sequencing. RESULTS: We identified 35 brain cell clusters in male and female brains, and 15 of those clusters were assigned to known cell types. Identified cell types include glia (astrocytes), Kenyon cells, (ventral) projection neurons, monoaminergic neurons, medulla neurons, and proximal medulla neurons. In addition, the cell type compositions of male and female brains were compared to each other showing that they were quantitatively distinct, as 17 out of 35 cell clusters varied significantly in their cell type proportions. Overall, the transcriptomes from each cell cluster looked very similar between the male and female brain as only up to 25 genes were differentially expressed in these clusters. The sex determination factor Nix was highly expressed in neurons and glia of the male brain, whereas doublesex (dsx) was expressed in all neuron and glia cell clusters of the male and female brain. CONCLUSIONS: An initial cell atlas of the brain of the mosquito Ae. aegypti has been generated showing that the cellular compositions of the male and female brains of this hematophagous insect differ significantly from each other. Although some of the rare brain cell types have not been detected in our single biological replicate, this study provides an important basis for the further development of a complete brain cell atlas as well as a better understanding of the neurobiology of the brains of male and female mosquitoes and their sexually dimorphic behaviors.

BACKGROUND: Nanopore sequencing technology has revolutionized the field of genome biology with its ability to generate extra-long reads that can resolve regions of the genome that were previously inaccessible to short-read sequencing platforms. Over 50% of the zebrafish genome consists of difficult to map, highly repetitive, low complexity elements that pose inherent problems for short-read sequencers and assemblers. RESULTS: We used long-read nanopore sequencing to generate a de novo assembly of the zebrafish genome and compared our assembly to the current reference genome, GRCz11. The new assembly identified 1697 novel insertions and deletions over one kilobase in length and placed 106 previously unlocalized scaffolds. We also discovered additional sites of retrotransposon integration previously unreported in GRCz11 and observed the expression of these transposable elements in adult zebrafish under physiologic conditions, implying they have active mobility in the zebrafish genome and contribute to the ever-changing genomic landscape. CONCLUSIONS: We used nanopore sequencing to improve upon and resolve the issues plaguing the current zebrafish reference assembly, GRCz11. Zebrafish is a prominent model of human disease, and our corrected assembly will be useful for studies relying on interspecies comparisons and precise linkage of genetic events to disease phenotypes.

BACKGROUND: DNA methylation has an important role in intergenerational inheritance. An increasing number of studies have reported evidence of germline inheritance of DNA methylation induced by nutritional signals in mammals. Vitamins and minerals as micronutrients contribute to growth performance in vertebrates, including Atlantic salmon (Salmo salar), and also have a role in epigenetics as environmental factors that alter DNA methylation status. It is important to understand whether micronutrients in the paternal diet can influence the offspring through alterations of DNA methylation signatures in male germ cells. RESULTS: Here, we show the effect of micronutrient supplementation on DNA methylation profiles in the male gonad through a whole life cycle feeding trial of Atlantic salmon fed three graded levels of micronutrient components. Our results strongly indicate that micronutrient supplementation affects the DNA methylation status of genes associated with cell signalling, synaptic signalling, and embryonic development. In particular, it substantially affects DNA methylation status in the promoter region of a glutamate receptor gene, glutamate receptor ionotropic, NMDA 3A-like (grin3a-like), when the fish are fed both medium and high doses of micronutrients. Furthermore, two transcription factors, histone deacetylase 2 (hdac2) and a zinc finger protein, bind to the hyper-methylated site in the grin3a-like promoter. An estimated function of hdac2 together with a zinc finger indicates that grin3a-like has a potential role in intergenerational epigenetic inheritance and the regulation of embryonic development affected by paternal diet. CONCLUSIONS: The present study demonstrates alterations of gene expression patterns and DNA methylation signatures in the male gonad when Atlantic salmon are fed different levels of micronutrients. Alterations of gene expression patterns are of great interest because the gonads are supposed to have limited metabolic activities compared to other organs, whereas alterations of DNA methylation signatures are of great importance in the field of nutritional epigenetics because the signatures affected by nutrition could be transferred to the next generation. We provide extensive data resources for future work in the context of potential intergenerational inheritance through the male germline.

BACKGROUND: Meiotic recombination plays an important role in reproduction and evolution. The individual global recombination rate (GRR), measured as the number of crossovers (CO) per gametes, is a complex trait that has been shown to be heritable. The sex chromosomes play an important role in reproduction and fertility related traits. Therefore, variants present on the X-chromosome might have a high contribution to the genetic variation of GRR that is related to meiosis and to reproduction. RESULTS: We herein used genotyping data from 58,474 New Zealand dairy cattle to estimate the contribution of the X-chromosome to male and female GRR levels. Based on the pedigree-based relationships, we first estimated that the X-chromosome accounted for 30% of the total additive genetic variance for male GRR. This percentage was equal to 19.9% when the estimation relied on a SNP-BLUP approach assuming each SNP has a small contribution. We then carried out a haplotype-based association study to map X-linked QTL, and subsequently fine-mapped the identified QTL with imputed sequence variants. With this approach we identified three QTL with large effect accounting for 7.7% of the additive genetic variance of male GRR. The associated effects were equal to + 0.79, - 1.16 and + 1.18 CO for the alternate alleles. In females, the estimated contribution of the X-chromosome to GRR was null and no significant association with X-linked loci was found. Interestingly, two of the male GRR QTL were associated with candidate genes preferentially expressed in testis, in agreement with a male-specific effect. Finally, the most significant QTL was associated with PPP4R3C, further supporting the important role of protein phosphatase in double-strand break repair by homologous recombination. CONCLUSIONS: Our study illustrates the important role the X-chromosome can have on traits such as individual recombination rate, associated with testis in males. We also show that contribution of the X-chromosome to such a trait might be sex dependent.

BACKGROUND: Nucleotide-binding and leucine-rich repeat (NLR) genes have attracted wide attention due to their crucial role in protecting plants from pathogens. SMRT-RenSeq, combining PacBio sequencing after resistance gene enrichment sequencing (RenSeq), is a powerful method for selectively capturing and sequencing full-length NLRs. Haynaldia villosa, a wild grass species with a proven potential for wheat improvement, confers resistance to multiple diseases. So, genome-wide identification of the NLR gene family in Haynaldia villosa by SMRT-RenSeq can facilitate disease resistance genes exploration. RESULTS: In this study, SMRT-RenSeq was performed to identify the genome-wide NLR complement of H. villosa. In total, 1320 NLRs were annotated in 1169 contigs, including 772 complete NLRs. All the complete NLRs were phylogenetically analyzed and 11 main clades with special characteristics were derived. NLRs could be captured with high efficiency when aligned with cloned R genes, and cluster expansion in some specific gene loci was observed. The physical location of NLRs to individual chromosomes in H. villosa showed a perfect homoeologous relationship with group 1, 2, 3, 5 and 6 of other Triticeae species, however, NLRs physically located on 4VL were largely in silico predicted to be located on the homoeologous group 7. Fifteen types of integrated domains (IDs) were integrated in 52 NLRs, and Kelch and B3 NLR-IDs were found to have expanded in H. villosa, while DUF948, NAM-associated and PRT_C were detected as unique integrated domains implying the new emergence of NLR-IDs after H. villosa diverged from other species. CONCLUSION: SMRT-RenSeq is a powerful tool to identify NLR genes from wild species using the baits of the evolutionary related species with reference sequences. The availability of the NLRs from H. villosa provide a valuable library for R gene mining and transfer of disease resistance into wheat.

BACKGROUND: Lentinula edodes (Berk.) is the second most productive mushroom in the world. It contains compounds effective for antiviral, antitumor, antioxidant and immune regulation. Although genomes have previously been reported for this species, a high-quality chromosome-level reference for L. edodes is unavailable. This hinders detailed investigation of population genetics, breeding history of strains and genes related to environmental stress responses. RESULTS: A high-quality chromosome-level genome was constructed. We separated a monokaryon from protoplasts of the commercial L. edodes strain L808 and assembled the genome of L. edodes using PacBio long-read and Illumina short-read sequencing, along with the high-throughput chromatin conformation capture (Hi-C) technique. We assembled a 45.87 Mb genome, and 99% of the sequences were anchored onto 10 chromosomes. The contig and scaffold N50 length were 2.17 and 4.94 Mb, respectively. Over 96% of the complete Benchmarking Universal Single-Copy Orthologs (BUSCO) were identified, and 9853 protein-coding genes were predicted. We performed population genome resequencing using 34 wild strains and 65 commercial cultivars of L. edodes originating from China, Japan, the United States and Australia. Based on whole-genome variants, we showed substantial differences in the Chinese wild population, which divided into different branches according to the main areas of their geographical distribution. We also determined the breeding history of L. edodes at the molecular level, and demonstrated that the cultivated strains in China mainly originated from wild strains from China and Northeast Asia. Phenotypic analysis showed that 99 strains exhibited differences on the Cd accumulation. Three significant loci in the of L. edodes genome were identified using the genome-wide association study (GWAS) of Cd accumulation traits. Functional genes associated with Cd accumulation traits were related to DNA ligase and aminoacyl tRNA synthetase, indicating that DNA damage repair and in vivo protein translation may be responses to Cd stress. CONCLUSIONS: A high-quality chromosome-level genome and population genetic data of L. edodes provide genetic resources for functional genomic, evolutionary and artificial breeding studies for L. edodes.

BACKGROUND: As the major source of sugar in moderate climates, sugar-producing beets (Beta vulgaris subsp. vulgaris) have a high economic value. However, the low genetic diversity within cultivated beets requires introduction of new traits, for example to increase their tolerance and resistance attributes - traits that often reside in the crop wild relatives. For this, genetic information of wild beet relatives and their phylogenetic placements to each other are crucial. To answer this need, we sequenced and assembled the complete plastome sequences from a broad species spectrum across the beet genera Beta and Patellifolia, both embedded in the Betoideae (order Caryophyllales). This pan-plastome dataset was then used to determine the wild beet phylogeny in high-resolution. RESULTS: We sequenced the plastomes of 18 closely related accessions representing 11 species of the Betoideae subfamily and provided high-quality plastome assemblies which represent an important resource for further studies of beet wild relatives and the diverse plant order Caryophyllales. Their assembly sizes range from 149,723 bp (Beta vulgaris subsp. vulgaris) to 152,816 bp (Beta nana), with most variability in the intergenic sequences. Combining plastome-derived phylogenies with read-based treatments based on mitochondrial information, we were able to suggest a unified and highly confident phylogenetic placement of the investigated Betoideae species. Our results show that the genus Beta can be divided into the two clearly separated sections Beta and Corollinae. Our analysis confirms the affiliation of B. nana with the other Corollinae species, and we argue against a separate placement in the Nanae section. Within the Patellifolia genus, the two diploid species Patellifolia procumbens and Patellifolia webbiana are, regarding the plastome sequences, genetically more similar to each other than to the tetraploid Patellifolia patellaris. Nevertheless, all three Patellifolia species are clearly separated. CONCLUSION: In conclusion, our wild beet plastome assemblies represent a new resource to understand the molecular base of the beet germplasm. Despite large differences on the phenotypic level, our pan-plastome dataset is highly conserved. For the first time in beets, our whole plastome sequences overcome the low sequence variation in individual genes and provide the molecular backbone for highly resolved beet phylogenomics. Hence, our plastome sequencing strategy can also guide genomic approaches to unravel other closely related taxa.

BACKGROUND: The formation and functioning of muscles are fundamental aspects of animal biology, and the evolution of 'muscle genes' is central to our understanding of this tissue. Feeding-fasting-refeeding experiments have been widely used to assess muscle cellular and metabolic responses to nutrition. Though these studies have focused on vertebrate models and only a few invertebrate systems, they have found similar processes are involved in muscle degradation and maintenance. Motivation for these studies stems from interest in diseases whose pathologies involve muscle atrophy, a symptom also triggered by fasting, as well as commercial interest in the muscle mass of animals kept for consumption. Experimentally modelling atrophy by manipulating nutritional state causes muscle mass to be depleted during starvation and replenished with refeeding so that the genetic mechanisms controlling muscle growth and degradation can be understood. RESULTS: Using amphioxus, the earliest branching chordate lineage, we address the gap in previous work stemming from comparisons between distantly related vertebrate and invertebrate models. Our amphioxus feeding-fasting-refeeding muscle transcriptomes reveal a highly conserved myogenic program and that the pro-orthologues of many vertebrate myoblast fusion genes were present in the ancestral chordate, despite these invertebrate chordates having unfused mononucleate myocytes. We found that genes differentially expressed between fed and fasted amphioxus were orthologous to the genes that respond to nutritional state in vertebrates. This response is driven in a large part by the highly conserved IGF/Akt/FOXO pathway, where depleted nutrient levels result in activation of FOXO, a transcription factor with many autophagy-related gene targets. CONCLUSION: Reconstruction of these gene networks and pathways in amphioxus muscle provides a key point of comparison between the distantly related groups assessed thus far, significantly refining the reconstruction of the ancestral state for chordate myoblast fusion genes and identifying the extensive role of duplicated genes in the IGF/Akt/FOXO pathway across animals. Our study elucidates the evolutionary trajectory of muscle genes as they relate to the increased complexity of vertebrate muscles and muscle development.

BACKGROUND: Novel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 μg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits. RESULTS: We compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits' fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection. CONCLUSIONS: The enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.