High-confidence Genes Research Articles

Genome-Wide CRISPR/Cas9 Screen Identifies New Genes Critical for Defense Against Oxidant Stress in Toxoplasma gondii

Toxoplasma gondii is one of the most widespread apicomplexans and can cause serious infections in humans and animals. Its antioxidant system plays an important role in defending against oxidant stress imposed by the host. Some genes encoding the antioxidant enzymes of T. gondii have been identified; however, critical genes that function in response to reactive oxygen species (ROS) stress are still poorly understood. Here, we performed genome-wide CRISPR/Cas9 loss-of-function screening in the T. gondii RH strain to identify potential genes contributing to the ROS stress response. Under hydrogen peroxide treatment, 30 single guide RNAs targeting high-confidence genes were identified, including some known important antioxidant genes such as catalase and peroxiredoxin PRX3. In addition, several previously uncharacterized genes were identified, among which five hypothetical protein-coding genes, namely, HP1–HP5, were selected for further functional characterization. Targeted deletion of HP1 in T. gondii RH led to significant sensitivity to H2O2, suggesting that HP1 is critical for oxidative stress management. Furthermore, loss of HP1 led to decreased antioxidant capacity, invasion efficiency, and proliferation in vitro. In vivo results also revealed that the survival time of mice infected with the HP1-KO strain was significantly prolonged relative to that of mice infected with the wild-type strain. Altogether, these findings demonstrate that the CRISPR/Cas9 system can be used to identify potential genes critical for oxidative stress management. Furthermore, HP1 may confer protection against oxidative damage and contributes to T. gondii virulence in mice.

The chromosome-level Stevia genome provides insights into steviol glycoside biosynthesis

Stevia (Stevia rebaudiana Bertoni) is well known for its very sweet steviol glycosides (SGs) consisting of a common tetracyclic diterpenoid steviol backbone and a variable glycone. Steviol glycosides are 150–300 times sweeter than sucrose and are used as natural zero-calorie sweeteners. However, the most promising compounds are biosynthesized in small amounts. Based on Illumina, PacBio, and Hi-C sequencing, we constructed a chromosome-level assembly of Stevia covering 1416 Mb with a contig N50 value of 616.85 kb and a scaffold N50 value of 106.55 Mb. More than four-fifths of the Stevia genome consisted of repetitive elements. We annotated 44,143 high-confidence protein-coding genes in the high-quality genome. Genome evolution analysis suggested that Stevia and sunflower diverged ~29.4 million years ago (Mya), shortly after the whole-genome duplication (WGD) event (WGD-2, ~32.1 Mya) that occurred in their common ancestor. Comparative genomic analysis revealed that the expanded genes in Stevia were mainly enriched for biosynthesis of specialized metabolites, especially biosynthesis of terpenoid backbones, and for further oxidation and glycosylation of these compounds. We further identified all candidate genes involved in SG biosynthesis. Collectively, our current findings on the Stevia reference genome will be very helpful for dissecting the evolutionary history of Stevia and for discovering novel genes contributing to SG biosynthesis and other important agronomic traits in future breeding programs.

Chromosome-level genome assembly and manually-curated proteome of model necrotroph Parastagonospora nodorum Sn15 reveals a genome-wide trove of candidate effector homologs, and redundancy of virulence-related functions within an accessory chromosome

BackgroundThe fungus Parastagonospora nodorum causes septoria nodorum blotch (SNB) of wheat (Triticum aestivum) and is a model species for necrotrophic plant pathogens. The genome assembly of reference isolate Sn15 was first reported in 2007. P. nodorum infection is promoted by its production of proteinaceous necrotrophic effectors, three of which are characterised – ToxA, Tox1 and Tox3.ResultsA chromosome-scale genome assembly of P. nodorum Australian reference isolate Sn15, which combined long read sequencing, optical mapping and manual curation, produced 23 chromosomes with 21 chromosomes possessing both telomeres. New transcriptome data were combined with fungal-specific gene prediction techniques and manual curation to produce a high-quality predicted gene annotation dataset, which comprises 13,869 high confidence genes, and an additional 2534 lower confidence genes retained to assist pathogenicity effector discovery. Comparison to a panel of 31 internationally-sourced isolates identified multiple hotspots within the Sn15 genome for mutation or presence-absence variation, which was used to enhance subsequent effector prediction. Effector prediction resulted in 257 candidates, of which 98 higher-ranked candidates were selected for in-depth analysis and revealed a wealth of functions related to pathogenicity. Additionally, 11 out of the 98 candidates also exhibited orthology conservation patterns that suggested lateral gene transfer with other cereal-pathogenic fungal species. Analysis of the pan-genome indicated the smallest chromosome of 0.4 Mbp length to be an accessory chromosome (AC23). AC23 was notably absent from an avirulent isolate and is predominated by mutation hotspots with an increase in non-synonymous mutations relative to other chromosomes. Surprisingly, AC23 was deficient in effector candidates, but contained several predicted genes with redundant pathogenicity-related functions.ConclusionsWe present an updated series of genomic resources for P. nodorum Sn15 – an important reference isolate and model necrotroph – with a comprehensive survey of its predicted pathogenicity content.

Peptide-tiling screens of cancer drivers reveal oncogenic protein domains and associated peptide inhibitors.

Gene fragments derived from structural domains mediating physical interactions can modulate biological functions. Utilizing this, we developed lentiviral overexpression libraries of peptides comprehensively tiling high-confidence cancer driver genes. Toward inhibiting cancer growth, we assayed ~66,000 peptides, tiling 65 cancer drivers and 579 mutant alleles. Pooled fitness screens in two breast cancer cell lines revealed peptides, which selectively reduced cellular proliferation, implicating oncogenic protein domains important for cell fitness. Coupling of cell-penetrating motifs to these peptides enabled drug-like function, with peptides derived from EGFR and RAF1 inhibiting cell growth at IC50s of 27-63μM. We anticipate that this peptide-tiling (PepTile) approach will enable rapid de novo mapping of bioactive protein domains and associated interfering peptides.

SARS-CoV-2 gene content and COVID-19 mutation impact by comparing 44 Sarbecovirus genomes

Despite its clinical importance, the SARS-CoV-2 gene set remains unresolved, hindering dissection of COVID-19 biology. We use comparative genomics to provide a high-confidence protein-coding gene set, characterize evolutionary constraint, and prioritize functional mutations. We select 44 Sarbecovirus genomes at ideally-suited evolutionary distances, and quantify protein-coding evolutionary signatures and overlapping constraint. We find strong protein-coding signatures for ORFs 3a, 6, 7a, 7b, 8, 9b, and a novel alternate-frame gene, ORF3c, whereas ORFs 2b, 3d/3d-2, 3b, 9c, and 10 lack protein-coding signatures or convincing experimental evidence of protein-coding function. Furthermore, we show no other conserved protein-coding genes remain to be discovered. Mutation analysis suggests ORF8 contributes to within-individual fitness but not person-to-person transmission. Cross-strain and within-strain evolutionary pressures agree, except for fewer-than-expected within-strain mutations in nsp3 and S1, and more-than-expected in nucleocapsid, which shows a cluster of mutations in a predicted B-cell epitope, suggesting immune-avoidance selection. Evolutionary histories of residues disrupted by spike-protein substitutions D614G, N501Y, E484K, and K417N/T provide clues about their biology, and we catalog likely-functional co-inherited mutations. Previously reported RNA-modification sites show no enrichment for conservation. Here we report a high-confidence gene set and evolutionary-history annotations providing valuable resources and insights on SARS-CoV-2 biology, mutations, and evolution.

Genetic analysis of grain protein content and dough quality traits in elite spring bread wheat (Triticum aestivum) lines through association study

Wheat quality parameters are complex traits predominantly influenced by the genetic background of varieties. A total of 802 spring bread wheat breeding lines were tested at Marchouch station, Morocco in 2018 aimed at mining invaluable QTNs for grain protein content (PR), gluten content (GL), alveograph strength (W) and zeleny sedimentation test (Z). A single-locus (MLM) and multi-locus (FarmCPU) GWAS models were implemented using 5,095 SNP markers generated from the genotyping-by-sequencing (GBS) platform. A total of 36 and 92 QTNs were detected for the four quality traits from the MLM and FarmCPU GWAS models, respectively. The FarmCPU model detected markers on homoeologous chromosome group one having highly significant QTNs with PR, GL, W and Z traits. The two implemented GWAS models detected significant QTNs on the short arm of homoeologous chromosome groups 2 and 6 for GL, PR, W and Z traits. FarmCPU model detected SNP_615302 marker on chromosome 3B with highly significant MTAs and concurrent effect for PR, GL and W traits. Moreover, significant MTAs were detected on chromosomes 3D, 4A, 4B, 5A, 5B, 7A, 7B and 7D for the four studied quality traits across the two GWAS models. The in-silico gene annotation for QTNs using the high confidence gene models detected genes involved in numerous functions including regulating plant growth, development and tolerance to biotic and abiotic stresses. The identified markers could potentially be useful tool for quality improvement through marker-assisted selection in future breeding programs.

Role of CNTNAP2 in autism manifestation outlines the regulation of signaling between neurons at the synapse

BackgroundAutism is characterized by high heritability and a complex genetic mutational landscape with restricted social behavior and impaired social communication. Whole-exome sequencing is a reliable tool to pinpoint variants for unraveling the disease pathophysiology. The present meta-analysis was performed using 222 whole-exome sequences deposited by Simons Simplex Collection (SSC) at the European Nucleotide Archive. This sample cohort was used to identify causal mutations in autism-specific genes to create a mutational landscape focusing on the CNTNAP2 gene.ResultsThe authors account for the identification of 15 high confidence genes with 24 variants for autism with Simons Foundation Autism Research Initiative (SFARI) gene scoring. These genes encompass critical autism pathways such as neuron development, synapse complexity, cytoskeleton, and microtubule activation. Among these 15 genes, overlapping variants were present across multiple samples: KMT2C in 167 cases, CNTNAP2 in 192 samples, CACNA1C in 152 cases, and SHANK3 in 124 cases. Pathway analysis identifies clustering and interplay of autism genes—WDFY3, SHANK2, CNTNAP2, HOMER1, SYNGAP1, and ANK2 with CNTNAP2. These genes coincide across autism-relevant pathways, namely abnormal social behavior and intellectual and cognitive impairment. Based on multiple layers of selection criteria, CNTNAP2 was chosen as the master gene for the study. It is an essential gene for autism with speech-language delays, a typical phenotype in most cases under study. It showcases nine variants across multiple samples with one damaging variant, T589P, with a GERP rank score range of 0.065–0.95. This unique variant was present across 86.5% of the samples impairing the epithelial growth factor (EGF) domain. Established microRNA (miRNA) genes hsa-mir-548aq and hsa-mir-548f were mutated within the CNTNAP2 region, adding to the severity. The mutated protein showed reduced stability by 0.25, increased solvent accessibility by 9%, and reduced depth by 0.2, which rendered the protein non-functional. Secondary physical interactors of CNTNAP2 through CNTN2 proteins were mutated in the samples, further intensifying the severity.ConclusionCNTNAP2 has been identified as a master gene in autism manifestation responsible for speech-language delay by impairing the EGF protein domain and downstream cascade. The decrease in EGF is correlated with vital autism symptoms, especially language disabilities.

Connecting tree-ring phenotypes, genetic associations and transcriptomics to decipher the genomic architecture of drought adaptation in a widespread conifer.

As boreal forests face significant threats from climate change, understanding evolutionary trajectories of coniferous species has become fundamental to adapting management and conservation to a drying climate. We examined the genomic architecture underlying adaptive variation related to drought tolerance in 43 populations of a widespread boreal conifer, white spruce (Picea glauca [Moench] Voss), by combining genotype–environment associations, genotype–phenotype associations, and transcriptomics. Adaptive genetic variation was identified by correlating allele frequencies for 6,153 single nucleotide polymorphisms from 2,606 candidate genes with temperature, precipitation and aridity gradients, and testing for significant associations between genotypes and 11 dendrometric and drought‐related traits (i.e., anatomical, growth response and climate‐sensitivity traits) using a polygenic model. We identified a set of 285 genes significantly associated with a climatic factor or a phenotypic trait, including 110 that were differentially expressed in response to drought under greenhouse‐controlled conditions. The interlinked phenotype–genotype–environment network revealed eight high‐confidence genes involved in white spruce adaptation to drought, of which four were drought‐responsive in the expression analysis. Our findings represent a significant step toward the characterization of the genomic basis of drought tolerance and adaptation to climate in conifers, which is essential to enable the establishment of resilient forests in view of new climate conditions.

Chromosome-scale genome assembly of Japanese pear (Pyrus pyrifolia) variety 'Nijisseiki'.

We analyzed the genome sequence of a Japanese pear (Pyrus pyrifolia) to facilitate its genetics and genomics as well as breeding programs, in which a variety 'Nijisseiki' with superior flesh texture has been used as a parent for most Japanese pear cultivars. De novo assembly of long sequence reads covered 136× of the Japanese pear genome and generated 503.9 Mb contigs consisting of 114 sequences with an N50 value of 7.6 Mb. Contigs were assigned to Japanese pear genetic maps to establish 17 chromosome-scale sequences. In total, 44,876 high-confidence protein-encoding genes were predicted, 84.3% of which were supported by predicted genes and transcriptome data from Japanese pear relatives. As expected, evidence of genome-wide duplication was observed, consistent with related species. This is the first chromosome-scale genome sequence analysis reported for Japanese pear, and this resource will support breeding programs and provide new insights into the physiology and evolutionary history of Japanese pear.

A high-quality genome assembly of the eggplant provides insights into the molecular basis of disease resistance and chlorogenic acid synthesis.

The eggplant (Solanum melongena L.) is one of the most important Solanaceae crops, ranking third for total production and economic value in its genus. Herein, we report a high-quality, chromosome-scale eggplant reference genome sequence of 1155.8Mb, with an N50 of 93.9Mb, which was assembled by combining PacBio long reads and Hi-C sequencing data. Repetitive sequences occupied 70.1% of the assembly length, and 35,018 high-confidence protein-coding genes were annotated based on multiple sources. Comparative analysis revealed 646 species-specific families and 364 positive selection genes, conferring distinguishing traits on the eggplant. We performed genome-wide comparative identification of disease resistance genes and discovered an expanded gene family of bacterial spot resistance in eggplant and pepper, but not in tomato and potato. The genes involved in chlorogenic acid synthesis were comprehensively characterized. Highly similar chromosomal distribution patterns of polyphenol oxidase genes were observed in the eggplant, tomato, and potato genomes. The eggplant reference genome sequence will not only facilitate evolutionary studies of the Solanaceae but also facilitate their breeding and improvement.

Co-expression networks in Chlamydomonas reveal significant rhythmicity in batch cultures and empower gene function discovery.

The unicellular green alga Chlamydomonas reinhardtii is a choice reference system for the study of photosynthesis and chloroplast metabolism, cilium assembly and function, lipid and starch metabolism, and metal homeostasis. Despite decades of research, the functions of thousands of genes remain largely unknown, and new approaches are needed to categorically assign genes to cellular pathways. Growing collections of transcriptome and proteome data now allow a systematic approach based on integrative co-expression analysis. We used a dataset comprising 518 deep transcriptome samples derived from 58 independent experiments to identify potential co-expression relationships between genes. We visualized co-expression potential with the R package corrplot, to easily assess co-expression and anti-correlation between genes. We extracted several hundred high-confidence genes at the intersection of multiple curated lists involved in cilia, cell division, and photosynthesis, illustrating the power of our method. Surprisingly, Chlamydomonas experiments retained a significant rhythmic component across the transcriptome, suggesting an underappreciated variable during sample collection, even in samples collected in constant light. Our results therefore document substantial residual synchronization in batch cultures, contrary to assumptions of asynchrony. We provide step-by-step protocols for the analysis of co-expression across transcriptome data sets from Chlamydomonas and other species to help foster gene function discovery.

Tailoring the resolution of single-cell RNA sequencing for primary cytotoxic T cells

Single-cell RNA sequencing in principle offers unique opportunities to improve the efficacy of contemporary T-cell based immunotherapy against cancer. The use of high-quality single-cell data will aid our incomplete understanding of molecular programs determining the differentiation and functional heterogeneity of cytotoxic T lymphocytes (CTLs), allowing for optimal therapeutic design. So far, a major obstacle to high depth single-cell analysis of CTLs is the minute amount of RNA available, leading to low capturing efficacy. Here, to overcome this, we tailor a droplet-based approach for high-throughput analysis (tDrop-seq) and a plate-based method for high-performance in-depth CTL analysis (tSCRB-seq). The latter gives, on average, a 15-fold higher number of captured transcripts per gene compared to droplet-based technologies. The improved dynamic range of gene detection gives tSCRB-seq an edge in resolution sensitive downstream applications such as graded high confidence gene expression measurements and cluster characterization. We demonstrate the power of tSCRB-seq by revealing the subpopulation-specific expression of co-inhibitory and co-stimulatory receptor targets of key importance for immunotherapy.

Wheat heat tolerance is impaired by heightened deletions in the distal end of 4AL chromosomal arm.

SummaryHeat stress (HS) causes substantial damages to worldwide crop production. As a cool season crop, wheat (Triticum aestivum) is sensitive to HS‐induced damages. To support the genetic improvement of wheat HS tolerance (HST), we conducted fine mapping of TaHST1, a locus required for maintaining wheat vegetative and reproductive growth under elevated temperatures. TaHST1 was mapped to the distal terminus of 4AL chromosome arm using genetic populations derived from two BC6F6 breeding lines showing tolerance (E6015‐4T) or sensitivity (E6015‐3S) to HS. The 4AL region carrying TaHST1 locus was approximately 0.949 Mbp and contained the last 19 high confidence genes of 4AL according to wheat reference genome sequence. Resequencing of E6015‐3S and E6015‐4T and haplotype analysis of 3087 worldwide wheat accessions revealed heightened deletion polymorphisms in the distal 0.949 Mbp region of 4AL, which was confirmed by the finding of frequent gene losses in this region in eight genome‐sequenced hexaploid wheat cultivars. The great majority (86.36%) of the 3087 lines displayed different degrees of nucleotide sequence deletions, with only 13.64% of them resembling E6015‐4T in this region. These deletions can impair the presence and/or function of TaHST1 and surrounding genes, thus rendering global wheat germplasm vulnerable to HS or other environmental adversities. Therefore, conscientious and urgent efforts are needed in global wheat breeding programmes to optimize the structure and function of 4AL distal terminus by ensuring the presence of TaHST1 and surrounding genes. The new information reported here will help to accelerate the ongoing global efforts in improving wheat HST.

Cell Type-Specific Predictive Models Perform Prioritization of Genes and Gene Sets Associated With Autism.

Bulk transcriptomic analyses of autism spectrum disorder (ASD) have revealed dysregulated pathways, while the brain cell type-specific molecular pathology of ASD still needs to be studied. Machine learning-based studies can be conducted for ASD, prioritizing high-confidence gene candidates and promoting the design of effective interventions. Using human brain nucleus gene expression of ASD and controls, we construct cell type-specific predictive models for ASD based on individual genes and gene sets, respectively, to screen cell type-specific ASD-associated genes and gene sets. These two kinds of predictive models can predict the diagnosis of a nucleus with known cell type. Then, we construct a multi-label predictive model for predicting the cell type and diagnosis of a nucleus at the same time. Our findings suggest that layer 2/3 and layer 4 excitatory neurons, layer 5/6 cortico-cortical projection neurons, parvalbumin interneurons, and protoplasmic astrocytes are preferentially affected in ASD. The functions of genes with predictive power for ASD are different and the top important genes are distinct across different cells, highlighting the cell-type heterogeneity of ASD. The constructed predictive models can promote the diagnosis of ASD, and the prioritized cell type-specific ASD-associated genes and gene sets may be used as potential biomarkers of ASD.

Genome-wide association analysis uncovers stable QTLs for yield and quality traits of spring bread wheat (Triticum aestivum) across contrasting environments

Heat and drought are the foremost abiotic stress factors that significantly diminish both grain yield and quality parameters of bread wheat. Genetic improvement of varieties withstanding such stresses and able to give sufficient grain yield with appropriate quality is very essential. A Genome-wide association study was conducted with 15 K wheat SNP assay for grain yield and quality traits of 189 spring bread wheat genotypes from ICARDA tested at two heat-stressed locations of Dongula and Wad Medani (Sudan) and the drought-stressed location at Marchouch (Morocco). Analysis of variance revealed a significant variation among genotypes for grain yield and quality traits. GWAS with a mixed linear model (MLM) revealed 192 marker-trait associations (MTAs) across the three environments on 17 chromosomes. Highest MTAs were scored for grain yield (50) followed by zeleny sedimentation test (39) and grain protein content (37). Gluten content and alveograph strength scored 29 and 25 MTAs, respectively. The SNP markers RAC875_c24504_119 and BS00063946_51 on chromosome 3B exhibited highly significant and stable MTAs for zeleny sedimentation test across the three environments. The BS00063624_51 marker on chromosome 3B had also revealed a robust and stable MTA for both zeleny sedimentation test and alveograph strength. Highly significant multi-MTAs were detected for grain yield on chromosome 5A and for both grain protein content and gluten content on the short arm of chromosome 1A across the three contrasting environments. The high confidence gene models identified for SNP markers with significant MTAs were involved in encoding genes for various functions including regulating plant growth, development and resistance to both biotic and abiotic stresses. The identified SNP markers with strong MTAs for grain yield and quality traits could potentially applicable for marker-assisted breeding.

Down-regulation of SORL1 is associated with Alzheimer's disease through activating ABC transporter pathway.

Alzheimer's disease (AD) is a common neurodegenerative disease with high morbidity among elderly people. A genetic attribution has been extensively proved. Here, we propose to further prioritize genes that harbor single nucleotide variation (SNV) or structural variation (SV) for AD and explore the underlying potential mechanisms through exploiting their expression and methylation spectra. A high-confidence AD-associated candidate gene list was obtained from the ClinVar and Human Gene Mutation Database (HGMD). Genome-wide methylation and expression profiles of AD and normal subjects were downloaded from the Gene Expression Omnibus (GEO). Through comprehensive comparison of expression and methylation levels between AD and normal samples, as well as different stages of AD samples, SORL1 was identified as the most plausible gene for AD incidence and progression. Gene Set Enrichment Analysis (GSEA) revealed significant activation of the ABC (ATP binding cassette) transporter with the aberrant up-regulation of SORL1 within AD samples. This study unfolds the expression and methylation spectra of previously probed genes with SNV or SV in AD for the first time, and reports an aberrant activation of the ABC transporter pathway that might contribute to AD progression. This should shed some light on AD diagnosis and precision treatment.

The chromosome-level genome sequence and karyotypic evolution of Megadenia pygmaea (Brassicaceae).

Karyotypic changes in chromosome number and structure are drivers in the divergent evolution of diverse plant species and lineages. This study aimed to reveal the origins of the unique karyotype (2n=12) and phylogenetic relationships of the genus Megadenia (Brassicaceae). A high-quality chromosome-scale genome was assembled for Megadenia pygmaea using Nanopore long reads and high-throughput chromosome conformation capture (Hi-C). The assembled genome is 215.2Mb and is anchored on six pseudochromosomes. We annotated a total of 25,607 high-confidence protein-coding genes and corroborated the phylogenetic affinity of Megadenia with the Brassicaceae expanded lineage II, containing numerous agricultural crops. We dated the divergence of Megadenia from its closest relatives to 27.04 (19.11-36.60) million years ago. A reconstruction of the chromosomal composition of the species was performed based on the de novo assembled genome and comparative chromosome painting analysis. The karyotype structure of M. pygmaea is very similar to the previously inferred proto-Calepineae karyotype (PCK; n=7) of the lineage II. However, an end-to-end translocation between two ancestral chromosomes reduced the chromosome number from n=7 to n=6 in Megadenia. Our reference genome provides fundamental information for karyotypic evolution and evolutionary study of this genus.

Oxidative Stress Responses and Nutrient Starvation in MCHM Treated Saccharomyces cerevisiae.

In 2014, the coal cleaning chemical 4-methylcyclohexane methanol (MCHM) spilled into the water supply for 300,000 West Virginians. Initial toxicology tests showed relatively mild results, but the underlying effects on cellular biology were underexplored. Treated wildtype yeast cells grew poorly, but there was only a small decrease in cell viability. Cell cycle analysis revealed an absence of cells in S phase within thirty minutes of treatment. Cells accumulated in G1 over a six-hour time course, indicating arrest instead of death. A genetic screen of the haploid knockout collection revealed 329 high confidence genes required for optimal growth in MCHM. These genes encode three major cell processes: mitochondrial gene expression/translation, the vacuolar ATPase, and aromatic amino acid biosynthesis. The transcriptome showed an upregulation of pleiotropic drug response genes and amino acid biosynthetic genes and downregulation in ribosome biosynthesis. Analysis of these datasets pointed to environmental stress response activation upon treatment. Overlap in datasets included the aromatic amino acid genes ARO1, ARO3, and four of the five TRP genes. This implicated nutrient deprivation as the signal for stress response. Excess supplementation of nutrients and amino acids did not improve growth on MCHM, so the source of nutrient deprivation signal is still unclear. Reactive oxygen species and DNA damage were directly detected with MCHM treatment, but timepoints showed these accumulated slower than cells arrested. We propose that wildtype cells arrest from nutrient deprivation and survive, accumulating oxidative damage through the implementation of robust environmental stress responses.

The Aegilops ventricosa 2NvS segment in bread wheat: cytology, genomics and breeding

Key messageThe first cytological characterization of the 2NvS segment in hexaploid wheat; complete de novo assembly and annotation of 2NvS segment; 2NvS frequency is increasing 2NvS and is associated with higher yield.The Aegilops ventricosa 2NvS translocation segment has been utilized in breeding disease-resistant wheat crops since the early 1990s. This segment is known to possess several important resistance genes against multiple wheat diseases including root knot nematode, stripe rust, leaf rust and stem rust. More recently, this segment has been associated with resistance to wheat blast, an emerging and devastating wheat disease in South America and Asia. To date, full characterization of the segment including its size, gene content and its association with grain yield is lacking. Here, we present a complete cytological and physical characterization of this agronomically important translocation in bread wheat. We de novo assembled the 2NvS segment in two wheat varieties, ‘Jagger’ and ‘CDC Stanley,’ and delineated the segment to be approximately 33 Mb. A total of 535 high-confidence genes were annotated within the 2NvS region, with > 10% belonging to the nucleotide-binding leucine-rich repeat (NLR) gene families. Identification of groups of NLR genes that are potentially N genome-specific and expressed in specific tissues can fast-track testing of candidate genes playing roles in various disease resistances. We also show the increasing frequency of 2NvS among spring and winter wheat breeding programs over two and a half decades, and the positive impact of 2NvS on wheat grain yield based on historical datasets. The significance of the 2NvS segment in wheat breeding due to resistance to multiple diseases and a positive impact on yield highlights the importance of understanding and characterizing the wheat pan-genome for better insights into molecular breeding for wheat improvement.

ASDmiR: A Stepwise Method to Uncover miRNA Regulation Related to Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a class of neurodevelopmental disorders characterized by genetic and environmental risk factors. The pathogenesis of ASD has a strong genetic basis, consisting of rare de novo or inherited variants among a variety of multiple molecules. Previous studies have shown that microRNAs (miRNAs) are involved in neurogenesis and brain development and are closely associated with the pathogenesis of ASD. However, the regulatory mechanisms of miRNAs in ASD are largely unclear. In this work, we present a stepwise method, ASDmiR, for the identification of underlying pathogenic genes, networks, and modules associated with ASD. First, we conduct a comparison study on 12 miRNA target prediction methods by using the matched miRNA, lncRNA, and mRNA expression data in ASD. In terms of the number of experimentally confirmed miRNA–target interactions predicted by each method, we choose the best method for identifying miRNA–target regulatory network. Based on the miRNA–target interaction network identified by the best method, we further infer miRNA–target regulatory bicliques or modules. In addition, by integrating high-confidence miRNA–target interactions and gene expression data, we identify three types of networks, including lncRNA–lncRNA, lncRNA–mRNA, and mRNA–mRNA related miRNA sponge interaction networks. To reveal the community of miRNA sponges, we further infer miRNA sponge modules from the identified miRNA sponge interaction network. Functional analysis results show that the identified hub genes, as well as miRNA-associated networks and modules, are closely linked with ASD. ASDmiR is freely available at https://github.com/chenchenxiong/ASDmiR.