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BackgroundGene annotation in eukaryotes is a non-trivial task that requires meticulous analysis of accumulated transcript data. Challenges include transcriptionally active regions of the genome that contain overlapping genes, genes that produce numerous transcripts, transposable elements and numerous diverse sequence repeats. Currently available gene annotation software applications depend on pre-constructed full-length gene sequence assemblies which are not guaranteed to be error-free. The origins of these sequences are often uncertain, making it difficult to identify and rectify errors in them. This hinders the creation of an accurate and holistic representation of the transcriptomic landscape across multiple tissue types and experimental conditions. Therefore, to gauge the extent of diversity in gene structures, a comprehensive analysis of genome-wide expression data is imperative.ResultsWe present FINDER, a fully automated computational tool that optimizes the entire process of annotating genes and transcript structures. Unlike current state-of-the-art pipelines, FINDER automates the RNA-Seq pre-processing step by working directly with raw sequence reads and optimizes gene prediction from BRAKER2 by supplementing these reads with associated proteins. The FINDER pipeline (1) reports transcripts and recognizes genes that are expressed under specific conditions, (2) generates all possible alternatively spliced transcripts from expressed RNA-Seq data, (3) analyzes read coverage patterns to modify existing transcript models and create new ones, and (4) scores genes as high- or low-confidence based on the available evidence across multiple datasets. We demonstrate the ability of FINDER to automatically annotate a diverse pool of genomes from eight species.ConclusionsFINDER takes a completely automated approach to annotate genes directly from raw expression data. It is capable of processing eukaryotic genomes of all sizes and requires no manual supervision-ideal for bench researchers with limited experience in handling computational tools.

Following the publication of the original article [1], the authors noted several typesetting errors which are noted in this Correction article.

BACKGROUND: Plants encounter pathogenic and non-pathogenic microorganisms on a nearly constant basis. Small RNAs such as siRNAs and miRNAs/milRNAs influence pathogen virulence and host defense responses. We exploited the biotrophic interaction between the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), and its diploid host plant, barley (Hordeum vulgare) to explore fungal and plant sRNAs expressed during Bgh infection of barley leaf epidermal cells. RESULTS: RNA was isolated from four fast-neutron immune-signaling mutants and their progenitor over a time course representing key stages of Bgh infection, including appressorium formation, penetration of epidermal cells, and development of haustorial feeding structures. The Cereal Introduction (CI) 16151 progenitor carries the resistance allele Mla6, while Bgh isolate 5874 harbors the AVRa6 avirulence effector, resulting in an incompatible interaction. Parallel Analysis of RNA Ends (PARE) was used to verify sRNAs with likely transcript targets in both barley and Bgh. Bgh sRNAs are predicted to regulate effectors, metabolic genes, and translation-related genes. Barley sRNAs are predicted to influence the accumulation of transcripts that encode auxin response factors, NAC transcription factors, homeodomain transcription factors, and several splicing factors. We also identified phasing small interfering RNAs (phasiRNAs) in barley that overlap transcripts that encode receptor-like kinases (RLKs) and nucleotide-binding, leucine-rich domain proteins (NLRs). CONCLUSIONS: These data suggest that Bgh sRNAs regulate gene expression in metabolism, translation-related, and pathogen effectors. PARE-validated targets of predicted Bgh milRNAs include both EKA (effectors homologous to AVRk1 and AVRa10) and CSEP (candidate secreted effector protein) families. We also identified barley phasiRNAs and miRNAs in response to Bgh infection. These include phasiRNA loci that overlap with a significant proportion of receptor-like kinases, suggesting an additional sRNA control mechanism may be active in barley leaves as opposed to predominant R-gene phasiRNA overlap in many eudicots. In addition, we identified conserved miRNAs, novel miRNA candidates, and barley genome mapped sRNAs that have PARE validated transcript targets in barley. The miRNA target transcripts are enriched in transcription factors, signaling-related proteins, and photosynthesis-related proteins. Together these results suggest both barley and Bgh control metabolism and infection-related responses via the specific accumulation and targeting of genes via sRNAs.

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.

The barley powdery mildew fungus, Blumeria hordei (Bh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley nucleotide‐binding leucine‐rich‐repeat (NLR) receptor MLA1 and therefore is designated AVRA1. Here, we show that AVRA1 and the sequence‐unrelated Bh effector BEC1016 (CSEP0491) suppress immunity in barley. We used yeast two‐hybrid next‐generation interaction screens (Y2H‐NGIS), followed by binary Y2H and in planta protein–protein interactions studies, and identified a common barley target of AVRA1 and BEC1016, the endoplasmic reticulum (ER)‐localized J‐domain protein HvERdj3B. Silencing of this ER quality control (ERQC) protein increased Bh penetration. HvERdj3B is ER luminal, and we showed using split GFP that AVRA1 and BEC1016 translocate into the ER signal peptide‐independently. Overexpression of the two effectors impeded trafficking of a vacuolar marker through the ER; silencing of HvERdj3B also exhibited this same cellular phenotype, coinciding with the effectors targeting this ERQC component. Together, these results suggest that the barley innate immunity, preventing Bh entry into epidermal cells, requires ERQC. Here, the J‐domain protein HvERdj3B appears to be essential and can be regulated by AVRA1 and BEC1016. Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host NLR receptors. Previous work has shown that AVRA1 is directly recognized in the cytosol by the immune receptor MLA1. We speculate that the AVRA1 J‐domain target being inside the ER, where it is inapproachable by NLRs, has forced the plant to evolve this challenging direct recognition. [ABSTRACT FROM AUTHOR]

Next-generation whole-genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost-efficient establishment of powerful genomic information for many species.

BackgroundA typical genetical genomics experiment results in four separate data sets; genotype, gene expression, higher-order phenotypic data and metadata that describe the protocols, processing and the array platform. Used in concert, these data sets provide the opportunity to perform genetic analysis at a systems level. Their predictive power is largely determined by the gene expression dataset where tens of millions of data points can be generated using currently available mRNA profiling technologies. Such large, multidimensional data sets often have value beyond that extracted during their initial analysis and interpretation, particularly if conducted on widely distributed reference genetic materials. Besides quality and scale, access to the data is of primary importance as accessibility potentially allows the extraction of considerable added value from the same primary dataset by the wider research community. Although the number of genetical genomics experiments in different plant species is rapidly increasing, none to date has been presented in a form that allows quick and efficient on-line testing for possible associations between genes, loci and traits of interest by an entire research community.DescriptionUsing a reference population of 150 recombinant doubled haploid barley lines we generated novel phenotypic, mRNA abundance and SNP-based genotyping data sets, added them to a considerable volume of legacy trait data and entered them into the GeneNetwork http://www.genenetwork.org. GeneNetwork is a unified on-line analytical environment that enables the user to test genetic hypotheses about how component traits, such as mRNA abundance, may interact to condition more complex biological phenotypes (higher-order traits). Here we describe these barley data sets and demonstrate some of the functionalities GeneNetwork provides as an easily accessible and integrated analytical environment for exploring them.ConclusionBy integrating barley genotypic, phenotypic and mRNA abundance data sets directly within GeneNetwork's analytical environment we provide simple web access to the data for the research community. In this environment, a combination of correlation analysis and linkage mapping provides the potential to identify and substantiate gene targets for saturation mapping and positional cloning. By integrating datasets from an unsequenced crop plant (barley) in a database that has been designed for an animal model species (mouse) with a well established genome sequence, we prove the importance of the concept and practice of modular development and interoperability of software engineering for biological data sets.

Even in the absence of excisional loss of the associated Mu transposons, some Mu-induced mutant alleles of maize can lose their capacity to condition a mutant phenotype. Three of five Mu-derived rf2a alleles are susceptible to such Mu suppression. The suppressible rf2a-m9437 allele has a novel Mu transposon insertion (Mu10) in its 5' untranslated region (UTR). The suppressible rf2a-m9390 allele has a Mu1 insertion in its 5' UTR. During suppression, alternative transcription initiation sites flanking the Mu1 transposon yield functional transcripts. The suppressible rf2a-m8110 allele has an rcy/Mu7 insertion in its 3' UTR. Suppression of this allele occurs via a previously unreported mechanism; sequences in the terminal inverted repeats of rcy/Mu7 function as alternative polyadenylation sites such that the suppressed rf2a-m8110 allele yields functional rf2a transcripts. No significant differences were observed in the nucleotide compositions of these alternative polyadenylation sites as compared with 94 other polyadenylation sites from maize genes.

Rf8 is a newly described nuclear gene that can substitute for Rf1 to partially restore pollen fertility to male-sterile, T-cytoplasm maize. Families segregating for Rf8 were used to investigate the mechanism of this fertility restoration and to compare it to the restoration conditioned by Rf1. Although Rf8 is unlinked to the rf1 locus, it also alters T-urf13 mitochondrial transcript accumulation and reduces the accumulation of the URF13 protein. Like the 1.6- and 0.6-kilobase (kb) T-urf13 transcripts that accumulate in T-cytoplasm plants carrying Rf1, 1.42- and 0.42-kb transcripts accumulate in plants that are partially restored by Rf8. A survey of T-cytoplasm maize lines, inbreds, and [F.sub.1] hybrids by mitochondrial RNA gel blot analyses revealed that Rf8 is rare in maize germplasm. These surveys revealed the presence of another rare, weak restorer factor, [Rf.sup.*], which is uniquely associated with the accumulation of 1.4- and 0.4-kb T-urf13 transcripts. Primer extension analyses position the 5[prime] termini of the 1.42/0.42-kb and 1.4/0.4-kb transcripts at +137 and +159 nucleotides, respectively, 3[prime] of the AUG initiation codon of the T-urf13 reading frame. The conserved motif, 5[prime]-CNACNNU-3[prime], overlaps the 5[prime] termini of the Rf1-, Rf8-, and [Rf.sup.*]associated transcripts and the 380 nucleotide, Rf3-associated orf107 transcript from cytoplasmic male sterility sorghum. These results demonstrate that multiple unlinked, nuclear genes can have similar but distinct effects on the expression of the unique T-urf13 mitochondrial coding sequence to restore pollen fertility to T-cytoplasm maize.