Genomic resources for a historical collection of cultivated two-row European spring barley genotypes.

Barley genomic resources are increasing rapidly, with the publication of a barley pangenome as one of the latest developments. Two-row spring barley cultivars are intensely studied as they are the source of high-quality grain for malting and distilling. Here we provide data from a European two-row spring barley population containing 209 different genotypes registered for the UK market between 1830 to 2014. The dataset encompasses RNA-sequencing data from six different tissues across a range of barley developmental stages, phenotypic datasets from two consecutive years of field-grown trials in the United Kingdom, Germany and the USA; and whole genome shotgun sequencing from all cultivars, which was used to complement the RNA-sequencing data for variant calling. The outcomes are a filtered SNP marker file, a phenotypic database and a large gene expression dataset providing a comprehensive resource which allows for downstream analyses like genome wide association studies or expression associations.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

Identifying plant genes shaping microbiota composition in the barley rhizosphere.

A prerequisite to exploiting soil microbes for sustainable crop production is the identification of the plant genes shaping microbiota composition in the rhizosphere, the interface between roots and soil. Here, we use metagenomics information as an external quantitative phenotype to map the host genetic determinants of the rhizosphere microbiota in wild and domesticated genotypes of barley, the fourth most cultivated cereal globally. We identify a small number of loci with a major effect on the composition of rhizosphere communities. One of those, designated the QRMC-3HS, emerges as a major determinant of microbiota composition. We subject soil-grown sibling lines harbouring contrasting alleles at QRMC-3HS and hosting contrasting microbiotas to comparative root RNA-seq profiling. This allows us to identify three primary candidate genes, including a Nucleotide-Binding-Leucine-Rich-Repeat (NLR) gene in a region of structural variation of the barley genome. Our results provide insights into the footprint of crop improvement on the plant's capacity of shaping rhizosphere microbes.

BaRTv2: a highly resolved barley reference transcriptome for accurate transcript-specific RNA-seq quantification.

Accurate characterisation of splice junctions (SJs) as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data, and enables detailed investigations of transcriptional and post-transcriptional regulation. Using novel computational methods and a combination of PacBio Iso-seq and Illumina short-read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset from the European 2-row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the SJs and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared with an earlier version, BaRTv1.0. The accuracy of transcript level quantification, SJs and transcript start and end sites have been validated extensively using parallel technologies and analysis, including high-resolution reverse transcriptase-polymerase chain reaction and 5'-RACE. BaRTv2.18 contains 39 434 genes and 148 260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high-quality resource for advanced transcriptomic analyses, including both transcriptional and post-transcriptional regulation, with exceptional resolution and precision.

The value of genotype-specific reference for transcriptome analyses in barley.

It is increasingly apparent that although different genotypes within a species share "core" genes, they also contain variable numbers of "specific" genes and different structures of "core" genes that are only present in a subset of individuals. Using a common reference genome may thus lead to a loss of genotype-specific information in the assembled Reference Transcript Dataset (RTD) and the generation of erroneous, incomplete or misleading transcriptomics analysis results. In this study, we assembled genotype-specific RTD (sRTD) and common reference-based RTD (cRTD) from RNA-seq data of cultivated Barke and Morex barley, respectively. Our quantitative evaluation showed that the sRTD has a significantly higher diversity of transcripts and alternative splicing events, whereas the cRTD missed 40% of transcripts present in the sRTD and it only has ∼70% accurate transcript assemblies. We found that the sRTD is more accurate for transcript quantification as well as differential expression analysis. However, gene-level quantification is less affected, which may be a reasonable compromise when a high-quality genotype-specific reference is not available.

Characterisation of barley landraces from Syria and Jordan for resistance to rhynchosporium and identification of diagnostic markers for Rrs1Rh4.

KEY MESSAGE: Diagnostic markers for Rrs1Rh4 have been identified by testing for associations between SNPs within the Rrs1 interval in 150 barley genotypes and their resistance to Rhynchosporium commune isolates recognised by lines containing Rrs1. Rhynchosporium or barley scald, caused by the destructive fungal pathogen Rhynchosporium commune, is one of the most economically important diseases of barley in the world. Barley landraces from Syria and Jordan demonstrated high resistance to rhynchosporium in the field. Genotyping of a wide range of barley cultivars and landraces, including known sources of different Rrs1 genes/alleles, across the Rrs1 interval, followed by association analysis of this genotypic data with resistance phenotypes to R. commune isolates recognised by Rrs1, allowed the identification of diagnostic markers for Rrs1Rh4. These markers are specific to Rrs1Rh4 and do not detect other Rrs1 genes/alleles. The Rrs1Rh4 diagnostic markers represent a resource that can be exploited by breeders for the sustainable deployment of varietal resistance in new cultivars. Thirteen out of the 55 most resistant Syrian and Jordanian landraces were shown to contain markers specific to Rrs1Rh4. One of these lines came from Jordan, with the remaining 12 lines from different locations in Syria. One of the Syrian landraces containing Rrs1Rh4 was also shown to have Rrs2. The remaining landraces that performed well against rhynchosporium in the field are likely to contain other resistance genes and represent an important novel resource yet to be exploited by European breeders.

Characterisation of barley resistance to rhynchosporium on chromosome 6HS.

KEY MESSAGE: Major resistance gene to rhynchosporium, Rrs18, maps close to the telomere on the short arm of chromosome 6H in barley. Rhynchosporium or barley scald caused by a fungal pathogen Rhynchosporium commune is one of the most destructive and economically important diseases of barley in the world. Testing of Steptoe × Morex and CIho 3515 × Alexis doubled haploid populations has revealed a large effect QTL for resistance to R. commune close to the telomere on the short arm of chromosome 6H, present in both populations. Mapping markers flanking the QTL from both populations onto the 2017 Morex genome assembly revealed a rhynchosporium resistance locus independent of Rrs13 that we named Rrs18. The causal gene was fine mapped to an interval of 660 Kb using Steptoe × Morex backcross 1 S2 and S3 lines with molecular markers developed from Steptoe exome capture variant calling. Sequencing RNA from CIho 3515 and Alexis revealed that only 4 genes within the Rrs18 interval were transcribed in leaf tissue with a serine/threonine protein kinase being the most likely candidate for Rrs18.

Genome Sequencing of Xanthomonas vasicola Pathovar vasculorum Reveals Variation in Plasmids and Genes Encoding Lipopolysaccharide Synthesis, Type-IV Pilus and Type-III Secretion Effectors.

Xanthomonas vasicola pathovar vasculorum (Xvv) is the bacterial agent causing gumming disease in sugarcane. Here, we compare complete genome sequences for five isolates of Xvv originating from sugarcane and one from maize. This identified two distinct types of lipopolysaccharide synthesis gene clusters among Xvv isolates: one is similar to that of Xanthomonas axonopodis pathovar citri (Xac) and is probably the ancestral type, while the other is similar to those of the sugarcane-inhabiting species, Xanthomonas sacchari. Four of six Xvv isolates harboured sequences similar to the Xac plasmid, pXAC47, and showed a distinct Type-IV pilus (T4P) sequence type, whereas the T4P locus of the other two isolates resembled that of the closely related banana pathogen, Xanthomonas campestris pathovar musacearum (Xcm). The Xvv isolate from maize has lost a gene encoding a homologue of the virulence effector, xopAF, which was present in all five of the sugarcane isolates, while xopL contained a premature stop codon in four out of six isolates. These findings shed new light on evolutionary events since the divergence of Xvv and Xcm, as well as further elucidating the relationships between the two closely related pathogens.