Discovery of the REN11 Locus From Vitis aestivalis for Stable Resistance to Grapevine Powdery Mildew in a Family Segregating for Several Unstable and Tissue-Specific Quantitative Resistance Loci

Race-specific resistance loci, whether having qualitative or quantitative effects, present plant-breeding challenges for phenotypic selection and deciding which loci to select or stack with other resistance loci for improved durability. Previously, resistance to grapevine powdery mildew (GPM, caused by Erysiphe necator) was predicted to be conferred by at least three race-specific loci in the mapping family B37-28 × C56-11 segregating for GPM resistance from Vitis aestivalis. In this study, 9 years of vineyard GPM disease severity ratings plus a greenhouse and laboratory assays were genetically mapped, using a rhAmpSeq core genome marker platform with 2,000 local haplotype markers. A new qualitative resistance locus, named REN11, on the chromosome (Chr) 15 was found to be effective in nearly all (11 of 12) vineyard environments on leaves, rachis, berries, and most of the time (7 of 12) stems. REN11 was independently validated in a pseudo-testcross with the grandparent source of resistance, “Tamiami.” Five other loci significantly predicted GPM severity on leaves in only one or two environments, which could indicate race-specific resistance or their roles in different timepoints in epidemic progress. Loci on Chr 8 and 9 reproducibly predicted disease severity on stems but not on other tissues and had additive effects with REN11 on the stems. The rhAmpSeq local haplotype sequences published in this study for REN11 and Chr 8 and 9 stem quantitative trait locus (QTL) can be used directly for marker-assisted selection or converted to SNP assays. In screening for REN11 in a diversity panel of 20,651 vines representing the diversity of Vitis, this rhAmpSeq haplotype had a false positive rate of 0.034% or less. The effects of the other foliar resistance loci detected in this study seem too unstable for genetic improvement regardless of quantitative effect size, whether due to race specificity or other environmental variables.

Genome Sequence Resource for Erysiphe necator NAFU1, a Grapevine Powdery Mildew Isolate Identified in Shaanxi Province of China

Erysiphe necator is an economically important biotrophic fungal pathogen responsible for powdery mildew disease on grapevine. Currently, genome sequences are available for only a few Erysiphe necator isolates from USA. Based on the combination of Nanopore and Illumina sequencing technologies, we present here the complete genome assembly for an isolate of E. necator NAFU1 identified in China. We acquired a total of 15.93 Gb raw reads. These reads were processed into a 61.12 Mb genome assembly containing 73 contigs with the N50 of 2.06 Mb and a maximum length of 6.05 Mb. Combining the results of three gene-prediction modules, i.e. an evidence-based gene modeler (EVidenceModeler or EVM), an ab initio gene modeler, and a homology-based gene modeler, we predicted 7235 protein-coding genes in the assembled genome of E. necator NAFU1. This information will facilitate studies of genome evolution and pathogenicity mechanisms of E. necator and other powdery mildew species through comparative genome sequence analysis and other molecular genetic tools.

Comparison of Short-Read Sequence Aligners Indicates Strengths and Weaknesses for Biologists to Consider

Aligning short-read sequences is the foundational step to most genomic and transcriptomic analyses, but not all tools perform equally, and choosing among the growing body of available tools can be daunting. Here, in order to increase awareness in the research community, we discuss the merits of common algorithms and programs in a way that should be approachable to biologists with limited experience in bioinformatics. We will only in passing consider the effects of data cleanup, a precursor analysis to most alignment tools, and no consideration will be given to downstream processing of the aligned fragments. To compare aligners [Bowtie2, Burrows Wheeler Aligner (BWA), HISAT2, MUMmer4, STAR, and TopHat2], an RNA-seq dataset was used containing data from 48 geographically distinct samples of the grapevine powdery mildew fungus Erysiphe necator. Based on alignment rate and gene coverage, all aligners performed well with the exception of TopHat2, which HISAT2 superseded. BWA perhaps had the best performance in these metrics, except for longer transcripts (>500 bp) for which HISAT2 and STAR performed well. HISAT2 was ~3-fold faster than the next fastest aligner in runtime, which we consider a secondary factor in most alignments. At the end, this direct comparison of commonly used aligners illustrates key considerations when choosing which tool to use for the specific sequencing data and objectives. No single tool meets all needs for every user, and there are many quality aligners available.

Transcriptomic Profiling of Acute Cold Stress-Induced Disease Resistance (SIDR) Genes and Pathways in the Grapevine Powdery Mildew Pathosystem

Temperatures from 2 to 8°C transiently induce quantitative resistance to powdery mildew in several host species (cold stress-induced disease resistance [SIDR]). Although cold SIDR events occur in vineyards worldwide an average of 14 to 21 times after budbreak of grapevine and can significantly delay grapevine powdery mildew (Erysiphe necator) epidemics, its molecular basis was poorly understood. We characterized the biology underlying the Vitis vinifera cold SIDR phenotype—which peaks at 24 h post–cold (hpc) treatment and results in a 22 to 28% reduction in spore penetration success—through highly replicated (n = 8 to 10) RNA sequencing experiments. This phenotype was accompanied by a sweeping transcriptional downregulation of photosynthesis-associated pathways whereas starch and sugar metabolism pathways remained largely unaffected, suggesting a transient imbalance in host metabolism and a suboptimal target for pathogen establishment. Twenty-six cold-responsive genes peaked in their differential expression at the 24-hpc time point. Finally, a subset of genes associated with nutrient and amino acid transport accounted for four of the eight most downregulated transcripts, including two nodulin 1A gene precursors, a nodulin MtN21 precursor, and a Dynein light chain 1 motor protein precursor. Reduced transport could exacerbate localized nutrient sinks that would again be transiently suboptimal for pathogen growth. This study links the transient cold SIDR phenotype to underlying transcriptional changes and provides an experimental framework and library of candidate genes to further explore cold SIDR in several systems, with an ultimate goal of identifying novel breeding or management targets for reduced disease.

A High-Throughput Phenotyping System Using Machine Vision to Quantify Severity of Grapevine Powdery Mildew

Powdery mildews present specific challenges to phenotyping systems that are based on imaging. Having previously developed low-throughput, quantitative microscopy approaches for phenotyping resistance to Erysiphe necator on thousands of grape leaf disk samples for genetic analysis, here we developed automated imaging and analysis methods for E. necator severity on leaf disks. By pairing a 46-megapixel CMOS sensor camera, a long-working distance lens providing 3.5× magnification, X-Y sample positioning, and Z-axis focusing movement, the system captured 78% of the area of a 1-cm diameter leaf disk in 3 to 10 focus-stacked images within 13.5 to 26 seconds. Each image pixel represented 1.44 μm2 of the leaf disk. A convolutional neural network (CNN) based on GoogLeNet determined the presence or absence of E. necator hyphae in approximately 800 subimages per leaf disk as an assessment of severity, with a training validation accuracy of 94.3%. For an independent image set the CNN was in agreement with human experts for 89.3% to 91.7% of subimages. This live-imaging approach was nondestructive, and a repeated measures time course of infection showed differentiation among susceptible, moderate, and resistant samples. Processing over one thousand samples per day with good accuracy, the system can assess host resistance, chemical or biological efficacy, or other phenotypic responses of grapevine to E. necator. In addition, new CNNs could be readily developed for phenotyping within diverse pathosystems or for diverse traits amenable to leaf disk assays.