A multitiered haplotype strategy to enhance phased assembly and fine mapping of a disease resistance locus.

Fine mapping of quantitative trait loci (QTL) to dissect the genetic basis of traits of interest is essential to modern breeding practice. Here, we employed a multitiered haplotypic marker system to increase fine mapping accuracy by constructing a chromosome-level, haplotype-resolved parental genome, accurate detection of recombination sites, and allele-specific characterization of the transcriptome. In the first tier of this system, we applied the preexisting panel of 2,000 rhAmpSeq core genome markers that is transferable across the entire Vitis genus and provides a genomic resolution of 200 kb to 1 Mb. The second tier consisted of high-density haplotypic markers generated from Illumina skim sequencing data for samples enriched for relevant recombinations, increasing the potential resolution to hundreds of base pairs. We used this approach to dissect a novel Resistance to Plasmopara viticola-33 (RPV33) locus conferring resistance to grapevine downy mildew, narrowing the candidate region to only 0.46 Mb. In the third tier, we used allele-specific RNA-seq analysis to identify a cluster of 3 putative disease resistance RPP13-like protein 2 genes located tandemly in a nonsyntenic insertion as candidates for the disease resistance trait. In addition, combining the rhAmpSeq core genome haplotype markers and skim sequencing-derived high-density haplotype markers enabled chromosomal-level scaffolding and phasing of the grape Vitis × doaniana 'PI 588149' assembly, initially built solely from Pacific Biosciences (PacBio) high-fidelity (HiFi) reads, leading to the correction of 16 large-scale phasing errors. Our mapping strategy integrates high-density, phased genetic information with individual reference genomes to pinpoint the genetic basis of QTLs and will likely be widely adopted in highly heterozygous species.

Effects of Nighttime Applications of Germicidal Ultraviolet Light Upon Powdery Mildew (Erysiphe necator), Downy Mildew (Plasmopara viticola), and Sour Rot of Grapevine.

Nighttime applications of germicidal ultraviolet were evaluated as a means to suppress three diseases of grapevine. In laboratory studies, UV-C light (peak 254 nm, FWHM 5 nm) applied during darkness strongly inhibited the germination of conidia of Erysiphe necator, and at a dose of 200 J/m2, germination was zero. Reciprocity of irradiance and duration of exposure with respect to conidial germination was confirmed for UV-C doses between 0 and 200 J/m2 applied at 4 or 400 s. When detached grapevine leaves were exposed during darkness to UV-C at 100 J/m2 up to 7 days before they were inoculated with zoospores of Plasmopara viticola, infection and subsequent sporulation was reduced by over 70% compared to untreated control leaves, indicating an indirect suppression of the pathogen exerted through the host. A hemicylindrical array of low-pressure discharge UV-C lamps configured for trellised grapevines was designed and fitted to both a tractor-drawn carriage and a fully autonomous robotic carriage for vineyard applications. In 2019, in a Chardonnay research vineyard with a history of high inoculum and severe disease, weekly nighttime applications of UV-C suppressed E. necator on leaves and fruit at doses of 100 and 200 J/m2. In the same vineyard in 2020, UV-C was applied once or twice weekly at doses of 70, 100, or 200 J/m2, and severity of E. necator on both leaves and fruit was significantly reduced compared to untreated controls; twice-weekly applications at 200 J/m2 provided suppression equivalent to a standard fungicide program. None of the foregoing UV-C treatments significantly reduced the severity of P. viticola on Chardonnay vines compared to the untreated control in 2020. However, twice-weekly applications of UV-C at 200 J/m2 to the more downy mildew-resistant Vitis interspecific hybrid cultivar Vignoles in 2021 significantly suppressed foliar disease severity. In commercial Chardonnay vineyards with histories of excellent disease control in Dresden, NY, E. necator remained at trace levels on foliage and was zero on fruit following weekly nighttime applications of UV-C at 200 J/m2 in 2020 and after weekly or twice-weekly application of UV-C at 100 or 200 J/m2 in 2021. In 2019, weekly nighttime applications of UV-C at 200 J/m2 also significantly reduced the severity of sour rot, a decay syndrome of complex etiology, on fruit of 'Vignoles' but not the severity of bunch rot caused by Botrytis cinerea. A similar level of suppression of sour rot was observed on 'Vignoles' vines treated twice-weekly with UV-C at 200 J/m2 in 2021. Nighttime UV-C applications did not produce detectable indications of metabolic abnormalities, phytotoxicity, growth reduction, or reductions of fruit yield or quality parameters, even at the highest doses and most frequent intervals employed.

Construction of a high-density linkage map and QTL detection of downy mildew resistance in Vitis aestivalis-derived 'Norton'.

KEY MESSAGE: A major QTL for downy mildew resistance was detected on chromosome 18 (Rpv27) in Vitis aestivalis-derived 'Norton' based on a high-resolution linkage map with SNP and SSR markers as well as 2 years of field and laboratory phenotyping data. Grapevine downy mildew caused by the oomycete Plasmopara viticola is one of the most widespread and destructive diseases, particularly in humid viticultural areas where it damages green tissues and defoliates vines. Traditional Vitis vinifera wine grape cultivars are susceptible to downy mildew whereas several North American and a few Asian cultivars possess various levels of resistance to this disease. To identify genetic determinants of downy mildew resistance in V. aestivalis-derived 'Norton,' a mapping population with 182 genotypes was developed from a cross between 'Norton' and V. vinifera 'Cabernet Sauvignon' from which a consensus map was constructed via 411 simple sequence repeat (SSR) markers. Using genotyping-by-sequencing, 3825 single nucleotide polymorphism (SNP) markers were also generated. Of these, 1665 SNP and 407 SSR markers were clustered into 19 linkage groups in 159 genotypes, spanning a genetic distance of 2203.5 cM. Disease progression in response to P. viticola was studied in this population for 2 years under both laboratory and field conditions, and strong correlations were observed among data sets (Spearman correlation coefficient = 0.57-0.79). A quantitative trait loci (QTL) analysis indicated a resistance locus on chromosome 18, here named Rpv27, explaining 33.8% of the total phenotypic variation. Flanking markers closely linked with the trait can be further used for marker-assisted selection in the development of new cultivars with resistance to downy mildew.

Discovery and genome-guided mapping of REN12 from Vitis amurensis, conferring strong, rapid resistance to grapevine powdery mildew.

Powdery mildew resistance genes restrict infection attempts at different stages of pathogenesis. Here, a strong and rapid powdery mildew resistance phenotype was discovered from Vitis amurensis 'PI 588631' that rapidly stopped over 97% of Erysiphe necator conidia, before or immediately after emergence of a secondary hypha from appressoria. This resistance was effective across multiple years of vineyard evaluation on leaves, stems, rachises, and fruit and against a diverse array of E. necator laboratory isolates. Using core genome rhAmpSeq markers, resistance mapped to a single dominant locus (here named REN12) on chromosome 13 near 22.8-27.0 Mb, irrespective of tissue type, explaining up to 86.9% of the phenotypic variation observed on leaves. Shotgun sequencing of recombinant vines using skim-seq technology enabled the locus to be further resolved to a 780 kb region, from 25.15 to 25.93 Mb. RNASeq analysis indicated the allele-specific expression of four resistance genes (NLRs) from the resistant parent. REN12 is one of the strongest powdery mildew resistance loci in grapevine yet documented, and the rhAmpSeq sequences presented here can be directly used for marker-assisted selection or converted to other genotyping platforms. While no virulent isolates were identified among the genetically diverse isolates and wild populations of E. necator tested here, NLR loci like REN12 are often race-specific. Thus, stacking of multiple resistance genes and minimal use of fungicides should enhance the durability of resistance and could enable a 90% reduction in fungicides in low-rainfall climates where few other pathogens attack the foliage or fruit.

High throughput saliency-based quantification of grape powdery mildew at the microscopic level for disease resistance breeding.

Imaging-based high throughput phenotyping (HTP) systems have demonstrated promising solutions to enhance genetic understanding of grapevine powdery mildew (PM) resistance and have accelerated PM-resistant cultivar breeding. The accuracy and throughput of extracting phenotypic traits from images are still the bottleneck of modern HTP systems, especially at the microscopic level. The goal of this study was to develop a saliency-based processing pipeline for the quantification of PM infection in microscopic images and comprehensively evaluate its performance for genetic analyses. An input image was segregated into subimages that were classified as infected or healthy by a pretrained CNN classifier. Saliency maps from the classification were generated post-hoc and used for the quantification of PM infection in the input image at the pixel level without the use of mask annotations. A total of seven phenotypic traits were extracted from images collected for a biparental population. Experimental results showed that optimal combinations of convolutional neural network and saliency methods achieved strong measurement correlations (r = 0.74 to 0.75) with human assessments at the image patch level, and the traits calculated by the saliency-based processing pipeline were highly correlated (r = 0.87 to 0.88) with reference PM infection ratings at the leaf image level. The high quantification accuracy of the saliency-based pipeline led to the increased explanation of phenotypic variance and reliable identification of quantitative trait loci. Therefore, the saliency-based processing pipeline can be used as an effective and efficient analysis tool for PM disease research and breeding programs in the future, especially agricultural and life science studies requiring microscopic image analysis.

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.