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Powdery mildew, caused by the fungus Erysiphe necator , is one of the primary causes of grape yield loss across the globe. While numerous resistance loci have been identified in various grapevine species, the genetic determinants of susceptibility to E. necator remain largely unexplored. Understanding the genetics of susceptibility for pathogenesis is equally important for developing durable resistance grapevines against this pathogen. To identify these factors in Vitis interspecific hybrid 'Chambourcin', a controlled leaf disc assay was conducted for two years using an automated microscopy phenotyping system with 273 F1 genotypes from a cross of 'Chambourcin' and V. vinifera 'Cabernet Sauvignon'. Additionally, a high-resolution linkage map using the same number of 'Chambourcin'-derived hybrids was constructed with 355 simple sequence repeats (SSR) and 1,394 RNaseH2-dependent amplicon sequencing (rhAmpSeq)-derived haplotype markers that clustered into 19 linkage groups. A quantitative trait locus (QTL) analysis identified a susceptibility locus (here named Sen2 ) on linkage group (LG) 7 explaining 8.90 % - 12.57 % of the total phenotypic variance. The markers associated with this susceptibility locus were used to identify 78 accessions in the USDA-ARS cold hardy Vitis collection at Geneva, NY that carry Sen2 and can be used to selectively exclude susceptible progenies. Additionally, 6 accessions carry the alternative haplotype encoding recessive resistance and can be used for resistance breeding. The identification of powdery mildew susceptibility loci is crucial for identifying genes that could be targeted for gene knock-out, gene editing, RNA interference (RNAi), or selection among breeding genotypes to enhance sustainable protection against pathogens.

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., Competing Interests: Conflict of interest statement. None declared., (Published by Oxford University Press on behalf of American Society of Plant Biologists 2023.)

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/m 2 , 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/m 2 applied at 4 or 400 s. When detached grapevine leaves were exposed during darkness to UV-C at 100 J/m 2 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/m 2 . In the same vineyard in 2020, UV-C was applied once or twice weekly at doses of 70, 100, or 200 J/m 2 , and severity of E. necator on both leaves and fruit was significantly reduced compared to untreated controls; twice-weekly applications at 200 J/m 2 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/m 2 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/m 2 in 2020 and after weekly or twice-weekly application of UV-C at 100 or 200 J/m 2 in 2021. In 2019, weekly nighttime applications of UV-C at 200 J/m 2 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/m 2 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., Competing Interests: The author(s) declare no conflict of interest.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Published by Oxford University Press on behalf of Nanjing Agricultural University 2023.)

Plant disease evaluation is crucial to pathogen management and plant breeding. Human field scouting has been widely used to monitor disease progress and provide qualitative and quantitative evaluation, which is costly, laborious, subjective, and often imprecise. To improve disease evaluation accuracy, throughput, and objectiveness, an image-based approach with a deep learning-based analysis pipeline was developed to calculate infection severity of grape foliar diseases. The image-based approach used a ground imaging system for field data acquisition, consisting of a custom stereo camera with strobe light for consistent illumination and real time kinematic (RTK) GPS for accurate localization. The deep learning-based pipeline used the hierarchical multiscale attention semantic segmentation (HMASS) model for disease infection segmentation, color filtering for grapevine canopy segmentation, and depth and location information for effective region masking. The resultant infection, canopy, and effective region masks were used to calculate the severity rate of disease infections in an image sequence collected in a given unit (e.g., grapevine panel). Fungicide trials for grape downy mildew (DM) and powdery mildew (PM) were used as case studies to evaluate the developed approach and pipeline. Experimental results showed that the HMASS model achieved acceptable to good segmentation accuracy of DM (mIoU > 0.84) and PM (mIoU > 0.74) infections in testing images, demonstrating the model capability for symptomatic disease segmentation. With the consistent image quality and multimodal metadata provided by the imaging system, the color filter and overlapping region removal could accurately and reliably segment grapevine canopies and identify repeatedly imaged regions between consecutive image frames, leading to critical information for infection severity calculation. Image-derived severity rates were highly correlated (r > 0.95) with human-assessed values, and had comparable statistical power in differentiating fungicide treatment efficacy in both case studies. Therefore, the developed approach and pipeline can be used as an effective and efficient tool to quantify the severity of foliar disease infections, enabling objective, high-throughput disease evaluation for fungicide trial evaluation, genetic mapping, and breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Gold, Combs, Cadle-Davidson and Jiang.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Grapevine is one of the most widely-planted fruit crops in the world, and is the most economically important fruit crop in the state of New York, USA. Symptoms of anthracnose on grapevine are similarly widely-reported on grapevine fruit and foliage, and such symptoms are commonly attributed to Elsinöe ampelina (Wilcox et al., 2015). However, similar symptoms, if not identical, to those associated with E. ampelina have been sporadically attributed to various species in the genus Colletotrichum. In September 2021, a survey was conducted in three research vineyards at Cornell AgriTech in Geneva, NY. Symptoms of anthracnose werebserved on four Vitis interspecific hybrid breeding lines in a 1 ha vineyard. Leaves, fruit, and petioles showing symptoms of anthracnose, i.e., sunken necrotic lesions with grayish centers and brownish margins, were collected. Symptomatic and healthy portions of surface-sterilized tissues were placed on PDA medium and incubated at 23oC for 7 days. Several petiole samples yielded colonies of white to greyish mycelium, with some red to orange pigmentation (Fig. 1A and 1B), similar to those described by Chowdappa et al. (2009) for Colletotrichum species isolated from grapevine in India. Cultures were allowed to sporulate. Slides from cultures were prepared and examined at 400X magnification. Conidia from cultures were cylindrical with rounded ends, 13.5-15.2 μm in length and 7.6-9.0 μm in width (Fig. 1C). Koch's postulates were fulfilled by inoculating detached healthy leaves of V. vinifera 'Chardonnay' that had been surface sterilized in 10% sodium hypochlorite and triple-rinsed in sterile distilled water. Drop inoculation was used from a suspension of 105 conidia/ml from the foregoing pure cultures as five 2 µL droplets per leaf. Inoculated detached leaves were maintained on water agar in a Petri dish at 23oC. Four days after inoculation, symptoms were observed and compared with the originally collected samples. Inoculated leaves displayed symptoms typically found on the collected tissues, and the original pathogen, as confirmed by colony morphology and conidial characteristics and dimensions, was reisolated from inoculated leaves, and not from non-inoculated controls. For molecular characterization, fungal DNA was isolated by using Qiagen DNeasy kit and amplified using the following primer pairs: ITS1/ITS4, TEF (Hyun et al., 2009), E. ampelina F/R (Santos et al. 2018), TUB2, ACT, HIS3, GAPDH and CHS1 (Damm et al., 2001). PCR products were purified using ExoSAP-IT, and samples were Sanger sequenced. Sequences were analyzed using Geneious Prime software, and the resulting sequences (NCBI accessions OL720215, OL720216, OL720217, OL720218, OL853836, OM982612, OM982613, OM982614, OM982615 and OM982616) had 94 to 100% identity to Colletotrichum fioriniae NCBI accessions MN944922.1, MK646015.1, MN944922.1, MN856415.1, KU847413.1, MN520490.1, MN544294.1, KY695259.1, MN535117.1 and MN544295.1. Symptoms of grapevine anthracnose caused by Colletotrichum species have been reported from India (Chowdappa et al., 2009) and Korea (Kim et al., 2021). To our knowledge this is the first report of grapevine anthracnose caused by C. fioriniae Anthracnose and ripe rot are diseases of increasing importance, particularly as new grapevine cultivars with resistance to powdery mildew or downy mildew are adopted. Taxonomy of the causal agents (E. ampelina and Colletotrichum spp.) has undergone considerable revision. Consequently, distribution and relative prevalence of the various taxa will require further study.

Increased map density and transferability of markers are essential for the genetic analysis of fruit quality and stress tolerance in interspecific grapevine populations. We used 1449 GBS and 2000 rhAmpSeq markers to develop a dense map for an interspecific F 2 population (VRS-F 2 ) that was derived by selfing a single F 1 from a Vitis riparia x 'Seyval blanc' cross. The resultant map contained 2519 markers spanning 1131.3 cM and was highly collinear with the Vitis vinifera 'PN40024' genome. Quantitative trait loci (QTL) for berry skin color and flower type were used to validate the map. Four rhAmpSeq transferable markers were identified that can be used in pairs (one pistillate and one hermaphroditic) to predict pistillate and hermaphrodite flower type with ≥99.7% accuracy. Total and individual anthocyanin diglucoside QTL mapped to chromosome 9 near a 5-O-GLUCOSYLTRANSFERASE candidate gene. Malic acid QTL were observed on chromosome 1 and 6 with two MALATE DEHYRDROGENASE CYTOPLASMIC 1 and ALUMINUM-ACTIVATED MALATE TRANSPORTER 2-LIKE ( ALMT ) candidate genes, respectively. Modeling malic acid identified a potential QTL on chromosome 8 with peak position in proximity of another ALMT. A first-ever reported QTL for the grassy smelling volatile (E)-2-hexenal was found on chromosome 2 with a PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE candidate gene near peak markers.

Malate is a major contributor to the sourness of grape berries ( Vitis spp.) and their products, such as wine. Excessive malate at maturity, commonly observed in wild Vitis grapes, is detrimental to grape and wine quality and complicates the introgression of valuable disease resistance and cold hardy genes through breeding. This study investigated an interspecific Vitis family that exhibited strong and stable variation in malate at ripeness for five years and tested the separate contribution of accumulation, degradation, and dilution to malate concentration in ripe fruit in the last year of study. Genotyping was performed using transferable rhAmpSeq haplotype markers, based on the Vitis collinear core genome. Three significant QTL for ripe fruit malate on chromosomes 1, 7, and 17, accounted for over two-fold and 6.9 g/L differences, and explained 40.6% of the phenotypic variation. QTL on chromosomes 7 and 17 were stable in all and in three out of five years, respectively. Variation in pre-veraison malate was the major contributor to variation in ripe fruit malate (39%), and based on two and five years of data, respectively, their associated QTL overlapped on chromosome 7, indicating a common genetic basis. However, use of transferable markers on a closely related Vitis family did not yield a common QTL across families. This suggests that diverse physiological mechanisms regulate the levels of this key metabolite in the Vitis genus, a conclusion supported by a review of over a dozen publications from the past decade, showing malate-associated genetic loci on all 19 chromosomes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

The foliage of the native grape species Vitis riparia and certain cold-hardy hybrid grapes are particularly susceptible to the insect pest phylloxera, Daktulosphaira vitifoliae Fitch. A previous study using a cold-hardy hybrid grape biparental F1 population (N~125) detected the first quantitative trait locus (QTL) for foliar resistance on chromosome 14, designated as resistance to Daktulosphaira vitifoliae 3 (Rdv3). This locus spans a ~7-Mbp (10-20 cM) region and is too wide for effective marker-assisted selection or identification of candidate genes. Therefore, we fine mapped the QTL using a larger F1 population, GE1783 (N~1023), and genome-wide rhAmpSeq haplotype markers. Through three selective phenotyping experiments replicated in the greenhouse, we screened 184 potential recombinants of GE1783 using a 0 to 7 severity rating scale among other phylloxera severity traits. A 500-kb fine mapped region at 4.8 Mbp on chromosome 14 was identified. The tightly linked rhAmpSeq marker 14_4805213 and flanking markers can be used for future marker-assisted breeding. This region contains 36 candidate genes with predicted functions in disease resistance (R genes and Bonzai genes) and gall formation (bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase). Disease resistance genes suggest a traditional R-gene-mediated resistance mechanism often accompanied by a hypersensitive response, which has been widely studied in the plant pathology field. A novel resistance mechanism, non-responsiveness to phylloxera gall formation is proposed as a function of the bifunctional dehydratase gene, which plays a role in gallic acid biosynthesis and is important in gall formation. This study has implications for improvement of foliar phylloxera resistance in cold-hardy hybrid germplasm and is a starting place to understand the mechanism of resistance in crops to gall-forming insects., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved.)

Vitis has two subgenera: Euvitis, which includes commercially important Vitis vinifera and interspecific hybrid cultivars, and Muscadinia. Of note, the market for Muscadinia grapes remains small, and only Muscadinia rotundifolia is cultivated as a commercial crop. To establish a basis for the study of Muscadinia species, we generated chromosome-level whole-genome sequences of Muscadinia rotundifolia cv. Noble. A total of 393.8 Mb of sequences were assembled from 20 haploid chromosomes, and 26 394 coding genes were identified from the sequences. Comparative analysis with the genome sequence of V. vinifera revealed a smaller size of the M. rotundifolia genome but highly conserved gene synteny. A genome-wide association study of 12 Muscadinia berry-related traits was performed among 356 individuals from breeding populations of M. rotundifolia. For the transferability of markers between Euvitis and Muscadinia, we used 2000 core genome rhAmpSeq markers developed to allow marker transferability across Euvitis species. A total of 1599 (80%) rhAmpSeq markers returned data in Muscadinia. From the GWAS analyses, we identified a total of 52 quantitative trait nucleotides (QTNs) associated with the 12 berry-related traits. The transferable markers enabled the direct comparison of the QTNs with previously reported results. The whole-genome sequences along with the GWAS results provide a new basis for the extensive study of Muscadinia species., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Hydroxycinnamylated anthocyanins (or simply 'acylated anthocyanins') increase color stability in grape products, such as wine. Several genes that are relevant for anthocyanin acylation in grapes have been previously described; however, control of the degree of acylation in grapes is complicated by the lack of genetic markers quantitatively associated with this trait. To characterize the genetic basis of anthocyanin acylation in grapevine, we analyzed the acylation ratio in two closely related biparental families, Vitis rupestris B38 × 'Horizon' and 'Horizon' × Illinois 547-1, for 2 and 3 years, respectively. The acylation ratio followed a bimodal and skewed distribution in both families, with repeatability estimates larger than 0.84. Quantitative trait locus (QTL) mapping with amplicon-based markers (rhAmpSeq) identified a strong QTL from 'Horizon' on chromosome 3, near 15.85 Mb in both families and across years, explaining up to 85.2% of the phenotypic variance. Multiple candidate genes were identified in the 14.85-17.95 Mb interval, in particular, three copies of a gene encoding an acetyl-CoA-benzylalcohol acetyltransferase-like protein within the two most strongly associated markers. Additional population-specific QTLs were found in chromosomes 9, 10, 15, and 16; however, no candidate genes were described. The rhAmpSeq markers reported here, which were previously shown to be highly transferable among the Vitis genus, could be immediately implemented in current grapevine breeding efforts to control the degree of anthocyanin acylation and improve the quality of grapes and their products.

Plant pathogens usually originate and diversify in geographical regions where hosts and pathogens co-evolve. Erysiphe necator, the causal agent of grape powdery mildew, is a destructive pathogen of grapevines worldwide. Although Eastern US is considered the centre of origin and diversity of E. necator, previous reports on resistant native wild and domesticated Asian grapevines suggest Asia as another possible origin of the pathogen. By using multi-locus sequencing, microsatellites and a novel application of amplicon sequencing (AmpSeq), we show that the population of E. necator in Israel is composed of three genetic groups: Groups A and B that are common worldwide, and a new group IL, which is genetically differentiated from any known group in Europe and Eastern US. Group IL showed distinguished ecological characteristics: it was dominant on wild and traditional vines (95%); its abundance increased along the season; and was more aggressive than A and B isolates on both wild and domesticated vines. The low genetic diversity within group IL suggests that it has invaded Israel from another origin. Therefore, we suggest that the Israeli E. necator population was founded by at least two invasions, of which one could be from a non-East American source, possibly from Asian origin., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Karn, Zou, Brooks, Fresnedo-Ramírez, Gabler, Sun, Ramming, Naegele, Ledbetter and Cadle-Davidson.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Musich, Cadle-Davidson and Osier.)

Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine ( Vitis vinifera L. ssp. vinifera ). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Segregation for leaf trichome density was observed in a cold-hardy hybrid grape population GE1025 ( N = ∼125, MN1264 × MN1246) that was previously used to detect a quantitative trait locus (QTL) underlying foliar phylloxera resistance on chromosome 14. Our hypothesis was that high trichome density was associated with resistance to phylloxera. Existing literature found trichome density QTL on chromosomes 1 and 15 using a hybrid grape population of "Horizon" × Illinois 547-1 and suggested a few candidate genes. To validate the reported QTL and our hypothesis, interval mapping was conducted in GE1025 with previous genotyping-by-sequencing (GBS) single nucleotide polymorphism (SNP) genotype data and phenotypic scores collected using a 0-6 trichome density scale at several leaf positions. Evaluations were done on replicated forced dormant cuttings in 2 years and on field-grown leaves in 1 year. There was no strong relationship between trichome density and phylloxera resistance except for a Pearson's correlation (r) of about -0.2 between a few trichome density traits and phylloxera severity traits at 2 and 3 weeks after infestation. Two genetic regions were repeatedly detected for multiple trichome density traits: from 10 to 20.7 Mbp (∼10 Mbp) on chromosome 1 for ribbon and simple density traits and from 2.4 to 8.9 Mbp on chromosome 10 for ribbon density traits, explaining 12.1-48.2 and 12.6-27.5% of phenotypic variation, respectively. To fine map, we genotyped a larger population, GE1783 ( N = ∼1,023, MN1264 × MN1246), with conserved rhAmpSeq haplotype markers across multiple Vitis species and phenotyped 233 selected potential recombinants. Evaluations were conducted on field-grown leaves in a single year. The QTL for ribbon trichome density on adaxial vein and adaxial leaf and simple density on abaxial vein was fine mapped to 12.63-13.38 Mbp (747 kb) on chromosome 1. We found variations of MN1264 and MN1246 at candidate genes NAC transcription factor 29, EF-hand protein, and MYB140 in this region and three other surrounding candidate genes proposed previously. Even though no strong relationship between foliar phylloxera resistance and trichome density was found, this study validated and fine mapped a major QTL for trichome density using a cold-hardy hybrid grape population and shed light on a few candidate genes that have implications for different breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yin, Karn, Cadle-Davidson, Zou, Underhill, Atkins, Treiber, Voytas and Clark.)

Powdery mildews can be controlled by brief exposure to ultraviolet (UV) radiation with devastating effect on their developmental stages including conidia germination. The treatment effect can be impaired by subsequent exposure to UV-A/blue light. UV-A/blue light-activated photolyase may be responsible for this and therefore we tested the function of three cryptochrome/photolyase family (CPF)-like genes (OINE01015670_T110144, OINE01000912_T103440, and OINE01005061_T102555) identified in the obligate biotrophic fungus Pseudoidium neolycopersici , the cause of tomato powdery mildew. A photolyase-deficient mutant of Escherichia coli transformed with coding sequence of OINE01000912_T103440 and exposed to brief (UV)-C treatment (peak emission at 254 nm) showed photoreactivation and cell survival when exposed to subsequent blue light, indicating complementation of photolyase activity. In contrast, the same photolyase-deficient E. coli transformed with the coding sequences of other two CPF-like genes did not survive this treatment, even though their expression were confirmed at protein level. This confirmed that OINE01000912_T103440 is a gene encoding photolyase, here named PnPHR1 , with functionality similar to the native photolyase in E. coli , and classified as a class I cyclobutane pyrimidine dimer (CPD) photolyase. Modeling of the 634-amino acid sequence of PnPHR1 suggested that it is capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). However, spectroscopic data of the protein produced in an E. coli expression system could only reveal the presence of a reduced form of FAD, i.e., FADH - as an intrinsic chromophore. Within the tested wavelength range of 365-525 nm, the survival of photolyase-deficient mutant E. coli transformed with PnPHR1 s howed a broad action spectrum from 365 to 454 nm. This was very similar to the previously characterized action spectrum for survival of P. neolycopersici conidia that had been treated with UV-C. Quantitative RT-PCR revealed that the expression of PnPHR1 in P. neolycopersici conidia was induced by UV-C, and peak expression occurred 4 h after brief UV-C treatment. The expression of PnPHR1 was repressed when incubated in red light after the UV-C treatment, but not when incubated in UV-A/blue light. The results may explain why the disease-reducing effect of short wavelength UV is impaired by exposure to UV-A and blue light., (Copyright © 2020 Pathak, Ergon, Stensvand, Gislerød, Solhaug, Cadle-Davidson and Suthaparan.)

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.

Transferable DNA markers are essential for breeding and genetics. Grapevine (Vitis) breeders utilize disease resistance alleles from congeneric species ~20 million years divergent, but existing Vitis marker platforms have cross-species transfer rates as low as 2%. Here, we apply a marker strategy targeting the inferred Vitis core genome. Incorporating seven linked-read de novo assemblies and three existing assemblies, the Vitis collinear core genome is estimated to converge at 39.8 Mb (8.67% of the genome). Adding shotgun genome sequences from 40 accessions enables identification of conserved core PCR primer binding sites flanking polymorphic haplotypes with high information content. From these target regions, we develop 2,000 rhAmpSeq markers as a PCR multiplex and validate the panel in four biparental populations spanning the diversity of the Vitis genus, showing transferability increases to 91.9%. This marker development strategy should be widely applicable for genetic studies in many taxa, particularly those ~20 million years divergent.

Isolating DNA from microbes on the surface of a grape berry is a challenge due to their adhesion to the thick berry skin and cuticle, making studies of the grape microbiome challenging. We developed a field-to-lab DNA extraction procedure that starts in the vineyard, disrupts the grape berry surface while en route to the lab through agitation, and efficiently extracts microbial DNA from the surface of the grape. It is cost effective and utilizes commonly available laboratory chemicals with low toxicity (Table 1). This protocol allows researchers to extract DNA from the grape berry surface in the field, therefore undergoing minimal manipulation of those microbiomes before DNA extraction., (© 2019 Published by Elsevier Ltd.)

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 μ m 2 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., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2019 Andrew Bierman et al.)

Amplicon sequencing (AmpSeq) is a practical, intuitive strategy with a semi-automated computational pipeline for analysis of highly multiplexed PCR-derived sequences. This genotyping platform is particularly cost-effective when multiplexing 96 or more samples with a few amplicons up to thousands of amplicons. Amplicons can target from a single nucleotide to the upper limit of the sequencing platform. The flexibility of AmpSeq's wet lab methods make it a tool of broad interest for diverse species, and AmpSeq excels in flexibility, high-throughput, low-cost, accuracy, and semi-automated analysis. Here we provide an open science framework procedure to output data out of an AmpSeq project, with an emphasis on the bioinformatics pipeline to generate SNPs, haplotypes and presence/absence variants in a set of diverse genotypes. Open-access tutorial datasets with actual data and a containerization open source software instance are provided to enable training in each of these genotyping applications. The pipelines presented here should be applicable to the analysis of various target-enriched (e.g., amplicon or sequence capture) Illumina sequence data.

Sour rot is a disease complex produced by an interaction between grape berries and various species of yeast and acetic acid bacteria in the presence of Drosophila fruit flies. While yeast and bacteria are consistently found on healthy grape berries worldwide, we explored whether the composition of these epiphytic communities differed depending on the presence or absence of sour rot symptoms. Using high-throughput sequencing, we characterized the microbiome of sour rot-affected grapes from two geographical areas across two years. In 2015 and 2016, both healthy and sour rot-affected berries were collected from commercial and research vineyards in Geneva, NY and commercial vineyards in Tasmania, AUS. In this experiment, all associated organisms grouped together primarily by location, and not by presence/absence of symptoms or cultivar. The predominant difference between asymptomatic and symptomatic samples, regardless of location, was the abundance of Acetobacter species, which were significantly more plentiful in the symptomatic samples. Yeast genera such as Candida, Hanseniaspora, Pichia and Saccharomyces were abundant in both sets of samples, but varied by region. The consistent presence of yeast species and the increased abundance of acetic acid-generating bacteria is consistent with our understanding of their etiological role in sour rot development. In 2016, diseased grapes also were collected from vineyards in Fredonia, NY, and Modesto, CA. Consistent with our comparison study, all associated organisms again grouped together primarily by location. Yeast genera such as Candida, Hanseniaspora, Pichia and Saccharomyces were abundant in both sets of samples, but varied by region. The consistent presence of yeast species and the abundance of acetic acid-generating bacteria in both experiments is consistent with our understanding of their etiological role in sour rot development., Competing Interests: The authors have declared that no competing interests exist.

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.

Sour rot, a disease affecting berries of cultivated Vitis spp. worldwide, has not been clearly defined. Reported symptoms of the disease include browning of the berry skin, oozing of disintegrated berry pulp, and the smell of acetic acid, all in the presence of fruit flies (Drosophila spp.). We determined acetic acid concentrations in multiple collections of symptomatic berries, isolated and identified microbes from them, and inoculated commonly isolated organisms into healthy berries with and without concurrent exposure to wild-type or axenic Drosophila melanogaster. Coinoculations combining one of several yeasts (Metschnikowia spp., Pichia spp., and a Saccharomyces sp.) plus an acetic acid bacterium (an Acetobacter sp. and Gluconobacter spp.) reproduced sour rot symptoms, defined here as decaying berries with a loss of turgor and containing acetic acid at a minimum of 0.83 g/liter, based on observed field levels. Symptoms developed only in the presence of D. melanogaster, either wild type or axenic, indicating a nonmicrobial contribution of these insects in addition to a previously suggested microbial role. We conclude that sour rot is the culmination of coinfection by various yeasts, which convert grape sugars to ethanol, and bacteria that oxidize the ethanol to acetic acid, and that this process is mediated by Drosophila spp.

Key Message: Downy mildew resistance across days post-inoculation, experiments, and years in two interspecific grapevine F 1 families was investigated using linear mixed models and Bayesian networks, and five new QTL were identified. Breeding grapevines for downy mildew disease resistance has traditionally relied on qualitative gene resistance, which can be overcome by pathogen evolution. Analyzing two interspecific F 1 families, both having ancestry derived from Vitis vinifera and wild North American Vitis species, across 2 years and multiple experiments, we found multiple loci associated with downy mildew sporulation and hypersensitive response in both families using a single phenotype model. The loci explained between 7 and 17% of the variance for either phenotype, suggesting a complex genetic architecture for these traits in the two families studied. For two loci, we used RNA-Seq to detect differentially transcribed genes and found that the candidate genes at these loci were likely not NBS-LRR genes. Additionally, using a multiple phenotype Bayesian network analysis, we found effects between the leaf trichome density, hypersensitive response, and sporulation phenotypes. Moderate-high heritabilities were found for all three phenotypes, suggesting that selection for downy mildew resistance is an achievable goal by breeding for either physical- or non-physical-based resistance mechanisms, with the combination of the two possibly providing durable resistance.

Key Message: Rapid characterization of novel NB-LRR-associated resistance to Phomopsis cane spot on grapevine using high-throughput sampling and low-coverage sequencing for genotyping, locus mapping and transcriptome analysis provides insights into genetic resistance to a hemibiotrophic fungus. Phomopsis cane and leaf spot, caused by the hemibiotrophic fungus Diaporthe ampelina (syn = Phomopsis viticola), reduces the productivity in grapevines. Host resistance was studied on three F 1 families derived from crosses involving resistant genotypes 'Horizon', Illinois 547-1, Vitis cinerea B9 and V. vinifera 'Chardonnay'. All families had progeny with extremely susceptible phenotypes, developing lesions on both dormant canes and maturing fruit clusters. Segregation of symptoms was observed under natural levels of inoculum in the field, while phenotypes on green shoots were confirmed under controlled inoculations in greenhouse. High-density genetic maps were used to localize novel qualitative resistance loci named Rda1 and Rda2 from V. cinerea B9 and 'Horizon', respectively. Co-linearity between reference genetic and physical maps allowed localization of Rda2 locus between 1.5 and 2.4 Mbp on chromosome 7, and Rda1 locus between 19.3 and 19.6 Mbp of chromosome 15, which spans a cluster of five NB-LRR genes. Further dissection of this locus was obtained by QTL mapping of gene expression values 14 h after inoculation across a subset of the 'Chardonnay' × V. cinerea B9 progeny. This provided evidence for the association between transcript levels of two of these NB-LRR genes with Rda1, with increased NB-LRR expression among susceptible progeny. In resistant parent V. cinerea B9, inoculation with D. ampelina was characterized by up-regulation of SA-associated genes and down-regulation of ethylene pathways, suggesting an R-gene-mediated response. With dominant effects associated with disease-free berries and minimal symptoms on canes, Rda1 and Rda2 are promising loci for grapevine genetic improvement.

Quantitative phenotyping of downy mildew sporulation is frequently used in plant breeding and genetic studies, as well as in studies focused on pathogen biology such as chemical efficacy trials. In these scenarios, phenotyping a large number of genotypes or treatments can be advantageous but is often limited by time and cost. We present a novel computational pipeline dedicated to estimating the percent area of downy mildew sporulation from images of inoculated grapevine leaf discs in a manner that is time and cost efficient. The pipeline was tested on images from leaf disc assay experiments involving two F 1 grapevine families, one that had glabrous leaves (Vitis rupestris B38 × 'Horizon' [RH]) and another that had leaf trichomes (Horizon × V. cinerea B9 [HC]). Correlations between computer vision and manual visual ratings reached 0.89 in the RH family and 0.43 in the HC family. Additionally, we were able to use the computer vision system prior to sporulation to measure the percent leaf trichome area. We estimate that an experienced rater scoring sporulation would spend at least 90% less time using the computer vision system compared with the manual visual method. This will allow more treatments to be phenotyped in order to better understand the genetic architecture of downy mildew resistance and of leaf trichome density. We anticipate that this computer vision system will find applications in other pathosystems or traits where responses can be imaged with sufficient contrast from the background.

Quantitative trait locus (QTL) identification in perennial fruit crops is impeded largely by their lengthy generation time, resulting in costly and labor-intensive maintenance of breeding programs. In a grapevine (genus Vitis ) breeding program, although experimental families are typically unreplicated, the genetic backgrounds may contain similar progenitors previously selected due to their contribution of favorable alleles. In this study, we investigated the utility of joint QTL identification provided by analyzing half-sib families. The genetic control of powdery mildew was studied using two half-sib F 1 families, namely GE0711/1009 (MN1264 × MN1214; N  = 147) and GE1025 (MN1264 × MN1246; N  = 125) with multiple species in their ancestry. Maternal genetic maps consisting of 1077 and 1641 single nucleotide polymorphism (SNP) markers, respectively, were constructed using a pseudo-testcross strategy. Ratings of field resistance to powdery mildew were obtained based on whole-plant evaluation of disease severity. This 2-year analysis uncovered two QTLs that were validated on a consensus map in these half-sib families with improved precision relative to the parental maps. Examination of haplotype combinations based on the two QTL regions identified strong association of haplotypes inherited from 'Seyval blanc', through MN1264, with powdery mildew resistance. This investigation also encompassed the use of microsatellite markers to establish a correlation between 206-bp (UDV-015b) and 357-bp (VViv67) fragment sizes with resistance-carrying haplotypes. Our work is one of the first reports in grapevine demonstrating the use of SNP-based maps and haplotypes for QTL identification and tagging of powdery mildew resistance in half-sib families., Competing Interests: The authors declare no conflict of interest. Ethical standards This article does not contain any studies with human participants or animal performed by any of the authors.

The genomics era brought unprecedented opportunities for genetic analysis of host resistance, but it came with the challenge that accurate and reproducible phenotypes are needed so that genomic results appropriately reflect biology. Phenotyping host resistance by natural infection in the field can produce variable results due to the uncontrolled environment, uneven distribution and genetics of the pathogen, and developmentally regulated resistance among other factors. To address these challenges, we developed highly controlled, standardized methodologies for phenotyping powdery mildew resistance in the context of a phenotyping center, receiving samples of up to 140 grapevine progeny per F 1 family. We applied these methodologies to F 1 families segregating for REN1- or REN2-mediated resistance and validated that some but not all bioassays identified the REN1 or REN2 locus. A point-intercept method (hyphal transects) to quantify colony density objectively at 8 or 9 days postinoculation proved to be the phenotypic response most reproducibly predicted by these resistance loci. Quantitative trait locus (QTL) mapping with genotyping-by-sequencing maps defined the REN1 and REN2 loci at relatively high resolution. In the reference PN40024 genome under each QTL, nucleotide-binding site-leucine-rich repeat candidate resistance genes were identified-one gene for REN1 and two genes for REN2. The methods described here for centralized resistance phenotyping and high-resolution genetic mapping can inform strategies for breeding resistance to powdery mildews and other pathogens on diverse, highly heterozygous hosts.

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had <10% missing data, and read depth had a median of 220-244×. Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, low-cost and easily automated analysis.

RNA sequencing (RNA-seq) and genotyping-by-sequencing (GBS) were used for single nucleotide polymorphism (SNP) identification from two economically important obligate plant pathogens, Pseudoperonospora cubensis and P. humuli. Twenty isolates of P. cubensis and 19 isolates of P. humuli were genotyped using RNA-seq and GBS. Principle components analysis (PCA) of each data set showed genetic separation between the two species. Additionally, results supported previous findings that P. cubensis isolates from squash are genetically distinct from cucumber and cantaloupe isolates. A PCA-based procedure was used to identify SNPs correlated with the separation of the two species, with 994 and 4,231 PCA-correlated SNPs found within the RNA-seq and GBS data, respectively. The corresponding unigenes (n = 800) containing these potential species-specific SNPs were then annotated and 135 putative pathogenicity genes, including 3 effectors, were identified. The characterization of genes containing SNPs differentiating these two closely related downy mildew species may contribute to the development of improved detection and diagnosis strategies and improve our understanding of host specificity pathways.