Lessons from a Phenotyping Center Revealed by the Genome-Guided Mapping of Powdery Mildew Resistance Loci

Abstract

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 family. We applied these methodologies to F 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.

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

“…Thus, with appropriate lighting, as employed by the APS system, the 4-5 휇m in diameter fungal hyphae in nascent colonies of E. necator can be resolved on live samples, using 3× magnification within 48 hours after inoculation. The hyphal transect method represents the previous gold standard for manual quantification of grapevine powdery mildew disease severity [21] and aside from throughput and repeated measures, there are strengths and weaknesses in data quality compared to the APS developed here. The primary weakness of hyphal transects comes from subsampling only along the vertical and horizontal transects, thus missing any fungal growth that occurs away from these lines.…”

“…As a result, a major effort is underway to genetically map host resistance loci for introgression from wild Vitis into domesticated V. vinifera [17][18][19][20]. In previous studies of host resistance to E. necator and pathogen resistance to fungicides, controlled phenotyping of E. necator on grape leaf tissue used 1-cm diameter circular leaf disks cut from living grape leaves, arrayed on agar within Petri dishes or glass trays [21][22][23]. For host resistance assessment at 2 to 10 days after inoculation, the disks were destructively sampled by bleaching the leaf samples and then staining with a dye to make the hyphae more visible for phenotypic analysis under brightfield microscopy at 100× to 400× [21].…”

“…In previous studies of host resistance to E. necator and pathogen resistance to fungicides, controlled phenotyping of E. necator on grape leaf tissue used 1-cm diameter circular leaf disks cut from living grape leaves, arrayed on agar within Petri dishes or glass trays [21][22][23]. For host resistance assessment at 2 to 10 days after inoculation, the disks were destructively sampled by bleaching the leaf samples and then staining with a dye to make the hyphae more visible for phenotypic analysis under brightfield microscopy at 100× to 400× [21]. Severity of infection was estimated by hyphal transects, a pointintercept method adapted from vegetation analysis, where the number of hyphal interceptions of axial transects in the field of view was recorded as the response variable.…”

SNP markers tightly linked to root knot nematode resistance in grapevine (Vitis cinerea) identified by a genotyping-by-sequencing approach followed by Sequenom MassARRAY validation

“…The C2-50 and Riesling SNP sets were reduced to 367 and 403 SNPs, respectively, as described in the material methods and R/OneMap was used to curate and construct the genetic maps. For each SNP set, 19 linkage groups (LGs) were produced and the final size for the C2-50 and Riesling genetic maps were 1587.3 and 1706.4 cM, respectively ( S5 and S6 Tables), which is similar in size to other Vitis genetic maps produced by next generation sequencing [ 23 , 25 , 26 , 28 ]. The map density or average distance between SNP markers for C2-50 and Riesling genetic maps was 4.3 and 4.2 cM, respectively.…”

Emergent Ascomycetes in Viticulture: An Interdisciplinary Overview

“…Partial resistance is conferred by major QTLs found on different chromosomes. Ren2 on chromosome 14 confers race-specific resistance in V. cinerea (Dalbó et al, 2001; Cadle-Davidson et al, 2016). Ren 3 on chromosome 15–derived from an undetermined American Vitis species–was localized in the variety Regent (Welter et al, 2007) and recently found to determine race-specific hypersensitive response by two different regions on that chromosome; in fact, Zendler et al (2017) defined the Ren3 limit and identified ex novo the distal Ren9 locus.…”