The long-term goal of this project is to develop grape varieties thatpossess effective and durable resistance to powdery mildew (PM). Stacking resistance genes from multiple resistant genetic backgrounds and with the least functional redundancy is a proven breeding strategy to improve both durability and level of resistance. This strategy requires (a) the identification of multiple sources of resistance, (b) the functional characterization of the mechanisms of resistance to prioritize optimal genetic combination, and, finally, (c) marker assisted breeding to introduce the selected genes into elite varieties. The specific objectives of the 2014-2015 funding period consisted of (i) the genetic mapping of the two PM resistance loci in V. piasezkii and development of molecular markers for marker assisted breeding and (ii) the functional characterization of resistance responses in a panel of V. vinifera accessions (Ren1, Ren6, Ren7). We have successfully completed the genetic map of the Chinese species V. piasezkii and identified two new loci, Ren6 and Ren7 that manifest complete and partial resistance to powdery mildew, respectively. Both of these loci are present on two chromosomes that were not reported before in earlier studies. The identification of these two loci has enhanced the repertoire of resistance loci for grape powdery mildew breeding. We have developed closely linked markers that will be used to facilitate the combining of resistance from V. piasezkii to other advance powdery mildew resistant selections in our grape breeding program for durable field resistance. To be able to select which specific accessions to use for further breeding we initiated the characterization of the resistance responses using RNA sequencing approaches (RNA-seq). RNA-seq was successfully applied to study early (1 day post infection, dpi) and late (5 dpi) responses to powdery mildew associated with Ren1 resistance. Comparisons of 7 V. vinifera accessions carrying the Ren1 locus led to the identification of sets of constitutive andinducible defense related genes associated with Ren1-dependent resistance. We also successfully produced replicated PM infections in control environment for individual genotypes segregating for Ren6, Ren7, or both Ren6/Ren7, for which sequencing libraries are being sequenced. Though breeding for genetic resistance is ongoing and new fungicides are being developed, little is known regarding how E. necator evolves to become resistant to fungicides or how it overcomes host immune responses.
In this grant review period we applied DNA sequencing to study the genomes of five E. necator isolates, and RNA-seq and comparative genomics to predict and annotate the protein-coding genes in the E. necator genome. Our results show that the E. necator genome is exceptionally large and repetitive and suggest that transposable elements are responsible for genome expansion. Frequent structural variations were found between isolates and included copy number variation in EnCYP51, the target of the commonly used sterol demethylase inhibitor (DMI) fungicides. A panel of 89 additional E. necator isolates collected from diverse vineyard sites was screened for copy number variation in the EnCYP51 gene and for presence/absence of a point mutation (Y136F) known to result in higher fungicide tolerance. We show that an increase in EnCYP51 copy number is significantly more likely to be detected in isolates collected from fungicide-treated vineyards. Increased EnCYP51 copy numbers were detected with the Y136F allele, suggesting that an increase in copy number becomes advantageous only after the fungicide-tolerant allele is acquired. We also show that EnCYP51 copy number influences expression in a gene-dose dependent manner and correlates with fungal growth in the presence of a DMI fungicide.