Location: Grape Genetics Research Unit (GGRU)
Project Number: 8060-21220-007-000-D
Project Type: In-House Appropriated
Start Date: Apr 1, 2018
End Date: Mar 31, 2023
Objective:
Objective 1: Characterize host and pathogen genetic factors applicable to grapevine disease management, with primary emphasis on powdery mildew.
Sub-objective 1.A. Elucidate the genetic basis of host resistance via QTL mapping and genome editing.
Sub-objective 1.B. Identify and target pathogen genes required for infection of grapevine for improved disease management.
Objective 2: Dissect and elucidate the genetic, genomic, and physiological mechanisms of grapevine abiotic stress tolerance and environmental adaptation.
Sub-objective 2.A. Elucidate the physiological basis of temperature sensing in grapevine and develop a rigorous set of phenotypes for cold hardiness and chilling requirement traits.
Sub-objective 2.B. Determine the genetic architecture of winter survival mechanisms in grapevine through genetic mapping, gene expression, and candidate gene studies.
Objective 3: Generate new germplasm, tools, and strategies for improving grapevine fruit quality and other traits.
Sub-objective 3.A. Develop the CRISPR-Cas9 based genome editing tool for improving fruit quality and other traits in elite grape cultivars.
Sub-objective 3.B. Elucidate genetic control of red-flesh pigmentation in grape berries through genetic mapping and functional analysis.
Objective 4: Intergrate key tratis and QTLs into breeding germplasm. Objective 4 will be coordinated with research on genetics/genomics of host-plant resistance to disease and plant tolerance to abiotic stress for an integrated, systems approach to grapevine improvement. Anticipated products include trait selection for resistance to powdery mildew disease; tolerance to stress from adverse drought and cold grape growing conditions; and understanding genetic factors affecting grape quality.
Approach:
Sub-objective 1.A. Collect multi-year vineyard foliar ratings and conduct detailed analysis by controlled inoculation for representative populations. The isolate-specific, quantitative resistance data will improve the reproducibility and precision of QTL mapping, uncovering novel resistance and susceptibility QTL. Pursuit of clonal improvement of existing varieties by editing two powdery mildew susceptibility genes: MLO and a Pectate lyase-like (PLL) gene.
Sub-objective 1.B. Characterize how powdery mildew adapts resistance to fungicides and Candidate Secreted Effector Proteins (CSEPs) that may interact with R-genes released in future cultivars. Use AmpSeq primers for the multiplexed genotyping of known fungicide resistance gene target sites in E. necator. Sequencing of the mating type loci to confirm that selective advantages are occurring with even distribution across mating types and sequence SSRs to monitor for shifts in the population biology of the fungus.
Sub-objective 2.A. Develop new methods of phenotyping supercooling ability, acclimation/de-acclimation, and chilling requirements using a combination of studies in programmable chambers and under field conditions, as well as through deployment of replicated, winter-kill experiments with mapping populations made between highly cold-resistant and cold-sensitive grapevine genotypes. Assay traits using dormant buds collected from field grown vines and potted greenhouse plants. Total vine cold hardiness assayed as winter survival by planting mapping populations constructed between highly tolerant and highly sensitive cultivars. Sub-objective 2.B. Search for genetic loci associated with supercooling, rapid acclimation, delayed de-acclimation, and budburst control through the use of mapping populations and QTL analysis. Examine genome patterns of methylation, differential gene expression analysis of phenotypically diverse “sensitive” and “resistant” phenotypes to identify pathways and downstream candidate genes. Use transgene technology to overexpress and delete the function of key cold stress response genes.
Sub-objective 3.A. Use of a VvMybA gene as a target to develop a CRSPR-Cas9 genome editing tool for grapevine improvement. Adaptation of existing and/or develop new protocols for generating embryogenic callus from target varieties, building various configurations of expression vectors, transforming these vectors into embryogenic callus, and evaluating the transformed cells for successful editing. Pursuit of two additional approaches to generate genome edits without stable integration: a) bombard plasmid DNA transiently expressing both CRISPR and Cas9 components in grape cells to facilitate the editing process; and b) deliver in vitro preassembled complexes of both components (Cas9–gRNA ribonucleoproteins) into grape cells to execute genome editing activities. Sub-objective 3.B. Conduct QTL mapping in bi-parental populations segregating for flesh color, RT-PCR analysis of expression profiles of VymybA genes in skin and flesh tissues of developing berries, and functional analysis of allelic sequence variation in the promoter region of the key VvmybA gene responsible for red flesh.
