Project Number: 2094-21220-003-000-D
Project Type: In-House Appropriated
Start Date: Mar 17, 2022
End Date: Mar 16, 2027
The long-term objective of this program is to formulate effective and economically sustainable methods for management of tree fruit diseases in high value production systems. Utilization of host resistance presents an economically effective, ecologically desirable, and durable disease control strategy. Additionally, horticultural traits that allow for disease avoidance, such as dwarfing of the scion that allows for better spray and light penetration of canopies, have yet to be widely incorporated into pear rootstocks. For this purpose, it is a necessary first step to understand the molecular mechanisms underlying resistance/avoidance traits in apple and pear roots by identifying key genes regulating root resistance responses and other relevant physiological processes. This research plan will make it possible to develop molecular tools for accurately and efficiently incorporating resistance traits into new apple and pear rootstocks. Objective 1: Discover rootstock resistance traits and architectural features in deciduous tree fruits involved in disease avoidance. Sub-objective 1.A: Characterize genotype-specific variations in biochemical and metabolic features linked to apple root resistance to P. ultimum. Sub-objective 1.B: Determine the connection between pear root architecture, disease resistance, and dwarfing. Sub-objective 1.C: Identify genetic components involved in rootstock-mediated dwarfing of pear scions. Sub-objective 1.D: Develop a rapid-cycle breeding tool for use in breeding pear rootstocks. Objective 2: Identify genotype-specific expression patterns of candidate genes underlying disease resistance and disease avoidance traits. Sub-objective 2.A: Conduct bioinformatic analysis of the sequence features of selected candidate genes identified in previous transcriptome analyses. Sub-objective 2.B: Characterize genotype-specific expression patterns for selected candidate genes between resistant and susceptible genotype groups. Sub-objective 2.C: Transgenic manipulation of selected apple candidate genes using CRISPR/Cas9 tool and in planta expression analysis.
A combination of current and emerging genetic and genomic techniques will be applied in this research plan. Various methodologies in phenotyping and molecular analysis including genomics, transcriptomics genetics, biochemistry, breeding and microscopy will be utilized to identify the genetic elements and unravel the regulation mechanisms underlying apple root disease resistance and pear architectural features. Such studies will improve knowledge of molecular mechanisms underlying important traits and genome annotation, facilitate the development of germplasm and establishment of a rapid-cycle breeding tool to enhance our understanding of how rootstocks confer traits to scions. Experiments will be conducted using previously identified apple rootstock germplasm pairs with contrasting resistant versus susceptible phenotypes, such as O3R5-#161 vs # 132; #58 vs #47 and/or #164 vs #1, respectively. Expression analysis of selected candidate genes and biochemical or enzymatic assays will provide experimental evidence connecting genes and trait during apple root response to P. ultimum infection. De novo sequencing of specific genomic fragments containing the genes of interest will identify variations at gene structure and sequences between resistant and susceptible O3R5 genotype groups, which may provide valuable reference for their functional roles in defense activation. The knockout (KO) transgenic line will be generated using established in-house protocols including plasmid construction, transformation of E. coli and Agrobacterium, subsequent callus induction and individual transgenic line establishment/propagation by tissue culture. For research related to pear architectural traits, parental genotypes will be established in tissue culture, rooted, acclimated, and grown to similar sizes before moving to different phenotyping methods during the first year. Data analysis will compare differential gene expression between the most and least dwarfing individuals across tissue type and time, allowing us to understand more about dynamic gene activity on the scion and rootstock sides of the graft union, as well as in the root system and how the rootstock is affecting scion leaf tissue. A major limitation for breeding new rootstocks is the long juvenility period of pear (reaching up to ~10 years in European pear), especially when stacking multiple traits is the desired goal. DNA-informed breeding speeds the selection process, but juvenility periods remain long, meaning variety releases are 20-40 years from initial crosses, depending on species and breeding scheme. To shorten this process, development of a rapid-cycle breeding tools by modifying flowering gene expression can greatly reduce amount of time allows for stacking multiple loci or traits.