Location: Physiology and Pathology of Tree Fruits Research
2022 Annual Report
Objectives
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.
Approach
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.
Progress Report
This is a new project which replaced expired project 2094-21220-002-000D, titled, "Utilization of the Rhizosphere Microbiome and Host Genetics to Manage Soil-borne Diseases”. The following report provides the progress that has been made so far in fiscal year (FY) 22.
Research in support of Objective 1 includes the completion of establishing and optimizing a procedure of lignin detection in apple root tissue to compare cell wall lignification among apple rootstock genotypes. Other biochemical assays, including for phenylalanine ammonia-lyase (PAL) and selected kinases, are currently being tested (Sub-objective 1A). Protocols/resources for investigating the role of select compounds in susceptibility/tolerance to replant disease are also in development. Test panel organisms (fungi/oomycetes previously isolated from apple roots) have been revived from water stocks and metabolites have been obtained. Assays are currently underway to identify the best compounds to advance to the next stage. Initial trials using the disk diffusion assay resulted in difficult to quantify/unexpected growth patterns. A protocol for measuring growth inhibition in liquid culture is being developed. Germplasm for the 8 diverse parental genotypes were obtained and two of them have begun to be propagated in tissue cultures, along with industry standard controls. Rooting protocols are underway to determine the best rooting conditions for each cultivar, as they vary significantly in their hormone responses (Sub-objective 1B). Pear tree architecture-related genes have been identified and located within the Bartlett and Anjou genomes. A workflow was developed to identify gene family members in non-model species genomes, a typically difficult task due to poor genome annotations. Pear genome annotation was improved, and a more confident list of architecture genes was obtained. Further, individuals in a pear population segregating for dwarfing and precocity traits were identified, adventitious shoot material collected from these plants, and this tissue is being propagated for use in transcriptome experiments on dwarfing. All pear tissues except roots and young stems, have been collected for an expression atlas Bartlett and Anjou (Sub-objective 1C). Initial accessions have been established in tissue culture and transformations to deliver the flowering construct into these plants have been initiated (Sub-objective 1D).
For Objective 2, several groups of apple candidate genes were identified for their potential roles in an effective defense activation and contributing to the resistance traits to P. ultimum infection. Members of the apple laccase gene family, MdLac3, MdLac5, MdLac7a and MdLac7b, have been repeatedly identified by transcriptome analyses for their role in cell wall fortification of apple root during P. ultimum infection. Based on recent transcriptome data from our research, progress was made on identifying and retrieving the sequences of four laccase encoding genes from apple genome, and analyzing their sequence features relevant to their proposed functions using various bioinformatics analyzing tools. The presence of specific sequence domains and motifs in the promoter of these genes revealed new insights on their functional roles in the biological context of defense activation. For example, the existence of binding sites of several plant hormones and transcription factors in the promoter region of these genes suggested the involvement of other cellular factors modulating the functionality of selected laccase genes. MdLAC7b appeared to be a primary responsible gene implicating in effective defense activation based on the genotype-specific expression patterns during the process of infection using quantitative Reverse transcription PCR. The genomic copies of these genes were amplified, cloned and sequenced, to examine the potential sequence variation between the target materials (apple rootstock O3R5 genotypes) and those of the model apple genome sequences. Such accurate sequences information is crucial in designing “guide sequences” for subsequent genetic manipulation using tools such as CRISPR. Similar bioinformatic analyses were also performed on other selected genes including transcription factor families of WRKY containing WRKYQK (protein domain) and MYB (myeloblastosis oncogene), as well as MLO (Mildew resistance Locus susceptibility gene) gene family (Sub-objective 2A).
Accomplishments
