CAP: FAST SELECTION, ASSESSMENT, AND DELIVERY OF HLB-RESISTANT/TOLERANT CITRUS VARIANTS AS NEW SCION AND ROOTSTOCK CULTIVARS

Huanglongbing (HLB) is the most devastating disease of citrus in the U.S. Economic losses over the past five years in FL alone amount to over $1 billion per year, with nearly 5,000 jobs lost annually. The U.S. citrus industry is in dire need of new citrus cultivars with stable and broad-spectrum resistance/tolerance to HLB as well as desirable horticultural characteristics, crop yield, and fruit quality. The other major citrus states are at high risk for a similar disastrous situation if suitable remedies are not forthcoming soon. Neither a simple cure nor successful management strategies have been identified even after 15+years of the disease' presence in Florida.Previous efforts to create new HLB resistant/tolerant cultivars were focused on conventional hybrid breeding or the development of transgenic plants. Unfortunately, the approaches used in those projects are inadequate to meet the current crisis because of the lack of sources of resistance in citrus, time constraints associated with citrus breeding and genetic engineering, and regulatory obstacles for transgenic citrus. Meanwhile, more than 15 years of HLB epidemics and tens of millions of infected trees in groves across Florida provide an unprecedented opportunity and high probability for selecting new mutant scions that are resistant or at least highly tolerant. Over the past years, our group has selected multiple citrus varieties with field resistance or tolerance to HLB from Florida groves. A recent study showed that HLB is a pathogen-triggered immune disease. These natural variants may have HLB resistance/tolerance through evasion of the immune response or loss of susceptibility genes. Although the exploitation of natural resistance/tolerance provides a key solution to developing new HLB-resistant/tolerant citrus scions and rootstocks, it still requires a significant amount of time to confirm stable resistance of the selected varieties. It is critical to develop rapid methods to shorten the selection process, to provide solutions within a shorter time frame to ensure the survival of the citrus industry, especially in Florida.Our previous research shows that transposon activities are one of the important drivers of diversification in sweet oranges. By monitoring the transposon activities in selected varieties, we may be able to determine their genetic stability, and then facilitate selection. Furthermore, there are many available sequences of citrus genomes, including several HLB-resistant/tolerant citrus plants and their counterparts that our group and others have sequenced. It is critical that we can integrate all genome-based information to understand the HLB-resistant/tolerant or susceptibility-related genes. With more sequences of HLB-resistant/tolerant citrus plants and their counterparts in this project, we can build a pangenome that includes all HLB-resistant/tolerant related variants. This should help identify HLB-resistant/tolerant or susceptibility-related genes.This project addresses the "Development of commercial citrus varieties (rootstocks and scions) for both fresh and processed markets with genetic tolerance and resistance to HLB using traditional breeding techniques and/or gene editing", which is one of the ECDRE focus areas.In this proposal, we first present a systematic approach with emphasis on the rapid selection, assessment, and delivery of new HLB-resistant/tolerant citrus variants as new scion and rootstock cultivars. Second, we will develop a novel pangenome to identify HLB resistance/tolerance or susceptibility-related genes. Moreover, with the HLB susceptibility-related genes identified in our previous study, we will develop new varieties via gene editing using CRISPR-Cas and test their resistance to HLB. We will conduct multistate field trails of our previous selected varieties and evaluate their horticultural performance and economic impacts. Meanwhile, we will conduct active outreach and extension to disseminate our project results to growers, stakeholders, and the public.Our practical methods for rapidly selecting, assessing, and delivering stable and broad-spectrum HLB-resistant/tolerant citrus variants will allow HLB-resistant/tolerant scion and rootstock cultivars to be developed and deployed much quicker without regulatory constraints. The integrated citrus pangenome can help identify the genetic elements associated with HLB resistance/tolerance or susceptibility. The identified HLB resistance/ tolerance or susceptibility related genes will provide a foundation for understanding the molecular mechanisms of Las infection and HLB symptom progression. Precise gene editing based on the CRISPR system will enable targeted disruption of citrus genes that are essential to Las infection, multiplication, and pathogenesis, production of loss-of-function mutants, and development of new HLB resistant/tolerant citrus cultivars that are free of GMO fingerprints and potentially free of regulatory obstacles to the market. Horticulture and socio-economics-based support tools will be developed for the industry to adopt new cultivars with confidence. The selected citrus varieties with HLB resistance/tolerance are expected to be the most durable, eco-friendly, and cost-effective solution to this devastating disease.The successful achievement of our goals and objectives will broadly impact the entire US citrus industry, by maintaining the viability of industries not yet fully impacted as in Florida, and by providing the plant materials that will encourage Florida growers to replant with confidence. The combined economic impacts of our US citrus industries will be measured in billions of dollars. The many communities throughout the country having economies built around viable citrus production will avoid the major disturbances that will come from the economic disasters that HLB incurs. The success of this project will set up a model for the citrus community across the globe to follow and solve the HLB problems in the not-too-distant future.

Methods1, Methods for Objective 11.1 Developing an assay to evaluate the resistance of citrus variants using a simultaneous multi-strain inoculation: We will graft the selected bud-sticks onto severely HLB-affected plants of different genetic backgrounds, and measure how quickly Las bacteria invade, multiply, and cause HLB in the new growth from these bud-sticks. We will propagate the selected citrus bud-sports on different rootstocks and stick-graft inoculate them with multiple Las strains simultaneously. Inoculated plants will be scored by symptom development periodically and Las titer detection and quantification will be conducted at 3, 6, 9 and 12 months after inoculation using 16S rDNA and rRNA based RT-qPCR to measure Las bacterial titers and their relative cellular activities.1.2 Developing enzyme-linked immunoassay (ELISA)-based method to screen the uniformity of progeny of selected scions/rootstocks: We will collect newly expanded leaves of similar maturity, and extract in PBS buffer (1:2 [w/v]) in a FastPrep machine (MP Biomedical, Irvine, CA). The supernatants are the sources of total soluble proteins. The ELISA assay will be conducted by using a femtoELISA HRP kit with 1:1000 dilution of primary antibody and 1:3000 dilution of the secondary antibody. Absorbance of each reaction will be read at 450 nm on a FLUO Star Optima microplate reader. All tests will be replicated three times.1.3. Bud sport selection and evaluation: We propose more extensive searches of bud sport and controlled inoculation. in addition to current selections of grapefruit, sweet orange, and pummelo, we will expand our selection to mandarins, lemons, and other important citrus plants.2. Methods for Objective 22.1 Resequencing experiments and analysis:We will sequence the genomes by a service provider. We will assemble the genomes using PECAT or Hifiasm, selecting one of the parent genomes as the reference. For large SV detection, we will align re-sequenced and reference genomes using Mummer. Genetic variant annotation and functional effect prediction for the small variants will be predicted by SnpEff, and genes overlapping the SVs analyzed with BEDTools.2.2 Constructing Citrus Pangenome for identifying HLB-resistance related genetic variants: We will use Minigraph-Cactus to create the haplotype-aware citrus pangenome and develop statistical methods to predict the impact of each variant, and identify genes with moderate or high impacts.2.3 Experimental validation of selected genes and genetic variations: Candidate SNPs, indels, SVs, CNVs and segment deletions will be validated using PCR followed by sequencing.3. Methods for Objective 33.1 Determine types and frequencies of mutations: The targeted regions will be amplified from each of the 173 plants in high-fidelity PCR and subjected to deep amplicon sequencing. Amplicon sequence reads will be analyzed, to determine the types and frequencies of mutations.3.2 Propagate mutants and produce clonal plants and evaluate their HLB resistance: Plants with high frequencies of mutations will be propagated by grafting and cuttings to produce at least 20 clonal plants required for each selected mutant, and eight clonal plants will be inoculated with Las. Inoculated plants will be evaluated in eight randomized complete blocks in the approved/secured greenhouses and fields. Levels of HLB resistance will be determined and assessed every 3 months.3.3 Evaluate horticultural characteristics and potential side effects: Clonal plants will be planted in a secure field in Florida, in eight randomized complete blocks, and exposed to natural inoculation of Las by ACPs. Data on tree, shoot, leaf, flower, and fruit characteristics will be taken 2-3 times a year over 3 years.3.4 Identify potential off-target mutations: Mutants with strong HLB resistance will be subjected to genome re-sequencing. The genome of each selected mutant will be sequenced to 100x coverage or more and compared to the genome of wildtype citrus to identify off-target mutations.4 Methods for Objective 44.1 Field Trials: First, four selected scion selections that produce fruits of grapefruit, mandarin, and sweet orange and two rootstock varieties will be included in the field trial in all three states using randomized complete block design and 8 replicates per genotype/rootstock combination. These six genotypes were previously sent to CCPP in CA. Trials will be managed using standard irrigation, insecticide applications, fertilization and weed management practices. In CA, the scion selections will be grafted on Carrizo and field trial will be in the UC Riverside fields; in TX, we will propagate the scion selections on sour orange and plant in the TAMU Citrus Center experimental fields; in FL, the scions will be grafted on US942 and planted in Lake Alfred. The rootstock selections will be evaluated by grafting three scion cultivars (Washington navel orange, Tango mandarin and Lisbon lemon) on each of the two rootstock varieties. At least 8 replicates per rootstock/scion combination will be planted.4.2 Horticultural and disease evaluations: We will score tree height, canopy volume, appearance, density, trunk diameters using standard procedures - at planting, after six months and then annually. For disease evaluations, we will collect leaf samples every six months and conduct qPCRs to quantify Las titers. We will document disease symptoms. When the field trees are fruiting, we will collect data on fruit yield, size, external and internal coloration, percent juice, Brix, acid percent, and Brix/acid ratio.4.3 Economic analysis: We will conduct economic assessment using scenario analysis and simulation based on yield scenarios of the cultivar and price data from USDA and other sources.?5 Methods for Objective 55.1 Organization and engagement of advisory committee: An advisory committee will be organized to provide oversight on the relevancy of progress made by the research team. This panel will include key stakeholders, and research and extension scientists, who will meet at least once a year. The team will report progress made during the year and challenges encountered, and the panel will evaluate progress against the proposed milestones of each objective.5.2 Extension program and stakeholder panel: We will have grower visits to established trials and greenhouse experiments. We will conduct surveys to collect baseline data for project metric evaluation mechanisms. We will include pre- and post-workshop questionnaires to determine the program's effectiveness in disseminating knowledge and determine stakeholder engagement and opinion of project activities and progress. After each activity, the group will meet with project personnel to discuss survey and questionnaire results and to review the activities' success. The stakeholder group will meet at least once per year, to allow for meetings that focus on project progress on research activities. Stakeholder groups will include the historical citrus grower meetings in FL and CA.5.3 Dissemination of project results: Results will be communicated to stakeholders, researchers, and the public through multiple channels, including presentations at trade/professional/stakeholder advisory meetings, scholarly journals and conferences, project websites and social media accounts, and on-site visits (field days and workshops).