The goal of this proposal is to develop a new generation of citrus cultivars that not only survive but are also profitable in the presence of HLB. Over the past years, we have successfully demonstrated that HLB-resistant/tolerant citrus plants can be selected in commercial groves and breeding orchards. The first generation of HLB-resistant/tolerant bud-sports and seedling variants that have been cleaned up by the Division of Plant Industry (DPI), Florida, which includes grapefruit, Valencia sweet orange, pummelo, and sour orange (as rootstock), will be subjected to advanced field trials. However, the selection and evaluation process still requires a long time. In this proposal, taking advantage of the success of our previous projects, we will develop practical methods for rapidly selecting, assessing, and delivering stable and broad-spectrum HLB-resistant/tolerant citrus variants as new scion and rootstock cultivars. We will pursue five interrelated research objectives in this project.
Objective 1: Establish and optimize methods for rapid selection, assessment, and delivery of HLB-resistant/tolerant citrus bud-sports and seedling variants. Currently, transforming a selected citrus mutant into cultivars still takes a long time to make sure that it is stabilized, uniform, and resistant to HLB variants. Here, we will develop two methods to accelerate this process to allow fast selection and evaluation of citrus HLB-resistant/tolerant mutants. First, uniformity of propagations from individual scions/rootstocks is critical for their HLB resistance/tolerance performance. We will develop an enzyme-linked immunoassay (ELISA)-based method to assess performance uniformity. Second, we demonstrated that stick-grafting is much more successful than bud-grafting for inoculation, leading to the recovery of a higher percentage of HLB-resistant/tolerant mutants. Therefore, we will develop an assay to evaluate the resistance of variants using a simultaneous multi-strain inoculation strategy to ensure broad-spectrum HLB resistance/tolerance. Finally, in addition to current selections of grapefruit, sweet orange, and pummelo, we will expand our selection to mandarins, lemons, and other important citrus plants. Co-PI Duan and Gmitter each will lead two postdocs (1.5 FTE/year) and one technician (0.5 FTE/year) to develop the fast selection and evaluation methods. Co-PI Mattia and Grosser each will lead one technician (0.25 FTE/year) to select mutant citrus.
Objective 2: Identify genetic variants and genes related to HLB resistance/tolerance. Understanding the genetic mechanism of selected HLB-resistant/tolerant mutants is critical for HLB-resistant/tolerant citrus breeding. We will re-sequence five resistant/tolerant and five susceptible siblings of representative plants and their parents. Bioinformatics and comparative genomics analysis will be used to identify HLB resistance/tolerance or susceptibility-related segment deletions, copy number variations (CNVs), structural variations (SVs), SNPs, and indels in each citrus line. 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 integrate all genome-based information to understand the HLB-resistant/tolerant or susceptibility-related genes. We will build a citrus pangenome by integrating our previous with newly sequenced genomes and applying the pangenome to identify genes with moderate/high impact variants. The identified candidate genes will be experimentally validated in a wide range of citrus species and relatives that display either resistant/tolerant or susceptible phenotypes. Co-PIs Duan, Gmitter, Mattia, and Grosser will provide the RNA samples of the citrus trees. PI Luo (0.06 FTE/year) and one postdoc (1 FTE/year) will perform bioinformatics and functional genomic analysis.
Objective 3: Validate gene-edited citrus mutants for HLB resistance. Previously we edited two genes in two cultivars (�Duncan� grapefruit and Carrizo citrange) and produced more than a dozen novel dmr6 and sweet1 mutants. Recently, 173 novel mutant candidate plants (140 from �Duncan� grapefruit and 33 from Kuharske citrange) were generated to target four other S genes singly or in pairs. These new mutants will be grown, propagated, inoculated, and evaluated for HLB resistance, potential changes in horticultural characteristics, potential side effects, and potential off-target mutations. Completing these evaluations will enable us to select the best target genes to edit in commercial citrus cultivars using mature citrus tissues to produce non-juvenile, non-transgenic HLB-resistant mutants as the next-generation citrus cultivars that can grow in HLB-endemic regions. Co-PI Gmitter, Deng, and Chater will lead one graduate student (0.5 FTE/year) to work on evaluating the gene-edited trees.
Objective 4: Multistate field evaluations of HLB-resistant/tolerant citrus scion and rootstock cultivars. Based on preliminary selections of scion and rootstock cultivars with superior HLB resistance/tolerance attributes and fruit quality, a systematic evaluation of these novel varieties in multi-state field trials will be beneficial to the citrus industries. We propose to conduct field evaluations of 6 genotypes in Florida and Texas to monitor HLB tolerance and horticultural performance. In California, we will evaluate 3 of these selections that are available through the Citrus Clonal Protection Program (CCPP) in Riverside. Gene-edited selections were generated by the team and may be very valuable to the industry based on the preliminary data obtained from the field trees in Florida. The success of developed cultivars will ultimately be determined by their economic performance. We will conduct economic assessment using scenario analysis and simulation. Co-PI Chater, Ramadugu, and Alabi will each lead one technician (0.15-0.25 FTE) on field evaluation. Co-PI Guan (0.1 FTE) will lead one student (0.2 FTE) perform the economic analysis and report information on economic aspects in the fifth year.
Objective 5: Outreach and dissemination of project results to stakeholders and the public. Outreach and dissemination of project results will keep stakeholders and the public informed and engaged with our research, which is critical to the success of the project. We will assemble a stakeholder advisory committee. Extension demonstration and informational activities will be held at the UF-CREC, the Citrus Expo, the Florida Citrus Show, and relevant grower organization meetings in CA or TX. The availability of these resistant/tolerant cultivars will be communicated during these extension events. We will establish replicated stage 2 field trials with these rootstocks across the US citrus production regions, during and after the project. A stakeholder advisory committee will be established with membership from FL, TX, and CA. A website will provide updates from this project to the public. Progress updates will be published in industry and peer-reviewed journals. Co-PI Chater, Ritenour, Ramadugu, and Alabi will lead the outreach in FL, CA, and TX.