Source: UNIVERSITY OF CALIFORNIA, RIVERSIDE submitted to NRP
CAP: EVALUATION OF PROMISING, HLB-RESISTANT/TOLERANT CITRUS SCION HYBRIDS GENERATED BY BREEDING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031583
Grant No.
2023-70029-41315
Cumulative Award Amt.
$3,281,160.00
Proposal No.
2023-06840
Multistate No.
(N/A)
Project Start Date
Sep 15, 2023
Project End Date
Sep 14, 2028
Grant Year
2023
Program Code
[ECDRE]- Emergency Citrus Disease Research and Extension Program
Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
(N/A)
Non Technical Summary
Citrus huanglongbing (HLB) is considered one of the most difficult challenges for several citrus industries worldwide. The financial impacts of HLB are staggering and may result in citrus being replaced by other fruit trees and alternative crops. In Florida, where the disease has been well established for over ten years, the yield of citrus trees has been reduced to yields reflecting the Florida industry 70-100 years ago. The reduced yield is reflected in the high price of orange juice for the morning breakfast. The disease threatens the citrus crop in other citrus-growing states like California, Texas, and Arizona. Finding long-term, sustainable solutions is imperative for continued citriculture.Citrus HLB is a complex disease associated with a non-cultivable bacterial pathogen, spread by a psyllid insect vector that is difficult to control, and the disease has no known cure; infected trees decline, produce unmarketable fruit, and die in a few years. Management of the disease by preventing spread, control of the insect vector, and application of anti-microbial substances are good practices - but of limited value for a tree crop that can live for several decades. Despite intensive research for the past fifteen years, tangible solutions to control HLB are not yet available. Novel approaches are needed to find practical methods to manage HLB in the citrus-growing states of the USA. Current methods of disease management are not financially affordable. Excessive use of pesticides to control the psyllid populations is environmentally unsafe; pesticides will lose effectiveness after prolonged use. Ecologically friendly biological control mechanisms that reduce the psyllid population will not be adequate to manage HLB since a single insect vector carrying the pathogen can spread the disease in a citrus grove. When the citrus processing plants and juice factories are closed because of low yield and diseased fruit, it will be very difficult to revive this industry infrastructure. Long-term strategies for disease management are urgently needed to continue citrus cultivation in areas where the insect vector and the pathogen have been established.Citrus is propagated mainly through grafting. The vegetative mode of propagation of a limited number of cultivars has resulted in innumerable cultivated types, but there is little genetic diversity in cultivated citrus. When new pathogens arrive, this genetic uniformity can result in disease epidemics and dire consequences for the crop. In addition, there is no documented resistance to HLB in cultivated citrus. However, other citrus-related wild plants may have resistance traits to HLB. Based on our previous work, we determined that certain non-citrus taxa that are closely related to citrus have disease-resistance traits and these may be utilized to impart the much-needed HLB resistance to cultivated citrus types. Our approach is to generate novel hybrids of citrus using conventional breeding methods with sexually compatible citrus relatives having genetic resistance to the HLB pathogen. Towards this goal, we have conducted a breeding program since 2013 by crossing citrus accessions with disease-resistant/tolerant Australian lime species belonging to the genus Microcitrus. Crop relatives are utilized as sources of beneficial traits in other cultivated plants. Citrus is sexually compatible with many closely related genera; this biological peculiarity of cultivated citrus types was exploited in our breeding program to generate hundreds of intergeneric hybrids.Citrus breeding is considered very difficult and time-consuming since the generation time can be 4 to 5 years. Since our breeding program has been in progress for ten years now, we have generated progeny from several different types of crosses. Evaluation of the novel hybrids in the field and in the greenhouse determined that HLB resistance traits can be inherited by a small proportion of the breeding progeny. However, since one parent of the cross bears fruit that is barely edible, many hybrids of the first-generation crosses have fruits that may not be acceptable as substitute varieties, even though they have disease-resistance traits. We have now generated hybrids of the second generation by crossing the promising hybrids from the first generation to commercial types of citrus. A small percentage of the advanced hybrids is expected to have retained the HLB resistance traits and may have fruit qualities similar to commercial citrus types. We propose to utilize molecular approaches to develop markers that will aid in the selection of the breeding progeny from the second generation of hybrids to identify individuals with promising HLB resistance. If such hybrids can resist HLB in field conditions and if they have fruit traits that are deemed acceptable by the public, we will have novel hybrids with disease resistance and acceptable organoleptic attributes. Cultivar development of promising individuals will be required before large-scale cultivation can be adopted.If novel hybrids with genetic resistance to HLB are developed, it will be possible to cultivate citrus despite having the pathogen and the insect vector in the area. Management of the groves that have resistant cultivars will be similar to the grove management done before HLB arrived. Financially it will be feasible to cultivate citrus if elaborate, expensive management practices are not necessary. Another advantage is that the novel hybrids may have new flavors that may enhance the known citrus flavors that are generally associated with citrus fruits. We will look for the metabolomics profile of novel hybrids to identify the flavor-enhancing compounds in the hybrid fruits and compare them with standard citrus. Replacement of presently cultivated citrus varieties with novel hybrids will be beneficial to manage HLB. The molecular markers we propose to develop in the project can be used in the future to make pre-selections from our breeding program in California. We envisage the development of multiple varieties resembling mandarins, sweet oranges, grapefruit, and lemons soon. The availability of HLB-resistant hybrids with citrus-like flavors (and some novel flavors) will be extremely beneficial to restoring the citrus industry in the United States. Citrus fruits provide valuable nutrients and constitute an essential part of a healthy diet. Restoring the citrus industries with useful, disease-resistant hybrids will result in job creation and improvement of economic situations in citrus-growing regions.
Animal Health Component
35%
Research Effort Categories
Basic
35%
Applied
35%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2010920104030%
2020999108120%
2122499108030%
2064010106020%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 206 - Basic Plant Biology; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0920 - Orange; 0999 - Citrus, general/other; 4010 - Bacteria; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1081 - Breeding; 1080 - Genetics;
Goals / Objectives
The overarching goal of our program is to generate novel citrus hybrids with durable, stable, broad-spectrum, genetic resistance to citrus huanglongbing. We aim to achieve this goal through genome-assisted, traditional breeding approaches. The critical needs that are addressed include 'Development of commercial citrus varieties (rootstocks and scions) for both fresh and processed markers with genetic tolerance and resistance to HLB using traditional breeding techniques and/or gene editing', and, 'large-scale evaluation of resistant breeding lines for horticultural performance, consumer acceptance and economics', identified by the Citrus Disease subcommittee. For the current project, we are utilizing hybrids already generated and evaluating them for HLB resistance and fruit quality. The two main goals are, development of HLB resistance and, identifying hybrids that will produce commercially acceptable fruit quality traits. A related objective is to develop molecular tools to identify promising candidates in the breeding population. Identification of the quantitative trait loci (QTL) associated with resistance will help to understand the basis of resistance. We also aim to evaluate fruits derived from advanced hybrids with metabolomics studies and taste panels.Since there is no documented resistance to HLB in the genus Citrus, for the past ten years, we conducted breeding with closely related Australian limes since they are good sources of resistance. New varieties generated by breeding will be acceptable by the public without regulatory hurdles. HLB resistant citrus hybrids with acceptable fruit quality will provide for a financially sustainable, long-term solution for citrus cultivation in the presence of the HLB- associated pathogen and the psyllid vector.The four proposed objectives are outlined below:Obj. 1. Evaluation of 24 novel citrus scion hybrids in Florida, Texas and California for HLB tolerance/resistance (UA, ER, MK, RK, and CR). From our previously generated breeding population, we have selected 24 hybrids (4 first generation, F1 hybrids and 20 second generation, advanced hybrids) for field trials. Preliminary selections were based on HLB disease response against two isolates of CLas in the contained research facilities in CA. Conducting field trials in regions where HLB is endemic and the trees are constantly re-inoculated with multiple field isolates of CLas through psyllid feeding will provide a good test for the hybrids. A similar field trial in TX conducted in the Citrus center, Weslaco will be useful to measure disease response against the prevalent CLas isolates in south TX. The field trial in CA where the pathogen does not exist in the field will be for evaluating horticultural traits only. Horticultural evaluations will be done in all three states; in years four/five, we will conduct metabolomic analysis of the fruit juice obtained from the 24 selected hybrids in CA. The usefulness of the hybrids will also depend on their susceptibility/tolerance to other citrus pathogens found locally. We will utilize a citrus pathogen detection array we have developed previously and analyze samples from mature trees from the field trial during year four to evaluate general resistance to other pathogens.Obj. 2: Analysis of resistance genes and targeted gene expression in 24 hybrids through PacBio DNA sequencing and singe-cell RNA sequencing (CD, MS, MK, CR). Identification of candidate genes or QTLs associated with resistance is essential to understand the mechanism of resistance and will provide molecular tools for selecting breeding progeny with maximum potential. We propose a combination of techniques to achieve this objective including PacBio sequencing of the 24 hybrids and their siblings (with different levels of resistance/susceptibility). Identification of genetic differences between the hybrids showing varied responses (structural variants, deletions, insertions, translocations, etc.) will be useful to identify the genomic architecture associated with the desired traits. We propose to conduct limited single-cell sequencing to identify genes expressed in target cells of the phloem where the CLas resides, generate total transcriptomic data and identify genes expressed in target cells. Information on resistance genes in Microcitrus used in the breeding program is available from our previous genome sequencing and analysis. We propose to build a project-specific pangenome using genome sequence data of the three Microcitrus parents, three citrus parents and selected hybrids sequenced earlier. We will generate three new citrus sequences in the current project and PacBio sequences for 32 hybrids. We will integrate the data generated, conduct analysis using the pangenome tools and identify QTLs associated with resistance traits in the current set of hybrids. Since diverse Microcitrus and citrus accesions were used in breeding, the project-specific pangenome is essential in identifying key regions of the genome associated with HLB resistance.The third sub-objective is to generate a qPCR-based array for the identification of genomic fragments derived from the Microcitrus parent in advanced hybrids showing resistance. The array would have primers and probes designed approximately at 5MB intervals in the 280-320 MB genome of Microcitrus species using single nucleotide polymorphisms conserved only in Microcitrus. Since the resistance in the hybrids is derived from Microcitrus parentage, identification of genomic fragments with Microcitrus DNA and correlation of previously identified resistance-associated genes in the hybrid will be valuable. Using a combination of the three approaches described in this objective, we will identify crucial QTLs useful for hybrid selection.Obj. 3: Metabolomic analysis and assessment of fruit quality to identify hybrids with potential for cultivar development (MA and CR). The characteristic flavors associated with different citrus types are dependent on the types of primary and secondary metabolites, volatile compounds, sugars and acids present in the fruit. Evaluation of organoleptic properties of fruit juice obtained from the hybrids is an important objective that helps to determine their suitability as substitute citrus varieties. Since the breeding was conducted with citrus relative genera that do not produce flavorful, citrus-like fruits, it is essential to ensure that the new hybrids selected have palatable fruits, contain the sugars, flavonoids, terpenes, etc. that are characteristic of commercial citrus and lack off-flavors imparted from the Microcitrus parents. Activities associated with objective 3 will be useful to identify promising hybrids that may be suitable for cultivar development (if they have resistance associated genomic fragments).Obj. 4: Extension and Outreach (UA, MK, CR). The main goal of the project is to develop HLB-resistant citrus varieties with fruit flavor that is acceptable to the citrus industry. Interaction with the growers and industry personnel is at the core of our activities. Since the hybrids are generated in CA, the fruit tasting events will be done primarily in CA. During the course of the project, we propose to conduct >10 taste panels to obtain feedback regarding the acceptability of the novel hybrid fruits. Extension activities in FL will include the presentation of research data to growers in citrus industry events, publishing articles in trade journals and interaction with growers at all major citrus industry events. Similar activities will be conducted in TX and CA. We will design a project-specific website to post project information and research updates.
Project Methods
Obj. 1. Evaluation of 24 novel citrus scion hybrids in FL, TX and CA for HLB tolerance/resistance.1.1. Obtain permits, interstate transport of hybrids, field planting. Transport budwood from CA under valid APHIS permits. Propagations on Kuharske rootstock (FL), Sour orange (TX), and Carrizo (CA) to test16 replicate trees per hybrid in a randomized complete block design at Immokalee and Fort Pierce, FL, at the Texas A&M citrus center, Weslaco, TX, and in UCR fields (CA, 272 trees per state).1.2. Greenhouse and field evaluations. Multiple CLas isolates will be used in greenhouse tests. Horticultural performance of field trees will be documented annually (tree growth, canopy health, rootstock compatibility, etc.). HLB resistance/tolerance evaluated annually on a scale of 1-10 (10 is resistant); CLas titers measured by qPCR. In-depth analysis of fruit traits and metabolomics study will be done in CA.1.3. Evaluate susceptibility to other diseases. In years 4/5, we will collect leaves from field trees in all 3 states, extract RNA, reverse transcribe and test for 26 citrus pathogens using a qPCR-based pathogen detection array. We determine resistance and horticultural performance. We will detect susceptibility to 20 other diseases by the citrus pathogen array.Obj. 2: Analysis of resistance genes and targeted gene expression in 24 hybrids through PacBio DNA sequencing and singe-cell RNA sequencing.Previously, we sequenced four Australian limes and three citrus parents of F1s; we will sequence four more citrus parents and construct a pan-genome to identify important QTLs and R genes. Single-cell sequencing technique help understand the resistance mechanism by profiling gene expression in target phloem cells. The information will identify genomic regions associated with resistance.2.1. Generate phased genomes of four additional citrus parents. Use PacBio HiFi sequences and Phase Hi-C data, generate contigs and scaffold into chromosomes using HifiAsm and 3dDNA. Annotate using Braker2, leverage available citrus annotations and our RNA Seq data sets.2.2. PacBio sequencing of 24 hybrids; structural variant (SV) analysis. Long-read PacBio sequences will be compared with parents and with each other to track haploblock inheritance and identify SV.2.3. Identify genomic fragments using Microcitrus array. Hybrid DNA will be analyzed by qPCR using the Microcitrus array having primers and probes to detect regions of Microcitrus. Validate against the PacBio data to identify specific genomic fragments.2.4. Single-cell sequencing and transcriptome analysis of four representative hybrids. Using infected and control samples, do limited single-cell RNA sequencing to study gene expression in target cells. In a complementary approach, we will do a transcriptome analysis for the same samples. Twist Biosciences multiplex DNA library prep kits will be used to sequence samples. Bioinformatics will identify, and separate data from infected vs. uninfected cells. Coupled with transcriptome data, we will identify transcripts associated with resistance.2.5. Pan genome and identification of resistance genes. Using the four Australian lime genome sequences and seven citrus sequences used in breeding, we will construct a project-specific pan genome to identify resistance genes common to Microcitrus. Practical haplotype graph (PHG) provides a comprehensive tool set to build a haplotype-based pangenome. We will delineate haplotype blocks from hybrid PacBio sequences, and integrate them with TASSEL for genetic trait association and breeding data (BrAPI). Perform low-coverage whole genome sequencing of populations of hybrids, and identify QTLs.Generating high-quality, phased genomes will provide genomic resources to analyze HLB disease resistance. PacBio sequencing and SV analysis identifies genetic variants correlated with resistance. Microcitrus array identifies regions associated with resistance and undesirable flavors. This breeding tool will help in hybrid screening and selection. Together, single cell sequencing and transcriptome will help to identify key genes expressed in resistant hybrids. A project-specific pan genome will help to identify common regions from HLB-resistant plants. Current data will complement previously generated R gene data from Microcitrus, provide experimental proof, and identify resistance genes.Obj. 3: Metabolomic analysis and assessment of fruit quality to identify hybrids with potential for cultivar development.Novel hybrids generated by crossing citrus with Australian limes are valuable if they are resistant and have citrus-like flavors. The advanced hybrids were generated by crossing F1 with citrus to introgress resistance traits. While fruit tasting sessions assess the overall acceptability of the fruits, metabolomics provides the scientific basis for classifying the hybrid fruits.3.1. Documenting fruit traits from advanced hybrids. We expect fruit from 100 novel, advanced hybrids from the field trees in CA. We will document data from the hybrid fruits including soluble solids content (SSC or °BRIX), titratable acidity, juice percentage, limited taste evaluation, and develop a scoring system for flavor acceptability. On a scale of 1-10 (10 being the most acceptable), hybrids scoring 7 or more will be considered.3.2. Metabolomic analysis of juice from hybrid fruit. Metabolomics core in UCR will identify the different metabolites present in 50 advanced hybrids. Comparison of the metabolomic profiles with standard citrus will identify promising individuals. Juice samples will be analysed by LC-MS. We will use open-source software, mass spectral metabolite databases - Metlin, Mass Bank of North America, and an in-house database to identify compounds.3.3. Taste panels for the hybrid fruits. Based on preliminary taste evaluations and fruit availability, we will do taste panels in CA. Twelve selections will be evaluated in two sessions per year with 60 panelists. Test design and data collection will be done with the cloud sensory software Compusense®.We have 800 advanced hybrids in the fields;100 will fruit in the next four years. Analysis of fruit traits identifies candidates suitable for future cultivar development. Metabolomic analysis provides scientific basis for the taste profile. The HLB-resistant novel hybrids generated in our program will be useful if the fruit quality is satisfactory. The taste panels will provide data on the acceptability. A rating of 7 or above (for HLB resistance phenotype, fruit trait evaluation, and consumer acceptability) will select candidates for cultivar development.Obj. 4: Extension and Outreach.4.1. Extension to disseminate project results. We will conduct fruit tasting events, present project results at scientific and grower meetings, and conduct meetings with advisory board members representing different citrus industries.4.2. Outreach to educate the citrus industry about the project. We plan to generate project-specific website to disseminate project related information, distribute educational materials, engage citrus industry personnel in fruit tasting events and other activities.4.3. Economic analysis to evaluate the benefits of novel hybrids. If certain hybrids are deemed suitable for commercialization, we will recruit an economist to conduct economic analysis.Since our breeding approach uses relatives of citrus, there will be concerns regarding the organoleptic properties of fruit from the novel hybrids. Extension work gives us an opportunity to share results of disease resistance and flavor from the hybrids directly with growers and scientists. In outreach, educational materials will be developed and shared with interested parties and apprehensions about fruit palatability will be addressed.

Progress 09/15/23 to 09/14/24

Outputs
Target Audience:The target audience comprises citrus growers, industry personnel, breeders, molecular biologists, early career scientists, students, and technicians. Our project aims to develop novel hybrids using molecular information developed on existing citrus hybrids of the F1 and advanced generations. HLB pathosystem is complicated and economically sustainable, and long-term solutions are not available. Hence, several researchers worldwide are trying to develop disease-resistant citrus hybrids using Australian limes as the source of HLB tolerance/resistance. The data we are generating regarding resistance-associated genes and flavor profiles of hybrids generated with Australian limes is of immense interest to many research groups. We present our results in several scientific and grower meetings like the International Citrus Huanglongbing conference (held during alternate years in Riverside, California), Plant and Animal genomics meetings (annual events held at San Diego, CA), American Society of Horticultural Sciences and specific citrus-related conferences in other countries (India, South Korea, etc). Many scientists following similar HLB management approaches consider the data presented valuable. We also conduct extension activities in California during the annual Citrus Day and Citrus Conference events (organized by the Citrus Research Board). We provide fruits of selected hybrids for tasting to assess the acceptability of novel hybrids. Co-PI Kunta conducts extension activities in Texas, and Ramadugu conducts outreach mainly in California (since the hybrids that are offered to the public for tasting are primarily located in California). Because of the nature of the project, we closely interact with citrus growers and obtain feedback. The discussions with citrus industry personnel benefit the researchers since they provide perspective and shed light on the most important traits we must focus on. Changes/Problems:We have modified objectve one of this project. Objective one was to evaluate 24 advanced hybrids in Florida, Texas, and California field conditions. We have now modified this objective with permission from the NIFA Awards Management Division. According to the revised plan, we will evaluate 250 hybrids in the field. We will plant only three replicates of each hybrid and assess the disease response when exposed to the psyllid populations in the fields in Florida and Texas. In California, we will plant the trees to determine horticultural traits. The reasons for this modification are: We were not satisfied with the greenhouse evaluations of the 24 hybrids in the contained research facilities (BSL3) in Riverside. The facility needs repairs, and we were unable to complete the evaluations. More importantly, we recognized the importance of field evaluations before the hybrid accessions are deemed tolerant or resistant. In the field conditions, disease pressure is severe, and only hybrids with a significant resistance/tolerance will survive. In Fort Peirce, we now have some F1 hybrids that have been in the field for about ten years and are thriving with good canopy. Evaluating a larger number of advanced hybrids (with a lower percentage of the Australian lime genome fragments and fruits similar to the commercial citrus genotypes) may be prudent since selecting the most promising hybrids is needed for the project to succeed. Based on parentage and other molecular information generated, about 250 hybrids are deemed most promising and need field evaluations. The disease pressure in Florida is very high, and frequent hurricanes contribute to severely compromised trees. We decided that evaluating a large germplasm base may be prudent during the disease epidemic. Since the hybrids are already available, we modified the first objective and evaluated 250 hybrids. What opportunities for training and professional development has the project provided?Through this project, we have trained postdoctoral scientists and early career professional researchers in conducting PACBIO long read sequencing and analysis, use of new gene identification pipelines like FindPlantNLRs to identify novel resistance-associated gene candidates in the Australian limes used to confer HLB resistance to the hybrid populations. Several Ph.D. students and technicians were involved in conducting routine analysis of Illumina sequences generated from the hybrid populations, conducting HLB pathogen assays in contained research facilities and in field conditions. We typically employ undergraduate students from underprivileged sections of society as part-time lab helpers to assist in the project activities. These individuals are taught essential plant maintenance work, citrus grafting, gathering field data, collecting hybrid fruits for documentation of properties and analysis, obtaining fruit juice for metabolomics, documentation of data using Microsoft Excel, PowerPoint, etc., for recording essential information generated in the project. Because of the nature of the work conducted in the project ranging from maintaining plants, pathogenicity assays, field evaluations, fruit characterizations, DNA extractions, sequencing, analysis of molecular data, assembling long read Pacbio sequences into chromosome-scale scaffolds, de novo assembly of the genomes, identification of novel genes, construction of pangenomes, etc. all the personnel involved in the project work have opportunities to learn various concepts and techniques that are useful in conducting plant research. How have the results been disseminated to communities of interest?List of activities for disseminating essential information to communities of interest: Presentations at the International scientific conference - 'Plant and Animal Genome meeting' held at San Diego in January 2024. Ramadugu and the research team at Riverside presented three posters at this meeting. They are: a). Singh et al. "Decoding the citrus defense arsenal: genome-wide identification of phloem protein 2 (PP2) and Callose synthase (CalS) in Citrus and wild Australian limes for enhanced insights and control of huanglongbing (HLB)"; b). Agarwal et al. "Metabolic profiling of fruit juice from novel citrus hybrids with Microcitrus parentage using untargeted metabolomics analysis"; and c). Zhang et al. "Chromosome-scale genomes for scion hybrids to accelerate Citrus huanglongbing resistance breeding". Presentations at the International Research Conference on Citrus Huanglongbing, held at Riverside, CA, March 2024. Ramadugu and the team members of the project from various organizations (Appalachian Fruit Research Station, WV; Dept of Entomology and Plant Pathology, TN; National Clonal Germplasm Repository for Citrus and Dates, USDA-ARS, CA; Texas A&M University, TX, USDA lab, Parlier, CA, Dept of Botany and Plant sciences, Univ of CA Riverside, CA, Univ of Florida, FL, and Washington State University, WA) had two oral presentations and five posters covering various aspects of the project. The titles were: da Silva et al. "Cultivating Novel Microcitrus Hybrids as Scion for HLB Management in Disease-Endemic Growing Conditions"; Liu et al. "Analysis of R-genes in Australian Limes in Comparison to Commercial Citrus Cultivars"; Huff et al. "Citrus Pan-Genome: The Search for Genes of Resistance in Australian Limes"; Agarwal et al. "Comparative metabolic profiling of fruit juice from Australian limes and Citrus hybrids with Microcitrus parentage"; Singh et al. "Navigating the Genetic Bottleneck of HLB: Exploring the Genomes of Australian Limes for development of disease resistance"; Park et al. "Generating de novo transcriptome assemblies of Australian native lime species using various tissue types and its use as a reference for transcriptome data analysis"; Ramadugu and Roose "Genome-assisted breeding to incorporate huanglongbing resistance in citrus". Ramadugu made a presentation on March 3, 2024 at the Citrus Day event held in Riverside, CA titled "Generating Novel Hybrids with Citrus Huanglongbing Resistance". Displayed a poster and hybrid fruits generated in the program to about 105 people from the California citrus industry. Selected hybrid fruits were provided to the public for tasting. Ramadugu gave the keynote address of the 78th Annual Conference of Subtropical Agriculture and Environments Society, South Padre Island, Texas. Organized by the Subtropical Agriculture and Environments Society. Feb 9, 2024. "Developing science-based solutions for a citrus disease". Meeting attended by about 150 people. Ramadugu presented a talk on "Citrus huanglongbing - Can Indian Germplasm provide solutions?". International Webinar of "Citrus Horizons: Genomics Tales, Germplasm Utilization and Cultivar Chronicles". Indian Council of Agricultural Research - Citrus Research Institute, Nagpur, Maharashtra, India. December 2023. Chandrika Ramadugu, 2023. Presented an invited keynote lecture on: "Citrus Huanglongbing: strategies to develop disease-resistant varieties through breeding". Asian Citrus Congress 2023. Organized jointly by the Indian Society of Citriculture, Nagpur, India; Indian Council of Agricultural Research - Central Citrus Research Institute, Nagpur, India and Asia-Pacific Association of Agricultural Research Institutions, Bangkok, Thailand. At Nagpur, Maharashtra, India, October 2023. Presented at the Asian Citrus Congress 2023, October 28-30th, 2023 at Nagpur, India. The event was attended by citrus scientists and industry personnel from India and 15 Asian countries. We had a project-specific meeting during the International Conference on Citrus Huanglongbing held at Riverside in March 2024. Five advisory board members and 15 project personnel attended this meeting. This was a fruitful activity since the discussions that ensued during and after the meeting were helpful to the project personnel. What do you plan to do during the next reporting period to accomplish the goals? We plan to start field evaluations of 250 hybrid plants in Florida, Texas, and California (a total of about 2000 trees (800 in Florida, 500 in Texas, and 700 in California) for field evaluations. The propagation will be done by Nate Jameson (BriteLeaf Nursery, Florida), Kunta's team (Texas), and Ramadugu's team (California). Publish manuscript describing novel R genes in Australian lime species used in our breeding program. Make progress in conducting Pangenome analysis of breeding parents and selected hybrids showing various disease response phenotypes. Conduct long-read sequences of additional hybrids and analyze the data to identify specific genomic architecture associated with disease-resistant hybrid populations. Publish two manuscripts describing the metabolomics profiles of the fruit juice from the parents used in our breeding program (selected citrus types and Australian limes) and a manuscript on the metabolomics profile of F1 hybrids. Conduct fruit juice anlysis of advanced hybrids that are now available in the program. Identify hybrid accessions with acceptable profiles. Correlate various R genes, metabolomics profiles and genomic variations in the different hybrids with disease resistance phenotype. Conduct extension activities in California and Texas. These will include presentations at the Citrus conference in Visalia, CA, at the UCR Citrus Day event in Riverside, presentations at an International meeting in Jeju island, South Korea (November 2024), at a citrus conference in Australia (March 2025), presentations at the Plant and Animal Genome meeting in San Diego, CA, and other venues. We will conduct fruit-tasting events at Parlier, CA, and Riverside (assuming we have no quarantine issues). During 2023-2024, Southern California had special quarantine regulations imposed due to fruit fly quarantine; hence, proper testing of the hybrid fruit was not feasible. As of August 11, 2024, the fruit fly quarantine has lifted. We plan to conduct a project-related meeting and invite all the project collaborators and advisory board members to attend the meeting so that information exchange can occur and the Advisory board members can provide their input for the project.

Impacts
What was accomplished under these goals? Obj. 1. Evaluation of 24 novel citrus scion hybrids in Florida, Texas, and California for HLB tolerance/resistance. Initially, we proposed to evaluate 24 novel citrus scion hybrids in field conditions in FL, TX, and CA. We modified this objective with approval from the NIFA grants management division. The reason for the change is because: a). The 24 selected hybrids could not be evaluated entirely in the Riverside BSL3 facility as planned since the USDA revoked the license as the Riverside BSL3 building did not meet the quarantine expectations of the permit department. b). Field evaluations in Florida are probably essential in selecting hybrids that will resist the intense disease pressure in Florida. To increase our chances of detecting hybrids with field resistance, we propose to evaluate 250 novel hybrids (three replicates each) in Florida and Texas. We have now made arrangements for budwood testing (Division of Plant Industry in FL), propagation of hybrids (BriteLeaf nursery, FL), and field planting (at Alico Inc., Arcadia, FL; US Horticultural research lab in Fort Pierce, FL; and at Immokalee, FL). For evaluation in Texas, we have arranged with co-PI Kunta to do propagations and planting (3 replicates of each hybrid) at the Citrus Center in Weslaco, TX. We are also planting the hybrids in CA for horticultural evaluations. Based on parentage and preliminary molecular data, we have identified 300 putatively valuable hybrids for this objective. Identifying the most promising hybrids is crucial for the project's success. Obj. 2: Analysis of resistance genes and targeted gene expression in 24 hybrids through PacBio DNA sequencing and single-cell RNA sequencing. Identification of resistance genes associated with HLB tolerance/resistance is valuable since the information is essential to pre-select hybrids from the breeding population that we generate (breeding in progress for the past 11 years) and for future biotechnological manipulation of elite citrus cultivars to incorporate resistance traits. We used a recently developed pipeline - FindPlant NLR - to identify and annotate NBARC type R (resistance) genes from five citrus types (three Australian limes showing various disease resistance levels and two susceptible citrus cultivars). Using this methodology, our team identified several new R genes in the Australian lime species. Disease resistance depends on pathogen recognition by host R genes containing a nucleotide binding site (NBS), a C-terminal leucine-rich repeat region (LRR), and other essential domains. The presence of specific domains is likely to be correlated with disease resistance. We have completed this study of R genes, and the manuscript has been submitted for publication (Liu et al., under review). We are conducting an analysis of 24 plants (mainly first-generation hybrids used in the study) showing various levels of disease resistance. Using long-read Pacbio sequences, we compare the genomic fragments and the presence of structural variants in the genomes of the selected hybrids to identify the genomic architecture associated with the desired resistance traits. We have generated long-read sequences for three mandarins used in the breeding project. We have assembled the genomes and using the information for the comparative study. We are also generating a project-specific pan-genome using the parents of the hybrids and selected hybrid accessions showing different disease responses when exposed to the HLB pathogen. Obj. 3: Metabolomic analysis and assessment of fruit quality to identify hybrids with potential for cultivar development. The presence of certain compounds in the fruit pulp of the hybrid fruits is essential for the fruit flavors to be similar to commercial citrus cultivars. Also, the absence of specific metabolites (derived from the citrus relative parent) is desirable for acceptable organoleptic properties. We have conducted metabolomic studies on the citrus and Microcitrus parents used for generating hybrids to identify the differences in the metabolomic profile and to assist in the pre-selection of the F1 hybrids for further rounds of breeding. Microcitrus genotypes are rich in coumarins that may be associated with the undesirable, bitter taste of the fruit of Australian limes and some of the F1 progeny. The study will be published shortly (Agarwal et al. 2024). We have conducted a similar analysis using selected F1 hybrids and advanced hybrids of the second generation (F1 hybrids are a result of the cross between a mandarin and a Microcitrus; the second-generation hybrids are generated by crossing selected F1 hybrids and a citrus cultivar). We measured the rind color of the fruits using a hand-held spectrophotometer (CR-600d, Konica Minolta, Japan), and the citrus color index of the fruit flesh was calculated. Juice from fruits of selected hybrid accessions was used to determine the soluble solids content (SSC) using a digital refractometer (Atago, Tokyo, Japan), and the titratable acidity was calculated using an automatic titrator (model T50, Mettler, Columbus, OH, USA). The juice was analyzed using standard solid-phase microextraction methods followed by gas chromatography (Agilent 7890A gas chromatography equipment). Untargeted secondary metabolites were studied by LC-MS using a Waters Synapt G2-Si quadrupole mass spectrometer (Waters Corporation, Milford, MA. USA). We obtained help from the UCR metabolomics core to conduct the assays and the subsequent data analysis. Taste panel evaluations were done with a few individuals in Riverside in March 2024. Standardized methods will be used to conduct taste evaluations in 2025 in the Parlier (central CA) facility with collaboration from Co-PI Arpaia. As mentioned in objective one, putatively valuable genotypes will be evaluated in Florida and Texas to determine the disease response phenotype when exposed to the HLB pathogen. Obj. 4: Extension and Outreach. We have conducted extension activities in CA and TX. PI Ramadugu presented the research to several groups of researchers and growers at many scientific meetings and grower groups in California and Texas. The section on "How the results have been disseminated to communities of interest" details the activities performed.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Khushwant Singh, Matthew Huff, Jianyang Liu, Jong-Won Park, Tara Rickman, Manjunath Keremane, Robert R. Krueger, Madhurababu Kunta, Mikeal L. Roose, Chris Dardick, Margaret Staton and Chandrika Ramadugu. 2024. Chromosome-scale, de novo, phased genome assemblies of three Australian Limes: Citrus australasica, C. inodora, and C. glauca. Plants 2024, 13, 1460. https://doi.org/10.3390/plants13111460.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Manjunath Keremane, Khushwant Singh, Chandrika Ramadugu, Robert R. Krueger and Todd H. Skaggs. 2024. NextGen Sequencing, and Development of a pipeline as a tool for the detection and discovery of citrus pathogens to facilitate safer germplasm exchange. Plants 2024, 13, 411. https://doi.org/10.3390/plants13030411
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: de Leon, V. S., J. Chen, G. McCollum, J.-W. Park, E. S. Louzada, M. S�tamou and M. Kunta. 2024. Diversity of Candidatus Liberibacter asiaticus strains in Texas revealed by prophage sequence analyses. Plant Disease 108: 1455-1460. https://doi.org/10.1094/PDIS-09-23-1994-SR
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Chandrika Ramadugu and Mikeal Roose, 2024. Breeding HLB-resistant citrus and field evaluation of novel hybrids. Citrograph Summer 2024. Volume 15, Number 3. pages 52-56.