Source: The Regents of University of California submitted to NRP
DEVELOPMENT OF HUANGLONGBING RESISTANT/TOLERANT CITRUS THROUGH GENOMIC APPROACHES.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1018373
Grant No.
2019-70016-29068
Cumulative Award Amt.
$3,941,090.00
Proposal No.
2018-08808
Multistate No.
(N/A)
Project Start Date
Jun 1, 2019
Project End Date
May 31, 2024
Grant Year
2019
Program Code
[CDRE]- Citrus Disease Research and Extension Program
Recipient Organization
The Regents of University of California
200 University Office Building
Riverside,CA 92521
Performing Department
College of Nat & Agr Sciences
Non Technical Summary
New cultivars of crop plants are often needed to address emerging challenges, to meet the needs of farmers, to develop sustainable crop cultivation methods, and to protect the environment. The Citrus industry in the United States of America is faced with a deadly disease, Huanglongbing (HLB) or citrus greening that has ravaged many citrus growing regions in the country and has caused significant concern in other areas where the disease is now spreading. The slow development of the disease, the un-culturable nature of the associated causal organism, and the complexity of the pathosystem make controlling this disease extremely difficult. A review published by the National Academy of Science in 2018 summarized the results of about 12 years of research conducted on finding solutions to the citrus HLB problem and declared that HLB may be the greatest challenge faced by the citrus industries in the Western world. Since citrus is economically and culturally very important for both Florida and California, we need to develop solutions for long term cultivation of this important fruit crop. The disease is associated with Candidatus Liberibacter asiaticus, an unculturable Alpha-proteobacterium and is spread by the Asian citrus psyllid. Efficient disease mitigation will be feasible with adequate vector control. Implementing management techniques for disease mitigation will also result in increased costs for cultivating citrus and some of the approaches practiced now may result in damage to the environment. According to some estimates, in Florida where the disease has established for about twelve years, the cost for caring of an acre of citrus has increased from an average of $769 in the year 2000 to about $2376 in 2017. Environmentally and economically sustainable solutions for citrus cultivation in presence of HLB will require development of disease resistance. Availability of commercial varieties that are resistant will provide long-term, eco-friendly, and cost effective solutions for this devastating disease. There is no report of resistance to HLB in citrus. Because of long cultivation and vegetative propagation methods used routinely, new genetic material is often not introduced into cultivated citrus and the currently grown varieties have a narrow genetic base. However, certain related genera in the citrus family are HLB resistant or highly tolerant. We have explored the genetic diversity in close relatives of citrus and identified sources of HLB resistance in Australian limes. Using these sexually compatible disease resistant taxa, we have generated hybrids of citrus by breeding with HLB resistant citrus relatives. Many of the hybrid plants that we have developed are very promising since they do not harbor the HLB pathogen for extended periods (more than two years). Our hybrids are now undergoing field evaluations. One caveat is that the hybrid plants inherit undesirable fruit characters along with resistance traits from the citrus relative parent. Traditional breeding to eliminate unwanted fruit characters while maintaining resistance will be feasible but time consuming. Citrus has a generation time of 4-7 years (depending on cultivars). Using a breeding population of hybrid plants that are either resistant or susceptible, we propose to utilize modern genetics methods to generate molecular tools that will be useful in developing commercial citrus cultivars resistant to HLB. Novel innovative methods of crop improvement are now possible with advanced and affordable sequencing technologies, gene editing procedures and ability to pyramid useful genes in transformed plants. Identification of target genes and availability of resistance-associated genes will be necessary to utilize these technologies to our advantage. Advances in genetics and genomics make it possible to develop an understanding of the basis of resistance and identify genes that can then be introduced into specific cultivars so that HLB resistance can be developed in commercial cultivars without altering the desirable horticultural traits. In this project, we propose to utilize hybrid populations of citrus (derived from crossing citrus with disease resistant relative genera) to generate sequence information that will lead to the identification of genomic regions associated with HLB resistance. Using a combination of several genomic approaches (whole genome sequencing, low depth skim sequencing of HLB resistant and susceptible F1 populations, pooled genome approach, transcriptome profiling, qPCR analysis for determining gene expression profiles, and bioinformatic analysis), we propose to identify genes that are associated with HLB resistance. For proof of concept, we will clone the putative resistance associated genes into suitable plant transformation vectors and transform a model citrus plant, Duncan grapefruit. The transformed plant will be tested for presence of transgene, expression levels, stability, and evaluated for HLB resistance. Useful constructs/genes/information about resistant genes will be beneficial for generating disease resistant citrus cultivars using many novel technologies. Development of genomics-based tools that can be used to select potentially resistant progeny will also accelerate traditional breeding approaches. Genes for HLB resistance, once identified and validated by the University of California, Riverside, will be made available through the appropriate University technology transfer mechanisms, including patenting and licensing of intellectual property (IP), inter-institutional, material transfer, and confidential disclosure agreements, so that others can incorporate the HLB resistant genes into various citrus cultivars to expeditiously translate the research results for commercial application to support the citrus industry. The project has a multi-state advisory panel consisting of a citrus scientist, a citrus nurseryman, an apple breeder and a citrus pathologist. The advisory board will provide guidance on goals, approaches and future directions.
Animal Health Component
30%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2020999108020%
2012499116050%
2124010108130%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

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

Field Of Science
1160 - Pathology; 1081 - Breeding; 1080 - Genetics;
Goals / Objectives
The overall goal of this project is to develop tools for generating HLB resistant citrus. Identification of candidate genes associated with HLB resistance will be useful to the industry for development of disease resistant commercial cultivars. We have identified sources of resistance to citrus HLB in Australian citrus relative genera that are sexually compatible with citrus, conducted wide crosses and generated populations of novel hybrids. We have both resistant and susceptible individuals that we propose to use for this project in order to identify genes associated with HLB resistance. Advanced technologies are now available for introduction of multiple genes in plant transformation experiments. Sophisticated gene editing technologies using CRISPR/Cas9 methods can now be used to generate non-transgenic, disease resistant citrus cultivars - if genes associated with HLB resistance are identified, evaluated and made available to the scientific community. Identification of resistance traits will also be valuable in traditional plant breeding since progeny selection can be guided by genomics-based tools.This SREP proposal specifically addresses a topic identified in the RFP and considered by the Citrus Disease Sub-committee to be an area of highest priority: 'Development of tolerance or resistance in commercial citrus in all production areas with a focus on new cultivars (or rootstocks and scions) using all available strategies, with consideration of pathogen genetic diversity'. The five objectives identified in the proposal are outlined below:1. Sequencing genomes of HLB resistant parent accessions and disease resistant/susceptible hybrid progeny (Dardick, Roose and Ramadugu).The genome sequences of Australian citrus relative genera used in the current study differ from citrus genomes that are currently publicly available. Generating a good quality reference genome sequence that is fully annotated is a requirement for conducting other genomic and transcriptomic analyses of hybrids of citrus generated by crossing with Australian citrus relative genera. Since we have both resistant and susceptible progeny from the breeding populations having similar parentage, a comparative genomics approach will be useful in identifying gene fragments that are constantly associated with disease resistance. We also propose to generate low depth skim sequencing of about one hundred hybrids with known HLB disease response patterns.2. Identification of candidate genes associated with resistance (Dardick, Kunta and Ramadugu).We will utilize two additional approaches (along with low-depth whole genome skim sequencing) for identifying resistance genes: a). Pooled genome analysis (P-nome) of selected samples to map and identify candidate genes associated with disease resistance traits; b). Transcriptome analysis to examine the gene expression profiles of HLB -resistant and -susceptible lines to provide a better understanding of the molecular changes in hybrids exposed to the HLB pathogen.3. Identification of molecular markers for off-flavors associated with resistance traits (Arpaia and Obenland).During the next four years, we expect to obtain fruit from about 50 novel hybrids that are generated from our breeding program and are already in the field. We will conduct fruit quality analysis of these hybrids to determine if certain genes present in the hybrid populations contribute towards generating unacceptable flavor profiles. This study will help us to select resistance associated genes present in fruiting hybrid trees that do not co-segregate with undesirable fruit quality traits.4. Development of HLB resistant citrus cultivars through plant transformations (Ramadugu and Roose).To generate proof of concept, we propose to transform certain citrus cultivars with resistance - associated genes identified in this project, raise transformed plants and evaluate them for HLB resistance by exposing them to the CLas pathogen either in FL or in CA (preferably in both states, pursuant to regulatory approval).5. Outreach and economic analysis (Kahn, Arpaia and Jetter).There is a need to educate both growers and the public about our efforts to incorporate resistance in commercial citrus so that there will be public acceptance of the transformed citrus cultivars. Our outreach and extension efforts will be focused on sharing information and knowledge developed, educating citrus growers, and obtaining feedback to improve our project. We will conduct economic analysis in year four to assess the financial benefits emanating from this research project.
Project Methods
Objective one: Sequencing genomes of HLB resistant parent accessions and disease resistant/susceptible hybrid progeny (Dardick, Roose and Ramadugu).1.1: To develop high quality phased reference genome sequences of three HLB resistant citrus relative accessions. We propose to sequence three Australian citrus types used as pollen parents to generate hybrid populations. Eremocitrus glauca, Microcitrus inodora and M. australasica will be sequenced to about 180X. Using a combination of short-read sequencing and linked reads (100 Kb fragments; 10X genomics), we will generate near chromosome length scaffolds with phasing. Discovery of haplotypes within each parental combination will be necessary for constructing sequence-based genetic maps. The sequences will be assembled using DeNovoMAGIC 3.0 software and pseudochromosomes will be constructed.1.2, 1.3: Low coverage skim sequencing of 100 F1 hybrids and certain F2/backcross hybrids with resistance/susceptibility to HLB pathogen. We will use a population of F1 hybrids that has been phenotyped for HLB disease response (Hall and McCollum). Chromosomal regions of the Australian citrus relative parent present consistently in resistant hybrids will be identified. A genetic map will be generated for each parent separately that will show the inheritance pattern of its haplotypes across all progenies and will be used to order parental physical assemblies into two distinct phased haplotypes. The correlation of HLB disease response data to the genetic/physical map will define the haplotype regions that accounts for the phenotypic variance for the resistance trait. Similar strategy will be used to analyze other available hybrids with Eremocitrus and Microcitrus parentages.Objective two: Identification of candidate genes associated with resistance (Dardick, Kunta and Ramadugu).2.1: Pooled genome analysis for mapping HLB resistance. A minimum of eight samples (resistant and susceptible) derived from each type of cross will be included in the pool. Genomic DNA libraries of target insert size of 500 bp will be constructed using an Illumina Truseq DNA PCR-free library preparation kit and sequencing done to a depth of 4X. We will utilize the genomic sequences of citrus relative genera generated in objective 1.1 and re-sequenced genomes available from citrus genome database site, as needed, for aligning sequences. We will identify patterns that show strong association with the resistance and/or susceptible traits. The most tightly linked molecular markers will also be useful in breeding and selection of the progeny for the resistance traits.2.2: RNAseq analysis. HLB resistant and susceptible parents, and selected hybrid genotypes will be used for RNAseq analysis. Plants will be graft inoculated with HLB-positive budwood in CA. Total RNA extractions from each genotype of selected plants will be done and RNAseq data generated using Illumina HiSeq 4000 platform at 10X depth. Gene expression value of unigenes in each hybrid and parental line will be determined. Comparisons will be done between samples collected before and after inoculations, resistant and susceptible types, and between hybrids and their parents.2.3: Analysis of results of whole genome sequencing, P-nome and RNAseq to identify resistant genes. The availability of genomic sequences from parental and hybrid populations coupled with P-nome and RNAseq data will enable the identification of a single, or a small number of high priority candidate genes associated with one of more or the HLB resistance traits. Collectively these data will provide information necessary to systematically identify high priority candidate genes for molecular marker design and as candidates for engineering resistance into commercial citrus.Objective three: Molecular markers for off-flavors associated with resistant hybrids (Arpaia and Obenland).3.1: Assess quality of fruit from HLB resistant hybrid trees. Hybrids that inherit resistance traits may also inherit undesirable fruit characters from the Australian citrus parent. We expect to obtain fruits from about 50 hybrid trees during the course of this project. We will collect fruit juice and determine soluble solids and titratable acidity. Volatiles will be tested by solid phase micro-extraction and using mass spectrometer and compared with Wiley/NBS library spectra. Sugars and organic acids will be analyzed by HPLC with a UV detector. For metabolomics analysis, LC-MS will be done on a Synapt G2-Si quadrupole time of flight mass spectrometer coupled to an I-class UPLC system. Data will be processed using Progenesis Qi. Principal component analysis will be performed using SIMCA (Umetrics).3.2: Correlation of Genetic markers with off-flavors. As part of objective 1, we will generate genomic information for many hybrids that are currently fruiting or will fruit in the next two years. Association of the genotypic data with fruit quality analysis will enable identification of markers associated with off-flavors.Objective four: Development of HLB resistant citrus cultivars through plant transformations (Ramadugu and Roose).4.1: Identification and evaluation of target genes. Based on the results of genomic and transcriptomic study, we will generate a list of candidate genes, do KEGG metabolic pathway mapping to understand the probable function. Gene expression levels will be measured by qPCR of candidate unigenes in resistant and susceptible hybrids. Selected genes will be used for plant transformation.4.2: Citrus transformations. We will transform Duncan grapefruit using pCAMBIA vector with transgene through Agrobacterium-mediated transformation. Presence of transgene and copy number will be evaluated by PCR and Southern blots. Selected transgenic plants will be evaluated for HLB resistance. We will also attempt mature citrus transformation using Tango mandarin, Hamlin orange and Valencia using the most promising resistance genes. We will collaborate with other labs conducting plant transformations for pyramiding useful genes.4.3: Evaluation of transformed plants for HLB resistance trait. The transgenic plants will be propagated and challenged with CLas in BLS3 facilities in CA. The plants will be maintained for several months, evaluated periodically for symptom development and bacterial titer. If transgenic plants show promise, we will move them to Florida (under permit) and evaluate.Objective five: Outreach and Economic analysis (Kahn, Arpaia and Jetter).5.1: Fruit tasting events. As we harvest hybrid fruits from the fields, we will conduct sensory testing with panelists seated in individual booths as per standard procedures. Ratings for acceptability, sweetness, tartness, bitterness and degree of off-flavor will be recorded. This is likely to take place in years three and four when sufficient fruits will be available from the novel hybrids generated.5.2: Outreach and extension: Information about resistance genes will be disseminated to other scientists for development of sufficient number of HLB resistant cultivars. Extension with citrus growers will be done for increasing public awareness and promote acceptance of genetically modified citrus. We will post information on UC ANR websites, publish articles in grower magazines and conduct extension.5.3: Economic analysis. In year four, using the available data, we will conduct economic analysis to assess the impact of our project at many levels. The first assessment will be at the farm level through changes in the life expectancy of orchards using traditional varieties versus resistant varieties. We will also evaluate the financial impact based on the rate of adoption of the resistant varieties. Utilizing market models, we propose to estimate the net benefits to consumers and producers.

Progress 06/01/19 to 05/31/24

Outputs
Target Audience:The target audience for this project consists of citrus growers, industry personnel, scientists, citrus breeders, molecular biologists, early career scientists, students, and technicians. The project aims to develop genomic tools for evaluating disease resistance to HLB and to develop information to identify potentially valuable germplasm with Microcitrus-ancestry. Our project is of immense interest to several groups of citrus breeders worldwide who are developing disease-tolerant/resistant germplasm suitable for various citrus industries compromised by the introduction and spread of citrus huanglongbing. Citrus scientists in other countries are interested in understanding our approach to developing resistant cultivars and have invited the project team to present this research in various scientific meetings. Citrus growers are interested in our study as many short-term solutions developed for mitigating the huanglongbing disease have not succeeded. PI Ramadugu has presented this research at various scientific and grower meetings since there is enormous interest in developing long-term solutions for this problem. University of California Riverside hosts Citrus Day events yearly (except during Covid). PI Ramadugu was invited to give presentations regarding the progress of this research, and many citrus growers keenly follow the advances made by our team in the development of new varieties suitable for the needs of the disease-ravaged citrus industry. Changes/Problems:Because of COVID-19-related restrictions on hiring and conducting research, there were some delays in conducting the project work from 2020 to 2023. What opportunities for training and professional development has the project provided?We have provided professional development opportunities to several individuals who participated in the project work. Postdoctoral scientists and other professional researchers obtained training in conducting genome assemblies, genome annotations, and RNAseq data analysis since these were needed for the project work. Interaction with vendors who performed certain essential sequencing services also helped in teaching techniques to project personnel and led to professional development. Pathogenicity experiments in contained research facilities were done in UC Davis and UC Riverside greenhouses. About five individuals obtained training and learned the proper procedures for working in biological safety level 3 situations. Scoring test plants for HLB disease response, maintenance of experimental plants in limited facilities like BSL3 greenhouses, and proper methods of disease challenge were taught to the technicians and scientists working at the contained research greenhouses. Site-specific standard operating procedures were developed as needed by PI Ramadugu. We have employed several part-time student workers (undergraduate) in the work-study program offered by the University of California Riverside. These programs cater to students from underprivileged sections of the society. As part of the work involved, we have provided training in the STEM areas for underprivileged students. Many students selected science careers because of this experience. Co-PI Kunta employed several individuals from underprivileged Hispanic communities to conduct the project work in Texas. The hired technicians and students were trained in conducting plant work, grafting, sample collection, plant processing, and sometimes DNA extractions. Because of the varied experiments conducted in the project, all the involved workers obtained additional training in new areas. This included scientists, professional researchers, post-doctoral researchers, laboratory technicians, field assistants, greenhouse technicians, graduate, and undergraduate students. How have the results been disseminated to communities of interest?1. Presentation of project results at the International Research Conference on citrus huanglongbing (IRCHLB): During March 2024, the Citrus Research Board arranged an International meeting in Riverside, CA. This conference is focused on HLB research and was attended by about 250 scientists from all the prominent citrus-producing countries of the world. Five presentations (oral and poster) emanated from the research conducted for this project. These are: a). Jianyang Liu, Khushwant Singh, Matthew Huff, Jong-Wong Park, Tara Rickman, Manjunath Keremane, Robert Krueger, Ping Zheng, Jodi Humann, Madhurababu Kunta, Mikeal Roose, Dorrie Main, Margaret Staton, Chandrika Ramadugu, Chris Dardick. "Analysis of R-genes in Australian Limes in Comparison to Commercial Citrus Cultivars." Presented at the International Research Conference on Citrus Huanglongbing organized by Citrus research board, March 2024. b). Pallavi Agarwal, Anil Bhatia, Manhoi Hur, David Obenland, MaryLu Arpaia, Amancio de Souza, and Chandrika Ramadugu. Comparative metabolic profiling of fruit juice from Australian limes and Citrus hybrids with Microcitrus parentage. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024. c). Khushwant Singh, Matthew Huff, Jianyang Liu, Jong-Wong Park, Tara Rickman, Manjunath Keremane, Robert Krueger, Ping Zheng, Jodi Humann, Madhurababu Kunta, Mikeal Roose, Dorrie Main, Margaret Staton, Chris Dardick, Chandrika Ramadugu. Navigating the Genetic Bottleneck of HLB: Exploring the Genomes of Australian Limes for development of disease resistance. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024. d). Jong-Won Park, Khushwant Singh, Tara Rickman, Erik Burchard, Manjunath Keremane, Robert Krueger, Chris Dardick, Margaret Staton, Mikeal Roose, Chandrika Ramadugu, and Madhurababu Kunta. Generating de novo transcriptome assemblies of Australian native lime species using various tissue types and its use as a reference for transcriptome data analysis. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024. e). Chandrika Ramadugu and Mikeal L. Roose. Genome-assisted breeding to incorporate huanglongbing resistance in citrus. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024. 2. Publication of results in trade journals. Ramadugu and Roose published an article in the Citrograph Grower magazine titled "Breeding HLB-resistant citrus and field evaluation of novel hybrids". Citrograph Summer 2024. Volume 15, Number 3. pages 52-56. The article mainly focused on research conducted for a project funded by the Citrus Research Board. But, the results generated in the current USDA NIFA project were essential in developing and selecting the hybrids in the Citrus Research Board-funded project. 3. Presentation of project work at the Plant and Animal Genome (PAG) meeting, San Diego, CA. We presented three posters at this meeting in January 2024. About 2500 scientists attend the PAG meeting. These are: a). Singh, K., Keremane, M., Krueger, R. and Ramadugu, C. 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). Plant and Animal Genome Meeting. Jan 2024, San Diego, CA. b). Metabolic profiling of fruit juice from novel citrus hybrids with Microcitrus parentage using untargeted metabolomics analysis. Agarwal P., Hur Manhoi, de Souza Amancio, Bhatia Anil, Obenland David, Arpaia MaryLu and Ramadugu Chandrika.Plant and Animal Genome Meeting. Jan 2024, San Diego, CA. c). Wei Zhang, Khushwant Singh, and Chandrika Ramadugu. Chromosome-scale genomes for scion hybrids to accelerate Citrus huanglongbing resistance breeding. Plant and Animal Genome meeting 31. Jan 2024, San Diego, CA. 4. Presentation at the UCR Citrus Day event in March 2024. Every year, the University of California Riverside arranges a UCR Citrus Day event (in conjunction with either Citrus research board or CAPCA). About 105 people attended the grower meeting. Ramadugu presented a poster and distributed some hybrid fruits to interested growers and scientists for tasting. The UCR Citrus Day event is organized most years (except when there are restrictions due to Covid19). Ramadugu always gets an opportunity to do extension work during this event. 5. Keynote talk at the South Padre Island, TX meeting. Ramadugu gave the keynote address at 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." About 150 people attended the meeting. 6. Ramadugu presented a talk on "Indian citrus germplasm: toward solving huanglongbing (greening) problems." Workshop on "Citrus Genetic Resources of North East India: Futuristic Insight." Organized by the Indian Council of Agricultural Research Complex at Umiam, Meghalaya, India, and the National Bureau of Plant Genetic Resources, New Delhi, India. December 2023. 7. Chandrika Ramadugu "Developing HLB resistant varieties." Oral presentation at the Santa Barbara County Citrus Industry Workshop. June 15, 2023. Santa Barbara County Citrus Industry Workshop. Citrus Pest and Disease Prevention Program. June 2023. Ramadugu was invited to give an oral presentation to citrus growers in Santa Barbara. The presentation was made to a group of about 25 citrus growers from Santa Barbara, Ventura, and San Luis Obispo counties in central California. 8. Javelina News, Texas A&M University-Kingsville https://www.tamuk.edu/news/2024/06/Kunta-research-teams-article-featured-as-cover-story-in-Plants-publication.html 9. At the IRCHLB meeting conducted in March 2024, several scientists from Australia, Brazil, Spain, Texas, and Florida were interested in seeing the hybrids generated from Australian citrus. Ramadugu gave tours to several groups and had opportunities to discuss the project and obtain feedback from other researchers interested in developing HLB resistance. The above list consists of extension activities conducted during 2023-2024 only. Extension activities conducted in the past years are listed in this grant's previously submitted annual reports. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact Statement: Citrus is a billion-dollar industry in Florida, California, and Texas. In 2005, a new citrus disease, huanglongbing or citrus greening, was reported from FL and has now spread to most citrus groves in Florida. In about 20 years, HLB has compromised the FL citrus industry; citrus yield has been reduced by 90% due to HLB (and the hurricanes that weakened sick trees). Since there is no cure for HLB, infected plants gradually die. Sustainable short-term remedies are not available for HLB management. Continuous use of pesticides to control insect vectors pollutes the atmosphere and is expensive. Previously, we identified sources of HLB resistance in citrus relatives; we have shown that the resistance trait is heritable and can be incorporated into cultivated citrus. Citrus breeding is a lengthy process, and the selection of promising progeny is challenging. In the current era of genomics, if the sequence of the resistant sources can be determined and molecular research conducted on the novel resistant hybrids, it will be possible to do genome-assisted breeding and select putatively useful progeny. In the current project, we developed genomic resources by sequencing the resistant parent varieties and conducting molecular studies. The information developed is publicly available to the scientific community. Several citrus breeding labs can now utilize the resources that have been published and can develop disease-resistant citrus. Our team consists of breeders, plant pathologists, molecular biologists, and bioinformatics specialists who have worked cooperatively to develop genomic resources that are valuable in providing tools for developing novel, HLB-resistant varieties. Obj.1: Sequencing genomes of HLB-resistant parent accessions and disease-resistant/susceptible hybrid progeny. In this project, we have sequenced the genomes of three Australian limes, Microcitrus australasica, M. inodora, and Eremocitrus glauca, identified as resistant sources and used previously for generating disease-resistant progeny when crossed with citrus varieties. The haplotype sequences were assembled de novo (without using a reference sequence - to avoid assembly errors). The research was published in the Journal Plants (https://doi.org/10.3390/plants13111460). Primary and alternate haplotypes of each Australian lime genome, DNA-seq and RNA-seq (transcriptome) data were submitted to NCBI database: C. australasica: primary haplotype-PRJNA924094, alternate- PRJNA924095. C. inodora: primary- PRJNA924048, alternate-PRJNA924114. C. glauca: primary- PRJNA924119, alternate- PRJNA924121. Please note: Microcitrus and Eremocitrus are now classified under the genus Citrus and hence referred to as C.australasica, C. inodora, and C. glauca. Obj.2: Identification of candidate genes associated with resistance. We have identified the genes associated with resistance (R genes) in our three genomes. Disease resistance may depend on the recognition of the pathogens by R genes. Different classes of resistance genes were documented: 1. NLR (with a nucleotide binding site -NBS- and a C-terminal Leucine-rich repeat region - LRR); 2. CNL (with a coiled-coil domain [CC] and NIT-NB-LRR; 3. TNL with a coiled-coil domain and NIT-NB-LRR with a Toll/Interleukin receptor domains; in addition, RLKs (with extracellular LRR, a transmembrane domain, and a cytoplasmic kinase domain; RLP (similar to RLK without the kinase), and cytoplasmic enzymatic R-genes that contain neither LRR nor NBS groups. Through bioinformatic analysis, we have determined the exact locations of resistance-associated genes in the three sequenced Australian lime genomes. The results will be published shortly. We have conducted transcriptome analysis of selected first-generation hybrids generated with mandarins and Microcitrus to study the differences between resistant and susceptible types. We are now verifying the data using hybrids of the next generation with about 15-37% of the Microcitrus genome (derived from the resistant parent). Obj.3: We have analyzed 50 novel hybrids (mainly from the F1 generation) to determine the metabolomic compositionof the fruit juice. We have data for different types of compounds (flavonoids, coumarins, terpenes, etc) that are known to influence the flavor profile of the novel hybrid fruits. Fruit flavor depends on the type of compounds present and their relative concentrations. It is challenging to identify specific compounds associated with certain off-flavors. The preponderance of certain compounds may be related to Microcitrus-like flavors. The metabolomic study and associated analysis will be published shortly. During the course of this project work, we have also conducted fruit-tasting sessions (when we did not have restrictions due to COVID-19 or the recent fruit-fly quarantine in Riverside, CA). Obj.4: Development of HLB-resistant citrus through plant transformations. When we started this project, we envisaged a situation where specific R genes would be associated with HLB resistance and planned to generate transformed plants with candidate genes conferring resistance. Since resistance seems to be a multigenic trait, we have modified the strategy and developed methodologies to pre-select hybrids for the presence of specific genomic fragments containing genes of interest. Several hybrid lines are identified as putatively valuable. They are under observation to determine fruit quality in the field and resistance phenotype in contained research facilities (BSL3) where the individual plants are challenged with the HLB pathogen via psyllid challenge or graft inoculations. Greenhouse plants with promising phenotypes have been identified and will be further evaluated in the fields in Florida (co-PI Rosskopf). Obj.5: Outreach and Economic analysis. Project outreach was done in California and Texas. In California, the PI Ramadugu presented the results of the study in various grower meetings like UCR Citrus Day, in scientific conferences (Plant and Animal Genome meetings in San Diego, International Research Conference on citrus huanglongbing meetings in Riverside, other scientific meetings like the American Society of Horticultural Sciences, Citrus Research Board meetings in Visalia, Citrus Nursery Board meetings in California, International Society for Citrus HLB and phloem-colonizing bacterial pathosystem meeting in Clearwater, FL, etc. Ramadugu also presented the research in citrus meetings in India during 2023 (Oct. and Dec.). Fruit-tasting sessions were organized in Riverside, where 50-100 participants tasted novel hybrids selected based on molecular studies conducted during this five-year project. Co-PI Kunta conducted several meetings with local school boards in TX, provided tours for college students, and showcased the project work. The research was presented at the 77th Annual Meeting of Subtropical Agriculture and Environments (South Padre Island, TX) in February 2023 by Kunta and Park. Ramadugu deliveredthe keynote address at this meeting in February 2024 and described the project results to 125 participants. Details of the extension activities done during 2023-2024 are explained in the following sections. The previous extension activities are included in the progress reports that were submitted before. Economic analysis: Jetter (UC Davis) has upgraded a grower budget analysis spreadsheet that is based on the methods used by the Budget Planner software. This software has been used since the mid-1990s to develop the Sample Costs of Production Budgets for the University of California. This tool will allow Cooperative Extension agents to meet with growers to estimate the costs and benefits of adopting the new technology resulting from this project.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Ming Huang, Mikeal L. Roose, Qibin Yu, Ed Stover, David G. Hall, Zhanao Deng, Frederick G. Gmitter, 2023. Mapping of QTLs and candidate genes associated with multiple phenotypic traits for Huanglongbing tolerance in citrus. Horticultural Plant Journal, Volume 9, Issue 4, 2023, Pages 705-719. https://doi.org/10.1016/j.hpj.2022.10.008.
  • 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: 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: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Dardick, C., Gottschalk, C., Callahan, A. and Artlip, T. 2023. "Using Rapid Cycle Breeding Systems to Facilitate Biotech and Traditional Cultivar Development in Woody Crops." IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL. Vol. 59. ONE NEW YORK PLAZA, SUITE 4600, NEW YORK, NY, UNITED STATES: SPRINGER, 2023.
  • 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: Other 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.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Singh, K., Keremane, M., Krueger, R. and Ramadugu, C. 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). Plant and Animal Genome Meeting. Jan 2024, San Diego, CA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Metabolic profiling of fruit juice from novel citrus hybrids with Microcitrus parentage using untargeted metabolomics analysis. Agarwal P., Hur Manhoi, de Souza Amancio, Bhatia Anil, Obenland David, Arpaia MaryLu and Ramadugu Chandrika. Plant and Animal Genome Meeting. Jan 2024, San Diego, CA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Wei Zhang, Khushwant Singh and Chandrika Ramadugu. Chromosome-scale genomes for scion hybrids to accelerate Citrus huanglongbing resistance breeding. Plant and Animal Genome meeting 31. Jan 2024, San Diego, CA
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Jianyang Liu, Khushwant Singh, Matthew Huff, Jong-Wong Park, Tara Rickman, Manjunath Keremane, Robert Krueger, Ping Zheng, Jodi Humann, Madhurababu Kunta, Mikeal Roose, Dorrie Main, Margaret Staton, Chandrika Ramadugu, Chris Dardick. "Analysis of R-genes in Australian Limes in Comparison to Commercial Citrus Cultivars ". Presented at the International Research Conference on Citrus Huanglongbing organized by Citrus research board, March 2024.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Pallavi Agarwal, Anil Bhatia, Manhoi Hur, David Obenland, MaryLu Arpaia, Amancio de Souza, and Chandrika Ramadugu. Comparative metabolic profiling of fruit juice from Australian limes and Citrus hybrids with Microcitrus parentage. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Khushwant Singh, Matthew Huff, Jianyang Liu, Jong-Wong Park, Tara Rickman, Manjunath Keremane, Robert Krueger, Ping Zheng, Jodi Humann, Madhurababu Kunta, Mikeal Roose, Dorrie Main, Margaret Staton, Chris Dardick, Chandrika Ramadugu. Navigating the Genetic Bottleneck of HLB: Exploring the Genomes of Australian Limes for development of disease resistance. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Jong-Won Park, Khushwant Singh, Tara Rickman, Erik Burchard, Manjunath Keremane, Robert Krueger, Chris Dardick, Margaret Staton, Mikeal Roose, Chandrika Ramadugu, and Madhurababu Kunta. Generating de novo transcriptome assemblies of Australian native lime species using various tissue types and its use as a reference for transcriptome data analysis. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Chandrika Ramadugu and Mikeal L. Roose. Genome-assisted breeding to incorporate huanglongbing resistance in citrus. Presented at the International Research Conference on Citrus Huanglongbing. Organized by Citrus Research Board, Riverside, CA. 03/2024.


Progress 06/01/22 to 05/31/23

Outputs
Target Audience:The target audience for the project consists of citrus researchers, growers, bioinformaticians, molecular biologists, early career professionals, technicians, and students associated with citrus breeding programs. As we develop new hybrids with novel fruit qualities, we try to obtain feedback from the industry about the acceptability of the new hybrids by conducting taste panels. In the current year, we arranged two taste panels; the first panel had about 45 people and they were offered juice from three novel hybrids producing lemon-like fruits (along with a standard Persian lime for comparison). We also evaluated a novel mandarin-like hybrid with a standard mandarin type of citrus. In the second taste panel (70 participants), we provided juice from four lemon-like hybrids and a standard lemon. The analysis indicated that some of the hybrids were deemed to have about 70% acceptability rate providing us hope that newer hybrids generated in the project may be found suitable for consumption. Our project aims to develop genomic tools that can be useful in understanding resistance to HLB that is conferred by associated genes in Australian limes. Toward this goal, we have generated genomic resources for the three Australian limes and made them accessible to interested scientists by posting them on publicly available sites like NCBI GenBank. The target audience for using these resources are citrus scientists, bioinformaticians, and molecular biologists. We have communicated about our project to citrus growers, industry personnel, and interested scientists on many platforms like UCR citrus day (02/2023), the NIFA progress evaluation meeting conducted in Riverside (01/2023, attended by 45 members - co-PIs of the project, advisory board members, citrus industry members, and local citrus scientists), the American Society of Horticultural Society webinar (07/2022), at the joint Congress of California Citrus Nursery Society and International Society of Citrus Nurserymen, held in Visalia (10/2022), and at the California Citrus Conference held in Visalia (10/2022). We have presented our research at scientific conferences like the American Society for Horticultural Sciences (ASHS) annual meeting in Chicago (07/2022), oral presentation at the Professional interest group webinar for citrus arranged by ASHS (07/2022), at the Second meeting of the International Society for Citrus huanglongbing and phloem-colonizing bacterial pathosystems held in Clearwater (10/2022), at the XXX Plant and Animal Genome meeting, an International research conference held in San Diego (01/2023), at the Plant Biology meeting in Portland, Oregon (09/2022), and at the XIV International Citrus Congress held in Mersin, Turkey (10/2022). During some of the grower meetings, the project team gets to interact with growers and industry persons and have an opportunity to obtain valuable feedback about the project. In Texas, co-PI Kunta has communicated with the local community including citrus growers and industry personnel about the project and the progress being made at the 77th Annual Meeting of Subtropical Agriculture and Environments (02/2023). We have communicated through extension articles published by UC Davis, published brief articles in the citrus industry growers magazine (07/2022), and through participation in Citrus Research Board webinars (06/2022). Changes/Problems:We requested a no-cost extension for one year to complete the objectives of the project. Due to Covid-related restrictions, certain aspects of the project got delayed. It was difficult for some participating institutions to hire new personnel required to conduct project work. University regulations precluded having more than one individual in a small laboratory. This impacted the project's progress. We now have a one-year extension, Covid19 restrictions are lifted and we hope to complete other objectives. What opportunities for training and professional development has the project provided?Through this project, we have provided training and professional development opportunities to many people (undergraduate students interested in STEM fields, graduate students interested in early career professional guidance), postdoctoral researchers eager to learn new techniques and concepts that will be useful in their future scientific endeavors, scientists and professional researchers interested in advancing their scientific pursuits. When possible, we employ individuals from underprivileged categories to provide encouragement in pursuing science careers and to provide exposure to scientific fields that may be of interest to young researchers. Several Hispanic students, undergraduate individuals whose families did not get a college education, and recent high school graduates have worked with our research teams and in the process were exposed to valuable scientific concepts. We have hired many recent college graduates and trained them in DNA/RNA extractions, plant experiments, data entry, basic molecular biology laboratory techniques, etc. At least six individuals got training in using advanced bioinformatics tools because of their association with the project. The best opportunity for the project personnel has been interacting with the local citrus industry leaders and growers in various grower-organized meetings in the Riverside NIFA project progress meeting that we arranged in 01/2023. Such interactions also occurred at the Citrus conference held in Visalia, at the UCR citrus day event, and at the International Citrus Nursery meeting (part of the meeting was in Riverside and part in Visalia). The interaction between the researchers and the industry members was deemed to be very valuable since it provided an opportunity for the researchers, technicians, and undergraduate students to appreciate the need for this research in protecting the local citrus industry that is faced with an incurable disease like the citrus huanglongbing. How have the results been disseminated to communities of interest?Here is a list of activities that resulted in effective communication of research results to communities of interest: Posting genome sequence information files on the NCBI Genbank site: Please note that Microcitrus australasica is referred to as Citrus australasica based on the new taxonomy proposed and followed by many. Similarly, Microcitrus inodora and Eremocitrus glauca are referred to as C. inodora and C. glauca. Primary and alternate haplotypes of each Australian lime genome, DNA-seq, and RNA-seq (transcriptome) data were submitted to NCBI database for C. australasica: primary haplotype-PRJNA924094, alternate- PRJNA924095. C. inodora: primary- PRJNA924048, alternate- PRJNA924114. C. glauca: primary- PRJNA924119, alternate- PRJNA924121. Tracy Kahn was invited to present a talk at the Citrus Research Board webinar series held on June 28, 2022. The topic was "New citrus varieties for California". Progress made in the current project was mentioned to the audience consisting of citrus growers and industry personnel. Ramadugu was invited in July 2022 by the American Society for Horticultural Sciences (ASHS) to present her research in a webinar at the Professional Interest Group seminar series (on citrus). The title was: "Developing Resistance: A long-term solution to Combat HLB". The audience consisted of horticulturists, geneticists, breeders, and molecular biologists working on various crops. Khushwant Singh presented a poster at the American Society for Horticultural Sciences annual meeting held in Chicago, IL in August 2022. The title of the poster was: "Chromosome-scale reference genomes of three Australian limes". The event was attended by approximately 1000 scientists from different parts of USA. Ramadugu presented a poster at the Citrus Research Board meeting in Visalia, CA in 10/2022. The title was: "Update on breeding advanced citrus hybrids with tolerance/resistance to huanglongbing". The grower meeting was attended by citrus growers and industry personnel from California and local citrus scientists from Universities, the California Department of Food and Agriculture, and other related organizations. Ramadugu presented a talk on "Novel genomes of three Australian limes: towards the identification of the source of resistance against huanglongbing (HLB) disease" at the Second meeting of the International Society for Citrus Huanglongbing and phloem-colonizing bacterial pathosystems (IS-CHPP), Clearwater, FL.10/2022. The meeting was attended mostly by scientists from different parts of the USA and some International Scientists. A few growers and citrus industry members also attended the meeting. Kunta and Park presented project results to an International audience in a presentation titled: "Comparison of gene expression profiles of HLB-tolerant and -sensitive F1 hybrids derived from a cross between mandarins and an Australian lime". The event was XIV International Citrus Congress held in Mersin, Turkey in November 2022. Ramadugu and Khushwant Singh made an oral presentation at the XXX Plant and Animal Genome meeting on 01/2023. The title of the talk was "Finding genetic solutions for a serious citrus disease - huanglongbing". This International meeting is attended by over 2500 scientists from all over the world. Several scientists working on citrus and other crops (and also animal systems) attended the meeting. Keremane made an oral presentation on "NextGen sequencing as a tool for the detection of citrus pathogens to facilitate safe germplasm exchange" at the XXX Plant and Animal Genome meeting on 01/2023. This International meeting is attended by over 2500 scientists from all over the world. Several scientists working on citrus and other crops (and also animal systems) attended the meeting. Ramadugu arranged a two-day workshop in Riverside, CA to discuss the NIFA project progress on January 18th and 19th 2023. The meeting was attended by 47 people (33 in person and 14 by Zoom; consisted of scientific collaborators, growers, advisory board members, and Citrus industry leaders from CA, FL, and TX). Sixteen scientists and six growers/citrus industry leaders talked about the research work or industry perspectives on the citrus HLB situation in the USA. Chris Dardick and Jiang (USDA lab West Virginia) presented the work on genome analysis of the three Australian limes and identification of R genes in the three taxa; Erin Rosskopf (USHRL, Fort Pierce, FL) presented the laboratory and field HLB evaluations of pathogen-exposed novel hybrids (generated in our program) in Fort Pierce, FL; Jong Won Park and Madhu (Texas A&M) presented transcriptome analysis work done with hybrids; Karen Jetter (UC Davis) spoke about economic analysis; Khushwant Singh (UCR) presented genome sequencing work; Pallavi Agarwal (UCR) presented Meabolomics work; Ramadugu (UCR) presented an overview of the project and the progress made to the group. Keremane (USDA Riverside) presented the pathogen array developed to screen multiple citrus pathogens in a single 96-well plate array. With guidance from MaryLu Arpaia, Ramadugu provided juice from three lemon-like hybrids and one novel mandarin-like hybrid to about 45 people to assess the acceptability of novel hybrids generated in the breeding program. Obenland analyzed the taste evaluation data and presented it to the group. The discussions that ensued between the scientists and citrus industry personnel were very beneficial to the group. Kunta and Park presented a talk on "Molecular genetics approach for the improvement of citrus tolerance to HLB" at the 77th annual meeting of Subtropical Agriculture and Environments, Feb. 9-10, 2023, South Padre, TX. The meeting was attended by local growers, students, scientists, and citrus industry persons from Texas. Ramadugu was invited to deliver a talk on "Citrus huanglongbing: finding solutions for disease management and long-term cultivation" at the University of California Riverside Citrus Day event on March 3, 2023. The event was attended by about 140 growers, scientists, students, and other interested individuals. Ramadugu conducted a tasting panel and provided juice from four hybrid fruits to 70 individuals for obtaining data on the acceptability of novel hybrids as substitute lemons. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we hope to accomplish the following: Publish the sequencing results in a scientific journal. Continue genome analysis of the Australian lime sequences and compare them with other citrus genomes to identify novel genes that may have a role in resistance. Identification of specific target genes may facilitate the development of HLB resistance in existing commercial citrus cultivars. Complete the HLB disease response assays in hybrid plants that are in progress in Florida and Texas. Complete analysis of metabolomics data from F1 hybrids of mandarin X Microcitrus and publish the results. Texas collaborators will complete transcriptome analysis of selected F1 hybrids and publish the results. Conduct fruit-tasting events and evaluate hybrids currently available. Present research to citrus growers and scientists in meetings.

Impacts
What was accomplished under these goals? Obj.1. Sequencing genomes of HLB-resistant parent accessions and disease resistant/susceptible hybrid progeny. We have done long-read sequencing of the three Australian limes (Microcitrus australasica, M.inodora, and Eremociturs glauca; also referred to as Citrus species) with one SMRTcell for each accession in the Sequel II using the Circular Consensus Sequences - PACBIO platform. Approximately 100X coverage was obtained. De novo assemblies were constructed using Improved Phase assembly (IPA) and Hifiasm. Chromosome-scale scaffolds were generated using the Proximo Hi-C method. Juicebox was used to correct scaffolding errors. The final chromosome-scale scaffolds for each genome were ordered and oriented to correspond to the Citrus clementina genome. Genome assembly was analyzed using BUSCO. Synteny relationships between primary and alternate haplotypes were done using minimap. We used SyRI to generate .vcf files containing variant information for single nucleotide polymorphism, inversion, translocation, duplication, insertion, deletion, and highly diverged regions. RNA was extracted from various tissues, libraries were constructed and sequenced (Genewiz). RNA seq reads were mapped o the corresponding genomes. We identified repeat regions using RepeatModeler and Repeat Masker. Annotations were done using BRAKER v 2.1.6. Primary and alternate haplotypes of each Australian lime genome, DNA-seq and RNA-seq (transcriptome) data were submitted to NCBI database for C. australasica: primary haplotype-PRJNA924094, alternate- PRJNA924095. C. inodora: primary- PRJNA924048, alternate- PRJNA924114. C. glauca: primary- PRJNA924119, alternate- PRJNA924121. The genomes are now complete and the manuscript will be published shortly. Obj.2. Identification of candidate genes associated with resistance. Wild Australian citrus species are hypothesized to have R-genes that differ from commercial citrus species and are potentially responsible for HLB tolerance/resistance traits. To test this hypothesis, the R-genes in the three Australian species were identified and compared with three commercial species (sweet orange, Clementina orange, and mandarin orange) via a suite of bioinformatics tools. Australian wild species have similar numbers of R-genes as commercial oranges, but the chromosomal distribution and types of R-genes showed distinct differences. For example, Australian species had a higher percentage of NL-type R genes than the commercial species, but less in N-type R genes. No clades unique to Australian species were identified. We identified the top 10 motifs from each R-gene, most of which are essential to the functionality of disease resistance. We found a highly conserved motif in the three Australian species that is lacking in any of the commercial species. This motif is responsible for nucleotide binding and may confer a particular function in pathogen detection in the Australian species, and its exact roles warrant further analysis. We mapped the R genes onto the nine chromosomes of each species to reveal their chromosomal locations. The R-genes are distributed unevenly, with most R-genes located on only four chromosomes (1, 3, 5, and 7), and chromosome 6 having only a few or no R-genes. Many R-genes of the same or similar types formed large clusters, likely due to gene duplication during evolution or selective breeding. The identification of functional motif sequences that are unique to Australian species provides a potential mechanism by which Australian species may provide HLB resistance/tolerance and provide a resource for future breeding approaches aimed at mitigating HLB. Gene expression analysis done using resistant and susceptible F1 hybrids show elevated expression of genes involved in phloem/xylem histogenesis, hypersensitive response, PAMP detection, systemic resistance, and lateral root development. Transcript discovery was made through this analysis and genome annotation was updated with information generated from the hybrid RNA-seq data analysis. Obj.3: Identification of molecular markers for off-flavors associated with resistant traits. We collected fruit from 76-F1 hybrids (mandarinXMicrocitrus) (some repeated a second year to evaluate year-to-year variation), and collected juice from vesicles for metabolomic analysis. Fruit traits (BRIX, acidity, size, seed count, juice%, etc) were documented and fruits were photographed. Metabolomic profiling was done to compare flavonoids, coumarins, terpenoids, sugars, and amino acids in the juice samples. We illustrate the data using six F1 hybrids of Wilking mandarinXMicrocitrus australasica (fingerlime). Three hybrids (from trees #18, 33, and 38) had mandarin-like fruit flavors and three (#37, 49, and 54) had fingerlime-like flavors. Flavonoids: type of flavonoids and relative amounts of the different types contribute to fruit bitterness, astringency, and sourness. All six hybrids had nobiletin and tangeritin. Hybrid#33 and 38 had tangor-like flavors and had the highest amounts of these two compounds. #18 had lower levels of these compounds. Relative amounts of nobiletin, vicenin, neohesperidin, tangeritin, poncirin, zapotin, naringenin, naringin, artemetin, etc. were documented. Flavonoid content in Wilking was ~74% (among the untargeted compounds) and in fingerlime it was 9%. Mandarin-like hybrids had 43-46% while fingerlime-like hybrids had 26-30% flavonoids. Mandarins generally have low coumarin content -1% in Wilking; fingerlime had 34%; mandarin-like hybrids had 4-5% while fingerlime-like hybrids had 7-18%. We documented 66 coumarins and related compounds in the juice of hybrid fruits. Levels of many coumarins like heraclenol, oxypeucedanin, auraptene, methoxsalen, isoimperatorin, phellopterin, isopimpinellin, and scopoletin varied in the different samples and were documented. The fingerlime-like flavors may be due to the type of coumarins in the fruit juice. The sugar content was 26% in mandarin, 5% in fingerlime, 17-20% in mandarin-like hybrids, and 12-14% in fingerlime-like hybrids. Aminoacid content: 43% in Wilking, 23% in fingerlime. In the hybrids, it ranged from 20-64%. The flavor of the fruit juice varies based on the type and composition of the different compounds. We are analyzing the metabolomic profile and the resistance trait recorded for the hybrid accessions. Volatile content was analyzed from 69 fruit juice samples in year 2 and 81 samples in triplicate in year 3. Analysis of the data is in progress. Obj.4: Development of HLB-resistant citrus cultivars through plant transformations: We are in the process of identifying candidate genes through the validation of known resistant hybrids. When target candidates are verified, plant transformations will be feasible. Obj.5: Outreach and Economic analysis. Outreach was mainly in California since the hybrid fruits are available in CA and can be shared with interested growers. We conducted two taste panels (01/2023 and 03/2023). In the first panel, 45 subjects were given juice from three lemon-like hybrids and one mandarin-like hybrid. Standard lime (Persian lime) and standard mandarin (Scarlet emperor mandarin) were used for comparisons. In the second taste panel (03/23), we included four hybrid lemons and a standard lemon (Meyer lemon) and provided juice for taste analysis to about 70 subjects. Certain hybrids fared well when compared to the standard cultivars. Various presentations made to growers, scientific presentations at research meetings, etc. are listed in other sections (publications, other products), and in the section that describes how results have been disseminated to communities of interest. Jetter analyzed the economic costs of pests for oranges grown in San Joaquin Valley, CA, using a market model. The analysis estimated the costs to both producers and consumers. The data was presented at the NIFA conference in Riverside in Jan 2023 and in a poster presentation at the UC ANR statewide conference in April 2023.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Jessica Trinh, Tianrun Li, Jessica Franco, Tania Toruno, Danielle M. Stevens, Shree P. Thapa, Justin Wong, Rebeca Pineda, Emmanuel Avila de Dios, Tracy Kahn, Danelle K. Seymour, Chandrika Ramadugu and Gitta Coaker. 2023. Variation in microbial feature perception in the Rutaceae family with immune receptor conservation in citrus. Plant Physiology, kiad263, https://doi.org/10.1093/plphys/kiad263
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2023 Citation: Chandrika Ramadugu and David Karp. 2022. Citron Genomics. In "The Etrog Citron" edited by Eliezer Goldschmidt and Moshe Barr-Joseph. 33 manuscript pages. Will be published by Springer. Article in press.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Muzi Li, Kelsey Galimba, Yuwei Xiao, Chris Dardick, Stephen M Mount, Ann Callahan, Zhongchi Liu, 2022. Comparative transcriptomic analysis of apple and peach fruits: insights into fruit type specification. The Plant Journal. 109(6): 1614-1629.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: David Obenland and Mary Lu Arpaia. 2023. Managing postharvest storage issues in Shiranui mandarin. HorTechnology 33(1): 118-124.


Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Citrus researchers, growers, bioinformatics professionals, early career professionals, technicians and students interested in STEM programs. The project aim is to develop genomic tools that can be used to evaluate novel hybrids generated in the breeding program. An important aspect of this endeavor is to obtain feedback from the citrus community about the taste of novel fruits and identify potentially useful hybrids. During the current reporting period, we exhibited hybrid fruits to citrus growers, members of the community, citrus industry personnel and University students - about 70 participants during a Citrus day event outreach program organized by the University of California team in collaboration with CAPCA (California Association of Pest Control Advisers). Since the Citrus day event was organized during March of 2022 when tasting the hybrid fruits was not a valid option (the hybrid fruits are edible between October and February), fruit tasting event could not be conducted. Due to Covid-related restrictions, we could not do the fruit tasting event earlier than March. Certain citrus growers and University researchers were provided with hybrid fruits of novel varieties to obtain feedback regarding the palatability of the fruits, juice content, sweet/sour nature and other parameters important for determining the suitability of novel hybrid fruits. Information about the project was communicated to growers, industry personnel and scientists during the California Citrus Conference held in Visalia, CA during October 2021. Ramadugu was invited to give an oral presentation describing project progress for the breeding program by the California Nursery Society to an audience of about 50 citrus growers, industry personnel, scientists and stakeholders; the event was held in Riverside during November 2021. The citrus day event organized by co-PI Tracy Kahn provided an opportunity for scientists and citrus growers to interact, exchange information, present talks and posters related to citrus cultivation and also provided a much-needed platform for researchers to conduct extension work. Due to Covid-related restrictions, this annual event could not be held in 2021 and was delayed in 2022 (held in March instead of January). In Texas, Kunta has communicated with the growers and stakeholders about the current project and how it can contribute to the Texas citrus industry in a recent Subtropical Agriculture and Environments Society's annual meeting. Changes/Problems:There were no significant changes in the objectives of the project. A co-PI from US Horticultural Research Laboratory from Florida, Dr. Greg McCollum, took retirement in December of 2021. A new co-PI, Dr. Erin Rosskopf, kindly agreed to be the new co-PI in place of McCollum. Dr. Rosskopf works in the same organization where McCollum worked and currently supervises Greg McCollum's technician, James Salvatore. Since Salvatore helped conduct the project work earlier, the transition has been smooth. NIFA was notified of this change and has provided approval. Covid 19-associated restrictions did affect our project work during the current reporting period. Hiring new project personnel was impossible in specific participating organizations for several months since the USDA imposed a hiring freeze that lasted several months during the current reporting period. Access to laboratories, greenhouses, or field locations was severely limited in the USDA lab in Fort Pierce (US Horticultural Research Laboratory). This affected our ability to collect infected plant samples from the field for analysis. Crucial research activities and data collection were delayed. Due to Covid 19, the USDA laboratory in Fort Pierce did not allow autoclave maintenance technicians inside the facility in Fort Pierce. Hence, we could not obtain permits to transport budwood from experimental hybrids (located in Riverside, CA) to Florida, delaying our HLB disease response evaluations. The University of California had restrictions on allowing undergraduate students who generally work part-time on the projects and help with routine plant maintenance, fieldwork, and laboratory chores. Undergraduates were allowed on the University of California campus after November of 2021. This resulted in a lack of help in conducting routine, essential tasks. Covid 19-related delays affected the progress of the project. What opportunities for training and professional development has the project provided?In this project, we have provided opportunities for two postdoctoral scientists and a graduate student to get training for conducting bioinformatic analysis using HLB resistant Australian citrus taxa that were selected for genome sequencing and analyses. Several undergraduate students were able to participate in the project work. They learned essential greenhouse plant management, plant propagations by grafting, and field evaluation of hybrid trees for horticultural performance. The students also received training in basic microbiology techniques required in a molecular biology laboratory (like autoclaving). They were taught DNA extraction procedures, nucleic acid quantification methods, and gel electrophoresis techniques. At least four technicians obtained training to conduct DNA extractions (either using Qiagen MagAttract kits, DNeasy method, or high molecular weight DNA preparation using Circulomics kits). They learned to use Nanodrop and Qubit to quantify the DNA, determine the purity of extracted DNA and prepare samples for downstream sequencing applications. One technician mastered the technology to conduct sequencing using Oxford Nanopore technology. How have the results been disseminated to communities of interest? Tracy Kahn organized a 'Citrus day' event in March 2022 at Riverside, CA, with collaboration from CAPCA. The event was attended by about 70 citrus growers, industry personnel, pest control agents, scientists, research professionals, and undergraduate students. The event provided excellent opportunities for fruitful interactions where researchers could exchange notes with citrus industry personnel, obtain feedback on the various research projects, and communicate project goals and progress to the attendees. Ramadugu displayed three posters at the event, explained research projects to interested members, exhibited hybrid fruits, and conducted limited fruit tasting. By March, most hybrid fruits generated in our breeding program are not suitable for tasting since the optimal time for fruit tasting is November to February. Ramadugu gave a presentation to the California Nursery Society about the project in a talk describing research progress on the generation of citrus hybrids, genome sequencing, and characterization of resistant hybrids. The event was held in Riverside, CA, on November 17, 2021, as part of the annual meeting of the Citrus Nursery Society. It was attended by about 50 growers, scientists, and industry personnel. The meeting was also attended by a few citrus industry members from Florida. Three posters were presented by Khushwant Singh Sandhu, Madhurababu Kunta and Ramadugu at the Citrus conference organized by Citrus Research Board in Visalia, CA during October 2021. The research projects were explained to over one hundred participants of the meeting; discussions with industry members and other scientists ensued and were deemed beneficial to the project team. At the Plant and Animal genomes meeting (held virtually during January 2022 due to Covid- related regulations), our team presented three posters and posted their presentations on-line so that other interested researchers can access the research data. In Texas, Kunta has communicated with the growers and stakeholders about the project and how it can contribute to the Texas citrus industry in a recent Subtropical Agriculture and Environments Society's annual meeting. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan to accomplish the following: Complete genome assembly of the three Australian lime taxa. With help from experts in the area that collaborate with our research team, we will curate the assemblies and make them available to members of the research communities by posting them on the publicly available Citrus Genome database website. Develop genome annotation information of the assembled genomes using GenSas and other methods. Complementary annotation information will be generated using the transcriptome data already generated by RNAseq analysis of various tissues of the three taxa used for genome sequencing. We will complete the HLB disease response evaluation for selected 50 backcross hybrids generated in the breeding program by challenging the propagated hybrids with grafts from HLB infected plants under greenhouse conditions (Rosskopf, Florida). Identify potentially useful candidate genes associated with disease resistance through bioinformatics analyses of the genome sequence data generated. We will conduct RNAseq to do transcriptomic analysis of HLB-resistant and susceptible hybrids. This information will provide data on genomic fragments associated with disease resistance and will eventually lead to the identification of candidate genes required to confer disease resistance/tolerance in commercial citrus varieties. Conduct large-scale fruit tasting event (with at least 100 volunteers and about 6 pre-selected hybrid fruits, depending on the Covid situation in California). We aspire to present research data at several scientific conferences and grower meetings (if Covid-related restrictions are lifted and in-person meetings are feasible during the next reporting period).

Impacts
What was accomplished under these goals? Obj.1. Sequencing genomes of HLB resistant parent accessions and disease resistant/susceptible hybrid progeny. We have generated genomic sequences of Microcitrus australasica, M. inodora, and Eremocitrus glauca and improved the genome assemblies we reported earlier. We utilized Pacific Biosciences (PacBio) long-read sequences (using Improved Phase Assembly, IPA), and chromatin interaction capture (Hi-C) approaches to generate chromosome-scale genome assemblies of the three taxa. Contig polishing (Racon version: 1.4.3) and statistics on the assembled genomes (Bandage version: 0.8.1) were done. The genome of M. australasica has an assembly size of 333 Mb, N50 value of 29.0 Mb, GC content of 36.39%. Draft genome assembly of M. inodora: assembly size of 321 Mb, N50 value of 30.3 Mb, and GC content of 37.70% (after quality control). The genome assembly of Eremocitrus glauca: size of 376 Mb, N50 value of 37.6 Mb, and GC content of 36.50%. All three assembled genomes were scaffolded into nine chromosomes for each accession and constituted 85.33% to 91.64% of the genomes. The integrity of the assembled genomes was verified using BUSCO (eudicots, 31 genomes) and showed a high score (96.5-97.6%). High genome coverage (>92% - 95%) of the three assembled genomes was observed when compared with two reference genomes: Citrus clementina and Poncirus trifoliata. This is the first chromosome-scale de novo genome assembly of the three Australian limes and can serve as an essential resource for genetic, genomic, and molecular research in citrus to fight against HLB. Annotating the genome using GenSas and complementary methods and structural variant analysis with known, publicly available citrus genomes is in progress. We are also generating long-read PacBio sequences for 20 selected F1 hybrids, previously phenotyped for HLB disease response. Comparing assembled genomes of F1 hybrids with HLB resistant and susceptible phenotypes will provide valuable information useful to correlate resistance traits with specific genomic fragments. Gene prediction conducted previously will be redone using the improved assemblies. Obj.2. Identification of candidate genes associated with resistance. We conducted a transcriptome analysis of differentially expressed genes in HLB tolerant and susceptible hybrids during this reporting period. We prepared a list of 15 differentially expressed genes (DEGs) that could be associated with resistance and conducted gene ontology (GO) analysis. DEGs between resistant and susceptible F1 hybrids ranged from 4087 to 5693. Eight to ten percent of DEGs were up-regulated in the tolerant F1 hybrid (Log2 fold change ≥ 2 and FDR p-value ≤ 0.05). The up-regulated DEGs were analyzed by BlastX and verified using other databases. Gene ontology (GO) annotation of up-regulated DEGs was conducted by GO mapping and merged with Interpro scan data to improve the annotations. A GO biological process graph was generated to infer the potential biological functions of the annotated DEGs. We prepared a list of 19 genes involved in the biological process "Defense response to other organisms" for their in-planta functional analysis. Two DEGs encoded proteins that share homology with LRR receptor-like Ser/Thr protein kinase and a disease resistance protein (that confers resistance to Pseudomonas syringae). These two genes may be involved in detecting CLas followed by a hypersensitive response (HR), a critical step that may lead to HLB resistance. In addition, other DEGs involved in systemic acquired resistance (SAR) and positive regulators of innate immune response were up-regulated in the resistant hybrid. Comparing a second set of plants showed 26 DEGs involved in "Defense response to other organisms". Two DEGs share their sequence homology to senescence-specific cysteine protease and are involved in HR. DEGs encoding RPS4 and receptor-like protein (RLP) 20, (which function as positive regulators of an innate immune response) were identified. 3 DEGs that can act as negative regulators of defense response to the pathogen were up-regulated in the tolerant compared to the susceptible plant. This indicates that certain susceptible hybrids have down-regulation of negative regulators. The GO analysis of DEGs obtained from comparing a third set of plants indicated 19 DEGs involved in "Defense response to another organism". Six DEGs were involved in an innate immune response. DEGs involved in induced systemic resistance and systemic acquired resistance and those functioning as positive regulators of innate immune response were recognized. Similar analysis will be conducted with other resistant and susceptible hybrids to understand possible disease resistance mechanisms. Obj.3. Identification of molecular markers for off-flavors associated with resistance traits. During this reporting period, we have analyzed 70 F1 hybrids (44 hybrids were analyzed last year and reanalyzed to obtain data from multiple years). We documented fruit characters by photographs of the whole or cut fruit. Recorded fruit traits: percentage acidity, BRIX ratio, seed count, juice content, fruit size, shape, rind thickness, and preliminary taste evaluations (based on feedback from a limited number of tasters). Metabolomic analysis was conducted at the University of California Riverside core facility. Fruit juice from at least five fruits per hybrid (a larger number when available) was squeezed from the juice vesicles (excluding the rind), filtered to remove debris, and analyzed for volatile, non-polar, and polar compounds. An in-depth analysis of the data is in progress. This objective aims to associate undesirable flavors with the presence of specific patterns in the metabolomics data. During breeding, in addition to the resistance traits that we are trying to introgress, other undesirable genes in proximity to the QTL of interest are inherited from the non-citrus parent due to linkage drag. Identification of specific patterns will be useful to select hybrid populations at the seedling stage and retain potentially promising plants for further study. Since metabolomics analysis of fruit juice obtained from F1 hybrids of Microcitrus type of plants has not been reported extensively, generating data from many hybrid fruits will provide a baseline for making selections (for hybrids with Microcitrus parentage). Obj.4. Development of HLB-resistant citrus cultivars through plant transformations. We are in the process of identifying potentially valuable candidate genes capable of conferring resistance to susceptible citrus cultivars. When target candidates are available, we will transform specific genotypes of citrus with candidate genes. Obj.5. Outreach and economic analysis. Outreach was conducted mainly in California, where the hybrid fruits for the project are available to demonstrate to the industry and shared for tasting with interested growers. Due to Covid-related social distancing requirements, many outreach opportunities were unavailable to the researchers. We shared information regarding the projects with citrus growers, industry members, and scientists at four events: Plant and Animal Genomics meeting (held virtually during January 2022), Citrus conference (held in person in Visalia, CA during October 2021), Citrus Nursery Society Annual meeting at Riverside (held on November 17, 2021), and, Citrus day event held in March 2022 in Riverside, CA (by the University of California Riverside in collaboration with CAPCA). Oral presentations, posters (presented in person or virtually), and discussions with industry members and scientists were the mode of outreach. Economic analysis: Karen Jetter, the economist on the team, will conduct economic analysis during the last year of the project and is now gathering the required information.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Keremane, M. L., McCollum, T. G., Roose, M. L., Lee, R. F., & Ramadugu, C. (2021). An Improved Reference Gene for Detection of Candidatus Liberibacter asiaticus Associated with Citrus Huanglongbing by qPCR and Digital Droplet PCR Assays. Plants 2021, 10(10), 2111. https://doi.org/10.3390/plants10102111
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Snyder, J., Dickens, K.L., Halbert, S.E., Dowling, S., Russell, D., Henderson, R., Rohrig, E. and Ramadugu, C. 2022. The Development and Evaluation of insect traps for the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Psyllidae), vector of Citrus Huanglongbing. MDPI Insects 13:295. https://doi.org/10.3390/insects13030295
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Kunta, M., Park, J.-W., Braswell, W.E., da Graca, J.V, Edwards, P. (2021) Modern tools for detection and diagnosis of plant pathogens. In: Singh K.P., Jahagirdar S., Sarma B.K. (eds) Emerging trends in plant pathology. Springer, Singapore. doi.org/10.1007/978-981-15-6275-4_4.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Park, J.-W., da Graca, J.V. Gonzalez, M., Louzada, E.S., Olufemi J.A. and Kunta, M. (2022) First report of citrus virus A in Texas associated with oak leaf patterns in Citrus sinensis. Plant Dis. DOI: 10.1094/PDIS-03-21-0628-PDN
  • Type: Other Status: Published Year Published: 2021 Citation: El-Kereamy, A., Arpaia, M. L., Doohan, G., and Obenland, D. 2021. Preharvest mandarin rind disorder in California. Citrograph 12:54-58.
  • Type: Other Status: Published Year Published: 2021 Citation: Arpaia, Mary Lu, Cranney, James, Tebbets, Steve, Walse, Spencer, Obenland, David, 2021. Response of California citrus to postharvest phytosanitary treatments. Citrograph 12(2):30-37.
  • Type: Other Status: Published Year Published: 2021 Citation: Obenland, D., Cranney, J.R., Tebbets, S., Walse, S., Arpaia, M.L., 2021. Fumigating citrus with phosphine does not impact marketability or eating quality. Plant Health Progress 22:516-523.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Mattia, M. R., Du, D., Yu, Q., Kahn, T., Roose, M., Hiraoka, Y., Wang, Y., Munoz, P., and Gmitter, F.G., Jr. 2022. Genome-wide association study of healthful flavonoids among diverse mandarin accessions. Plants 11: 317. https://doi.org/10.3390/plants11030317


Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Target audience consists of citrus growers, citrus researchers and breeders. Unlike previous year, we were not able to conduct the planned outreach activities in person due to Covid19-imposed regulations in the various institutions. There was newspaper coverage regarding development of resistance in citrus by breeding approach. Similar articles intended for the public were published in several grower magazines. There were also some radio interviews that reached many citrus enthusiasts all over the country. Through email correspondence, we were able to interact with some citrus industry personnel and described our project goals and progress. Similarly, taste panels of novel hybrid fruit samples could not be conducted as in previous years. Our taste evaluations this year were restricted to four peoject personnel that were on location in Riverside and had opportunity to conduct tasing and provide feedback regarding the acceptability of the fruit flavours. Changes/Problems:The major challenge during this reporting period was caused due to Covid-related restrictions. We were not able to hire professional personnel, recruit undergraduate students (who perform routine sample collection and processing), or visit collaborating laboratories for in person discussions. Field trees of several novel hybrids that are included in the study yielded fruit. We were not able to provide fruit samples to volunteers for tasting. Due to social distancing protocols and general trepidation associated with the pandemic, people were not available to provide feedback on the acceptability of novel fruit types. Generally, we correlate metabolomic data with fruit taste panels. We were able to gather feedback from a very few individuals who are closely associated with the project and were available for fruit tasting. In addition, we were not able to provide fruit to the citrus growers who normally provide very useful feedback on the acceptability of the hybrids. Lastly, the major HLB related conferences were cancelled due to the pandemic depriving us of the ability to have in-person discussions with knowledgeable, interested individuals of the citrus community. What opportunities for training and professional development has the project provided?Postdoctoral researchers, technicians and other scientists involved in the project had an opportunity to learn about the citrus HLB pathosystem and the complexities of disease development. Since the disease is very difficult to control, a proper understanding of this disease is essential for devicing solutions to the problem. Genome assembly programs including Hifiasm, Canu, etc were learnt by postdoctoral scientists involved in the proejct. Several other programs required for Oxford nanopore sequencing, synteny analysis and genome comparison by dotplot were also mastered by the postdoctoral scientist and other professional researchers involved in this aspect. The technicians associated with the program mastered DNA extraction methods suitable for generating high molecular weight DNA required. Generally we have undergraduate students who get exposed to a variety of laboratory techniques and field methodologies that are required for sample preparation, fruit quality analysis, etc. However, this year, due to Covid restrictions, we were not able to hire undergraduate students. How have the results been disseminated to communities of interest?The main mode of communication this year was through news articles that described the novel hybrids generated in the program for developing HLB resistance. Following this coverage, many citrus growers communicated with the PI and in the process were educated about the breeding and analysis approach for generating disease resistant citrus hybrids. Discussions with the growers was helpful to understand the needs of the industry in resolving the citrus huanglongbing problem facing the different citrus cultivation areas in the US. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to validate our genome assemblies by comparing the different approaches used by our collaborating scientists. Gaps in the assembly will be filled by other accepted methods like PCR and Sanger sequencing (of smaller fragments) or by other bioinformatic methods used to compare the different reference assemblies available with the scaffolds generated in our project for the three parental species. We have generated transcriptome data for the three parental species by obtaining relevant samples for RNAseq analysis. We will include some more samples (like root tissue) that are required for a complete understanding of the transcriptome. We will be improving the genome assembly in the next year and use this information to reanalyze RNAseq data generated previously. We are in the process of generating additional CLas exposed hybrid plant samples for transcriptome analysis. Correlation of disease rating with transcriptome data will be useful in validating the putative candidates that are up-regulated or down-regulated in the diseased state - in both disease resistant and susceptible types, Regarding metabolomic analysis of fruit juices and leaf samples, we will increase the number of samples (per genotype) to obtain more consistent, statistically significant data. Metabolomic analysis of fruit samples will provide valuable information regarding the acceptability of the hybrid fruit and comparison of the metabolomic profile of the fruit juice and the leaf samples. This data may be helpful in preliminary selectioon of hybrid seedlings likely to have the right components. Comparison of leaf and fruit samples will be conducted for more samples. We hope that Covid situation will improve and the restrictions will be lifted. This will enable us to conduct extension activities, fruit tasting events, hire undergraduate student help so that the scientists can conduct other essential activities. Interaction with the citrus growers can be resumed, in person meetings can be arranged and valuable discussions will ensue.

Impacts
What was accomplished under these goals? Impact: Development of HLB-resistant citrus varieties will provide long term, sustainable, ecologically and economically feasible solutions. Using other resources, we have developed several types of citrus hybrids by crossing with disease resistant Australian citrus. Analysis of the genome structure of the resistant types and molecular comparison of resistant and susceptible hybrids will help to develop markers correlated with resistance. It will be possible select useful hybrids by screening in the seedling stage (molecular breeding). Increasing the efficiency of breeding projects is needed to generate commercially useful varieties. Obj.1. Sequencing genomes of HLB resistant parent accessions and disease resistant/susceptible hybrid progeny. The genomes of Australian citrus relative genera with resistance to the HLB pathogen (Microcitrus species and Eremocitrus glauca) are likely to have segments not present in standard citrus sequenced earlier. Hence, there is a need to conduct de novo assembly. We have generated long-read genomic sequences using PacBio with an average fragment length of 13 kb. In an alternate approach, we have generated long read sequences using Oxford nanopore technologies (ONT; an estimated DNA fragment length of 50 kb. The PacBio reads will have a low error rate while the ONT method produces long scaffolds (but with high error rate). The ONT library generated using SQK-LSK109 kit was loaded onto a R9 MinION flow cell and run on a MinION Mk1B sequencher. Base calling was done in real-time using minKNOW software. The draft assemblies of Australian limes obtained from NRGene were improved using nanopore reads with minimap2 (alignment program for long reads) and LRScaf (Long Reads Scaffolder). Final N50 values ranged from 26 to 28Mbp, arriving at near chromosome-level. BUSCO scores obtained were: 98.4% (Eremocitrus glauca), 98.5% (Microcitrus inodora) and 98.1% (M. australasica). Structural variants (SVs) were detected using NGMLR (long-read mapper designed to align PacBio and ONT sequences to reference) and Sniffles (structural variation caller). We are annotating using BRAKER2 with Citrus sinensis and C. clementina gene models used for training. Scaffolding using Unphased PacBio Reads and Raw ONT Reads: Raw nanopore reads were aligned to the PacBio scaffolds to generate input files to LRScaf that is optimized for Third-Generation Sequencing (TGS) technologies and performs scaffolding using long, noisy reads. Assessing the Quality of scaffolded assemblies was done using BUSCO scores, and by comparing number of SVs in different versions of assemblies. Assembly statistics: Eremocitrus glauca had 701 contigs, total length 371 Mb. Average contig length: 529,278. N50: 26,549,705. N90 was 1,656,766. Microcitrus inodora, had 139 contigs, total length 310 Mb. Average length of the contigs:2,236,969. N50:26,825,459. For M. australasica, the total length was 330 Mb and the number of contigs was 403. Average length of the contig was 819,510. N50:28,288,250. N90 was 1,863,641. Alternate approach: we conducted de novo assembly of the PacBio CCS reads using Hifiasm (de novo assembler for long high-fidelity reads to generate phased assemblies). We have conducted dotplot analysis of the scaffolds with reference genomes. Synteny analysis provided additional data for comparison. Plot coverage of the graphs identified gaps at different regions. In this analysis, N50 was M.inodora was 26Mb. Total length:318 Mb. Largest contig assembled:29 Mb. For E.glauca, the N50 was 20Mb. Sequence length:293 Mb. Largest scaffold:29MB. We have compared M. australasica assembly with reference citrus genomes. Transcriptome analysis of parental species: For RNA sequencing, we used various tissue samples (leaves, bark, flower, fruit; Illumina HiSeq 2X150 bp paired end sequencing approach). Trinity v2.5 de novo assembler generated one de novo assembled transcriptome per species. EMBOSS tools (getorf) identified ORFs within the de novo assembled transcriptome. The number of annotated contigs (using Diamond blastx) were: 222,783 (E.glauca), 205,374 (M.australasica) and 229,712 (for M.inodora). Outline of annotation: repetitive DNA masked by RepeatMasker v.4.0.9. Eukaryotic gene prediction by AUGUSTUS 'ab initio (Hidden Markov Models)' using Arabidopsis thaliana. Functional annotation was done by blast searches followed by gene ontology mapping of protein blast hits against GO annotated protein database v.2020.06; GO annotation and InterPro scan (IPS) followed by merging IPS to the GO annotation. Gene prediction for M. inodora: 41,706 gene predictions; average gene length:3,652 nucleotides; 2,264 genes could not be mapped to the current assembly; 5.1 exons per gene. In M. australasica, 45,500 genes predicted; average gene length:3,509; 5806 genes unmapped. In Eremocitrus glauca, 40,681 genes predicted, average gene length:3,558; average exons per gene:5.5; 3,192 genes mapped, not placed in the assembly. Obj.2. Identification of candidate genes associated with resistance. RNA seq analysis done for eight F1 hybrids [mandarinXAustralian citrus with a HLB disease rating of 0 to 4 ('0' indicates resistance; '5' indicates susceptibility). Two samples [F1 hybrid 1735.1 (Fortune mandarinXMicrocitrus inodora; disease rating of 0) and F1 1761.1 (Fortune mandarinXM. inodora; disease rating of 4)] were chosen for differential gene expression (DE) analysis. DE indicated 274 up-regulated and 227 down-regulated genes. GO analysis of DE showed genes related to cellular component organization/biogenesis and catabolic processes in the hybrid 1735.01. Genes involved in transmembrane transport, small molecule metabolism and intracellular localization were down-regulated. Consistency of the DE data will be evaluated by cross-checking the RNAseq data of other F1 hybrids. We are generating data on many hybrids using triplicate plants exposed to CLas to validate the preliminary results. Obj.3: Identification of molecular markers for off-flavors associated with resistance: In the previous year we generated metabolomics data for 32 types of hybrids by collecting juice from ripe fruits. We have now collected juice from 44 unique hybrids (from 77 plants) by the same technique. In addition, we collected leaf samples from the same field plants (that were used for metabolome analysis) and conducted analysis for polar and nonpolar metabolites as before. If certain undesirable flavors can be associated with specific metabolomic profiles in the fruit juice, it would be advantageous to detect it in the seedling stage from vegetative tissue. This would help preliminary selection of the hybrid progeny based on the leaf metabolome profiles. The results are now being analyzed. Obj.4. Development of HLB resistant cultivars through plant transformations. More progress in gene identification (obj.2) will result in identification of target genes. Obj.5. Outreach and economic analysis: Due to Covid restrictions, our outreach was limited to a few activities. PI Ramadugu was interviewed by a local CA newspaper showcasing HLB resistance development. Several other papers and grower magazines covered similar content leading to publicity for the resistance project. Many growers communicated with the PI by email and were educated on the project. Co-PI Kahn was involved in showcasing Finger-lime varieties through a citrus nursery-board organized webinar event. Jetter has updated sample costs for production of citrus fruits in the Southern San Joaquin valley. The information will be used to conduct farm level analysis of the benefits of developing HLB resistant citrus varieties.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 1. Eliezer S. Louzada and Chandrika Ramadugu. 2021. Grapefruit: History, use, and breeding. HortTechnology. Pp1-16. https://doi.org/10.21273/Horttech04679-20
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 2. Lee, R.F., Keremane, M.L. and Ramadugu, C. 2021. Use of young plans for biological indexing of graft transmissible pathogens of citrus. Crop Protection 143: 10524. https://doi.org/10.1016/jcroppro.2020.105524.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 3. Huang, C.Y., Niu, D., Kund, G., Jones, M., &Ramadugu, C. Jin, H. 2020. Identification of citrus immune regulators involved in defense against huanglongbing using a new functional screening system. Plant Biotechnology Journal 19: 757-766. https://doi.org/10.1111/pbi.13502.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 4. Stover, E., Ramadugu, C., Roose, M., Krystel, J., Lee, R.f. and Keremane, M. 2021. Incidence of Asiatic citrus canker on trifoliate orange and its hybrid accessions in a Florida field planting. HortScience 56: 525-531. https://doi.org/10.21273/HORTSCI15684-20.
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Xu, Q and Roose, M L. 2020. Citrus genomes: from sequence variations to epigenetic modifications. In: The Citrus Genome, Ed. A. Gentile, S. La Malfa, and Z. Deng. Springer. pp. 141-165.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 3) Hausch, B. J., Arpaia, M. L., Kawagoe, Z., Walse, S., and Obenland, D. 2020. Chemical Characterization of Two California-Grown Avocado Varieties (Persea americana Mill.) over the Harvest Season with an Emphasis on Sensory-Directed Flavor Analysis. J. Agric. Food Chem. 68:15301-15310.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Waite JM, Dardick C. The roles of the IGT gene family in plant architecture: past, present, and future. Curr Opin Plant Biol. 2021 Feb;59:101983. doi: 10.1016/j.pbi.2020.101983. Epub 2021 Jan 7. PMID: 33422965.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wisniewski M, Artlip T, Liu J, Ma J, Burchard E, Norelli J, Dardick C. Fox Hunting in Wild Apples: Searching for Novel Genes in Malus Sieversii. Int J Mol Sci. 2020 Dec 14;21(24):9516. doi: 10.3390/ijms21249516. PMID: 33327659; PMCID: PMC7765095.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kahn, T., Siebert-Wooldridge, T., and Trunnelle, K. 2020. Pigmented Citrus Cultivars Differentiate Market: Fruit Quality Evaluations of New Introduced Red-Fleshed Citrus Cultivars. Citrograph. 11(4):38-44.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Federici, C., Kupper, R., and Roose, M. Ventura County Lemon Rootstock Trial: Identifying Choices and Trade-offs. 2020. Citrograph, 11(4):52-57.


Progress 06/01/19 to 05/31/20

Outputs
Target Audience:The target audience consists of citrus growers, researchers and breeders. In our project we evaluate novel hybrids generated in our breeding program. We utilize opportunities available to us to share fruits from hybrid plants with the members of the industry so that we can obtain feedback from the public and stakeholders about the acceptability of these new varieties of fruits. We have conducted extension by presenting the goals of the project and displaying some of the hybrid fruits developed in our breeding program to growers, scientists and public on UCR citrus day held at Riverside, California. Six selected hybrid fruits were used for a taste panel with about 120 volunteers. Information on the palatability of hybrid fruits, juicy nature, sourness, sweetness and likabiity were collected from the survey. We utilize this information to select hybrid plants suitable for further breeding, associate acceptability and HLB resistance with the various types of markers we are generating in the project. We have also displayed information about the project to growers, industry personnel and scientists at the California Citrus Mutual Showcase held at Visalia, California In March 2020. We interact regularly with growers and stakeholders of the citrus industries in CA and TX and obtain feedback about the industry needs. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided many opportunities for training in bioinformatic methods, understanding sequence analysis pipelines, condcuting fruit quality evaluations, analysis of metabolomics data, assessment of hybrids for characters that are perceived as valuable by the public, etc. Technician was trained on relevant R package software tools including: WGCNA, GenSAS, GO term and other annotation pipelines, Cytoscape viewer and gene network exploration, creating circus plots for whole genome comparison, and comparison of various RNAseq analysis tools. How have the results been disseminated to communities of interest?We have presented our research work to stakeholders, growers, citrus industry personnel, researchers, and scientists, mainly in three activities. The first activity was a presentation made at the University of California Riverside Citrus day in January 2020. The event was attended by about 300 people. We had a booth dedicated to our project and displayed a poster describing the goals of the project, representative fruits generated in the breeding program, and discussed with interested individuals about the project. The event was organized as part of the annual UCR outreach activity by co-PI Kahn in collaboration with other individuals. Kahn introduced the project goals and activities to the audience, and Ramadugu participated by explaining the project to the people that visited the booth. More information can be found here: Inside UCR Article on Citrus Day: https://insideucr.ucr.edu/stories/2020/02/07/citrus-day-didnt-hit-single-sour-note The second activity was a fruit tasting event conducted by Ramadugu in collaboration with co-PIs MaryLu Arpaia and David Obenlanld. We conducted a taste panel using six representative fruits from the F1 progeny. Fruit pieces were provided to about 120 individuals for tasting and sensory evaluations. The fruit pulp was scored for overall acceptability, juiciness, tartness, and sweetness, on a scale of 1-9. This analysis was helpful in selecting parents for subsequent breeding conducted in 2020. The interaction with the stakeholders and the industry was very beneficial to the project team since it provided us with an opportunity to understand public perception regarding our project. The experience reiterated the need for HLB resistant hybrid plants with acceptable fruit quality for the citrus industry in the USA. The third educational outreach event was at Visalia, CA in March 2020. The event was California Citrus Mutual Citrus showcase attended by about 750 people. Co-PI Kahn had a booth and displayed a poster describing the project, had discussions with members of the industry, including growers, pest control agents, regulators, scientists, and the public that visited the booth at this event. Ramadugu has provided a tour of the fields where hybrid plants are grown to many interested growers and nurserymen through out the year. There is tremendous public interest in our project since the industry requires disease-resistant varieties from breeding programs. We interact with the industry personnel, obtain feedback, educate them regarding the project, and learn about what is essential to the industry. These outreach activities provided an opportunity to promote useful dialogue between members of the citrus industry and researchers from our team. What do you plan to do during the next reporting period to accomplish the goals? Complete genome annotation and assembly. Perform initial expression profiling analyses to compare genome expression profilies among the different Australian limes included in the study. Perform synteny analyses and assess genome content, identify disparate regions, and prepare genomes for p-nome analyses. Initiate p-nome mapping from select hybrid populations. Create genetic maps, identify QTL regions, and identify SNP markers for confirmation. Working with vendor (NRGene) to finalize genome assemblies and annotations for publication. Conduct transcriptomic analysis of selected hybrid progeny. Identification of genomic regions associated with resistance. Metabolomic analysis for understanding general composition of hybrid fruits and also for identifying specific polar, non-polar and volatile compounds that are known to be associated with flavor enhancement.

Impacts
What was accomplished under these goals? Obj. 1. Sequencing genomes of HLB resistant parent accessions and disease resistant/susceptible hybrid progeny. We have generated draft genomic sequence for three HLB resistant parent accessions - Eremocitrus glauca (now classified as Citrus glauca), Microcitrus australasica (or C. australasica) and M. inodora (or C. inodora) using NRgene services. These citrus relative genera were used as breeding parents to introgress useful HLB resistance traits into the hybrid plants. Generating a reliable, phased, fully annotated genome of the resistant parents is essential for understanding the basis of HLB resistance and identification of resistance-associated genes in the hybrid progeny. High molecular weight genomic DNA samples (>150 Kb fragments) were sequenced using short reads sequencing technology. We have total coverage of approximately 241X (Microcitrus inodora), 214X (for M. australasica), and 223X for Eremocitrus glauca using Illumina sequences. Paired-end, mate-pair, and linked reads sequencing libraries (10X genomics Chromium) were utilized to produce the raw data. The sequencing data were processed and assembled using DeNovo MAGIC assembler application version 3.0. The software creates a fully assembled, unphased genome in addition to a fully phased genome. The number of scaffolds, N50, and N90 will be accurately determined after long read PACBIO sequences are obtained. Pseudochromosome regions have been tentatively assigned and need to be confirmed after more data is collected. BUSCO analysis that indicates the integrity of the genic region, ploidy and zygosity characteristics of the assembled genome were satisfactory. The genetic maps that will be obtained from the segregating population genotype will provide a more accurate analysis. We are now generating information on the transcriptome using different tissues of the HLB resistant parents. Transcriptome data is required to annotate the sequenced genomes. Long-read PACBIO sequencing is in progress. Obj. 2. Identification of candidate genes associated with resistance. We are generating low depth sequencing data from breeding populations, and will conduct pooled genome analysis that is required for the identification of candidate genes. We are also generating transcriptomic data from known HLB resistant and susceptible hybrids. A fully annotated genome of the HLB resistant parents (Obj.1) is required to accomplish Obj.2. Obj. 3. Identification of molecular markers for off-flavors associated with resistance traits. During this year, we obtained fruits from 32 F1 hybrids (mandarins X Australian limes). Juice from 24 hybrids was obtained by hand squeezing only juice vesicles from five fruits of each hybrid and used for metabolomic analysis of nonpolar and polar compounds. Performed on a Synapt G2-Si quadrupole time-of-flight mass spectrometer (Waters) coupled to an I-class UPLC system (Waters). The MS was operated in positive ion mode (50 to 1200 m/z) with a 100 ms scan time. Source and desolvation temperatures were 150° C and 600° C, respectively. A quality control sample, generated by pooling equal aliquots of each sample, was analyzed every 4-6 injections to monitor system stability and performance. Targeted metabolomics of polar metabolites was performed on a TQ-XS triple quadrupole MS (Waters) coupled to an I-class UPLC system. Separations were carried out on a ZIC-pHILIC column (2.1 x 150 mm, 5 µM) (EMD Millipore). Untargeted data processing (peak picking, alignment, deconvolution, integration, normalization, and spectral matching) was performed in Progenesis Qi software. For identification of metabolites, we searched several mass spectral databases (Metlin, Mass Bank of North America, and an in-house database). Targeted data processing was performed with the open-source Skyline software. We detected and quantified 98 polar and 4489 nonpolar metabolites. We have a reliable identification for 261 nonpolar metabolites. As expected, the Australian lime parent had a much greater impact on the metabolomic profile than the mandarin parent (especially true for the nonpolar secondary metabolites and lipids). Several interesting patterns were observed for many of the metabolites analyzed. Relative abundance of the metabolites was measured by peak area and expressed as units. Bergamottin (a natural furanocoumarin found in many citrus fruits, especially in pummelos and grapefruit) was detected at various levels in our analysis. Four hybrids, standard lime, and lemon had 2100-5700 units. Bergamottin was not detectable in 16 of the 24 hybrid fruits tested and also in the mandarin parents. Neohesperidin, a flavanone glycoside common in citrus fruits has an intense sweet taste and is a flavor enhancer. In mandarin parents, this compound is present at high levels (120,000 to 210,000 units). Levels were lower in Microcitrus australasica (1200), limes and lemons (74000-84000), and in hybrids (1,000 to 65,000). Naringin is a flavonone associated with bitter taste characteristic of many citrus types; it inhibits drug-metabolizing enzymes that result in undesirable drug interactions. Varying levels of naringin were found in mandarins (200-2000 units), standard lime, and lemon (1500-1700). In 12 F1 hybrids, the level was 0-100; in six hybrids from 200-700; and in 7 hybrids, 1000-3500 units. Naringin, along with other derivatives -Narigenin-7-rutinoside, Naringenin-7-O-glucoside - affect the flavor profile. For analyzing volatiles, we conducted GCMS using an Agilent 7890A gas chromatograph and analyzed with an Agilent 5975C mass spectrometer. 35 volatile compounds were quantified from 23 hybrid fruits and six standard citrus fruits relevant to the study. Similar to the metabolomics study, only juice from the pulp was utilized. Many interesting patterns were observed. Limonene, a cyclic monoterpene common in citrus oils, is used as a dietary supplement to prevent illnesses and used in cosmetics. It was detected at low levels in mandarins and Persian lime - used as standards. Finger lime and six hybrid fruits had an increased level of limonene. Fifteen hybrids had low but detectable levels, while three hybrids had no detectable limonene. Hybrids RCF037 and 010 had high levels of 12-15 volatile compounds, and the fruits from these had an intense flavor as assessed by tasting. Obj. 4. Development of HLB resistant citrus cultivars through plant transformations. We need to identify candidate resistance-associated genes (Obj. 2) before doing plant transformations. Obj. 5. Outreach and economic analysis. Our team participated in educational and outreach activities geared to citrus growers and industry representatives during 2019-2020. The 9th annual UCR Citrus Day for Industry was held on January 29, 2020 on the University of California, Riverside campus. Co-PI Kahn was an organizer of this event that included many presentations and displays on citrus industry-oriented topics and was attended by growers, industry representatives, and researchers. Ramadugu displayed new hybrids, which were the result of breeding for HLB resistance. Another educational outreach event, the California Citrus Mutual Citrus Showcase in Visalia on March 4, 2020, had a booth (sponsored by the California Citrus Research Board), where we showcased our work. Co-PI Kahn displayed our research to growers and industry members from CA and FL. These outreach activities provided an opportunity to promote dialogue between members of the citrus industry and researchers from our team. Jetter performed an economic analysis to generate baseline information that will be useful to assess significant financial changes that will be apparent when the project is completed.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Ramadugu, C., Keremane, M.L., Lee, R.F., Hall, D.G., McCollum, T.G., and Roose, M.L. 2019. Novel citrus hybrids with HLB resistance. Citrograph 10: 60-64. http://www.citrusresearch.org/wp-content/uploads/CRB-Citrograph-Mag-Q2-Spring-2019-Web.pdf
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Hall, David, G., Ramadugu, Chandrika, Hentz, Mathew, G., Gmitter, Fred. G. and Stover, Ed. 2019. Survey of Poncirus trifoliata hybrids for resistance to colonization by Asian Citrus Psyllid. Florida Entomologist 103(3):635-637. https://doi.org/10.1653/024.102.0339
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Butelli, E., Licciardello, C., Ramadugu, C., Durand-Hulak, M., Celant, A., Recupero, G.R., Froelicher, Y., Martin, C. 2019. Noemi controls production of flavonoid pigments and fruit acidity and illustrates the domestication routes of modern citrus varieties. Current Biology 29: 158-164. https://doi.org/10.1016/j.cub.2018.11.040.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jessica Franco, Chandrika Ramadugu and Gitta Coaker. 2019. Developing immune assays for rapid citrus evaluation. Citrograph 10: 56-59. http://www.citrusresearch.org/wp-content/uploads/CRB-Citrograph-Mag-Q2-Spring-2019-Web.pdf
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kahn, T., Siebert-Wooldridge, T. and Stover, E. 2020. The Mandarin By Any Other Name Would Taste As Sweet: USDA Official Name and Release of US Superna. Citrograph. 11(1):46-47.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Bowman KD, McCollum G, Plotto A and Bai J. 2019. Minnie Finger Lime: A new novelty citrus cultivar. HortScience 54: 1425-1428. https://doi.org/10.21273/HORTSCI13622-18
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Simons, T J, McNeil C J, Pham, A D, Slupsky, C M, Roose, M L., and Guinard, J-X. 2019. Chemical, sensory, and consumer evaluations of DaisySL mandarins grafted onto three different rootstocks. HortSci. 54:1217-1222. doi.org/10/21273/HORTSCI14023-19
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Obenland, D., Arpaia, M.L., 2019. Effect of harvest date on off-flavor development in mandarins following postharvest wax application. Postharvest Biol. Technol. 149, 1-8.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Galimba K, Tosetti R, Loerich K, Michael L, Pabhakar S, Dove C, Dardick C, Callahan A. 2020. Identification of early fruit development reference genes in plum. PLoS One. 2020;15(4):e0230920.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Setamou, M., O. J. Alabi, M. Kunta, J. Dale, and J. da Graca. 2019. Distribution of Candidatus Liberibacter asiaticus in citrus and the Asian citrus psyllid in Texas over a decade. Plant Disease. https://doi.org/10.1094/PDIS-08-19-1779-RE.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Strazzer, P, Spelt, C E, Shuangjiang, L, Bliek, M, Federici, C T, Roose, M L, Koes, R, and Quattrocchio F M. 2019. Hyperacidification of Citrus fruits by a vacuolar proton-pumping P-ATPase complex. Nature Comm. Doi: 10.1038/s41467-019-08516-3.