Source: ARS-USDA submitted to NRP
ACCELERATING IMPLEMENTATION OF HLB TOLERANT HYBRIDS AS NEW COMMERCIAL CULTIVARS FOR FRESH AND PROCESSED CITRUS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1014825
Grant No.
2018-70016-27453
Cumulative Award Amt.
$2,922,114.00
Proposal No.
2017-08555
Multistate No.
(N/A)
Project Start Date
Jan 15, 2018
Project End Date
Jan 14, 2024
Grant Year
2018
Program Code
[CDRE]- Citrus Disease Research and Extension Program
Recipient Organization
ARS-USDA
950 COLLEGE STATION ROAD
ATHENS,GA 30605
Performing Department
US Department of Agriculture
Non Technical Summary
This research proposal aims to determine citrus scion hybrids with tolerance or resistance to citrus greening or huanglongbing (HLB) disease, that have acceptable commercial quality for fresh or processed fruit and fruit juice, respectively, and that can be classified as orange or grapefruit based on their aroma profiles. Citrus is the most economically important fruit tree crop in Florida, contributing substantially to employment, and providing cultural context for many generations of growers. Citrus had an estimated on-tree fruit value of $1.5 billion for 2006-7 prior to HLB disease, and declined to $0.82 billion for 2015-16. The value-added processed orange and grapefruit juice brings the industry value to $8-9 billion prior to this disease. Unfortunately, HLB disease came on the heels of a canker eradication effort and several hurricanes, which all together have reduced citrus production in Florida by around 70%. The disease has also been discovered in other citrus-growing states including Texas and California, and is caused by a phloem-restricted bacterium, Candidatus Liberibacter asiaticus (CLas) that is spread by the insect vector Diaphorina citri (Asian citrus pysllid or ACP). As a result of this yield reduction, packinghouse and processing facilities are closing and at some point this infrastructure will not be enough to support the citrus industry, and will be hard to replace once gone. In addition, the disease affects the flavor quality of the fruit and resulting juice in that it reduces sugars while increasing acids, bitter limonoids and astringent flavonoids and altering the aroma profile. It is hoped that the more resistant/tolerant hybrid fruit will have improved yield and flavor characteristics. Meanwhile, to mitigate symptoms, growers are using foliar nutrition applications, pesticide sprays to control the vector and bactericides to control the pathogen, which have increased grove costs by 2.5 times or more. The project will identify cultivars and hybrids with commercially useful resistance/tolerance to HLB for near term industry use, with good flavor and other attributes, some specifically for use in juice blends that resemble orange or grapefruit juice. This should improve yield, stabilize infrastructure and reduce grove costs. Molecular markers will be identified to increase confidence that selected cultivars truly are tolerant/resistant even though they lack long term testing.
Animal Health Component
50%
Research Effort Categories
Basic
20%
Applied
50%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20309991081100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1081 - Breeding;
Goals / Objectives
The overall objective of this proposed research is to identify orange- and grapefruit-like citrus hybrids that are tolerant or resistant to HLB, as determined by molecular biology, pathology, physiology, flavor chemistry and sensory science. There appears to be no true resistance in any conventional citrus scions. Some resistance or field tolerance to HLB within citrus and citrus relatives has been described over many years prior to finding HLB in the U.S. In Florida, we have identified several released cultivars that appear to have commercially useful levels of HLB resistance and these are predominately mandarin in their pedigrees. In more distant relatives, Poncirus has been the most important source of genetic resistance, with successful mapping of genetic markers and identification of gene candidates conferring the resistance. Trees of P. trifoliata are clearly less susceptible to HLB than cultivated citrus scion varieties. P. trifoliata and its hybrid, 'Carrizo' citrange developed less severe HLB symptoms and among the lowest titer of CLas among the genotypes evaluated in a greenhouse study with a Florida isolate. To determine tolerance or resistance to HLB, molecular markers will be identified to increase confidence that selected cultivars truly are tolerant/resistant even though they lack long term testing. These markers will expedite identification of additional HLB-tolerant/resistant scions for near term use and will contribute to longer term breeding efforts. Meanwhile on the flavor side, juice from tolerant hybrids will be analysed for sugars, acids, limonoids, flavonoids and aroma volatiles for comparison to commercial orange and grapefruit cultivars. Those hybrids with commercial acceptability for fresh fruit and juice quality will be selected for grower trials. Communication with stakeholders to identify obstacles to new cultivar acceptance is planned. It is also essential to disseminate information rapidly as it arises from this proposed research. This will be accomplished efficiently via websites, presentations at industry and scientific meetings as well as through our stakeholder members of the advisory board through biannual meetings.
Project Methods
Five to six years of growth in the field are needed to unequivocally demonstrate HLB resistance or tolerance. Validated markers for resistance/tolerance would increase confidence in near-term use of promising selections, would expedite selection of hybrids for medium-term use, and facilitate future breeding efforts. Marker-assisted breeding would allow assessment of tolerance/resistance at the seedling stage, greatly increasing throughput of hybrids. Growth, health, and pathogen titer will be the primary tolerance metrics in these field trials. Trees will be genotyped using high density SNP markers and genotyping-by-sequencing. RNA-seq will uncover expression of tolerance-associated loci. Comparisons will be made between HLB susceptible/tolerant materials with and without Poncirus in the pedigree, to identify markers to facilitate conventional breeding. Initially, replicated field plantings will be phenotyped for growth, health, cropping, HLB symptoms, and CLas titer, and this has been underway for several years. Growth will be assessed by measuring canopy volume. Health and HLB symptoms will be assessed on a five point subjective scale. Quantitative PCR will used to determine CLas titer levels from symptomatic and randomly sampled leaves. Many trees are entering fruit bearing age, so crop yields will be evaluated to determine the economic potential of HLB-resistance in the new selections compared to standard cultivars. Genotyping-by-sequencing will be used with our selections, along with high density SNP markers (50k and 700k) and possibly a pNOME approach (sequencing pooled samples of tolerant and susceptible genotypes). DNA for genotyping will be extracted from 3-6 fresh young leaves using the Qiagen DNeasy Plant Kit. DNA quality and quantity will be determined using Qubit. Identification of key gene expression differences associated with tolerance/resistance will provide guidance on specific genes associated with identified markers, and may provide more rapid and inexpensive screening of large populations. RNA-seq will be used to uncover these tolerance-associated loci that modify expression of key genes or alter the function of critical proteins, using several closely related hybrids with very different levels of HLB-susceptibility, which are included in the genotyping effort. For those hybrids that are fruiting, chemical analyses will be conducted in triplicate on frozen fruit juice including determination of soluble solids using a refractometer, and titratable acidity using a titrator. Individual sugars and organic acids will be analyzed with a high performance liquid chromatography (HPLC). Concentrations of limonids and flavonoids will be done by HPLC-mass spectrometry (MS). CLas titer will be done by PCR. Sensory analysis will involve 10-15 trained panelists who will evaluate juice from citrus hybrids and compare with orange, grapefruit or tangerine cultivars as in the "polarized sensory positioning" method. Juice will also be rated using a set of descriptors selected through panel discussion. Panelists have been trained for over 60 hours, and practiced citrus fruit/juice evaluation for over 5 years. All tasting will take place in isolated booths under red lighting. Data will be recorded using Compusense® Cloud (Compusense, Guelf, ON, Canada).

Progress 01/15/18 to 01/14/24

Outputs
Target Audience:The primary audience for our recent research is scientists in the field of citrus breeding. Our findings offer valuable insights for the development of new citrus varieties that are tolerant to HLB. Additionally, growers and the general public will benefit from the introduction and adoption of these new cultivars. Our investigation into creating HLB-tolerant citrus hybrids with orange flavor led to the discovery of a crucial gene, CsAAT1, which plays a key role in ester formation in oranges. We also developed a SNP-based DNA marker that can effectively predict the phenotypes related to orange flavor. This work significantly advances our understanding of the molecular mechanisms behind orange flavor and opens new avenues for breeding orange-like varieties. Our research has been published in "Science Advances," a leading academic journal, and has gained attention from various media outlets including USDA-ARS 2024 Research News (https://www.ars.usda.gov/news-events/news/research-news/2024/new-discovery-speeds-scientists-push-for-hlb-tolerant-citrus/), FOX Business (https://www.foxbusiness.com/economy/florida-orange-growers-hybrid-fruits-citrus-greening), NewScientist (https://www.newscientist.com/article/2419584-we-now-know-what-makes-oranges-taste-of-oranges/), and a newspaper in Spain (https://www.elperiodico.com/es/sociedad/20240324/proteger-naranjas-dragon-amarillo-99716891), highlighting the potential impact of our discovery in advancing the development of HLB-tolerant citrus varieties. With this new DNA marker, breeders now have the means to efficiently screen citrus hybrids for the desired orange flavor traits at an early stage, substantially reducing the waiting period for fruit production. Changes/Problems:1. Extension of the project: COVID-19 delayed the research project and led to applications for two no-cost extensions to January 14, 2024, from the original end-time of January 14, 2022. 2.Changes in PD and co-PDs a.USDA/ARS: Dr. Jinhe Bai replaced Dr. Elezabeth Baldwin (Retirement). b. University of Florida: Dr. Mark Ritenour replaced Dr. Rhuanito Ferrarezi (Relocation to UGA) c.USDA/ARS: Dr. Matthew Mattiareplaced Dr. Ed Stover (Retirement) d.New Mexico Consortium: The sub-award was completed/terminated in 2021, and Dr. Goutam Gupta left the research team since then. What opportunities for training and professional development has the project provided?Temporary Workers: Across the duration of the project, there were 12 different post-doctoral research associates, three senior biological scientists, six graduate students, five visiting research scholars, and three undergraduate interns who received hands-on training in various aspects of the research conducted in our labs, such as genomics and genetic analysis, citrus juice processing, chemical analysis and sensory panel. All individuals presented results in poster or oral presentations at national or international meetings, and/or published in peer-reviewed or non-peer-reviewed journals. National and International Conference: The professional training and development included the 2019, 2022 and 2023 Florida Citrus Show; 2019-2023 American Society for Horticultural Sciences annual meeting; Hawaii Tropical Fruit Growers Conference 2022; International Citrus and Beverage Conference 2019, 2022and 2023; International Research Conference on HLB 2019; Florida State Horticultural Society annual meeting, 2018-2023; American Chemical Society Spring meeting 2023. How have the results been disseminated to communities of interest? The websites below disseminate current and recently published data for Florida citrus growers and researchers. On the citrus rootstock selection webpage, there is an internal artificial intelligence software designed to help growers make the best choice of citrus rootstock depending on their desired horticultural traits, tolerances, and disease/pest incidences for citrus rootstock and scion compatibility. https://crec.ifas.ufl.edu/extension/citrus_rootstock/index.html Grower, processor, and consumer surveys were developed using Qualtrics and are accessible for the first time digitally via a QR code link at the 2023 Florida Citrus Show. The results of this project have been disseminated through a series of invited talks, research presentations, and white paper publications. In addition, three field days were held to display varieties with HLB tolerance and good fruit quality that were identified as a part of this project. Informally results have also been discussed with area extension agents and interested growers. The presentations are included in the publication list. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Develop markers to quickly identify hybrids with mandarin-derived and Poncirus-derived HLB tolerance. Under the first objective, collaboration between USDA-ARS Horticultural Research Laboratory (USHRL) Stover/Mattia team, University of Florida (UF) Gmitter team, and University of California, Riverside (UCR) Roose team focused on the development of markers to quickly identify hybrids with tolerance to Huanglongbing (HLB), derived from both mandarin and Poncirus species. The research targeted HLB tolerance in hybrid populations such as mandarin x mandarin, Citrus x Pancirus, and Citrus clementina x C. latipes. Mandarin Tolerance: The study included a significant evaluation of a population consisting of 678 seedlings from 78 hybrids of Fortune x Fairchild mandarin and 342 trees from 43 other mandarin types, mostly Ponkan. These were tested under field conditions in Ft. Pierce, assessing aspects such as growth, health, and HLB symptoms. This dataset was analyzed in conjunction with a high-density linkage map featuring 3475 unique markers, and SNP marker analysis was conducted to confirm the parentage of each seedling and to determine whether they were nucellar or zygotic. QTL mapping of HLB tolerance in the Fortune x Fairchild population identified potential QTLs linked to traits like canopy volume and tree health, though the effects of individual QTLs were generally weak. The presence of QTLs similar to those found in trifoliate orange hybrids was a novel discovery, suggesting a broader base of genetic tolerance than previously thought. The findings underscore substantial variability in HLB tolerance among mandarins, driven by multiple genes. Poncirus Tolerance: For Poncirus-derived tolerance, targeted populations for mapping were established, consisting of seedlings from Poncirus pedigree crossed with Poncirus pedigree material from both USDA and UC-Riverside. The trees underwent rigorous testing for traits such as ACP colonization, CLas titer, and overall growth and vigor. The initial linkage map for trifoliate orange was constructed using SNP data from five-year-old trees, which was later enhanced by a high-quality chromosome-scale genome assembly. This resulted in a high-density SNP-based genetic map with 1402 markers, identifying 26 quantitative trait loci (QTLs) across four linkage groups. The study highlighted several defense or immunity proteins among the 85 differentially expressed genes located within the QTL clusters, which showed significant transcriptional changes following CLas infection. C. latipes Tolerance: QTL mapping for HLB tolerance was also conducted using a field trial of an F1 segregating population of C. clementina x Khasi papeda (C. latipes). The trees in this trial were grown in a field for over six years, with many still testing PCR negative for CLas. Comprehensive data collection included phenotypic traits related to HLB diagnosis and growth evaluations. A high-density linkage map was constructed using 11,765 co-segregating high-quality SNP markers from 493 F1 hybrids. The mapping analysis identified nine QTLs across three different linkage groups, with one major QTL highlighted by integrating multiple overlapping QTLs and bulked segregant analysis (BSA). This study also involved high-throughput transcriptomic and metabolomic analyses to identify key genes and metabolites linked to HLB tolerance. True Sweet Orange Tolerance: The project also uncovered true sweet orange varieties exhibiting tolerance to HLB, including 'Donaldson' and 'Tobias', which were noted for their increased tolerance and are being propagated for extensive field trials under the Citrus Research and Field Trial (CRAFT) program. The findings from these studies are expected to help in the development and selection of citrus varieties with enhanced HLB tolerance. Objective 2. Utilization of HLB-tolerant citrus selection/cultivars in resynthesizing "orange-like" and "grapefruit-like" fresh fruits and juice products. This objective focused on using HLB-tolerant citrus selections to recreate "orange-like" and "grapefruit-like" fruits and juices. The USHRL Baldwin/Plotto/Manthey/Bai/Mattia team evaluated 58 accessions for qualities like acidity, Brix, sugars, acids, limonin, and sensory characteristics since the 2016-2017 season. They identified 26 key compounds for orange flavor, notably seven crucial esters. The discovery of the CsAAT1 gene, essential for ester production, led to the development of highly sensitive DNA markers. The team also tested blending various citrus varieties to replace more susceptible ones in commercial juice production. 'SunDragon' stood out, maintaining high juice quality and matching the sensory attributes of traditional varieties like 'Hamlin' and 'Valencia'. This variety's excellent flavor and HLB tolerance make it ideal for juice blends, potentially mitigating the impact of HLB. Blending experiments, such as Valencia:Hamlin (60:40) and Valencia:SunDragon (60:40), showed minimal flavor differences, even with untrained panelists noting little variance. Additional tests with mandarin and complex Poncirus hybrids confirmed consumer acceptance, with some blends favorably compared to pure sweet orange juice. Further sensory studies identified consumer-preferred attributes across various citrus types. USDA-5-51-2 emerged as a top performer with favorable HLB tolerance and preferred flavor. Pilot processing of early season varieties like 'Donaldson', 'Tresca', and 'SunDragon', and the late season 'Tobias', against standards like 'Hamlin' and 'Valencia', offered crucial data on the market feasibility of these hybrids. Extensive genomic analysis also provided insights into the genetic basis of traits, with analyses revealing how mandarin and pummelo ancestry influences juice quality and disease tolerance. UF Wang/Gmitter team analyzed fruit of 20 cultivars and selections which were identified as HLB-tolerant in the field, including mandarin, mandarin hybrids, and sweet orange. Sensory studies were conducted to understand the key sensory and consumer-liked attributes representing these fruit types. In addition, aroma and tastant compounds were determined using GC-MS and LC-MS. Chemicals representing key sensory attributes of mandarin, mandarin hybrids and sweet orange were identified. Objective 3.Education and outreach of the project theme, research progress and capabilities.: UF Ferrarezi/Ritenour team established and maintained two websites: 1). The Citrus Scion Selection Guide ( https://programs.ifas.ufl.edu/scion-guide/) has been periodically updated with current and relevant publications and presentations. 2) The Florida Rootstock Selection Guide 4th Edition (https://crec.ifas.ufl.edu/extension/citrus rootstock/index.html) has been periodically updated with relevant Florida citrus rootstock published data and has received 4-5 thousand views yearly from more than 20 countries globally. The top five countries that most visited the website are the USA, Brazil, Spain, Mexico, and India. Most users use the Windows operating system and Google Chrome to access and navigate the website, followed by Apple's Macintosh operating system (OS) and their Safari web browsers. We began developing the Florida Rootstock Selection Guide for deployment as an application on mobile devices.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Zhuo, X., Yu, Q., Russo, R., Zhang, Y., Wei, X., Wang, Y.Z., Holden, P.M., and Gmitter, F.G. Role of long non-coding RNA in regulatory network response to Candidatus Liberibacter asiaticus in citrus. Frontiers in Plant Science, 2023, 14. https://doi.org/10.3389/fpls.2023.1090711
  • Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Jeffries, K., Fan, Z., Sun, X., Olmedo, G., Zhao, W., Mattia, M., Stover, E., Manthey, J., Baldwin, E., Lee, S., Gmitter, F.G., Bai, J. and Plotto, A. Flavor and sensory evaluation of Huanglongbing-tolerant Citrus hybrids with/ Poncirus trifoliata in their pedigree. Frontiers in Plant Science.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Mattia, M.R. Florida Citrus Research Foundation Update. Florida Citrus Research Foundation Annual Meeting, Groveland, FL, January 12, 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Mattia M.R. New this Season: Citrus Scion Selections from the USDA, ARS Citrus Scion Breeding Program. The Florida Citrus Show, Ft. Pierce, FL, April 12,2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Mattia, M.R. Florida Citrus Research Foundation Update. Florida Citrus Research Foundation Annual Meeting, Groveland, FL, May 15, 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Mattia, M.R., Bai, J., Fan, Z., Jefferies, K., Olmedo, G., and Plotto, A. Donaldson: Characterization of a Rediscovered Variety for Use in Orange Juice. Florida Society of Horticultural Sciences, Daytona Beach, FL, June 8, 2023
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Fan, Z., Jeffries, K., Sun, X., Olmedo, G., Zhao, W., Mattia, M., Stover, E., Manthey, J., Baldwin, E., Lee, S., Gmitter, F.G., Plotto, A., and Bai, J. Chemical and genetic basis of orange flavor. Science Advances, 2024, 10, eadk2051. https://doi.org/10.1126/sciadv.adk2051
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Yu, Q., Dai, F., Russo, R., Guha, A., Pierre, M., Zhuo, X., Wang, Y.Z., Vincent, C., and Gmitter Jr., F.G. Phenotypic and genetic variation in morphophysiological traits in Huanglongbing-affected mandarin hybrid populations. Plants, 2023, 12(1), 42; https://doi.org/10.3390/plants12010042.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Mattia M.R. CUPS Varieties for Citrus Production. Florida Citrus Expo Tampa, FL, August 16, 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Mattia M.R. Citrus Breeding Grand Plan. Citrus Research and Development Foundation Meeting Lake Alfred, FL, December 2023
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Plotto A., Bai J, Mattia M., Manthey J., Zhao W., Stover, E., Gmitter, F., Baldwin, E., Jeffries, K., Fan, Z., Olmedo, G., Sun, X. Broadening the sweet orange definition to offset HLB. Florida Citrus Show, Fort Pierce, FL, April 2024.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Fan, Z., Jeffries, K.A., Olmedo, G., Zhao, W., Manthey, J., Stover, E., Mattia, M., Sun, X., Gmitter, F.G., Lee, S., Baldwin, E.A., Bai, J., Plotto, A. Sensory, chemical and genetic analyses unravel the basis of orange flavor. Flavor Summit, Orlando April 4-6, 2024.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Olmedo, G., Jeffries, K., Sun, X., Zhao, W., Fan, Z., Mattia, M., Stover, E., Baldwin, E., Manthey, J., Plotto, A., Bai J. Correlations between Pathogen DNA Titer and Sensory/Chemical Evaluations in Juice from Citrus Fruit Affected by HLB. ASHS, Orlando, FL, August 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Baldwin, E., Bai J, Manthey J., Plotto A., Stover, E., Mattia M., Jeffries, K., Fan, Z., Olmedo, G. Potential Citrus hybrids tolerant to HLB for orange juice. Florida Citrus Show, Fort Pierce, FL, April 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Jeffries, K.A., Fan, Z., Sun, X., Baldwin, E.A., Manthey, J., Zhao, W., Stover, E., Mattia, M., Bai, J., Plotto, A. A 5-year study of flavor and sensory evaluation of HLB-tolerant citrus hybrids. American Chemical Society Spring meeting, March 2023, virtual.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Fan, Z., Jeffries, K.A., Sun, X., Baldwin, E.A., Manthey, J., Zhao, W., Olmedo, G., Stover, E., Mattia, M., Plotto, A., Bai, J. Volatile and RNAseq analyses revealed ester biosynthesis pathway in citrus and Poncirus trifoliata. American Chemical Society Spring meeting, March 2023, virtual.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Baldwin, E., Bai J, Manthey J., Plotto A., Stover, E., Mattia M. Accelerating implementation of HLB tolerant hybrids as new commercial cultivars for fresh and processed citrus. Presented to the Florida Citrus Research Foundation, Bartow, FL, January 2023.


Progress 01/15/22 to 01/14/23

Outputs
Target Audience:Targeted audiences include citrus growers, packers and shippers; orange and grapefruit juice makers; food and agricultural students (undergraduate and gratulate) and postdoctorals, scientists and educator in research instituts and universities. Changes/Problems:Due to the COVID-19 facility closing, the research progress has been substantially delayed. We asked for another no-cost extension. Now the project ends on 1/14/2024. What opportunities for training and professional development has the project provided?The professional training and development included the 2022 Florida Citrus Show, 2022 American Society for Horticultural Sciences annual meeting, Hawaii Tropical Fruit Growers Conference 2022, International Citrus and Beverage Conference 2022. An Assistant Project Scientist received additional training in genomics and genetic analysis. A postdoc received a training in the International Fresh Produce Association/ Quality & Safety of Fresh-cut Vegetables and Fruits Multistate Research Project meeting.? How have the results been disseminated to communities of interest?Qualtrics grower, processor, and consumer surveys were developed and will be accessible for the first time digitally via a QR code link at the 2023 Florida Citrus Show. Results will be published and disseminated at the 2023 ASHS Orlando, FL and American Chemical Society 2023 annual Spring meeting. Two websites, The Citrus Scion Selection Guide (https://programs.ifas.ufl.edu/scion-guide/) and The Florida Rootstock Selection Guide 4th Edition (https://crec.ifas.ufl.edu/extension/ citrus_rootstock/index.html), continue to disseminate current and relevant data. Efforts into developing a standalone mobile application were initiated in the Fall of 2022. This mobile application will integrate the 'expert system' into a mobile friendly app for growers and users. In addition to this update, the citrus rootstock table will be revised and updated. The mobile application is expected to be released at the end of Q2 2023. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Develop markers to quickly identify hybrids with HLB tolerance. [USHRL] 1. Further developing the most promising HLB markers and applying them to segregating populations for validation. 2. There will be a major push on identifying, validating, and using HLB tolerance markers in the next years. Before assessing physiological/molecular metrics of tolerance, we wanted greater confidence in HLB-sensitivity and have conserved a large proportion of funds for the last two years of this four year grant plus a no-cost extension. 3. A targeted RNA-Seq component for maker identification has been done for seven year-old HLB infected plants to be screened for HLB health metrics and CLas titer. RNA was extracted and will be sent for sequencing and analysis. 4. Targeted differentially expressed genes from tolerant and susceptible individuals to generate markers that may be used to determine genes are differentially expressed and assess their efficacy as functional markers for HLB immune and infection response. 5. Field and greenhouse data will be compared to determine overlap between short and longer term HLB tolerance and potentially validate DEGs from the greenhouse study. 6. Poncirus pedigree x Poncirus pedigree material from USDA and UC-Riverside crosses will be replicated by grafting on unifoliate rootstocks to prepare for a replicated field trial in an HLB endemic environment. 7. Unifoliate rootstocks have been planted and are finally to the stage where they can be grafted to replicate this population. Grafting of these populations in triplicate will happen in the next few months. [UF] 1. Establish a replicated field trial of F2 Poncirus families described above, to enable further QTL mapping of HLB tolerant traits. This will be done in collaboration with Drs. Roose and Mattia (new post-doc taking over Dr. Stover's activities upon his retirement). 2. Using the current F2 backcross data to improve the resolution of published F1 linkage map. 3. Working on QTL mapping of phenotype traits measured in 2021 using F1 Poncirus hybrid populations. 4. Preparing manuscript of QTL mapping for HLB tolerance using the Citrus clementina × Khasi papeda (Citrus latipes) family. 5. Preparing manuscript of GWAS mapping multiple loci of physiological and morphological traits in mandarins associated with HLB tolerance. [UCR] 1. Mandarin Tolerance - A more sophisticated analysis that better accounts for the variation in genotype, phenotype and number of seedling trees of each hybrid is still needed. We also plan to conduct a complementary GWAS analysis. 2. Genome composition analysis: Essentially all accessions in the UCR Citrus Variety Collection and 814 accessions from USDA and UF breeding programs were analyzed with a 56K SNP array (mostly in previous years). Accessions with mandarin and pummelo ancestry (similar to sweet orange) have now been analyzed using D-statistics to determine "local ancestry" of each 0.5 Mb chromosome segment, that is, did the segment derive from mandarin, pummelo, or other species. This analysis enables testing the hypothesis that many characteristics of sweet orange and grapefruit are due to the particular chromosome segments derived from their ancestors. Phenotype analysis of these accessions is being collected in other projects and this data will be combined with this genome analysis to identify regions associated with sweet orange traits. 3. Haplotype determination: The physical organization of chromosome segments in sweet orange and many other citrus genotypes is also being determined by whole-chromosome haplotype analysis of whole-genome amplified DNA from about 5 single pollen grains each. Haplotypes of about 60 accessions have now been genotyped and most analyzed. We have also analyzed the species of origin of each 1 Mb chromosome segment in several genotypes and plan to extend this to all haplotypes. We can identify regions matching sweet orange haplotypes in different accessions and in some cases this reveals new information about the cultivar ancestry. For example, King mandarin, an important parent in the UCR breeding program, has often been considered as a possible tangor. Analysis of its haplotypes shows that, although many chromosomes contain some regions identical to sweet orange haplotypes, its ancestry is more complex. It does not contain whole chromosomes derived from sweet orange. Objective 2: Evaluate sensory and chemical quality of HLB-tolerant citrus selection. [USHRL] In 2022-23, the mandarin FF-5-51-2 and the hybrid SunDragon, as well as the standard 'Valencia' and 'Hamlin' fruit will be (were) harvested and processed in a pilot plant facility to represent commercial juice extraction followed with pasteurization. Blends will be performed as in 2021-22 and presented to the lab-wide untrained panelists. PCR analysis for the quantification of CLas DNA in juice harvested since the beginning of the study will be completed. Data will be compared with instrumental and sensory values to evaluate whether CLas can be used as a marker of HLB infection and juice quality. Furthermore, selected fruit were harvested from P. trifoliata, Citrus x P. trifoliata hybrids, grapefruit and other hybrids to determine the chemical composition of fruit with bitter taste. Full metabolomics and nutritional analysis by LC-MS/MS will be conducted, and the data will be used to analyze the mechanism of volatile formation and CLas-infection. [UF] The sensory dataset will be analyzed in conjunction with the instrumental quality attributes, as well as genetic information, such as, SNP marker, and GWAS mapping to confirm the hybrids with a potential to be high quality juice cultivars. Objective 3: Education and outreach. [UF] Design the mobile application for Android and iOS devices and gather data from numerous researchers to update the table. The 5th edition will add and remove rootstocks that have not yet undergone the typical field evaluation and/or not commercially available for growers. Specifically, the UFR series rootstocks and USDA rootstocks.

Impacts
What was accomplished under these goals? Objective 1. Develop markers to quickly identify hybrids with mandarin-derived and Poncirus-derived HLB tolerance. USDA-ARS Horticultural Research Laboratory (USHRL) Mattia/Shatters team, University of Florida (UF) Gmitter team and University of California, Riverside (UCR) Roose team are collaborating under this objective. The teams focused on HLB-tolerance of mandarin x mandarin, Citrus x Poncirus, and Citrus clementina x C. latipes hybrid populations. Most of this work is setting the stage for identification of tolerance markers. Mandarin Tolerance: A population seedlings derived from Fortune x Fairchild mandarin hybrids and other mandarins (mostly Ponkan types) tested under field conditions at Ft. Pierce was evaluated for growth, health and HLB symptoms. This dataset is being analyzed in conjunction with a high-density linkage map (3475 unique markers) and SNP marker analysis of each seedling to verify its parentage and determine whether it is a nucellar or zygotic seedling. Preliminary QTL mapping of HLB tolerance in this population was completed. Possible QTLs for canopy volume and tree health were identified, but QTL effects were weak. Poncirus Tolerance: Target populations for the Poncirus-derived tolerance map were identified. We verified hybrids in a group of seedlings of Poncirus pedigree x Poncirus pedigree material from USDA and UC-Riverside crosses and collected material for sequencing. The decision was made that data would be stronger for replicated grafted trees and plants have been potted to a larger size and rootstock liners are growing. C. latipes Tolerance: QTL mapping for HLB tolerance using a field trial of an F1 segregating population of C. clementina × Khasi papeda (C. latipes) has been conducted. RNA-seq and bulked segregant analysis (BSA) from HLB positive and negative trees were performed. At 4 years since planting, a large proportion of trees are healthy and vigorous and a significant proportion continue to be free of CLas. C. latipes is the only species in narrow-sense Citrus reported to be resistant to CLas. Objective 2: Utilization of HLB-tolerant citrus selection/cultivars in resynthesizing "orange-like" and "grapefruit-like" fresh fruits and juice products. USHRL Plotto/Manthey/Bai and Mattia/Stover team evaluated 58 genotypes, including orange-like hybrids, other promising hybrids, and commercial cultivars since 2016-2017 season with several harvests and multiple years for some genotypes. The evaluation included titratable acidity, soluble solids (Brix), individual sugars, individual acids, limonin and nomilin, volatiles, and sensory with trained panelists. Data analysis over the 6-year span (including 209 genotypes/harvest combinations) identified esters were necessary to classify flavor as "orange flavor". Further study from these results included differential gene expression analysis between ester producers and non-producers, and identified a novel alcohol acyltransferase (CsAAT1) responsible for ester production in orange. Sensory analysis combined with soluble solids (SSC- Brix), titratable acidity (TA), limonin and nomilin showed classification of hybrids based on their genotype (mandarins, with high sweetness and SSC, and mandarin flavor), oranges with orange flavor and greater in sourness than mandarins (including the two references, 'Valencia' and 'Hamlin'), and hybrids with Poncirus trifoliata in their background, with high TA and sourness, high limonin+nomilin and bitterness, and off flavor. Over the time span of this study, the Citrus x P. trifoliata hybrid US SunDragon maintained high juice quality with high orange flavor. SunDragon is tolerant to HLB and its maturity spans November to January. SunDragon was tested in blends with Valencia juice to evaluate whether it could replace Hamlin, highly susceptible to HLB. Blends were also made including mandarin hybrids (FF-1-51-2, Superna and SugarBelle), and one Citrus x P. trifoliata hybrid, FF-1-89-11. UF Wang team collected potential HLB-tolerant hybrids, analyzed the instrumental and sensory attributes. The data analysis is on-going. Objective 3: Education and outreach of the project theme, research progress and capabilitie. UF Ritenour team maintained two websites: 1). The Citrus Scion Selection Guide ( https://programs.ifas.ufl.edu/scion-guide/) is periodically updated with current and relevant publications and presentations, three of them from the year 2022; and 2. The Florida Rootstock Selection Guide 4th Edition (https://crec.ifas.ufl.edu/extension/ citrus_rootstock/index.html) is periodically updated with relevant Florida citrus rootstock published data and has received 5 thousand views in over a 12-month period (01.01.2022 - 12.31.2022) from approximately 25 countries globally. The top five countries that most visited the website are the USA, Brazil, Spain, Mexico, and India. Most users use Windows operating system and Google chrome to access and navigate the website, followed by Apple's Macintosh operating system (OS) and their Safari web browsers.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Bai, J., Jord�n, M.J., and Li, J. Metabolism of fruit volatile organic compounds. Frontiers in Plant Science. 13:873515. 2022. https://doi.org/10.3389/fpls.2022.873515
  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Cruz, M.A., Plotto, A., Ferrarezi, R.S, Leite Junior, R.R., and Bai, J.* Effect of Huanglongbing on volatile organic compound profile of fruit juice and peel oil in Ray Ruby grapefruit. Foods. 12:713. 2023. https://doi.org/10.3390/foods12040713
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Plotto, A., Bai, J., Manthey, J., Stover, E., Mattia, M., Driggers, R., Sun, X., Baldwin, E. Citrus hybrids tolerant to Huanglongbing as orange juice drinks. Poster presented at the University of Florida Flavor Summit, Orlando (FL), 23 February, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Plotto, A. and Wang, Y. Mandarin or Orange? They both taste so good! Talk presented at the International Citrus Beverage Conference, Clearwater (FL), 23 September, 2022. PowerPoint Presentation (ufl.edu)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Roose, ML. 2022. Comparative genomics and evolution of Citrus. Keynote at International Citrus Congress 2022, November 7, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mattia, M.R. Accelerated Citrus Breeding: The Quest for Improved Varieties. Florida Citrus Show, Ft. Pierce, FL, January 25, 2022. Invited talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mattia, M.R. USDA Citrus Scion Breeding. Florida Citrus Commission, Lake Wales, FL, March 10, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Mattia, M.R. Florida Citrus Research Foundation Update. Florida Citrus Research Foundation Annual Meeting, Groveland, FL, January 12, 2023.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Parajuli S, Huo H, Gmitter FG, Duan Y, Luo F, Deng Z. Editing the CsDMR6 gene in citrus results in resistance to the bacterial disease citrus canker. Horticulture Research. 2022 Jan 5;9.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Mattia MR, Du D, Yu Q, Kahn T, Roose M, Hiraoka Y, Wang Y, Munoz P, Gmitter FG. Genome-wide association study of healthful flavonoids among diverse Mandarin accessions. Plants. 2022 Jan;11(3):317.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ribeiro C, Xu J, Hendrich C, Pandey SS, Yu Q, Gmitter F, Wang N. Seasonal transcriptome profiling of susceptible and tolerant citrus cultivars to citrus Huanglongbing. Phytopathology. 2022 Aug 24(ja).


Progress 01/15/21 to 01/14/22

Outputs
Target Audience:Citrus growers, packers and processors. In the Florida Citrus Show held at Ft. Pierce on May 12 and 13, 2021, Gmitter and Ritenour (both are co-PD) presented the research results obtained from this project. Due to COVID-19 pandemic, many meetings were held virtually as follows: Stover: Citrus Research and Development Foundation, July 21, 2021. American Society for Horticultural Sciences, August 6, 2021. · International Conference on Beverages and Citrus, September 23, 2021. · Florida Citrus Commission, Bartow, FL, August 24, 2021. Roose:· Plant and Animal Genome meeting, January 2022. Plotto:· American Society of Horticultural Sciences Bai:· Brown Technical Service Conference, September 26, 2022 Changes/Problems:1. COVID-19 caused delay and a potential application for a no-cost extension to 1/14/2024. The work conducted in the past year has been limited because of COVID-19 pandemic restrictions. 2. Changes of co-PDs a. University of Florida: Dr. Mark Ritenour replaced Dr. Rhuanito 'Johnny' Ferrarezi b. USDA/ARS: Dr. Robert Shatters replaced Dr. Ed Stover. c. New Mexico Consortium: Sub-award completed in 2021, and Dr. Goutam Gupta will no longer work on the project. What opportunities for training and professional development has the project provided?The professional training and development included the 2021 Florida Citrus Show, 2021 Florida Citrus Expo, 2021 University of Florida AG Safety Workers, and the 2021 ASHS Denver, Colorado. Technicians (1.2), postdocs (1.75) and non-student or faculty scientist/professional (2.85) were a part of one of our teams working on the project. How have the results been disseminated to communities of interest?The websites below disseminate current and recently published data for Florida citrus growers and researchers. On the citrus rootstock selection webpage, there is an internal artificial intelligence software designed to help growers make the best choice of citrus rootstock depending on their desired horticultural traits, tolerances, and disease/pest incidences for citrus rootstock and scion compatibility. In addition, results have been disseminated at 2021 Florida Citrus Grower Show Symposium, 2021 American Society for Horticultural Sciences (2021 Denver, Colorado), and at the 2021 Florida Citrus Expo (Fort Myers, FL) https://crec.ifas.ufl.edu/extension/citrus_rootstock/index.html https://programs.ifas.ufl.edu/scion-guide/ What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Develop markers to quickly identify hybrids with HLB tolerance. [USHRL] 1. Further developing the most promising HLB markers and applying them to segregating populations for validation. 2. There will be a major push on identifying, validating, and using HLB tolerance markers in the next two years. Before assessing physiological/molecular metrics of tolerance, we wanted greater confidence in HLB-sensitivity and have conserved a large proportion of funds for the last two years of this four-year grant plus a no-cost extension. 3. A targeted RNA-Seq component for maker identification will be done for seven-year-old HLB infected plants been screened for HLB health metrics and CLas titer. Targeted differentially expressed genes will be analyzed from tolerant and susceptible individuals to generate markers that may be used to determine. 4. Determine whichtargeted genes are differentially expressed and assess their efficacy as functional markers for HLB immune and infection response. 5. Poncirus pedigree x Poncirus pedigree material from USDA and UC-Riverside crosses will be replicated by grafting on unifoliate rootstocks to prepare for a replicated field trial in an HLB endemic environment. [UF] 1.Establish a replicated field trial of F2 Poncirus families described above, to enable further QTL mapping of HLB tolerant traits. This will be done in collaboration with Drs. Roose and Mattia (new post-doc taking over Dr. Stover's activities upon his retirement). 2. Using the current F2 backcross data to improve the resolution of the published F1 linkage map. 3. Working on QTL mapping of phenotype traits measured in 2021 using F1 Poncirus hybrid populations. 4. Preparing manuscript of QTL mapping for HLB tolerance using the Citrus clementina × Khasi papeda (C.latipes) family. 5. Preparing manuscript of GWAS mapping multiple loci of physiological and morphological traits in mandarins associated with HLB tolerance. [UCR] 1. QTL and GWAS analysis of HLB tolerance in the Fortune x Fairchild population will be completed and submitted for publication. Genetic control of fruit quality in this population will be analyzed using data on the Riverside parent trees to provide an integrated analysis of HLB tolerance and fruit quality traits. 2. Analysis of local ancestry of most accessions in the UCR citrus collection will be completed and published. 3. Haplotypes based on about 56000 SNPs in approximately 50 important citrus cultivars will be finalized and published. Objective 2: Evaluate the sensory and chemical quality of HLB-tolerant citrus selection. 1. We are developing the foundation for genomic-based selection of HLB-tolerant sweet-orange-like hybrids: Our work on sweet-orange-like hybrids has leveraged further resources as citrus was awarded "On-Ramp" status in the USDA/Cornell Breeding Insights (BI) program which focuses on bringing genomic breeding tools to specialty crops. We are working with BI staff and have designed and implemented high-throughput phenotyping with a strong artificial intelligence (AI) component. This procedure is being implemented in a replicated population of 50 accessions now in their seventh year in the field. 2. Continually conduct sensory evaluation and physicochemical attributes study for the promise selections. 3. Conduct citrus juice blending research to effectively use the hybrids with high sugar, low acid, and/or premium flavor note. 4. Transcriptomic and metabolomic research to reveal the flavor quality of Poncirus, mandarin, orange, and hybrids. Objective 3: Education and outreach. 2022 Florida Citrus Grower Show 'Variety Improvement' and 2022 Florida Citrus Expo, UF IFAS Extension Symposium, ASHS poster/abstract submission for 2022, 'Survey to identify key issues in accepting new HLB tolerant hybrids as new commercial cultivars for fresh and processed citrus 2nd Version.' A digital survey is being designed to migrate from the older printed copies of the survey to a digital online version that utilizes a QR code to link the survey to attendees for a more efficient platform for collecting feedback.

Impacts
What was accomplished under these goals? Objective 1. Develop markers to quickly identify hybrids with mandarin-derived and Poncirus-derived HLB tolerance. USDA-ARS Horticultural Research Laboratory (USHRL) Stover/Shatters team, University of Florida (UF) Gmitter team and University of California, Riverside (UCR) Roose team are collaborating under this objective. The teams focused on HLB-tolerance of mandarin x mandarin, Poncirus hybrid populations, and C. latipes hybrids. Most of this work is setting the stage for identification of tolerance markers. Mandarin Tolerance: Phenotyped mandarin hybrid/clonal population. The dataset is being analyzed in conjunction with a high density linkage map (about 14,000 markers) and SNP marker analysis of each seedling to verify its parentage and determine whether it is a new hybrid. Research showed that leaf area index is highly associated with HLB tolerance. Genome-wide association study was carried out to identify possible SNPs associated with HLB responses. Poncirus Tolerance: Research was focused on the "F2 backcross" population of Poncirus orange (TO; Poncirus trifoliata) consisted of two families: 1) 53 plants (Tahitian pummelo x Flying Dragon) x Towne TO; 2) 91 plants from Gainesville 71 TO x Carrizo or Troyer citrange. Collected the SNP dataset. The work halted at this point because there is no phenotypic data yet on these plants to justify any further mapping efforts. C. latipes Tolerance: QTL mapping for HLB tolerance using a field trial of an F1 segregating population of Citrus clementina × Khasi papeda (Citrus latipes) has been conducted. RNA-seq and bulked segregant analysis (BSA) from HLB positive and negative trees were performed. C. latipes is the only species in narrow-sense Citrus reported to be resistant to CLas. Objective 2: Utilization of HLB-tolerant citrus selection/cultivars in resynthesizing "orange-like" and "grapefruit-like" fresh fruits and juice products. USHRL Plotto, Manthey, Bai and Shatters/Stover team evaluated 50 orange-like hybrids and cultivars since 2018 allowed to eliminate poor performers, formulate harvest dates, and plan strategy for creating juice blends. New hybrids appearing tolerant to HLB continue to be evaluated each year, including hybrids produced in crosses of US SunDragon. Gathered data include soluble solids, titratable acidity, individual and total sugars and acids, volatile compounds, limonin, nomilin, sensory evaluation by a trained panel, and CLas titer in juice. USDA-5-51-2, USDA- 6-9-96 and USDA-6-32-67 are low acid hybrids with high SSC/TA ratio, potential candidates to improve flavor of Hamlin juice affected with HLB. Fruit for a large volume of US SunDragon juice was provided for blending experiments. UF Wang/Gmitter team conducted sensory and chemical analysis of 20 cultivars and selections which were identified as HLB-tolerant in the field. Chemicals representing key sensory attributes of mandarin, mandarin hybrids and sweet orange were identified. Objective 3: Education and outreach of the project theme, research progress and capabilitie. UF Ritenour team maintained the project website https://programs.ifas.ufl.edu/ (HLB TOLERANT HYBRIDS AS NEW CITRUS COMMERCIAL CULTIVARS), and properly updated the research project description, objectives, people and activities, publications, presentations and resulting products.

Publications

  • Type: Other Status: Published Year Published: 2021 Citation: Albrecht U., Alferez F., Zekri M. 2021. Florida Citrus Production Guide: Rootstock and Scion Selection. HS1301. Gainesville: University of Florida Institute of Food and Agriculture Sciences. https://doi.org/10.32473/edis-hs1308-2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Stover, E. 2021. Overview of USDA citrus scion breeding. New Varieties Development and Management Corporation via Zoom, Mar 22, 2021. March 22, 2021. Invited talk
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Stover, E., F.G. Gmitter, Jr., M.L. Roose, J.C. Motamayor, R. Bryant, A. Plotto, J. Bai, M. Sheehan, J. W. Grosser, N. Kretchman, W. Johnson, C. Nyinyi-Hatcher, and P. Ouimet. 2021 Topwork trial-foundation for genome-based breeding. Citrus Research and Development Foundation via Zoom, July 21, 2021. Invited talk
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Stover, E., F.G. Gmitter, Jr., M.L. Roose, J.C. Motamayor, R. Bryant, A. Plotto, J. Bai, M. Sheehan, J. W. Grosser, N. Kretchman, W. Johnson, C. Nyinyi-Hatcher, and P. Ouimet. 2021 Developing a genomic pipeline for citrus breeding. American Society for Horticultural Sciences via Zoom, August 6, 2021. Invited talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Stover, E., F.G. Gmitter, Jr., M.L. Roose, J.C. Motamayor, R. Bryant, A. Plotto, J. Bai, M. Sheehan, J. W. Grosser, N. Kretchman, W. Johnson, C. Nyinyi-Hatcher, and P. Ouimet. 2021 Developing a genomic pipeline for citrus breeding. International Conference on Beverages and Citrus via Zoom, September 23, 2021. Invited talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Stover, E. 2021. Overview of USDA citrus scion breeding. Florida Citrus Commission, Bartow, FL, August 24, 2021.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Deterre, S., T. McCollum, C. Leclaire, J. Manthey, J. Bai, E. Baldwin, S. Raithore, E. Stover, and A. Plotto. 2021. Effect of Poncirus trifoliata on chemical composition of fruits in pedigrees of Citrus scion hybrids. Sci. Hortic. 277: doi.org/10.1016/j.scienta.2020.109816.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Feng, S., Gmitter Jr, F. G., Grosser, J. W. and Wang, Y. Identification of key flavor compounds in citrus fruits: a flavoromics approach. ACS Food Science & Technology, 2021, doi: 10.1021/acsfoodscitech.1c00304.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Ferrante SP, Jia Z, Wu GA, and Roose ML. 2022. Complete parental haplotypes inference through whole genome amplification of single pollen grains. Poster at Plant and Animal Genome meeting (online), January 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Plotto, A., Sun, X., Baldwin, E., Driggers, R., Manthey, J., Stover, E., Bai, J. Orange or mandarin flavor? Determining components of orange quality in a breeding program.Abstract presented at the American Society of Horticultural Sciences (ASHS) annual meeting, August 5-9, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Bai, J., Plotto, A., Baldwin, E., Zhao, W., Manthey, J., Sun, X., Stover, E. Challenges of Orange/Grapefruit Juice Production in HLB era - Breeding and Processing. Brown Technical Service Conference, September 26, 2021


Progress 01/15/20 to 01/14/21

Outputs
Target Audience:Gitrus growers, packers and processors. In the Florida Citrus Show held at Fort Pierce on January 22-23, 2020, Drs. Stover and Ferrarezi (both are co-PD) presented the research results obtained from this project. Because of COVID-19 pandemic, many other meetings and activities were cancelled. Thus we had less audience in this year. Changes/Problems:The work conducted this past year has been limited because of COVID-19 pandemic restrictions. The major impacts for this project includes: 1. Develop markers to quickly identify hybrids with mandarin-derived and Poncirus-derived HLB tolerance 1.1. Genotyping-by-sequencing analysis of parents and hybrids (substantially delayed and will not be completed on time). 1.2. Genotyping using dense SNP markers in segregating F1, F2 and other hybrids (the major part has been accomplished but the task has not been completely finished). 1.3. RNA-seq to correlate with identified markers for disease resistance-related gens (substantially delayed and will not be completed on time). 1.4. Test range of hybrids using DNA and RNA markers (the task will not be able to complete on time). 1.5. Compile information on most promising selections for outreach (experiments has been interrupted). 2. Utilization of HLB-tolerant citrus selection/cultivars in resynthesizing "orange-like" and "grapefruit-like" fruits and juice products. 2.1. Collect fruit quality data on HLB tolerant/resistant varieties (lab work on fruit quality has been substantially delayed, and missed some key harvest dates). 2.2. Analyze volatile data to identify stand-alone processing types and blends like OJ and GFJ (juice blend work has not been started yet and samples for volatile chemical analysis are markedly delayed). 2.3. Taste-test juice and juice blends from identified tolerant/resistant selections (taste panel tasks have been completely stopped in the past 8 months). 2.4. Compile information on most promising selections for outeach (largely delayed). 3. Education and outreach of the project theme, research progress and capabilities 3.1. Share information at filed days, workshops, and conferences (all activities have been shifted to online, thus extensions based on field day and workshop were substantially impacted). Thus, we will request a 12-month, no-cost extension.? What opportunities for training and professional development has the project provided?UF Ferrarezi team provided a field tour with about 50 citrus grower attendees. However, we were not be able to provide more external training because of COVID-19 pandemic restrictions. Technicians (2), postdocs (4) and granulate students (2) were a part of one of our teams working on the project. How have the results been disseminated to communities of interest?The results have been disseminated by website, presentation/exhibition at stakeholder/grower extension meetings, presentation at academic meetings, and publication in peer-reviewed or trading journals although the virtual meetings under the pandemic somehow limited the effect to stakeholders and other attendees. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Develop markers to quickly identify hybrids with HLB tolerance. Mandarin Tolerance: Data were collected on KASP analysis and phenotypes HLB symptoms and will be assembled for analysis. Poncirus Tolerance: to genotype the 16 missed Poncirus hybrids for GBS in 2021; to construct parental linkage maps for the F2 backcross Poncirus population, and the Monreal Clementine x Citrus latipes family; to establish field experiment of F2 Poncirus hybrid plants. Tolerance Markers: To identify QTLs for HLB resistance within the Monreal Clementine x Citrus latipes family; to identify and use tolerance markers in the last two years (will request a one-year no-cost extension) in a larger population. A spin-off project with the Florida Citrus Processors Association, and the team for this grant, has resulted in establishment of a replicated trial of 200 diverse accessions topworked onto 5-year-old CLas-infected Valencis/Swingle trees. The accessions have been 25x sequenced and funds are being sight for extensive phenotyping and bioinformatics analyses to establish a foundation for Genomic Selection in citrus scion breeding Objective 2: Evaluate sensory and chemical quality of HLB-tolerant citrus selection. Based on the results from prior years and the data will be used to eliminate some poor performers, determine optimal harvest dates for new hybrids, and prepare to receive 25 selections for sensory and chemical evaluation in the 2020-21 season. Will conduct juice blending research to actively utilize the special hybrids with strong orange flavor or super sweet taste. Objective 3: Education and outreach. To enhance the website by uploading more presentations, publications, audios and videos to meet the needs from growers, packers and processers. Presentation of data, and grower observation of field trials relating to this project over the last several years, has compelled a major shift in the thinking of the Florida citrus industry. The scientists involved in this project, in coordination with several industry leaders, have created and circulated a white paper, made numerous presentations, and published a manuscript ("Rationale for reconsidering current regulations restricting use of hybrids in orange juice"). This change would result in ability to include hybrids that meet the final criteria in orange juice. This is predicted to greatly accelerate planting of new HLB-tolerant hybrids, allowing large scale grower/processor experimentation, and ultimately resulting in enhanced quality and sustainability of the Florida citrus industry. Associated with this process, the Florida Citrus Processors Association is sponsoring a series of six replicated trials of 60-200 selections that will be extensively phenotyped for HLB-tolerance and parameters relating to juice quality and quantity. All 200 selections were sequenced, and when combined with phenotypic data should provide a foundation for breeding citrus using genomic selection. Budwood was used to topwork the initial planting onto reasonably healthy but CLas-infected Valencia on Swingle, allowing rapid and uniform infection. Processors have evaluated US SunDragon juice and found it very promising for OJ blends. Through our efforts it became apparent that taxonomic names in regulations could impose severe restrictions on use of new hybrids. We have been working with the industry to consider development of "Citrus Cultivar Groups" as a more suitable system of nomenclature.

Impacts
What was accomplished under these goals? Objective 1. Develop markers to quickly identify hybrids with mandarin-derived and Poncirus-derived HLB tolerance. By closely working together of U.S. Department of Agriculture - Horticultural Research Laboratory (USDA-HRL) Stover team, University of Florida (UF) Gmitter team and University of California, Riverside (UCR) Roose team, efforts were made on mandarin tolerance, Poncirus tolerance, and tolerance markers. Mandarin Tolerance: Data were again collected on HLB symptoms and growth of a replicated mandarin x mandarin population for mapping. KASP analysis, to identify clonal vs new hybrid material, was repeated by the Roose team to correct possible errors. Complementary replicated plantings were also phenotyped. Poncirus Tolerance: With Gmitter team, target populations for the Poncirus-derived tolerance mapping were identified. Gmitter team verified hybrids in a group of seedlings of Poncirus pedigree x Poncirus pedigree material from USDA and UC-Riverside crosses and collected material for sequencing. Tolerance Markers: Key replicated plantings have likely reached "equilibrium" with the CLas pathogen this fall, permitting reliable characterization of relative HLB-sensitivity. Clementine x Tangelo cultivars/selections reflecting a range of sensitivity were replicated as clean plants, a subset was CLas ACP-inoculated with a complementary set fed-on by CLas-free ACP The plants were extensively tested over 10 months of disease progression for metrics of HLB-sensitivity, photosynthetic capacity and other physiological markers that may be closely related to CLas sensitivity. Tissue samples have been collected for RNAseq analysis to identify differentially expressed genes associated with HLB tolerance. The most promising of these markers will be applied to segregating populations for validation. Objective 2: Utilization of HLB-tolerant citrus selection/cultivars in resynthesizing "orange-like" and "grapefruit-like" fresh fruits and juice products. USDA-HRL Plotto, Manthey, Stover and Bai teams evaluated commercial and hybrid citrus fruits, and provided multiple harvests of fruit, representing >30 selections and cultivars. Analyses are described in other reports. Bai/Plotto/Stover teams, reviewed results from prior years and data were used to eliminate some poor performers, formulate harvest dates, and plan strategy for supplying fruit of selections for sensory and chemical evaluation in the 2020-21 season. Gathered data include soluble solids, titratable acidity, individual and total sugars and acids, volatile compounds, limonin, nomilin, sensory evaluation by a trained panel, and CLas titer in juice. So far, US SunDragon, SugarBelle and USDA-5-51-2 are the best performing cultivars/selections with good HLB-tolerance. Three cultivars produced in crosses of US SunDragon with potentially superior fruit quality and similar tolerance were newly selected and harvested for evaluations. Fruit for a large volume of US SunDragon juice was provided for blending experiments. UF Wang and Gmitter teams conducted sensory studies using juice of Hamlin and Valencia sweet orange blended with juice from HLB-tolerant LB8-9 Sugar Belle®, to expand on the previous season's work on blending to improve overall OJ quality. Objective 3: Education and outreach of the project theme, research progress and capabilitie. UF Ferrarezi team maintained the project website https://programs.ifas.ufl.edu/ (HLB TOLERANT HYBRIDS AS NEW CITRUS COMMERCIAL CULTIVARS), and properly updated the research project description, objectives, people and activities, publications, presentations and resulting products.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Stover, E., F.G. Gmitter, Jr., J. Grosser, E. Baldwin, G.A. Wu, J. Bai, Y. Wang, P. Chaires, and J.C. Motamayor. 2020. Rationale for reconsidering current regulations restricting use of hybrids in orange juice. Hort Res 7:38. https://doi.org/10.1038/s41438-020-0277-5
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Deterre, S., G. McCollum, C. Leclair, J.A. Manthey, J. Bai, E.A. Baldwin, S. Raithore, E. Stover, and A. Plotto. 2021. Effect of Poncirus trifoliata on the chemical composition of fruits in pedigrees of Citrus scion hybrids. Sci. Hortic. 277:109816. https://doi.org/10.1016/j.scienta.2020.109816.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Hiraoka, Y., Ferrante, S., and Roose, M. L. Citrus Diversity Study Using High-Density SNP Genotyping Array. Plant and Animal Genome Meeting poster. Jan. 2020, San Diego, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Roose, M. L. and Hiraoka, Y. Introgression Patterns at Chromosome Level in Citrus. Presentation to Citrus Germplasm Advisory Committee, April 14, 2020, Riverside, CA
  • Type: Websites Status: Published Year Published: 2020 Citation: https://programs.ifas.ufl.edu
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Baldwin, E., E. Stover, R., Driggers, J., Bai, J., Manthey, X., Sun, C., Ference, and A. Plotto. 2019. Accelerating implementation of Huanglongbing-tolerant hybrids as new commercial cultivars for fresh and processed citrus. Proc. Fla. State Hort. Soc. 132:177-181.
  • Type: Websites Status: Published Year Published: 2020 Citation: https://crec.ifas.ufl.edu/extension/citrus_rootstock/
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stover, E., et al. 2020. USDA progress toward developing HLB-tolerant/resistant Citrus scions. Florida Citrus Show. January 23, 2020. Ft. Pierce, FL. Invited talk
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stover, E., et al. 2020. USDA progress toward developing HLB-tolerant/resistant Citrus scions. American Society for Horticultural Sciences Webinar Series February 4, 2020. Invited talk
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang, Y., Gmitter, F., Grosser, J., Suh J.H., and Chaires, P. Top picks from citrus variety display days. Citrus Industry magazine, 2020, 101(6):22-25.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Gonzalez, A.; Eckman, M.; Stover, E.; Ferrarezi, R. S. 2020. Survey to identify key issues in accepting new HLB tolerant hybrids as new commercial cultivars for fresh and processed citrus. HortScience 55(9): S210 (Abstr.).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stover, E., et al. 2020. Update on USHRL Conventional Citrus Scion Breeding and Germplasm Evaluation.. Citrus Crop Germplasm Committee Meeting via Zoom. April 14, 2020. Invited talk
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stover, E.. 2020. Update on USHRL Conventional Citrus Scion Breeding.New Varieties Development and Management Committee Meeting via Zoom. April 22, 2020. Invited talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stover, E., F. Gmitter, E. Baldwin, and M. Schori. 2020. Rationale for Reconsidering Orange Juice Standards. International Fruit and Vegetable Juice Assoc. Zoom Meeting May 15, 2020. Invited talk
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Schori, M. and E. Stover. 2020. Taxonomic Revisions in Citrus and Why They Matter. Citrus Research and Development Foundation Zoom Conference. July 21, 2020. Invited talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stover, E.,. 2020. Huanglongbing disease putting the squeeze on OJ production in Florida. American Society for Horticultural Sciences. Annual Meeting via Zoom. August 4, 2020. Invited talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stover, E., et al. 2020. USDA progress toward developing HLB-tolerant/resistant Citrus scions. Hawaii Tropical Fruit Growers Recorded Seminar Series, August 26, 2020. Invited talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Schori, M., S. Lura, and E. Stover. 2020. Taxonomic Revisions in Citrus and Why They Matter. University of California Riverside Zoom Conference. September 1, 2020. Invited talk.


Progress 01/15/19 to 01/14/20

Outputs
Target Audience:?Major goals of the project The overall objective of this proposed research is to identify orange- and grapefruit-like citrus hybrids that are tolerant or resistant to HLB, as determined by molecular biology, pathology, physiology, flavor chemistry and sensory science. In Florida, we have identified several released cultivars that appear to have commercially useful levels of HLB resistance and these are predominately mandarin in their pedigrees. In more distant relatives, Poncirus has been the most important source of genetic resistance, with successful mapping of genetic markers and identification of gene candidates conferring the resistance. To determine tolerance or resistance to HLB, molecular markers will be identified to increase confidence that selected cultivars truly are tolerant/resistant even though they lack long term testing. These markers will expedite identification of additional HLB-tolerant/resistant scions for near term use and will contribute to longer term breeding efforts. Meanwhile on the flavor side, juice from tolerant hybrids will be analyzed for sugars, acids, limonoids, flavonoids and aroma volatiles for comparison to commercial orange and grapefruit cultivars. What was accomplished under the goals? Objective 1. Develop markers to quickly identify hybrids with HLB tolerance Greenhouse and field trials of various collections from U.S, Department of Agriculture (USDA), Horticultural Research Laboratory (HRL), University of California, Riverside (UCR) and University of Florida (UF) Citrus Research and Education Center (CREC) were conducted at USDA-HRL, UF, UCR and New Mexico Consortium (NMC). Plant samples were shared by team members. The USDA Stover team conducted and maintained replicated trials of hybrid in USDA and grower blocks relating to this project. Replicated plants were phenotyped for HLB response and growth, and leaf samples collected to assess CLas titer. For the mandarin tolerance study, data were collected on HLB symptoms and growth of mandarin x mandarin populations for mapping. KASP analysis, to identify clonal vs new hybrid material, was conducted by the UCR Roose team. For the Poncirus tolerance study, with UF Gmitter team, target populations for the Poncirus-derived tolerance mapping were identified. The Gmitter team verified hybrids in a group of seedlings of Poncirus pedigree x Poncirus pedigree material with putatively monoembryonic seed parents. For the tolerance marker study, key replicated plantings have likely reached "equilibrium" with the CLas pathogen this fall, permitting reliable characterization of relative HLB-sensitivity. NMC Gupta team used a multi-step system biology approach to examine how two CLas effectors, P235 and Effector 3, interacted with the citrus innate immune proteins. Objective 2: Evaluate sensory and chemical quality of HLB-tolerant citrus selection Drs. Plotto, Manthey, Bai and Baldwin, and breeder Dr. Stover at USDA-HRL worked closely to evaluate the sugars, acids, limonoids, flavonoids, aroma volatiles and sensory quality of selected candidates, and CLas titer in the fruit/juice was also assessed. To date, 118 fruit/juice samples (containing multiple harvests), representing 52 genotypes were analyzed for sugars, acids, limonoids, volatiles and sensory evaluation over a period of 3 years. The USDA recently released hybrid FTP-6-49-96, 'Sundragon', which is the closest to having orange juice quality, with consistent quality at harvests from end of November to end of January. Another quality evaluation team consist of Dr. Wang's food science group and Dr. Gmitter's breeding group at UF-CREC selected several new hybrids from breeding populations at the CREC. The team used Sugar Belle® mandarin hybrid and 'Valquarius' sweet orange to produce pasteurized juice and juice blends and conducted consumer preference tests. Sugar Belle® blends were the most preferred by consumers. Objective 3: Education and outreach UF Ferrarezi team has maintained the project website, https://programs.ifas.ufl.edu/ (HLB TOLERANT HYBRIDS AS NEW CITRUS COMMERCIAL CULTIVARS), and properly update the progress of the project. Some topics closely relate to the project are linked to the same platform. The team also applied the first series of surveys for consumers, growers and processors in the 2019 Citrus Show. What opportunities for training and professional development has the project provided? Technicians (2), postdocs (4) and granulate students (3) were funded by the project. The new hires have been trained in following lab, field and/or data analysis skills: population genomics analysis of array data and methods to identify trait-marker association; genome analysis; SSR marker genotyping data analysis; genetic association study between phenotype and genomic data to identify markers associated with HLB tolerant traits; chemical (instrument analysis of sugars, acids, flavonoids, limonoids, and volatiles) profile and sensory data analysis; and webpage maintenance. How have the results been disseminated to communities of interest? The results have been disseminated by website, presentation/exhibition at stakeholder/grower extension meetings, presentation at academic meetings, and publication in peer-reviewed or trading journals. One of the most important event was the 6th International HLB Research Conference. PD and most co-PDs (Drs. Baldwin, Stover, Bai, Ferrarezi, Gmitter, Wang, Roose and Gupta) presented their results and most advisory board members attended the conference. What do you plan to do during the next reporting period to accomplish the goals? Objective 1. Develop markers to quickly identify hybrids with HLB tolerance The USDA Stover team plan includes: Source Plants for Evaluations and Fruit: Continue to maintain, phenotype for HLB response and growth, and collect leaf samples to assess CLas titer; Mandarin Tolerance. They plan to continue to maintain, phenotype for HLB response and growth, and collect leaf samples to assess CLas titer. There will be a major effort to identify tolerance markers in the next two years. The Gmitter team will continue analyzing SSR markers to identify true hybrids in the Poncirus F2 backcross population. Additional KASP markers will be assessed to further confirm the SSR genotype results. Possible genotyping methods using genotype by sequencing will be finalized; Continuing phenotyping and genotyping from field populations. The UCR Roose team will analyze additional populations of trifoliate and trifoliate hybrid seedlings that have been planted in the field in Florida since 2012 with KASP markers to help interpret variation in tolerance. Additional populations from Florida will be assessed with the Axiom citrus genotyping arrays to identify markers associated with tolerance. Objective 2: Evaluate sensory and chemical quality of HLB-tolerant citrus selection Plotto/Manthey/Bai/Baldwin/Stover teams reviewed results from prior years and data were used to eliminate some poor performers, determine optimal harvest dates for new hybrids, and prepare to receive 25 selections for sensory and chemical evaluation in the 2019-20 season. Fruit for a large volume of US SunDragon juice was provided for blending experiments. For the UF Wang/Gmitter team plan, they will continue selection and testing of fruit from UF breeding populations for their potential use in juice products. Objective 3: Education and outreach UF Dr. Ferrarezi plans to enhance public and grower awareness to facilitate acceptance of alternative juice products and fresh citrus that are produced sustainably in the presence of endemic HLB, by presenting the results in technical and scientific events and assisting the other PIs/Co-PIs publishing the project outcomes in extension and scientific journals. Changes/Problems:Due to the retirement of Dr. Elizabeth Baldwin, we made arequesttoappointDr. Jinhe Bai as project director for the continuation of the project on July 1, 2019. All paperwork regarding this change have been submitted to NIFA OGFM. However, the request has not been completed, thus the progress report and other submissions have been delayed since then. What opportunities for training and professional development has the project provided?Technicians (2), postdocs (4) and granulate students (3) were funded by the project. The new hires have been trained in following lab, field and/or data analysis skills: population genomics analysis of array data and methods to identify trait-marker association; genome analysis; SSR marker genotyping data analysis; genetic association study between phenotype and genomic data to identify markers associated with HLB tolerant traits; chemical (instrument analysis of sugars, acids, flavonoids, limonoids, and volatiles) profile and sensory data analysis; and webpage maintenance.? How have the results been disseminated to communities of interest?The results have been disseminated by website, presentation/exhibition at stakeholder/grower extension meetings, presentation at academic meetings, and publication in peer-reviewed or trading journals. One of the most important event was the 6th International HLB Research Conference. PD and most co-PDs (Drs. Baldwin, Stover, Bai, Ferrarezi, Gmitter, Wang, Roose and Gupta) presented their results and most advisory board members attended the conference. What do you plan to do during the next reporting period to accomplish the goals??Objective 1. Develop markers to quickly identify hybrids with HLB tolerance The USDA Stover team plan includes: Source Plants for Evaluations and Fruit: Continue to maintain, phenotype for HLB response and growth, and collect leaf samples to assess CLas titer; Mandarin Tolerance. They plan to continue to maintain, phenotype for HLB response and growth, and collect leaf samples to assess CLas titer. There will be a major effort to identify tolerance markers in the next two years. The Gmitter team will continue analyzing SSR markers to identify true hybrids in the Poncirus F2 backcross population. Additional KASP markers will be assessed to further confirm the SSR genotype results. Possible genotyping methods using genotype by sequencing will be finalized; Continuing phenotyping and genotyping from field populations. The UCR Roose team will analyze additional populations of trifoliate and trifoliate hybrid seedlings that have been planted in the field in Florida since 2012 with KASP markers to help interpret variation in tolerance. Additional populations from Florida will be assessed with the Axiom citrus genotyping arrays to identify markers associated with tolerance. Objective 2: Evaluate sensory and chemical quality of HLB-tolerant citrus selection Plotto/Manthey/Bai/Baldwin/Stover teams reviewed results from prior years and data were used to eliminate some poor performers, determine optimal harvest dates for new hybrids, and prepare to receive 25 selections for sensory and chemical evaluation in the 2019-20 season. Fruit for a large volume of US SunDragon juice was provided for blending experiments. For the UF Wang/Gmitter team plan, they will continue selection and testing of fruit from UF breeding populations for their potential use in juice products. Objective 3: Education and outreach UF Dr. Ferrarezi plans to enhance public and grower awareness to facilitate acceptance of alternative juice products and fresh citrus that are produced sustainably in the presence of endemic HLB, by presenting the results in technical and scientific events and assisting the other PIs/Co-PIs publishing the project outcomes in extension and scientific journals.

Impacts
What was accomplished under these goals? Objective 1. Develop markers to quickly identify hybrids with HLB tolerance Greenhouse and field trials of various collections from U.S, Department of Agriculture (USDA), Horticultural Research Laboratory (HRL), University of California, Riverside (UCR) and University of Florida (UF) Citrus Research and Education Center (CREC) were conducted at USDA-HRL, UF, UCR and New Mexico Consortium (NMC). Plant samples were shared by team members. The USDA Stover team conducted and maintained replicated trials of hybrid in USDA and grower blocks relating to this project. Replicated plants were phenotyped for HLB response and growth, and leaf samples collected to assess CLas titer. For the mandarin tolerance study, data were collected on HLB symptoms and growth of mandarin x mandarin populations for mapping. KASP analysis, to identify clonal vs new hybrid material, was conducted by the UCR Roose team. For the Poncirus tolerance study, with UF Gmitter team, target populations for the Poncirus-derived tolerance mapping were identified. The Gmitter team verified hybrids in a group of seedlings of Poncirus pedigree x Poncirus pedigree material with putatively monoembryonic seed parents. For the tolerance marker study, key replicated plantings have likely reached "equilibrium" with the CLas pathogen this fall, permitting reliable characterization of relative HLB-sensitivity. NMC Gupta team used a multi-step system biology approach to examine how two CLas effectors, P235 and Effector 3, interacted with the citrus innate immune proteins. Objective 2: Evaluate sensory and chemical quality of HLB-tolerant citrus selection Drs. Plotto, Manthey, Bai and Baldwin, and breeder Dr. Stover at USDA-HRL worked closely to evaluate the sugars, acids, limonoids, flavonoids, aroma volatiles and sensory quality of selected candidates, and CLas titer in the fruit/juice was also assessed. To date, 118 fruit/juice samples (containing multiple harvests), representing 52 genotypes were analyzed for sugars, acids, limonoids, volatiles and sensory evaluation over a period of 3 years. The USDA recently released hybrid FTP-6-49-96, 'Sundragon', which is the closest to having orange juice quality, with consistent quality at harvests from end of November to end of January. Another quality evaluation team consist of Dr. Wang's food science group and Dr. Gmitter's breeding group at UF-CREC selected several new hybrids from breeding populations at the CREC. The team used Sugar Belle® mandarin hybrid and 'Valquarius' sweet orange to produce pasteurized juice and juice blends and conducted consumer preference tests. Sugar Belle® blends were the most preferred by consumers. Objective 3: Education and outreach UF Ferrarezi team has maintained the project website, https://programs.ifas.ufl.edu/ (HLB TOLERANT HYBRIDS AS NEW CITRUS COMMERCIAL CULTIVARS), and properly update the progress of the project. Some topics closely relate to the project are linked to the same platform. The team also applied the first series of surveys for consumers, growers and processors in the 2019 Citrus Show.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Stover, E., Gmitter, F., Grosser, J., Baldwin, L., Wu, G.A., Bai, J., Chaires, P., Motamayor, J.C., and Wang, Y. Rationale for reconsidering current regulations restricting use of hybrids in orange juice. Horticulture Research. In press.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Raithore, S., Keifl, J., Manthey, J.A., Plotto, A., Bai, J., Zhao, W., and Baldwin, E.A. Mitigation of off-flavor in huanglongbing-affected orange juice using natural citrus non-volatile compounds. Journal of Agricultural and Food Chemistry. 68:1038-1050. 2020. Available: https://pubs.acs.org/doi/pdf/10.1021/acs.jafc.9b07756
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Dala-Paula, B.M., Plotto, A., Bai, J., Manthey, J.A., Baldwin, E.A., Ferrarezi, R.S., and Gloria, M.B.A. Effect of Huanglongbing or greening disease on orange juice quality, a review. Frontiers in Plant Science. 9:1976. 2019. Available: https://www.frontiersin.org/articles/10.3389/fpls.2018.01976/full
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Deng, H., Achor, D., Etxeberria, E., Yu, Q., Du D., Stanton, D., Liang, G., Gmitter, F. G. Phloem regeneration is a mechanism for Huanglongbing-tolerance of Bearss lemon and LB8-9 Sugar Belle� mandarin. Frontiers in Plant Science, 2019. DOI:10.3389/fpls.2019.00277.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yao LX, Yu QB, Huang M, Hung WL, Grosser J, Chen SC, Wang Y, Gmitter FG: Proteomic and metabolomic analyses provide insight into the off-flavour of fruits from citrus trees infected with 'Candidatus Liberibacter asiaticus'. Horticulture Research 2019, 6:31.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, M., Roose, M. L., Yu, Q., Du, D., Yu, Y., Deng, Z., Stover, E., Gmitter Jr., F. G. Construction of high-density genetic maps and detection of QTLs associated with Huanglongbing tolerance in citrus. Frontiers in Plant Science, 2018, https://doi.org/10.3389/fpls.2018.01694.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, Y., Sims, C., Gao, Z., House, L., Gmitter, F., and Grosser, J. Sugar Belle� shows potential in orange juice processing. Citrus Industry magazine, 2019, 100(9):14-17.
  • Type: Websites Status: Published Year Published: 2019 Citation: Castle, W. S.; Bowman, K. D.; Grosser, J. W.; Ferrarezi, R. S.; Futch, S. H.; S, Rogers. 2019. Florida Citrus Rootstock Selection Guide, 4th Edition. EDIS Publication, UF/IFAS Extension, Horticultural Sciences Department, # SP248. URL: https://edis.ifas.ufl.edu/hs1260)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Roose, M. Application of KASP markers to improve studies of HLB tolerance. 6th International Research Conference on HLB. 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Stover, E. Update on USDA Citrus Scion Breeding. Florida Citrus Show. 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Stover, E. Identifying and Developing Resistance and Tolerance to HLB. 6th International Research Conference on Huanglongbing. 2019


Progress 01/15/18 to 01/14/19

Outputs
Target Audience:Citrus growers, packers and processors Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Due to hiring obstacles in the first year, the USDA Stover team has used minimal personnel funds from this grant, amounting $40K for part-time diversion of personnel primarily committed to other projects, but recently acquired a postdoc who is being trained in marker assisted breeding. The Baldwin lab had several students over the summer and has advertised for a postdoc. The UF-Indian River Research and Education Center (IRREC) Ferrarezi group has hired personnel to design and maintain the project webpage. A part time specialist was also hired to develop surveys. How have the results been disseminated to communities of interest?Ferrarezi's Citrus Horticulture lab at the UF-IRREC was able to hire a person to design the project webpage, currently available at https://programs.ifas.ufl.edu/scion-guide. The existing website lists the research project description, objectives, people and activities, and is ready to receive publications, presentations and any other resulting product from the project for massive distribution. The current format is based on the University of Florida's terminal four (T4), the world's leading digital marketing & web content management platform for Higher Education. The Ferrarezi lab started incorporating the UF/IFAS Citrus Rootstock Selection Guide to the same platform, and will be able to link both websites to facilitate the information access. Dr. Ferrarezi is migrating the Citrus Rootstock Selection Guide [by Drs. William S. Castle, Jude W. Grosser, Stephen H. Futch, James H. Graham (UF/IFAS CREC, Lake Alfred, FL) and Kim D. Bowman (USDA-ARS, Fort Pierce, FL)] from a paid server (kindly provided at no cost over the years by Dr. Steve Rogers and his company EcoStat Inc., Lakeland, FL) to the University of Florida's servers. The new system will be administered in house. We are also converting the expert system from Exsys Inc., Albuquerque, NM, which executes the query in a remote server and is currently hosted at https://crec.ifas.ufl.edu/extension/citrus_rootstock/templates/guide and http://rootstock.hostjava.net/CorvidCore/CorvidSR, to run client-side in the users' web browser, removing the need for any backend servers available at https://programs.ifas.ufl.edu/rootstock-guide. Making those necessary changes will prepare the system to receive future updates from Florida's Citrus Improvement Team, comply with UF requirements and minimize the need for annual license requirements and fees. The Ferrarezi lab also hired a part-time specialist to develop separate surveys for growers, processors and consumers to determine reaction to and acceptance of new HLB-tolerant citrus cultivars. The survey will be distributed at the 2019 Florida Citrus Show in January 23-24, 2019. The event is organized by Dr. Ferrarezi and his team, gathering approximately 800 growers, vendors, scientists and industry leaders. What do you plan to do during the next reporting period to accomplish the goals?Continue measurement of tree growth, health index and bacterial titer and disease symptom ratings of hybrids in the field. Continue genotype-by-sequencing analysis of parents and hybrids. Continue to conduct genotyping using dense SNP markers in segregating F1, F2 and other hybrids. Conduct RNA-seq analysis of HLB susceptible and tolerant hybrids to identify possible genes associated with resistance or tolerance to the disease. Test a range of hybrids using DNA and RNA markers. Continue to identify HLB tolerant/resistant hybrids for flavor quality analyses and collect fruit quality sensory and chemical data. Analyze aroma volatile data to identify stand-alone processing types for orange and grapefruit juice and develop juice blends. Continue to expend and maintain website containing interactive resources. Share information at field days, workshops and conferences.

Impacts
What was accomplished under these goals? To identify sources of citrus greening or huanglongbing (HLB) resistance or tolerance, leaf samples were collected by the U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), Horticultural Research Laboratory (USHRL) for KASP (genotyping assay) analysis of the mandarin x mandarin population for mapping, to identify clonal vs new hybrid material. Working with the University of Florida Citrus Research and Education Center (UF-CREC) Gmitter team, potential populations for the Poncirus-derived tolerance mapping have been assembled and are under discussion for finalizing the study group. Initial studies suggest that F1 hybrids between Poncirus and sweet orange were intermediate in HLB-response and ACP colonization, so the University of California, Riverside (UCR) Roose and USDA Stover teams collaborated to develop seedling populations from which Poncirus pedigree is derived from both parents, potentially recovering tolerance more similar to pure Poncirus. These data are still to be analyzed to determine potential for use as HLB-tolerance markers, although there currently no obvious responses distinguishing tolerant vs. susceptible types. Constructs have been created for plant expression of many effectors, so that transient expression can be used to elicit and assay potential tolerance markers. A literature search is underway to identify other potential tolerance markers that merit evaluation. Minimal grant funds have been used by the USDA team as they are designated for hiring personnel, and hiring obstacles continue in USDA, however one postdoc is on board and the other position has been announced. Looking for HLB tolerance in the field, analysis of open-pollinated seedling trees are being tested for HLB tolerance to evaluate tolerance of parent trees. Evaluation of the HLB tolerance of parents trees in mapping populations and of potential parent trees for use in breeding can be better accomplished if we determine whether each seedling tree tested in the field for HLB response originates as a nucellar seedlings, a zygotic seedling from self-pollination, or a zygotic seedlings from an outcrossing event. Leaf tissue samples were collected from 734 seedlings being evaluated in the field in Ft. Pierce Florida and analyzed by LGCGenomics for 15 SNP markers using the KASP marker technology at UCR. Markers selected for analysis were located on all 9 chromosomes and at distal locations. More than 97% of seedlings had calls for more than 90% of the markers. One marker locus could not be accurately called in about 30% of samples but the others had more than 99% calls. Interpretation of this data is still in progress but it is clear that some maternal plants produce almost entirely nucellar seedlings while others produce both types. For some source trees, all seedlings appear to result from outcrossing, an expected result if the maternal tree does not produce nucellar seedlings and is self-incompatible. Overall, the technology is working well. We plan additional studies of this sort on other populations under evaluation. Analysis of Triumph and Duncan grapefruit genotypes and haplotypes is also underway. Triumph (and its low-seeded derivative, Jackson) is among the most HLB tolerant Citrus varieties, whereas standard grapefruit such as Duncan are among the most susceptible. The precise ancestry of Triumph is not known. To better understand the basis of tolerance in Triumph and select additional tolerant grapefruit types, we are analyzing the Triumph genome to determine the origin of each chromosome segment, that is, whether it originates from pummelo or mandarin and, more specifically, whether current germplasm collections include accessions that could be the parents of Triumph. This analysis includes inference of haplotypes for these varieties based on genotyping of several single pollen grains from each. To test HLB tolerant hybrids for flavor quality, cropload status and visual HLB-responses were assessed and used in refining the harvest plan for USDA hybrids. Twenty-five selections were delivered to the USDA Baldwin food science group for chemical and sensory evaluation. The food science group at USDA-ARS-USHRL has analyzed USDA hybrids for the past two seasons, and are now collecting juice for this season. The Stover lab is screening the hybrids for HLB tolerance, and the Baldwin group is doing analysis of hybrids that show promise in that area. The Baldwin group did analysis of Brix, titratable acidity, individual sugars (glucose, fructose, sucrose), individual acids (citric, malic, ascorbic), aroma compounds and bitter limonoids (limonin and nomilin). If the hybrid had too much bitterness and levels of bitter compounds (mandarin can have delayed bitterness) they then rejected them for commercial juice. Multiple harvests were done for hybrid where there was no data for optimum harvest date, and determined optimum date based on ratio. All hybrids were rated by a sensory panel for sweetness, sourness, bitterness, off-flavor and aftertaste as well as "orange-like", "mandarin-like" or "grapefruit-like" characteristics. To date 25 hybrids were analyzed for two seasons with around 11 in multiple harvests. Of these seven were rated "orange-like" and eight, "grapefruit-like" in sensory panels. Of those rated "orange-like", four also rated high for "mandarin-like". Bottom line is there seem to be some interesting hybrids with HLB tolerance and some commercial possibilities.

Publications