Source: UNIVERSITY OF FLORIDA submitted to NRP
SP: RAPID GENERATION AND EVALUATION OF EREMOCITRUS-DERIVED POPULATIONS FOR HLB TOLERANCE AND FRUIT QUALITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031586
Grant No.
2023-70016-41308
Cumulative Award Amt.
$928,000.00
Proposal No.
2023-06863
Multistate No.
(N/A)
Project Start Date
Sep 15, 2023
Project End Date
Sep 14, 2026
Grant Year
2023
Program Code
[ECDRE]- Emergency Citrus Disease Research and Extension Program
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
Huanglongbing (HLB) or citrus greening disease is considered the biggest threat to the U.S. citrus industry. Given the loss of revenue being sustained by citrus growers due to HLB, there is a critical need for resistant and tolerant varieties to alleviate disease management costs, improve yield and offset production losses. Resistance has been identified in the related species Eremocitrus glauca; however introgression of resistance into commercial varieties typically takes decades due to the long juvenility of citrus. We have an E. glauca selection with precocity that flowers and sets fruit with viable seeds within a year of planting. Portions of F1 and BC1 hybrids with this selection also show precocity. We are proposing to create crosses and backcrosses (up to BC2) to generate varieties of commercial quality and resistance/tolerance to HLB using the precocious selection much faster than with previously available genotypes. We are also proposing to determine the HLB resistance, tolerance, early flowering anf fruit quality of these populations foth under greenhouse (graft-inoculated for with HLB) and field conditions (natural infection). We will also use previously E. glauca x Citrus BC1 populations characterized for the past 5 years for HLB tolerance/resistance to generate PCR molecular markers based on GBS analysis to facilitate the identification and future breeding of tolerant/resistant types. Similarly, precocious BC1 segregating populations will be used to produce markers for early flowering. Finaly, E. glauca x Poncirus trifoliata will be produced and evaluated for Phytophtora root rot and HLB tolerance to determine their potential use as rootstocks.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20109991081100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1081 - Breeding;
Goals / Objectives
The overall goalof this application is to move favorable genetic characteristics from the desert lime (E. glauca), such as HLB resistance or tolerance and early flowering, to citrus using conventional breeding techniques thus generating new HLB resistant/tolerant commercial citrus varieties that can be grown in the U.S. and be used for fresh fruit, juice or as rootstock.Objective 1. Generate second generation backcrosses (BC2) of sweet orange, mandarin and grapefruit using early flowering BC1 populations ((Eremocitrus x Citrus) x Citrus) to improve fruit quality while maintaining HLB tolerance.Objective 2. Evaluate the fruit quality of the BC2 plants.Objective 3. Evaluate the HLB tolerance/resistance of BC1 and BC2 plants using graft inoculation in the greenhouse and under field conditions.Objective 4. Use genotype by sequencing (GBS) and perform QTL analysis to identify genomic regions conferring HLB tolerance, early flowering, and fruit quality traits, to facilitate and accelerate the identification of superior progeny.Objective 5. Generate F1 E. glauca x Poncirus trifoliata hybrids to combine HLB resistance (from E. glauca) with Phytophthora root rot tolerance (from P. trifoliata) and evaluate the progeny for susceptibility to these diseases and potential use as rootstocks.
Project Methods
Objective 1 : ALL BC1 progeny establishedin soil in the greenhouse.BC2 populations generated by crossing the precociousBC1 progeny with grapefruit, mandarin and sweet orange. Seeds germinated in tissue culture to improve germination rates and speed up the process.When seedling epicotyls reach 1.5-2 cm and 3-4 true leaves transferred to soil and acclimated before transferring to greenhouse. The progeny will be grown in a 5,200 sq ft psyllid-proof greenhouse assigned to the PD exclusively for growing citrus plants. The progeny will be fertilized regularly and propagated by grafting onto 'Rangpur' lime (C. limonia) rootstock for further characterization (Objectives 2 and 3). Flowering times data will be collected for the BC1 and BC2 plants.2)Objective 2:. Early flowering individuals from the BC2 obtained in Objective 1 will be hand-pollinated every flowering season until the completion of the project. Fruit produced by precoious seedlings will be evaluated for shape, size, weight,and, peel and flesh color. Juice from a 10 fruit sample will be evaluated for juice color, TSS and TTA.3)Objective 3:BC1 obtained prior to the start of this project (18 individuals) and BC2 plants generated in Objective 1 will be clonally propagated by grafting onto 'Rangpur' lime (5 replicates per plant). They will be maintained in 2 gal pots in the psyllid proof greenhouse assigned to the PD and regularly fertilized. Once the graft has taken, 3 of the replicates will be graft inoculated with CLas-infected budwood and the other 2 will be planted in the field (Gainesville, FL). The budwood will be tested for HLB prior to grafting using real time PCR using our standard detection system which with TaqMan probes and primers for CLas and COX [37, 38] to ensure only HLB-infected is used for inoculations. The inoculated plants will be moved to a general-purpose greenhouse (1,020 sq ft) assigned to the PD. All experimental trees (greenhouse and field) will be evaluated for growth (stem diameter, height, canopy volume), canopy symptoms and CLas titers using real-time PCR twice a year to determine the resistance/tolerance to HLB in the segregating populations. E. glauca (non-hybrid) trees (16 plants, including a clone of the early flowering selection) also planted in the field since 2021 will be used as controls and also tested for CLas. As of the writing of this pre-proposal these trees remain HLB-negative. Additionally, 18 E. glauca x 'UFGlow' mandarin F1 hybrids (including those seven used as parents of the BC1 and BC2 populations analyzed here and obtained in Objective 1) will also be planted in the field in Year 1 to serve as controls and determine their field resistance/tolerance to HLB. These trees are currently in the process of being graft-propagated onto 'Rangpur' lime and should be ready on time for the start of this proposal.4) Objective 4:DNA will be extracted from all BC1 individuals (Families 1 and 2) and 3 parents (94 individuals) and sequenced. GBS will be used to perform segregation analysis to map the HLB resistance/tolerance locus/loci. Similarly, DNA will also be extracted from the early flowering BC1 populations composed of (E. glauca x C. reticulata) x C. paradisi and (E. glauca x C. reticulata) x C. reticulata progeny and all parents. Currently 59 plants are available but further crosses will be conducted to reach 100 plants. These populations, characterized for flowering times, will be used to map early flowering loci. The isolated DNA will be digested with ApeKI prior to the GBS library construction followed by sequencing. The obtained sequences will be aligned to the reference C. clementina genome to locate SNPs/Indels and QTL analyses performed for the traits of interest, by linking QTL clusters to the measured parameters (HLB tolerance and flowering time). Following the above analysis, polymorphic sites and their flanking regions will be used to design fluorescently tagged primer pairs for fragment size analysis by capillary electrophoresis to generate PCR-based markers. We estimate we will need to screen 100 primer pairs for HLB tolerance/resistance and another 100 for precocity. Initially, 6-8 individuals of contrasting phenotypes, including parents will be tested and about 20 of the most informative primer pairs per locus will be tested in the respective 100 tree population. Based on the BC1 segregation of HLB resistance/tolerance we estimate around 3 QTL will explain most of the phenotypic variation. For early flowering 2-3 loci are potentially involved in early flowering.5)Objective 5: Early flowering E. glauca will be crossed with P. trifoliata to generate F1 populations. The objective is to incorporate the HLB resistance found in E. glauca and the Phytophthora tolerance of P. trifoliata into one selection to be used as rootstock. The seedlings will be inoculated with Phytophthora [39] and those surviving will be graft inoculated with HLB. Symptomatology and CLas titers will be determined using real time PCR.Efforts:As mentioned previously, The two principal scientists teach two courses one at the undergraduate level (FRC 3212) and an advaced graduate level plant breeding course (HOS 6201). Results from the research will be incorporated into the FRC 3212 course indicating the impact of HLB on citrus production and the efforts being undertaken to develop new HLB tolerant and resistant citrus cultivars. The research methods and technology being used in the project will be presentedin the graduate level course. Results from the research will be presented at scientific conferences and published in professional journals. Annual Industry meetings and field days will be used as outreach venues to inform growers and industry representatives of progress in the project.Evaluation:Year 1Objective 1: BC2 crosses will be generated to obtain a minimum of 20 BC2 plants. Data on thepollination number, fruit harvested, number of seed/fruit and the number of seedlings recovered collected.Objective 3: The 18 E. glauca x 'UFGlow' mandarin F1 hybrids and their E. glauca parentwill be planted in the field 1 to serve as controls.Objective 4:DNA will be extracted from all BC1 individualsand the parents (94 individuals) and sequenced. GBS will be will be initiated.Objective 5: Crosses will be made to generate E. glauca x P. Trifoliata hybrids.Data on the number of pollinations made, number of fruit harvested, average number of seed/fruit and the number of seedlings recovered will be collected.Year 2Objective 1: BC2 seedlings estblished in soil. At least 20 BC2 seedlings survive.Objective 3: Clones of BC1 and BC2 progeny generated by grafting on Rangpur lime. BC1 and BC2 clones graft innoculated. Field test of HLB infection established. E. glauca parent and F1 hybrids tested for HLB.Objective 4: GBS analysis of populations finished and QTL loci identified for early flowering and HLB initiated. Development of precocity and HLB tolerance/resistance markers initiated.Objective 5: Hybrids transplanted to soil, inoculated with Phytophthora, survivors inoculated with HLB.Year 3Objective 2: Fruit produced by precocious BC2 evaluatedObjective 3: BC2 progeny graft innoculated.Greenhous and fieldBC1 and BC2 plants tested for HLB.Objective 4:Design and test of PCR markers finished.Objective 5: Inoculation finished and plants tested for HLB infection.

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

Outputs
Target Audience:A presentation was made at the annual citrus fruit tasting on October 27,2023 at the University of Florida Teaching Grove in Gainesville, Florida. A PowerPoint presentation on the use of Eremocitrus glauca for the breeding of HLB tolerant citrus cultivars was given. Attendees consisted of citrus industry representatves and citrus growersfrom the state of Florida. The Fruit Display was held on November 18, 2024. Prior to the tasting of fresh fruit samples and evaluation of juices, Presentations were made by Maria Belen Besilla, Rafael Montalt, Vicente Febres and Jose Chaparro. Ms. Besilla presented on progress in generating (Eremocitrus x Citrus) x Citrus backcross progeny. Dr. Montalt presented data on the analysis of the genotype by sequencing of BC1 Populations 1 and 2. Dr. Febres presented data on detection and titer determination of Candidatus Liberibacter asiaticus in the two BC1 populations using rtPCR. Dr. Chaparro presented an overview of the citrus breeding effort and the inportance of incorporating HLB resistance from E. glauca into commercial citrus germplasm. Changes/Problems:There has been a significant delay in the identification, hiring and VISA processing of the Post doc. It is likely that his start date will be delayed until June 2024. We have been unable to generate BC2 progeny from pollinations in 2024 and 2025. BC1 trees are being clonally propagatedd on Rangpur lime to increase the number of potential flowers for the sprring of 2026. What opportunities for training and professional development has the project provided?Mantoring was provided to Ms. Besilla in the preparation of a manuscript for submission to a scientific describing the photypic segregation in the BC1 progenies. Assitance was provided in the design of figures and presentation of graphical data. How have the results been disseminated to communities of interest?Progress in the project was presented at the citrus fruit tasting in November 2024. Presntations were made by Maria Belen Besilla on the phenotypic evaluation of BC1 progeny, Dr. Febres on the use of real time PCR for determining bacterial titer on citrus trees, Dr. Montalt on GBS data analysis, and by the PI on the use of Eremocitrus glauca as a source of HLB resistance. What do you plan to do during the next reporting period to accomplish the goals?October -December 2023 Objective 1. Generate second generation backcrosses (BC2) of sweet orange, mandarin and grapefruit using early flowering BC1 populations ((Eremocitrus x Citrus) x Citrus) to improve fruit quality while maintaining HLB tolerance. Additional BC1 plants are under observation for the presence of flower buds. Any BC1 plants that flower will be pollinated to generate additional BC2 progeny. Objective 3. Evaluate the HLB tolerance/resistance of BC1 and BC2 plants using graft inoculation in the greenhouse and under field conditions. The UF 08-76 × UF 09-13 BC1ramets will be graft inoculated with HLB to confirm their resistant phenotype. The clonal ramets of the 18 UF 18-12 ef X UF 10-02 F1 progenywill be planted in the spring of 2024 at the University of Florida Teaching Grove in Gainesville, FL for evaluation of HLB resistance under natural infection. Objective 4. Use genotype by sequencing (GBS) and perform QTL analysis to identify genomic regions conferring HLB tolerance, early flowering, and fruit quality traits, to facilitate and accelerate the identification of superior progeny. Once the Post doc comes on board, DNA will be extracted from the segregating populations and submitted for GBS. The Post doc will review and analyze the morphological data that has been collected to date. Objective 5. Generate F1 E. glauca x Poncirus trifoliata hybrids to combine HLB resistance (from E. glauca) with Phytophthora root rot tolerance (from P. trifoliata) and evaluate the progeny for susceptibility to these diseases and potential use as rootstocks. The flowers will be pollinated using sweet orange pollen to ensure fruit set and determine whether the seed produced by the hybrids are poly or monoembryonic. Clonal ramets of both genotypes will be generated by budding on to Rangpur lime rootstock. These ramets will be graft inoculated with HLB to determine if they are resistant to HLB. April-June 2025 1. Planting of clonal replicates in the field to monitor field resistance to HLB 2. Graft innoculation of clonal replicates to determine the resistance of individual BC1 progeny. 3. Design of PCR based markers for the putative QTLs that have been identified.

Impacts
What was accomplished under these goals? October -December 2023 Objective 1. At present, three out of the 15 of the ((Eremocitrus x Citrus) x Citrus) BC1 generated using Hudson grapefruit have flowered. Two of the trees are currently being pollinated with sweet orange pollen to generateBC2 seed. Additional BC1 plants are under observation for the presence of flower buds. Objective 3. The UF 08-76 × UF 09-13 BC1 population was successfully propagated onto 'Rangpur' lime rootstock with 13 out of the 14 BC1 progeny in this population having at least 2 ramets. Seedling BC1-45 is currently represented by only one ramet, however sprouting is expected from two additional grafts. These ramets will be graft inoculated with HLB to confirm their resistant phenotype. Two clonal ramets were generated for 16 of the 18 UF 18-12 ef X UF 10-02 F1 progeny. We obtained 1 successful ramet of seedling F1-1 in 10 attempts and no ramets of seedling F1-13 in 14 attempts. The poor take for these two F1 seedlings appears to result from their poor growth and lack of appropriate material for grafting. Objective 4. We are in the process of hiring a postdoctoral associate with the qualifications to perform the GBS/QTL analysis. Interviews were conducted in November-December 2023 and a suitable candidate has been selected. Currently the application material of the candidate is going through the University of Florida's Human Resources hiring process. A letter of offer will be sent once HR completes their evaluation. Objective 5. Two of the three existing E. glauca x P. trifoliata F1 seedlings have flowered. The flowers will be pollinated using sweet orange pollen to ensure fruit set and determine whether the seed produced by the hybrids are poly or monoembryonic. Clonal ramets of both genotypes will be generated by budding on to Rangpur lime rootstock. These ramets will be graft inoculated with HLB to determine if they are resistant to HLB. January -March 2024 Objective 1. Controlled pollinations were performed in the greenhouse to generate additional BC1 progeny. E. glauca x Mandarin F1s were pollinated with Hudson grapefruit, Mandarin and Marrs sweet orange pollen.Preliminary observations indicated that fruit set was obtained for all cross combinations. Three (E. glauca x Citrus) x Hudson grapefruit BC1 and a single (E. glauca x Citrus) x 06-07 grapefruit BC1 bloomed in February 2024.Flowers the BC1 flowers were morphologically normal and were pollinated with sweet orange pollen but fruitlets aborted. Objective 3. Budding ofE. glauca x Citrus F1 hybrids on Rangpur lime rootstocks initiated. Objective 4. DNA was extracted from leaf tissue of Family 1, Family 2, and the three parents. Objective 5. Pollinations were performed on E. glauca usingPoncirus trifoliatapollen. April - June 2024 Objective 3. The budding of E. glauca x Citrus F1s on Rangpur lime continued as more plants developed mature budwood. Objective 4. DNA of Family 1, Family 2, and the three parents submitted for sequencing. July-September 2024 Objective 1. A total of 159 putative BC1 seed were obtained using Hudson grapefruit, Mandarin and Marrs sweet orange pollen.Seed cultured in vitro for germination. Objective 3. The budding of E. glaucaxCitrus reticulataF1s on Rangpur was continued as more plants developed mature budwood and grafts were repeated of buds that did not take previously. Two clonal replicates of 17 differentE. glaucaxCitrus reticulataF1 hybrids budded on Rangpur lime were planted in the field at Gainesville Florida August 0f 2024. .Objective 4. DNA of Family 1,Family 2, and the three parents was sent for sequencing. Dr. Rafael Montalt was hired for the analysis of the GBS data. Phenotypic data such as chlorosis, tree defoliation, trunk diameter, tree height and bacterial titer were analyzed to identify the trait/s most indicative of susceptibility, tolerance, and resistance phenotypes in the progeny. Objective 5. Seeds fromE. glauca(Fla. 18-12) xPoncirus trifoliatacross harvested and planted in vitro. October -December 2024 Objective 1. The BC1 seed initiated germination.Germination was not uniform with some seed germinating precociously and others delayed or not germinating within this period.Germinated seedlings were transplanted into small pots and placed in the greenhouse under shade. Objective 3. The budding of existingE. glaucaxCitrus reticulataF1 hybrids on Rangpur lime rootstocks was continued as more plants developed mature budwood and grafts were repeated of buds that did not take previously.Any genotypes where not successful after three attempts were determined to be graft incompatible with Rangpur lime rootstock and no further attempts at clonal propagation were made. Objective 4. Preliminary analysis of the sequencing data was initiated.Sequence data was filtered to eliminate poor quality sequence data and regions were there was insufficient depth to allow for detection of sequence polymorphisms.The remaining high-quality reads were aligned to theCitrus_clementina_v1.0reference genome and the genome assemblies of E. glauca generated at the University of Queensland and University of California, Riverside. January - March 2025 Objective 1. Controlled pollinations were repeated in the greenhouse to generate additional BC1 progeny. Multiple E. glauca x Mandarin F1s were pollinated with Hudson grapefruit, Mandarin, Mediterranean sweet orange, and Pineapple sweet orange pollen.Preliminary observations indicated that fruit set was obtained for all cross combinations. Pollinations on BC1 progeny were attempted on two BC1 progeny that flowered. All fruit aborted shortly after pollination and nocrosses intended to generate BC2s were succesfull. However, pollinations were performed in the greenhouse usingE. glaucax Citrus F1s as female parents. The parents used included Hudson grapefruit, Pineapple sweet orange and Flame grapefruit. No further crosses were performed using Marrs Orange as a male parent due to the high mortality of the BC1 progeny. Pollinations were also performed on clonally propagatedE. glaucax Citrus F1s that were planted in the field in the fall of 2024. Objective 3. The budding of existingE. glaucaxCitrus reticulataF1 hybrids on Rangpur lime rootstocks was continued as more plants developed mature budwood and grafts were repeated of buds that did not take previously. The propagation of five clonal replicates of all theE. glaucaxCitrus reticulataF1 hybrids were budded on Rangpur lime to be used for controlled inoculation with theCandidatusLiberibacter asiaticus bacterium (3 trees) and for planting in the field to test for field resistance (2 trees). Progress is determined by the availability of useful budwood due to variability in seedling vigor.F1 and BC1 trees demonstrating graft incompatibility symptoms were removed from the experiment. Objective 4. Analysis of the phenotypic data (chlorosis, tree defoliation, trunk diameter, tree height and bacterial titer) revealed that bacterial titer data allowed the classification of progeny into Susceptible, Tolerant and Resistant groups. Analysis of the GBS data for Family 1:C. reticulatax (E. glaucaxC. maxima) (28 individuals), Family 2 (E. glaucaxC. maxima) xC. reticulata(65 individuals) allowed the inferring of theE. glaucaandC. maximahaplotypes. QTL analysis of the data identified multiple genomic regions controlling phenotypic variation for HLB resistance. Objective 5.E. glauca(Fla. 18-12) xPoncirus trifoliatapollinations were done again to generate additional F1 hybrids.Fruit will be harvested in Julyto ensure the survival of the hybrid seed. ExistingE. glauca(Fla. 18-12) xPoncirus trifoliatahybrids grew weakly during 2024 and did not produce any flowers.The F1 hybrids were clonally propagated on Rangpur lime rootstock in attempt to increase vegetative growth and the potential for flowers in the spring of 2026. ?

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