Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 999 - Citrus, general/other | 1081 - Breeding | 100% |
Huanglongbing (HLB) is presently the most devastating citrus disease worldwide. Because of the extensive spread and a lack of effective control measures for HLB, Florida's $9 billion citrus industry has experienced a decline in production of nearly 75%. As a result, the industry is presently fighting for its survival. The other major citrus states are at high risk for a similar disastrous situation if suitable remedies are not forthcoming soon. Neither a simple cure nor successful management strategies have been identified even after 10+years of the disease presence in Florida. Previous efforts to create new HLB tolerant/resistant cultivars focused on conventional breeding or development of transgenic plants. Unfortunately, the approaches used in those projects are inadequate to meet the current crisis because of the lack of sources of resistance in citrus and time constraints associated with citrus breeding. Furthermore, genetic engineering presents many regulatory and public acceptance obstacles for transgenic citrus. On the other hand, more than 10 years of HLB and tens of millions of infected trees in groves across Florida provide an unprecedented opportunity and high probability to select resistant natural mutants. Field resistance or tolerance to HLB has already been noted amongst the citrus cultivars and their relatives.This provides circumstantial evidence that natural variation exists regarding HLB tolerance/resistance within the citrus gene pool.Exploitation of this natural tolerance/resistance may provide a key solution, within a short time frame, to the survival of the citrus industry, especially in Florida.This project addresses the "Development of tolerance or resistance in commercial citrus in all production areas with a focus on delivery of new cultivars [or rootstocks and scions] using all available strategies", which is one of the priorities set forth by the Citrus Disease Sub-committee.In this proposal, we present a systematic approach with emphasis on skillful selection of natural mutants to develop new HLB tolerant/resistance citrus cultivars. First, we will select variant seedlings and bud sports with greater HLB tolerance/resistance in citrus groves across Florida and test them in field trials. With the obtained HLB tolerant/resistant bud sports/variant seedlings and their susceptible siblings, we will be able to apply transcriptome profiling and comparative genomics to identify HLB tolerance/resistant or susceptibility related genes. Moreover, with the HLB susceptibility related genes identified in our preliminary study and this project, we will develop new resistant varieties via gene editing using CRISPR-Cas9 on the identified target genes. The CRISPR engineered citrus plants do not include foreign genes and are currently not restricted in the US. Meanwhile, we will conduct active outreach and extension to disseminate our project results to growers, stakeholders and the public. The successful achievement of our goals and objectives will broadly impact the entire US citrus industry, by maintaining the viability of industries not yet fully impacted as in Florida, and by providing the plant materials that will encourage Florida growers to replant with confidence. The combined economic impacts of our US citrus industries will be measured in billions of dollars. The many communities throughout the country having economies built around viable citrus production will avoid the major disturbances that will come from the economic disasters that HLB incurs. Current approaches for HLB management are based on an over-reliance of chemical insect control, and more recently the widespread use of antibiotic compounds; the environmental impacts of these practices may prove to be unacceptable in the long run, and resistant cultivars can mitigate such damage. The selected citrus varieties with HLB resistance/tolerance are expected to be the most durable, eco-friendly and cost-effective solution to this devastating disease. The success of this project will set up a model for the citrus community across the globe to follow, and therefore solve the HLB problem.
The goal of this proposal aims to develop new generation of citrus cultivars that not only survive, but are also profitable in the presence of HLB. Huanglongbing (HLB) is presently the most devastating citrus disease worldwide. Because of the extensive spread and a lack of effective control measures for HLB, Florida's $9 billion citrus industry is presently fighting for its survival from the crippling effects of billions of dollars in lost revenue. In recent years, we have observed field resistance or tolerance to HLB in some citrus plants in commercial groves and breeding orchards, providing evidence that there is variation regarding HLB tolerance/resistance in the citrus gene pool. In this proposal, we take advantage of existing genetic variations and deliver HLB tolerant/resistant cultivars with desirable fruit traits through the screening of natural mutant citrus plants or bud sports of commercially grown cultivars with a greater tolerance/resistance. The selected HLB tolerant/resistant citrus variants are expected to be the most durable, eco-friendly and cost-effective solution to the menace of this very devastating disease. We will pursue five interrelated research objectives in this proposal.
Methods 1. Methods for Objective 11.1. Select, propagate and evaluate HLB tolerant/resistant scionMore graft inoculations will be performed on additional volunteer Ruby Red grapefruit seedlings from the Scott grove. The new plants will be grown in pots in the greenhouse located at the US Horticultural Research Laboratory under standard management protocols. Ten propagated plants from each VG plant will be inoculated via graft or psyllid inoculation with grapefruit plants from a commercial nursery serving as controls. The inoculation process will be repeated three times with a total of thirty propagated plants for each VG plant. The tolerance/resistance of individual plants will be assessed by monitoring symptom severity in addition to Las titers. The assessment of the trees will be performed at time 0, 3 months, 6 months, 9 months and 12 months after graft inoculation. The best performing line(s) will then be subjected to field trials. 1.2. Bud sport selection and evaluationWe propose more extensive searches of bud sport and controlled inoculation. In addition to our own searching, we will work with extension agents and growers to collect potential bud sports from commercial citrus varieties. The number of new plants from each sport will be prepared, inoculated, and tested as mentioned above. To accelerate the evaluation of tolerance/resistance, we also propagate the selected bud sticks on HLB-affected and symptomatic Duncan plants (recipient) via bud stick grafting, the resistant/tolerant bud sports should grow out and remain vigorous growth along with low or high Las titers. The sports with greater tolerance/resistance will be integrated into field trials.1.3. Field trial of selected trees for HLB resistanceOnce the most resistant/tolerant plants have been verified and propagated, we will conduct field trials in Florida to ensure the productivity of the selected trees in an area where HLB is endemic. The quantity and quality of the fruit produced during the field trial will also be evaluated by determining the overall fruit drop and conducting routine juice analysis (Brix, acid, percent juice).2. Methods for Objective 22.1. Transcriptome profiling design and experimentsWe will profile the transcriptomes of 30 HLB tolerant/resistant citrus trees and their susceptible siblings before and after HLB infection. All the transcriptomic experiments will be performed with appropriate biological replication.2.2. Bioinformatics and functional genomics analysis of transcriptome dataSequence reads will first be quality checked, then used to construct transcriptome. We will then perform the DE (differentially expressed), alternative isoform difference gene functionality, co-expression network and pathway analyses. 2.3. Experimental verification of differentially expressed genesWe will experimentally verify the expression of genes and isoforms found to be differentially expressed in citrus lines as well as in other HLB tolerant/resistant or susceptible citrus lines.3. Methods for Objective 33.1. Resequencing design and experimentsWe will re-sequence six tolerant/resistant and six susceptible sibling plants and their parents.3.2. Bioinformatics and comparative genomics analysis of re-sequencing dataAfter cleaning the raw reads (remove the low quality reads and adaptors), SNPs, Indels, SVs and CNVs will be identified, annotated and compared. 3.3. Experimental verification of genetic variationsCandidate SNPs, Indels, SVs, CNVs and segment deletions will be validated using PCR followed by sequencing.4. Methods for Objective 44.1. Choose target genes and target sitesWe propose to focus on 6 to 12 genes that are likely involved in HLB susceptibility in sweet orange and grapefruit.4.2. Design gRNAs and construct expression cassettesGuide RNAs (gRNAs) will be designed based on the whole genome sequence of sweet orange in two Citrus genome databases and the grapefruit genomic sequences from Objective 3.4.3. Test the functionality of expression constructs using a transient expression systemExpression cassettes containing gRNAs and other essential components will be first tested to find out if they will function, i.e. induce mutations, in citrus. This will be done using a transient expression system.4.4. Deliver expression constructs into citrus and regenerate mutant plantsAgrobacterium-mediated transformation will be performed using the protocol that has been optimized by Gmitter's team. Complete plantlets will be grown in a growth room and then in a secured greenhouse. 4.5. Validate sequence changes in putative mutant plantsTotal genomic DNA will be isolated from mutant lines and amplified in high fidelity PCRs with specific primers. The amplified products will be cloned and at least 30 randomly selected colonies per construct per citrus line will be sequenced. The sequences will be aligned with the genomic sequences of sweet orange and grapefruit to identify and verify nucleotide deletions, insertions, and/or changes. 4.6 Analyze gene expression levels in mutant plantsTotal RNA will be extracted from mutant and wildtype plants at multiple time points after Las infection. qRT-PCR will be performed on available real-time thermal cycler. The relative quantification of gene expression levels will be calculated.4.7. Assess HLB resistance of mutant plantsMutant lines with confirmed changes in nucleotide sequence and gene expression will be propagated to produce clonal plants for Las inoculation and HLB tolerance/resistance assessment. For each mutant line, two clonal plants will be mock-inoculated with HLB-free buds, and six clonal plants inoculated with HLB-positive buds. Mutant lines with no or mild HLB symptoms will be identified for field testing.4.8. Field testing of mutant plantsMutant lines with confirmed changes in nucleotide sequence and gene expression, and increased HLB tolerance/resistance will be planted in a secure citrus grove (USHRL, Pico's Road Farm). Mutant and wildtype plants will be arranged in the field in six completely randomized blocks and measured for shoot growth and rated for HLB severity as above described. Shoot growth (diameter and length) will be measured every three months, and HLB severity will be rated every three months.5. Methods for Objective 55.1. Organization and engagement of stakeholder advisory panelA stakeholder advisory panel will be organized to provide oversight on the relevancy of progress made by the research team. This panel will include university scientists, industry organization members, growers, and nurserymen. An annual meeting will be held. The panel will evaluate the progress against the proposed milestones of each objective.5.2. Dissemination of project outcomes via print, electronic and social mediaWritten materials will be disseminated via the project website, and other industry websites. In addition, articles and press release will be written and submitted to industry magazines. Project outcomes and events will be shared via Twitter and other social media platforms.5.3. Organization of seminar and workshopsWorking group will be organized for university extension specialists and farm advisors Project team members will be available to speak at local grower seminars and workshops organized by these extension personnel.5.4. Industry meetings and eventsThe outreach team will give presentations in industry organization meeting in each citrus producing state, thereby reaching a large audience of stakeholders.5.5. Field DaysField days will be organized for the growers and nurserymen to judge overall health and vigor of the tree, and evaluate the fruit characteristics and qualities.5.6. Project WebsiteA project website will be developed at the beginning of the project. Specifically, we will have a webpage allow growers to report the variant citrus plants for our team to evaluate.
Target Audience
Nothing Reported
Changes / Problems
Nothing Reported
Training & Professional Development
This is an interdisciplinary project that involves expertise in bioinformatics, citrus breeding, genetics, plant pathology, and extension service. The project has provided researchers in multiple disciplines an unprecedented opportunity to work together to conduct this challenging research. The PIs maintain regular communication/meetings to discuss the progress of their individual projects. The postdoc research associates, graduate students, technicians and students in the different groups can gain knowledge and expertise from members in other groups. The project supports postdocs, graduate students and undergraduates to attend citrus HLB meetings and other professional meetings.
Dissemination Streams
We have disseminated our results to communities of interest through social media, grower meetings, invited talks, and conference presentations. We continue to publicize the project goals and progress at speaking engagements including the monthly meetings of the Texas Citrus Pest and Disease Management Corporation and during engagements with individual growers and industry stakeholders. The team has published research results in prestigious journals, including Nature Communications, Horticulture Research.
Next Reporting Steps
Nothing Reported
Target Audience
Nothing Reported
Changes / Problems
Nothing Reported
Training & Professional Development
This is an interdisciplinary project that involves expertise in bioinformatics, citrus breeding, genetics, plant pathology, and extension service. The project has provided researchers in multiple disciplines an unprecedented opportunity to work together to conduct this challenging research. The PIs maintain regular communication/meetings to discuss the progress of their individual projects. The postdoc research associates, graduate students, technicians and students in the different groups can gain knowledge and expertise from members in other groups. The project supports postdocs, graduate students and undergraduates to attend citrus HLB meetings and other professional meetings.
Dissemination Streams
We have disseminated our results to communities of interest through social media, grower meetings, and conference presentations. We continue to publicize the project goals and progress at speaking engagements including the monthly meetings of the Texas Citrus Pest and Disease Management Corporation and during engagements with individual growers and industry stakeholders. The team has published research results in prestigious journals in the year 2021, including one paper in Nature Communications.
Next Reporting Steps
Based on our accomplishments in the first five years, our goals for the next reporting period will primarily focus on the following aspects: 1) continued screening of HLB resistant/tolerant citrus plants, seedlings and bud sports; 2) continued propagation of selected HLB resistant/tolerant seedlings or bud sports; 3) continued use of RNA-Seq and resequencing to identify genetic variants in selected HLB citrus plants; 4) combine RNA-seq and genome-resequencing to further explore and possibly identify genes or genomic structures leading to the apparent HLB tolerance of the mutant clones 5) continued develop CRISPR expression constructs for three to five additional HLB susceptibility genes (2nd group of target genes); 6) have more field trial in Florida, Texas and California; 7) have a Stakeholder Advisory Committee meeting to obtain feedback on our research progress; 8) continue dissemination of results to communities of interest through social media, grower meeting, conference presentation and the project website. <br><br>
<br>What was accomplished under these goals? The five years of the project have been fruitful. Below are summaries of accomplishments for each objective. Objective 1: We have evaluated 22 lines of volunteer seedlings (Ruby Red grapefruit) selected from Scott groves, Fort Pierce, Florida and 4 Duncan, one pumelo and one sweet seedling selection from US horticultural research laboratory (USHRL) greenhouse via graft inoculations. Propagates from the selected Duncan seedlings and bud sports of these seedlings were further evaluated either via graft inoculation or psyllid inoculation. The progenies (second generation) of the selected tolerant Duncan seedlings were further evaluated for their genetic inheritance of HLB tolerance via psyllid inoculation. The results showed some of these selections were better HLB resistant/tolerant than control or their siblings. Fifteen field selections of bud sports (14 grapefruit of 4 varieties and 10 sweet orange selections from Valencia) were propagated on three different rootstocks and evaluated via graft inoculation or directly grown in USHRL Picos' Research Farm with high HLB disease pressure. In addition, we have had several pomelo bud spots selected from radiation treatments. The results from field trials for 4-6 years indicated that several selections (volunteer seedling or bud sports) were better resistant/tolerant than their parental clones or siblings though all plants were infected by Candidatus Liberibacter asiaticus (Las) at the end of 2020. These selections not only showed their resistance/tolerance but also showed that their fruit quality was equal or better than their mother plants in consecutive two years. The selected sour orange rootstock is as good as the current best rootstock US-942 in terms of HLB disease index, and better fruit quality for scion grapefruits. Another important feature of the selected sour orange limited the tree canopy so that it will be suitable for a high-density planting for combatting HLB endemics. In addition, we have propagated and planted over 1200 grapefruit plants from 8 bud sport/seedling selections back to grower's groves from field trials. All the plantings in the field are under evaluation for their variations of HLB tolerance/resistance. we have also selected HLB-tolerant budsports with better fruit quality, such as higher brix and deeper red color. Four clones of the selected sour orange (rootstock), 4 clones of grapefruit from Scott groves, two clones of red grapefruits and two clones of Valencia sweet orange have gone through clean-up program by the Department of Plant Industry (DPI), Florida, and some of these clean-up selections are being propagated for large scale of field trials. We have added 10 more selections into the DPI cleaning up the pipeline, and are planning to expand our clean-up program for those selections that performed well under both greenhouse and field trials. In general, after several years of research, we have developed a rapid and effective selection and evaluation system for improved HLB resistant/tolerant commercial citrus varieties. We have developed a simple ELSIA evaluation system using our newly developed antibody. Using this system, we revealed the association between the HLB tolerance and levels of the targeted gene expression, and more importantly the uniformity of population within a variety/breeding line. We also observed that a couple of grapefruit bud-sports were not only tolerant to HLB but also resistant to Lasiodiplodia theobromae (Diplodia), and therefore had much less fruit drop. These selections are in the pipeline to release for growers to use without any regulatory constraints. Objective 2: We have carried out comparative transcriptome analysis for several budsport selections, and RT-PCR conformations for the identified differential expression of the genes that are related to HLB tolerance are underway. We performed RNAseq of all accessions and produced mRNA, sRNA and lncRNA sequences on a normal valencia sweet orange tree and three mutants from irradiation. The mRNA was used for annotation of the DVS genome assembly. A high average mapping rate of 96.02% was acquired using our new assembly as reference. A total of 1,749 DEGs (including 116 lncRNA) and 1,814 DEGs (including 148 lncRNA) were identified. The other RNA data sets remain to be analyzed further. Objective 3: We have re-sequenced a normal Valencia sweet orange tree and three mutants from irradiation using Pacibio sequencing. We assembled the normal Valencia tree as our reference. We are able to obtain the diploid genome sequence of Valencia with a total genome size of 607,657,665. We have completed a comparative analysis of genome sequencing data generated by the PACBIO Sequel II system from three mutant Valencia lines (T19, T78, SF-Unknown), and a non-irradiated common Valencia as control. All trees were planted in the field in 2000, but only T19 and SF-Unknown have exhibited HLB-tolerance until the current time. To make objective measures of tolerant versus sensitive tree performance, we measured leaf area index (LAI) using an AccuPAR LP-80 instrument (Meter Group, Pullman, WA, USA) near solar noon in June 2021. On average, 6-7 measurements were taken around each tree. For CLas titer measurements, DNA was extracted from leaf midribs and petioles of each tree. qPCR quantification of the CLas using 16S rRNA primers showed that three irradiated Valencia lines (T19, T78, SF-Unknown) and common Valencia in the field were all infected, and there were no differences in CLas titer. However, we found that LAI of mutant T19 was significantly greater than the common Valencia. Structural variants and candidate genes associated with INDELs identified between the radiation-induced mutants and common Valencia were confirmed by regular PCR. Twenty-three DEGs identified by RNA-seq and associated with HLB tolerance were selected for quantitative real-time PCR (qPCR) verification. Gene expressions of 23 candidate genes indicated that mutant sweet orange T19 had significantly higher expression levels than the control Valencia. Objective 4: Seven knock-out Carrizo citrange and Duncan grapefruit mutants have been selected for carrying frameshift mutations (100%) in one of two targeted genes and high resistance to Xanthomonas. The primary mutations in these knock-outs were deletions of one, two, four, seven or 10 bases, or insertions of one or two bases, resulting in frameshifts in the coding regions near the 5' end of the targeted gene. When these mutants were inoculated with Xanthomonas citri ssp. citri (Xcc), canker lesion sizes were reduced by 90% to 99% compared to wildtype Carrizo or Duncan grapefruit. Xcc bacterial cell populations were reduced by more than 99% compared to wildtype. A number of mutants were generated for the third candidate gene, and these mutants were assessed for resistance to Xcc as well. Interestingly, these mutants showed increased susceptibility to Xcc. Experiments are ongoing to assess the resistance of these mutants to citrus greening. Another major effort in 2021 has been to generate new mutants. Gene editing constructs were made to edit the fourth candidate gene. GFP-positive shoots have emerged. These shoots will be micro-grafted to produce complete plantlets. Several double mutants have been generated and established in containers. Experiments are ongoing to determine the type and frequency of mutations in these double mutants. Objective 5: An annual project progress meeting of the team was organized and held via Zoom on April 29, 2021. The meeting afforded each of the participating labs to give presentations on their research progress and plans. A Tweet of the meeting was posted on April 29, 2021 and has so far received 1,470 impressions. Updates on the efforts of the team and progress made were also provided at the business meetings of the Texas Citrus Pest and Disease Management Corporation. <br><br><b>Publications</b><br>
Target Audience
Nothing Reported
Changes / Problems
Dr. Marylou Polek retired in 2020 and now Dr. John E. Preece is the co-PI from USDA California.
Training & Professional Development
This is an interdisciplinary project that involves expertise in bioinformatics, citrus breeding, genetics, plant pathology, and extension service. The project has provided researchers in multiple disciplines an unprecedented opportunity to work together to conduct this challenging research. The PIs maintain regular communication/meetings to discuss the progress of their individual projects. The postdoc research associates, graduate students, technicians and students in the different groups can gain knowledge and expertise from members in other groups. The project supports postdocs, graduate students and undergraduates to attend citrus HLB meetings and other professional meetings.
Dissemination Streams
We continue to publicize the project activities through remotely held interactions with growers and citrus industry stakeholders. A project related Tweet posted on March 17, 2020 received 241 impressions and 11 engagements.
Next Reporting Steps
Based on our accomplishments in the first four years, our goals for the next reporting period will primarily focus on the following aspects: 1) continued screening of HLB resistant/tolerant citrus plants, seedlings and bud sports; 2) continued propagation of selected HLB resistant/tolerant seedlings or bud sports; 3) continued use of RNA-Seq and resequencing to identify genetic variants in selected HLB citrus plants; 4) combine RNA-seq and genome-resequencing to further explore and possibly identify genes or genomic structures leading to the apparent HLB tolerance of the mutant clones 5) continued develop CRISPR expression constructs for three to five additional HLB susceptibility genes (2nd group of target genes); 6) have more field trial in Florida, Texas and California; 7) have a Stakeholder Advisory Committee meeting to obtain feedback on our research progress; 8) continue dissemination of results to communities of interest through social media, grower meeting, conference presentation and the project website. <br><br>
<br>What was accomplished under these goals? The first four years of the project have been fruitful. Below are summaries of accomplishments for each objective. Objective 1: We have evaluated 22 lines of volunteer seedlings (Ruby Red grapefruit) selected from Scott groves, Fort Pierce, Florida and 4 Duncan seedling selections from US horticultural research laboratory (USHRL) greenhouse via graft inoculations. Propagates from the selected Duncan seedlings and bud sports of these seedlings were further evaluated either via graft inoculation or psyllid inoculation. The progenies (second generation) of the selected tolerant Duncan seedlings were further evaluated for their genetic inheritance of HLB tolerance via psyllid inoculation. Fifteen field selections of bud sports (14 grapefruit of 4 varieties and 10 sweet orange selections from Valencia) were propagated on three different root stocks and evaluated via graft inoculation or directly grown in USHRL Picos' Research Farm with high HLB disease pressure. In addition, we have had a number of pomelo bud spots selected from radiation treatments. The results from field trials for 3-5 years indicated that several selections (volunteer seedling or bud sports) were better resistant/tolerant than their parental clones or siblings though all plants were infected by HLB at the end of 2020. These selections not only showed their resistance/tolerance but also showed that they fruit quality were equal or better than their mother plants in consecutive two years. The selected sour orange root stock is as good as the current best root stock US-942 in terms of HLB disease index, and better fruit quality for scion grapefruits. Another important feature of the selected sour orange limited the tree canopy, so that it will be suitable for a high-density planting for combatting HLB endemics. In addition, we have propagated and plants over 1200 grapefruit plants from 8 bud sport/seedling selections back to grower's groves from field trials. All the plantings in the field are under evaluations for their variations of HLB tolerance/resistance. Four clones of the selected sour orange (rootstock), 4 clones of grapefruit from Scott groves, two clones of red grapefruits and two clones of Valencia sweet orange have gone through clean-up program by Department of Plant Industry (DPI), Florida, and some of these clean-up selections are being propagated for large scale of field trials. We have added 6 more selections into the DPI cleaning up pipeline, and are planning to expand our clean-up program for those selections that performed well under both greenhouse and field trials. Objective 2: We have carried out comparative transcriptome analysis for several budsport selections, and RT-PCR conformations for the identified differential expression of the genes that related to HLB tolerance are underway. Moreover, transcriptome (including both mRNA and lncRNA) profiling was carried out on a normal valencia sweet organe tree and three mutants from irradiation. A high average mapping rate of 96.02% was acquired using our new assembly as reference. A total of 1,749 DEGs (including 116 lncRNA) and 1,814 DEGs (including 148 lncRNA) were identified. Objective 3: We have re-sequenced a normal Valencia sweet orange tree and three mutants from irradiation using Pacibio sequencing. We assembled the normal Valencia tree as our reference. We obtain the diploid genome sequence of Valencia with total genome size of 607,657,665. 3 mitochondria contigs and 2 plastid contigs were further connected to form intact mitochondria and plastid genome. Most nuclear genomic regions (~ 601 Mbp) were assembled into haplo-contigs (already phased contigs), leaving only 42 diploid regions, adding up to a total length of ~ 6 Mbp. These diploid regions were re-assembled and phased by Falcon and Falcon-Unzip. 28 diploid regions were successfully phased to recover two haplotypes, and the rest 14 diploid regions were partially phased, leaving 3,873,743 diploid regions in the final assembly. Then, we assembled three mutant Valencia genomes and aligned them onto normal Valencia reference genome. We detected a total of 3,068 structure variation in the mutants. The 3,068 SVs from assembly comparison were further screened for high-quality ones according to their read support in corresponding SVs identified by mapping based method. As a result, 2,464 were filtered due to no or poor (<br><b>Publications</b><br>
Target Audience
* monthly meetings of the Texas Citrus Pest and Disease Management Corporation and during engagements with individual growers and industry stakeholders. * Joint 21st Conference of the International Organization of Citrus Virologists (IOCV) and the 6th International Research Conference on Huanglongbing (IRCHLB). * Stakeholders will be able to visit and assist in the evaluation of the field plots during events such as Citrus Day, California Citrus Nursery Society field tours and walk-throughs.
Changes / Problems
Nothing Reported
Training & Professional Development
This is an interdisciplinary project that involves expertise in bioinformatics, citrus breeding, genetics, plant pathology, and extension service. The project has provided researchers in multiple disciplines an unprecedented opportunity to work together to conduct this challenging research. The PIs maintain regular communication/meetings to discuss the progress of their individual projects. The postdoc research associates, graduate students, technicians and students in the different groups can gain knowledge and expertise from members in other groups. The project supports postdocs, graduate students and undergraduates to attend citrus HLB meetings and other professional meetings.
Dissemination Streams
We have disseminated our results to communities of interest through social media, grower meetings, and conference presentations. The Advisory Committee Progress Report Meeting was held during the sixth International Research Conference on Huanglongbing (IRCHLB VI). We continue to publicize the project goals and progress at speaking engagements including the monthly meetings of the Texas Citrus Pest and Disease Management Corporation and during engagements with individual growers and industry stakeholders. We have continuously publicized the project activities through social media, project website and grower and scientific meetings. Mulitple posters were presented at the Joint 21st Conference of the International Organization of Citrus Virologists (IOCV) and the 6th International Research Conference on Huanglongbing (IRCHLB) held March 10-16, 2019 at Riverside, California. A website about the project was developed and launched: http://www.bigdata.clemson.edu/citrus/home.html.
Next Reporting Steps
Based on our accomplishments in the first three years, our goals for the next reporting period will primarily focus on the following aspects: 1) continued screening of HLB resistant/tolerant citrus plants, seedlings and bud sports; 2) continued propagation of selected HLB resistant/tolerant seedlings or bud sports; 3) continued use of RNA-Seq and resequencing to identify genetic variants in selected HLB citrus plants; 4) combine RNA-seq and genome-resequencing to further explore and possibly identify genes or genomic structures leading to the apparent HLB tolerance of the mutant clones 5) continue to develop CRISPR expression constructs for three to five additional HLB susceptibility genes (2nd group of target genes); 6) have more field trial in Florida, Texas and California; 7) have a Stakeholder Advisory Committee meeting to obtain feedback on our research progress; 8) continue dissemination of results to communities of interest through social media, grower meeting, conference presentation and the project website. <br><br>
<br>What was accomplished under these goals? The first three years of the project have been fruitful. Below are summaries of accomplishments for each objective. Objective 1: We have evaluated 22 lines of volunteer seedlings (Ruby Red grapefruit) selected from Scott groves, Fort Pierce, Florida and 4 Duncan seedling selections from US horticultural research laboratory (USHRL) greenhouse via graft inoculations. Propagates from the selected Duncan seedlings and bud sports of these seedlings were further evaluated either via graft inoculation or psyllid inoculation. The progenies (second generation) of the selected tolerant Duncan seedlings were further evaluation for their genetic inheritance of HLB tolerance via psyllid inoculation. Fifteen field selections of bud sports (14 grapefruit of 4 varieties and 10 sweet orange selections from Valencia) were propagated on three different root stocks, and evaluated via graft inoculation or directly grown in USHRL Picos Research Farm with high HLB disease pressure. In addition, we have had a number of pomelo bud spots selected from radiation treatments. The results from field trials for 2-4 years indicated that several selections (volunteer seedling or bud sports) were better resistant/tolerant than their parental clones or siblings. These selections not only showed their resistance/tolerance but also showed that they fruit quality were equal or better than their mother plants. The selected sour orange root stock is as good as the current best root stock US-942 in terms of HLB disease index, and better fruit quality for scion grapefruits. In addition, we have propagated and plants over 1200 grapefruit plants from 8 bud sport/seedling selections back to grower's groves from field trials. All the plantings in the field are under evaluations, and variations of HLB tolerance/resistance. Four clones of the selected sour orange (rootstock), 4 clones of grapefruit from Scott groves, two clones of red grapefruits and two clones of Valencia sweet orange have gone through clean-up program by Department of Plant Industry (DPI), Florida, and all the cleared materials are ready for further propagations for large scale of field trials. We are planning to expand our clean-up program for those selections that performed well under both greenhouse and field trials. Objective 2: We have carried out comparative transcriptome analysis for several budsport selections, and RT-PCR conformations for the identified differential expression of the genes that related to HLB tolerance are underway. Moreover, transcriptome (including both mRNA and lncRNA) profiling was carried out on a normal valencia sweet organe tree and three mutants from irradiation. A high average mapping rate of 96.02% was acquired using our new assembly as reference. A total of 1,749 DEGs (including 116 lncRNA) and 1,814 DEGs (including 148 lncRNA) were identified. Objective 3: We have re-sequenced a normal Valencia sweet orange tree and three mutants from irradiation using Pacibio sequencing. We assembled the normal Valencia tree as our reference. We are able to obtain the diploid genome sequence of Valencia with total genome size of 607,657,665. 3 mitochondria contigs and 2 plastid contigs were further connected to form intact mitochondria and plastid genome. Most nuclear genomic regions (~ 601 Mbp) were assembled into haplo-contigs (already phased contigs), leaving only 42 diploid regions (regions where reads from both homologous chromosomes were assembled into one contig) adding up to a total length of ~ 6 Mbp. These diploid regions were re-assembled and phased by Falcon and Falcon-Unzip. 28 diploid regions were successfully phased to recover two haplotypes, and the rest 14 diploid regions were partially phased, leaving 3,873,743 diploid regions in the final assembly. A majority (3, 164, 294) of the left diploid regions belong to a single diploid region located at one end of Chromosome 2. Then, we assembled three mutant Valencia genomes and aligned them onto normal Valencia reference genome. We detected a total of 3,068 structure variation in the mutants. The 3,068 SVs from assembly comparison were further screened for high-quality ones according to their read support in corresponding SVs identified by mapping based method. As a result, 2,464 were filtered due to no or poor (<br><b>Publications</b><br>
Target Audience
Huanglongbing (HLB) is presently the most devastating citrus disease worldwide. Because of the extensive spread and a lack of effective control measures for HLB, Florida's $9 billion citrus industry has experienced a decline in production of nearly 75%. As a result, the industry is presently fighting for its survival. The other major citrus states are at high risk for a similar disastrous situation if suitable remedies are not forthcoming soon. Neither a simple cure nor successful management strategies have been identified even after 10+years of the disease presence in Florida. Previous efforts to create new HLB tolerant/resistant cultivars focused on conventional breeding or development of transgenic plants. Unfortunately, the approaches used in those projects are inadequate to meet the current crisis because of the lack of sources of resistance in citrus and time constraints associated with citrus breeding. Furthermore, genetic engineering presents many regulatory and public acceptance obstacles for transgenic citrus. On the other hand, more than 10 years of HLB and tens of millions of infected trees in groves across Florida provide an unprecedented opportunity and high probability to select resistant natural mutants. Field resistance or tolerance to HLB has already been noted amongst the citrus cultivars and their relatives. This provides circumstantial evidence that natural variation exists regarding HLB tolerance/resistance within the citrus gene pool. Exploitation of this natural tolerance/resistance may provide a key solution, within a short time frame, to the survival of the citrus industry, especially in Florida. This project addresses the "Development of tolerance or resistance in commercial citrus in all production areas with a focus on delivery of new cultivars [or rootstocks and scions] using all available strategies", which is one of the priorities set forth by the Citrus Disease Sub-committee. In this proposal, we present a systematic approach with emphasis on skillful selection of natural mutants to develop new HLB tolerant/resistance citrus cultivars. First, we will select variant seedlings and bud sports with greater HLB tolerance/resistance in citrus groves across Florida and test them in field trials. With the obtained HLB tolerant/resistant bud sports/variant seedlings and their susceptible siblings, we will be able to apply transcriptome profiling and comparative genomics to identify HLB tolerance/resistant or susceptibility related genes. Moreover, with the HLB susceptibility related genes identified in our preliminary study and this project, we will develop new resistant varieties via gene editing using CRISPR-Cas9 on the identified target genes. The CRISPR engineered citrus plants do not include foreign genes and are currently not restricted in the US. Meanwhile, we will conduct active outreach and extension to disseminate our project results to growers, stakeholders and the public. The successful achievement of our goals and objectives will broadly impact the entire US citrus industry, by maintaining the viability of industries not yet fully impacted as in Florida, and by providing the plant materials that will encourage Florida growers to replant with confidence. The combined economic impacts of our US citrus industries will be measured in billions of dollars. The many communities throughout the country having economies built around viable citrus production will avoid the major disturbances that will come from the economic disasters that HLB incurs. Current approaches for HLB management are based on an over-reliance of chemical insect control, and more recently the widespread use of antibiotic compounds; the environmental impacts of these practices may prove to be unacceptable in the long run, and resistant cultivars can mitigate such damage. The selected citrus varieties with HLB resistance/tolerance are expected to be the most durable, eco-friendly and cost-effective solution to this devastating disease. The success of this project will set up a model for the citrus community across the globe to follow, and therefore solve the HLB problem.
Changes / Problems
Nothing Reported
Training & Professional Development
This is an interdisciplinary project that involves expertise in bioinformatics, citrus breeding, genetics, plant pathology, and extension service. The project has provided researchers in multiple disciplines an unprecedented opportunity to work together to conduct this challenging research. The PIs maintain regular communication/meetings to discuss the progress of their individual projects. The postdoc research associates, graduate students, technicians and students in the different groups can gain knowledge and expertise from members in other groups. The project supports postdocs, graduate students and undergraduates to attend citrus HLB meetings and other professional meetings.
Dissemination Streams
We have disseminated our results to communities of interest through social media, grower meetings, and conference presentations. The Advisory Committee Progress Report Meeting was held Nov. 14-15, 2018 at the Indian River Research & Education Center, Fort Pierce, FL. Each investigator provided updates about the research/outreach progress. We continue to publicize the project goals and progress at speaking engagements including the monthly meetings of the Texas Citrus Pest and Disease Management Corporation and during engagements with individual growers and industry stakeholders. The project was also mentioned at invited talks such as one given at the 2018 Agriculture & Applied Economics Association (AAEA) Annual Meeting held Aug. 5-7, 2018 at Marriott Wardman Park, Washington, D.C., U.S.A. on the topic "HLB in Texas: Current Situation, Implications, Lessons Learned, and Economic Modeling". We utilize social media platforms for providing visibility to the project and team members. For instance, a photograph of the project team that was taken during the Advisory Committee Progress Report meeting and tweeted on November 15, 2018 made 82 impressions (i.e. number of times people viewed the Tweet on Twitter) and got 2 engagements (i.e. number of times people interacted the Tweet on Twitter). An outreach poster entitled "Developing tolerance/resistance in citrus cultivars to HLB" (Poster number IRCHLB-P14-150) was presented at the Joint Conference of the International Organization of Citrus Virologists (IOCV XXI) and the International Research Conference on Huanglongbing (IRCHLB VI) held March 10-15, 2019 at Riverside, California, U.S.A.; with all the project team members as co-authors. A website about the project was developed and launched: http://www.bigdata.clemson.edu/citrus/home.html.
Next Reporting Steps
Based on our accomplishments in the first two years, our goals for the next reporting period will primarily focus on the following aspects: 1) continued screening of HLB resistant/tolerant citrus plants, seedlings and bud sports; 2) continued propagation of selected HLB resistant/tolerant seedlings or bud sports; 3) continued use of RNA-Seq and resequencing to identify genetic variants in selected HLB citrus plants; 4) combine RNA-seq and genome-resequencing to further explore and possibly identify genes or genomic structures leading to the apparent HLB tolerance of the mutant clones 5) continued production of more stable transformants for DMR6-like gene and identification of sweet orange and grapefruit mutants with modified DMR6-like genes (1st group of CRISPR-induced mutants); 6) start to develop CRISPR expression constructs for three to five additional HLB susceptibility genes (2nd group of target genes); 7) have more field trial in Florida, Texas and California; 8) have a Stakeholder Advisory Committee meeting to obtain feedback on our research progress; 9) continue dissemination of results to communities of interest through social media, grower meeting, conference presentation and the project website. <br><br>
<br>What was accomplished under these goals? The first two years of the project have been fruitful. Below are summaries of accomplishments for each objective. Objective 1: We have evaluated 22 lines of volunteer seedlings (Ruby Red grapefruit) selected from Scott Groves, Fort Pierce, Florida and 4 Duncan seedling selections from the US Horticultural Research Laboratory (USHRL) greenhouse via graft inoculations. Propagations from the selected Duncan seedlings and bud sports of these seedlings were further evaluated either via graft inoculation or psyllid inoculation. Fifteen field selections of bud sports were propagated on different rootstocks and evaluated via graft inoculation or directly grown in the USDA research farm under high HLB disease pressure. In addition, we have selected several pummelo bud sports and two Valencia clones from radiation treatments. The results indicated that several selections (seedling or bud sports) were more tolerant than their parental clones or siblings. Four clones of the selected sour orange (rootstock), 4 clones of grapefruit from Scott Groves, two clones of red grapefruit and two clones of Valencia sweet orange were sent to the Department of Plant Industry (DPI), Florida for cleaning up, and all the four sour orange were cleared and received back at the USHRL greenhouse. These selected clones will serve as future field trial sources. Concurrently, we have planted ca. 1,000 propagations from these selected seedlings or bud sports on three different rootstocks in the USHRL Picos farm, ranging from 8 - 36 months old. Some of the selected plants look promising because of their very low infection rates or apparent tolerance. In addition to field evaluations being conducted in our research farm, over 150 propagations of 4 bud sport selections of different grapefruit are also being evaluated in the grower's grove. All the plantings in the field are being monitored for variations in their HLB tolerance/resistance, and we have observed differences in their disease response. It is important to note that different isolates of the HLB bacterium cause very different disease index both in the greenhouse and in the fields. Additional propagations on different rootstocks have been budded and grown in the greenhouse and are expected to be planted in two growers' groves within 3 months. Objective 2: We have used genomic resequencing, RNA-Seq and metabolomics to analyze three tolerant sweet orange lines identified in field plantings of the UF-CREC breeding program. These include an unnamed late maturing sweet orange clone (Kansu), and two Valencia clones (T19 and T78) derived from irradiation experiments. In total 3,105 differentially expressed genes (DEGs) were detected. Sub-network enrichment analysis revealed that the genes involved in defense response, plant defense, and hypersensitive response were highly expressed in the tolerant oranges. Some plant defense and defense response genes identified in T19 are also involved in the regulation of flower development. The results may help to explain the observed irregular flowering time in tolerant irradiated sweet orange. Global metabolomics profiling was performed on leaf extracts of Valencia (control) and three of its tolerant clones (T78, T19 and Kansu). Preliminary results suggested an abundance of some amino acids involved in the shikimate pathway and the phenylpropanoid cycle, which are implicated in plant defense, in the tolerant varieties. Transverse sections of the leaf lamina and midrib were examined. The result indicates that phloem regeneration may be one important aspect of HLB tolerance. To reveal the underlying mechanisms of the thornless mutant related to HLB tolerance, we compared the transcriptome of leaves of wild-type (W), Thorny (T) and Thornless (TL) HLB tolerant mutants. Significantly more genes (3,541) were differentially expressed between T and W than between TL and W (1,660 genes). These results suggested that the enhanced tolerance of T and TL could have been derived either from the pathways altered in both mutants, or from the up-regulation of different defense response genes in T and TL. Objective 3: We have re-sequenced the genomes of two HLB-tolerant pummelo trees using PacBio SMRT sequencing. One of the pummelo trees is thornless and the other is thorny. Both trees were propagated from the same HLB tolerant/resistant branch. We obtained 30X coverage of PacBio reads for each tree. A total of 1,241,138 and 1,418,586 putative SNVs and indels were identified in T and TL, respectively, of which 831,660 variations were specific to TL. And 9,840 putative SVs including 7,590 insertions and 2,250 deletions were specific to TL. In order to understand the genome variations in citrus bud sports, we re-sequenced 10 buds of one Duncan tree with HLB resistance/tolerance. We are currently performing bioinformatics analysis to understand the variations. Objective 4: We edited the DMR6 gene in 'Duncan' grapefruit and 'Carrizo' citrange. In citrus, the DMR6 gene was up-regulated after CLas infection, as revealed in multiple transcriptome profiling and gene expression analyses. We designed two gRNAs from a sweet orange DMR6 and cloned the gRNAs into a pCas9 plasmid that carries the spCas9 gene and a fusion green fluorescence protein (GFP) and neomycin phospho-transferase (nptII) gene. The plasmid was transformed into EHA101 Agrobacterium strain; citrus epicotyl segments were used for Agrobacterium-mediated transformation. Twenty-five lines (17 Carrizo and 8 grapefruit) were regenerated, micro-grafted onto rootstock, and are being grown in the greenhouse. Leaves were sampled from all the lines and pooled into five groups (five lines per group) for next-generation sequencing. Four of the pooled samples showed mutations at the DMR6 gene. Individual lines are being analyzed to determine the types and frequencies of nucleotide changes. To increase the efficiency of the current CRISPR/Cas9 system, we tested two sources of Cas9 protein, two types of citrus tissue, and heat treatments on the type and frequencies of CRISPR induced mutation. The tests were performed using a newly developed transient expression system and the PDS (phytoene desaturase) gene. Results showed that SpCas9 induced higher frequencies of mutation than the high-fidelity spCas9. Mutations were detected in citrus leaf and callus tissues 96 and 144 hours, respectively after Agrobacterium infiltration. Heat treatment at 37?C for 48 hours increased the gene mutation efficiency by several folds. Objective 5: We have a Stakeholder Advisory Committee meeting to hear the progress by the scientific team in November 2018. During 2018, a land request was submitted to the University of California, Riverside to establish a field evaluation plot. A field has been assigned and land preparation has begun. Rootstock materials have been generated and are being maintained within protective structures. Candidate plant materials developed by the Florida research teams of Gmitter (UFL) and Duan (USDA-ARS) will be sent to California to investigate their potential for use in California citrus production. Because of federal and state quarantine regulations, the potentially resistant/tolerant trees developed in Florida must go through a budwood clean up and testing process first, by the Florida Department of Plant Industry (FL DPI) and second, by either the USDA-ARS National Clonal Germplasm Repository for Citrus & Dates (NCGRCD) or the Citrus Clonal Protection Program (CCPP). Some materials (developed by Gmitter) are expected to be released by the CCPP early in 2019. Another batch of materials (developed by Duan) are currently undergoing the sanitation process at the FL DPI. Once scion materials have been released from quarantine status, they will be grafted onto rootstocks and planted in the field evaluation plot. We have continuously publicized the project activities through social media, project website and grower and scientific meetings. <br><br><b>Publications</b><br>
Target Audience
Citrus growers, crop consultants, county extension agents, extension and research faculty, agroindustry, regulators, and residents.
Changes / Problems
Nothing Reported
Training & Professional Development
This is an interdisciplinary project that involves expertise in bioinformatics, citrus breeding, genetics, plant pathology, and extension service. The project has provided researchers in multiple disciplines an unprecedented opportunity to work together to conduct this challenging research. The PIs maintain regular communication/meetings to discuss the progress of their individual projects. The postdoc research associates, graduate students, technicians and students in the different groups can gain knowledge and expertise from members in other groups. The experience and knowledge obtained in the first year will facilitate the research in second year and beyond.
Dissemination Streams
We have disseminated our results to communities of interest through social media, grower meeting, and conference presentation. A photograph of the project team was tweeted on March 15, 2017. The Tweet made 104 impressions (i.e. number of times people viewed the Tweet on Twitter). The project was publicized at the monthly meeting of the Texas Citrus Pest & Disease Management Corporation. Participants at these meetings include growers and other citrus industry stakeholder. The project was also mentioned during an invited keynote address on HLB that Dr. Olufemi Alabi gave at the 2nd Annual Materials Innovation for Sustainable Agriculture Symposium held Nov. 6-7, 2017 at the University of Central Florida, Orlando, FL. In Florida, PIs communicated with growers on a weekly basis, and updated the Florida Citrus Research and Development Foundation production committee members with the results. The PI Feng Luo has given a report presentation at the Citrus Disease Subcommittee meeting in Ft. Pierce, Florida on January 24 and 25, 2018.
Next Reporting Steps
Based on our accomplishments in the first year, our goals for the next reporting period will primarily focus on the following aspects: 1) continued screening of HLB resistant/tolerant citrus plants, seedlings and bud sports; 2) continued propagation of selected HLB resistant/tolerant seedlings or bud sports; 3) continued use of RNA-Seq and resequencing to identify genetic variants in selected HLB citrus plants; 4) continued production of more stable transformants for DMR6-like gene and identification of sweet orange and grapefruit mutants with modified DMR6-like genes (1st group of CRISPR-induced mutants); 5) start to develop CRISPR expression constructs for three to five additional HLB susceptibility genes (2nd group of target genes); 6) have a Stakeholder Advisory Committee meeting to obtain feedback on our research progress; 7) continue disseminating our results to communities of interest through social media, grower meeting, and conference presentation. <br><br>
<br>What was accomplished under these goals? The first year of our project has been fruitful. Here, we summarized our accomplishment for each objective. Objective 1: Select variant citrus plants or bud sports with HLB tolerance/resistance. We have evaluated 22 lines of volunteer Ruby Red grapefruit seedlings selected from Scott groves, Fort Pierce, Florida and 4 Duncan seedling selections from the US Horticultural Research Laboratory (USHRL ) greenhouse via graft inoculation. Propagations from the selected Duncan seedlings and their bud sports were further evaluated either via graft or psyllid inoculation. Twelve bud sports (11 grapefruit of 4 varieties and one sweet orange variety) were propagated on different rootstocks and evaluated via graft inoculation or directly grown in a field with high HLB disease pressure. In addition, two pummelopummelo bud sports and two Valencia clones were selected from radiation treatments. The results indicated that several selections (seedlings or bud sports) showed enhanced HLB tolerance compared with the parental clones or siblings. We have submitted 4 clones of the selected sour orange (rootstock), 4 clones of grapefruit from Scott groves and 2 clones of sweet orange to the Department of Plant Industry (DPI), Florida for shoot tip grafting (STG) and subsequent indexing and certification of pathogen testing status. Once completed, these promising clones will be available for propagation of trees for future field trials. Concurrently, we have planted 600 trees propagated from these selected seedlings or bud sports on three different rootstocks in the USHRL Picos farm. These trees are 2 to 18 months old. Preliminary assessments determined a very low infection rate among the 18 months old grapefruit trees. We are encouraged by these results. There are additional bud sports that will be ready to plant in a few months in the greenhouse. There are 110 Hamilton (Group 1), 90 Hamilton (Group 2), and 4 groups from Blue Goose totaling 198, all on R7T6, US-942, MM SO, or Rucks SO rootstocks. We are evaluating four bud sports selection from Packers with graft inoculation. All of these selections show better HLB tolerance than control grapefruit. Objective 2: Identify citrus HLB tolerance/resistance-related genes for potential citrus genome editing targets through transcriptome profiling. We have used RNA-Seq methodology with three tolerant sweet orange clones identified in field plantings of the UF-CREC breeding program. These include an unnamed late maturing sweet orange clone, and two Valencia clones derived from irradiation as part of a previous mutation breeding project. These three mutant clones were compared to standard HLB-sensitive Valencia orange trees growing in the same field locations in order to explore the underlying mechanisms leading to their apparent HLB tolerance. All trees were planted in the field 13 years ago, and three individual tree replications of each clone were sampled. Four mature leaves were collected randomly from every tree for characterization of CLas population levels by qPCR. Other healthy mature leaves were sampled for RNA extraction. mRNA was sequenced using the Illumina HiSeq platform. The reads were mapped to the Clementine genome V1.0, and the associated gene annotation (C. clementina 182) was used. Reads were also mapped to the CLas genome. Differentially expressed genes (DEGs) were identified using DESeq. Significant DEGs were selected based on a p-value <br><b>Publications</b><br>