This is the end of the second year plus a 6 month NCE for a currently funded multi-investigator, multi-institution project. Although many parts of this research were successful, it cannot be continued in its present form. The USDA group, who was receiving almost 50% of the funding, does not want to continue for a third year. The post-doc who was working on the project has left and it is difficult to get a new post-doc, particularly in Ft. Pierce, when only a year is left on the project. The group has published a paper on their successful efforts (Marutani-Hert, Mizuri, Evens, Terence,McCollum, Gregory, and Niedz, Randall. 2011. Bud emergence and shoot growth from mature citrus nodal stem segments. Plant Cell, Tissue and Organ Culture 106:81-91). The Moore laboratory is also making good progress on the use of cell penetrating peptides to get molecules into citrus without having to use Agrobacterium. In the past 6 months, we have been doing successful experiments with DNA as well as proteins. This is far from a mature technology but shows. promise.
This project sought the development of in vitro regeneration techniques for Murraya paniculata, a presumed host plant citrus relative highly favored by psyllids; these regeneration methods were then to be used to attempt first the genetic transformation of Murraya with marker genes, to optimize the transformation protocol. If successful, then insecticidal or psyllid-suppressive gene construct could be introduced. The ultimate objective was to attempt the development of a deadly trap plant for psyllids that could be deployed in citrus groves to potentially decrease psyllid populations and consequent inoculum potential. Further, such deadly trap plants could be used in the urban landscape to decrease the reservoir of uncontrolled CLas inoculum from commercial or residential areas impacting nearby citrus production areas. We were successful in developing a reasonably efficient regeneration protocol for Murraya via organogenesis, with defined levels of hormone and growth regulator supplementation as well as appropriate plant tissue management and handling techniques; a manuscript on this work is under preparation, the first ever report of in vitro regeneration of this citrus relative. We struggled, however, with the objective of achieving successful genetic transformation. One bottleneck was the unavailability of a reliable source of abundant and viable seed sources necessary to initiate the large-scale experiments that we wanted to conduct. Despite this, we explored various parameters for genetic transformation of Murraya, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments were conducted after testing various plasmids and Agrobacterium strains. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were likewise tested, and we established a standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth was monitored, as well as the stability and uniformity of GFP expression over time. Nearly all of these transformation events proved to be either chimeric or transient, so further production of new transgenic events was pursued. Though the project has ended, we have shared our results with ctrus transformation experts, and the work is continuing in collaboration now with the Core Citrus Transformation Facility at the UF-CREC, to attempt to further refine and improve our abilities to transform Murraya, and perhaps ultimately to produce, test, and deploy the deadly trap plants we aimed to develop, to test their value and utility as part of integrated approaches to manage HLB disease in Florida citrus.
More than two thousand transgenic plants have been produced containing various combinations of natural or synthetic genes and promoters; many of these are currently in field trial locations, greenhouse tests, or still being grown off. Additional transgenic plants have been propagated for new hot psyllid greenhouse tests, and for field planting. New candidate genes have been identified from citrus and other plants for HLB and canker resistance; vectors have been constructed for new rounds of transgenic plant production. Citrus-specific promoters, transcription factors, and other genetic elements have been identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. The sweet orange citrus genome sequence was mined to identify genes controlling anthocyanin expression, in an effort to develop visual, citrus-derived markers for genetic transformation. Several candidates genes were characterized and modified by in vitro sequence alteration techniques, and these will be tested first in grape and then citrus. More than 875 transgenic plants have now been planted with a collaborator in Martin county, and these are being monitored regularly, along with a second site in Indian River county. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests. New rootstock trials of advanced selections (> 15,000 trees) were planted, to assess their adaptation to advanced citrus production systems; data have been collected on their early performance. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests; rootstock hybrids are showing diverse responses when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance; four complex tetraploid rootstocks have shown some repression of HLB in greenhouse tests (one was symptom-free up to 22 months ). We continued the program to rotate new germplasm (rootstock and transgenic) through a ‘hot psyllid’ house to ensure HLB inoculation prior to approved field planting; two groups of 50 trees have been rotated through so far, scheduled for planting at a collaborators field site, under permit from DPI. A new hot greenhouse site, with SG Citrus, is now being used with >340 transgenic plants grown there in replication. More than 150 new rootstock candidates that produce nucellar seedlings have been identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora, and have been used to produce new trees for pending rootstock trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. Final data have been collected from a field trial of various Valencia somaclones and seedless Midsweet selections, and following final analysis the most consistently high yielding clones from each will be moved forward for release; most candidates have already moved through the DPI-Parent Tree Program. New pummelo-grapefruit seedless hybrids have been selected, some showing field tolerance to canker; their fruit have been assayed for furanocoumarin content and several with good fruit quality have been found FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Patents have been issued by the US-PTO for Valquarius (SF14W-62) and Valenfresh (N7-3), very early- and late- maturing Valencia selections respectively, and licensing is in process. Patent applications and documents for release were developed for 7 new cultivars, and these were approved by the UF-IFAS Cultivar Release Committee for release and commercialization according to UF-IFAS policy.
We continue to monitor HLB and canker resistance or tolerance among the more than two thousand transgenic lines we have produced thus far, in field trials (2 locations under permit) and in greenhouse tests at the CREC and with grower-collaborators. These transgenic lines contain various combinations of natural or synthetic genes and promoters. New candidate genes continue to be identified and cloned into vectors for testing. Additional citrus-specific promoters, transcription factors, and other genetic elements are being identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. A gene controlling anthocyanin expression in citrus plants (CMybA1) has been identified by mining the sweet orange citrus genome sequence and has been ‘repaired’ by in vitro sequence modification, and shown to be effective in transgenic grapevines; tests in citrus are underway. If successful, this element can be used as a natural citrus-derived marker fir genetic transformation, thereby eliminating the use of antibiotic or GFP genes, and increasing the potential acceptance of transgenic citrus products by consumers. More than 875 transgenic plants in a collaborators field site in Martin County are being monitored regularly, along with a second site in Indian River County. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests, including some containing a broad spectrum, ancient disease resistance gene from rice; the latter are being propagated for HLB challenge. Data are being collected on the early performance of new advanced selections in trials planted to assess adaptation to advanced citrus production systems. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests. Diverse responses of rootstocks are being noted when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance. Greenhouse experiments are underway examining interactions of rootstock and nutrients in severity of symptom expression. A new hot psyllid greenhouse facility is now being used with >340 transgenic plants grown there in replication, and infection rates are being determined by symptom expression and qPCR; the materials being tested represent our most advanced genetic constructs with phloem-limited promoter sequences and previously proven genes. More than 150 new rootstock candidates preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora, and have been used to produce new trees that were planted into new rootstock trials, or held for pending trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. More new pummelo-grapefruit seedless hybrids have been identified, some showing field tolerance to canker, good fruit quality, and FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Trees were propagated onto 30 new sour orange-like hybrid rootstocks, some already shown to be tolerant of CTV quick decline, for a new field trial. A new demonstration planting of advanced sweet orange selections and newly-released cultivars, selected for high yields and superior juice quality, has been established to assess production performance in the grove, but more importantly to demonstrate their performance and utility in commercial processing. Two additional trials are being prepared now with the same collaborator to be planted at other locations.
We have reported previously on the escape trees that have been identified and assessed in collaboration with colleagues in Guangdong and Guangxi provinces in China. There has been no change in the status of the trees and no additional testing has been carried out. the trees propagated remain in their respective locations. From the visit with Mr. Li, Jian, a citrus extension specialist from the Fujian Provincial Academy of Agricultural Sciences in October 2010, our search for survivors was expanded. Gmitter revisited the Pinghe County Guanximiyou (Chinese honey pummelo) production area in Fujian in summer 2011, and it was impressive to see how this region of the Chinese citrus industry adjusted their production and continued to remain profitable in response to HLB. Looking into many successful or failed situations in HLB management in China, we learned that psyllid control was the key to maintain as low HLB incidence as possible, and good nutrition and routine tree health management were considered vital. Further, the natural tolerance of this pummelo variety, and several other citrus varieties in other locations in China, also contribute to sustainable productivity and profitability. The key elements outlined to us were critically timed pesticide applications, use of pathogen-free planting materials, and maintenance of tree health through good nutrition, as we interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy orchards. These insights, unintended side benefits of this project, have been widely and repeatedly shared with citrus growers and researchers in Florida and elsewhere around the world.
Three healthy-looking trees were found in HLB ravaged orchards in Guangdong and Guangxi province, where all other trees planted at the same time or newly replanted were either dead or severely declining; these escape trees have been the focus of the follow-up efforts on this project. The two trees free of HLB symptoms from Guangdong had been propagated in the greenhouse at the Guangdong Institute of Fruit Tree Research facilities. Some were grafted with HLB affected branches for re-inoculation at the greenhouse, and others were planted in their research field to assess their reaction to natural inoculation with HLB. The other tree in Guangxi was transplanted to a protected location at the Guangxi Citrus Research Institute, and used to propagate more trees for inoculated by grafting infected material. Under observations for several months months, the propagated trees in the field surrounded by severe HLB disease and intense inoculum and vector pressure in Guangdong appeared not to show any HLB symptoms and no pathogen was detected by qPCR. Most of these propagated trees from the individual symptom-free tree found in Guangxi, inoculated in a protected greenhouse, were confirmed to be infected and some displayed HLB symptoms. Though they apparently were not resistant to inoculation, the question remains as to why the original source tree was not infected and symptom-free; the possibility of vector resistance in the host could be explored further. There are continuing observations on other grafting-inoculated trees propagated from the three trees at the protected greenhouse at both institutes, which could lead to a conclusion on the susceptibility, as well as propagation of enough materials for some transcriptome comparison to determine the molecular mechanism of survival. As a additional mission for this project, revisits to several commercial groves seen by us previously did demonstrate that psyllid control, together with other integrated management, is critical to minimize the HLB incidence rate and maintain the tree health and profitable production, from those successful and failed HLB management cases.
Three healthy-looking trees were found in HLB ravaged orchards in Guangdong and Guangxi province, where all other trees planted at the same time and newly replanted were either dead or severely declining; symptoms have been monitored and qPCR has been run multiple times, and thus far the original trees in the field remained free of HLB symptoms and CLas. After propagation and grafting inoculation, and observations over the past years, it was shown that the propagated trees from the one tree found in Guangxi could be re-infected by grafting in a protected greenhouse and expressed typical HLB symptoms. It is thought that this tree might possess some unknown mechanism for its survival in the field, perhaps ACP resistance or repellency. The two trees found in Guangdong appeared to remain healthy and uninfected in the field, which is interesting; however because of personnel changes in the institute, until now there have been no successful graft inoculations with infected budwood done in greenhouse. This greenhouse test is needed to determine its susceptibility. It is important to mention that propagated trees grown at the institute’s own field site, where HLB is widespread and barely managed, have remained asymptomatic. All the exchange of information with colleagues in China during this time period have been through emails, phone calls, and other meeting occasions when possible. No visits were made this year to the two institutes or the commercial groves, to directly observe the propagated and graft-inoculated trees, or to explore for new escape trees in abandoned or severely infected groves.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and data on disease progression have collected, through symptom expression and repeated PCR assays. We are now seeing striking differences in the rates of disease development as well as the severity of symptom expression; seedlings of several sources of germplasm have remained free of HLB symptoms and CLas detection. e=we have reinoculated these plants to continue to challenge them. Some accessions that were quickly infected, for example Daidai, have shown the ability to recover healthy growth flushes while retaining high titer values of CLas, as these plants were inoculated with CTV-free isolates in Carrizo citrange originally, they represent a new potential inoculum source for additional experiments in the future. New seedlings that have reached sufficient size have now also been inoculated with the original Carrizo HLB source. The Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges upon which we have done and will continue to do extensive genomic characterization, has been propagated and will soon be planted in a replicated field trial at the USDA-ARS farm on Picos Road, Ft. Pierce in collaboration with Dr. E. Stover. This population is of significant interest as the trifoliate orange and some of its hybrids have been shown to be very HLB-tolerant, and this experiment provides the opportunity to map genetic components responsible for the tolerance. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified and materials received for testing. Eleven crosses between 10 different susceptible and reputedly tolerant parents were made in spring 2010, and populations of at least 150 from each cross have been planted and now are growing in a DPI-certified propagation house, being prepared for replication and inoculation experiments. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
Two full genome sequences have been assembled and annotated, and made available to the citrus research community. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI and HudsonAlpha(US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. Both genome sequence assemblies along with annotation are available at the Phytozome portal at JGI, as well as Tree Fruit GDR (citrusgenomedb.org). The Clementine haploid genome has been substantially improved in its assembly and consequent gene model predictions, annotations, and utility to the research community, consisting of 9 pseudomolecules, representing the 9 basic chromosomes of the haploid genome; it has been named Clementine v. 1.0, and is being used in comparative genomics studies to result in a high-profile manuscript describing the phylogeny of sweet orange. New citrus genome sequences have been generated by the Machado lab in Brazil (Ponkan mandarin, 4x coverage, using 454 technology) and the Gmitter lab and UF-ICBR (low-acid pummelo, 25x coverage by Illumina technology). These new genome sequences are being integrated and compared with sweet orange and the reference haploid Clementine v. 1.0. Regions within the sweet orange genome have been identified that represent mandarin/mandarin haplotypes, as have mandarin/pummelo haplotype regions. Work has proceeded on the other objectives of this project. The candidate genes for silencing were sent to our collaborator in Spain, and constructs were prepared to initiate silencing experiments to provide proof of the gene’s specific involvement in development of HLB disease symptoms. However, in assessing the constructs it was found that use of the original pHellsgate12 resulted in unstable inserts, though at least 3 candidate gene silencing sequences were cloned into it, and infiltrated into plants. However, due to the difficulty in obtaining the double integration events using this vector, we acquired new destination vectors. A total of 6 candidates have been cloned successfully now in a silencing vector and the pipeline to handle recalcitrant plasmids and to check for positive clones has been developed, so more candidates may be explored in the future if the silencing is successful in planta. Infiltration experiments are set up now, and we hope to know if the system works in the next few months. We have mined, screened, and verified SNPs derived from the BAC end-sequences from Dvorak’s lab and the GoldenGate assay platform for hi-throughput genotyping has been produced. DNA samples are being prepared from ore than 150 individuals of a large mapping family, to integrate the sweet orange genome sequence with genetic and physical linkage maps, thus improving the quality of the orange genome sequence assembly and its annotation. Analysis of data from two microarray studies looking at differences in gene expression between sensitive and tolerant citrus types is proceeding.
Quarterly report October 2011: Two full genome sequences have been assembled and annotated, and made available to the citrus research community. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI and HudsonAlpha(US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. Both genome sequence assemblies along with annotation are available at the Phytozome portal at JGI, as well as Tree Fruit GDR (citrusgenomedb.org). The Clementine haploid genome is under revision for substantial improvements in its assembly and consequent gene model predicitons, annotations, and utility to the research community. The current assembly consists of 9 pseudomolecules, representing the 9 basic chromosomes of the haploid genome, and was developed by integrating and anchoring BAC end sequences from the haploid BAC library with the high-density genetic linkage map constructed by the ICGC collaboration. There are 1398 scaffolds harbored on these 9 chromosomes, representing 301.4 million bases (MB), representing an estimated 98.9% of the full genome. The scaffold N/L50 numbers, 4/31.4 MB, are substantial improvements over the version 0.9. Comparisons of the haploid Clementine genome with that of sweet orange are underway, to understand better the phylogeny of sweet orange. A manuscript is being drafted by the ICGC and the sequencing center scientists involved in the project to incorporate both of these sequences into a single work, enhancing the value and utility of each. Work has proceeded on the other objectives of this project. A preliminary list of candidate genes for silencing was sent to our collaborator in Spain, and constructs were prepared to initiate silencing experiments to provide proof of the gene’s specific involvement in development of HLB disease symptoms. However, in assessing the constructs it was found that use of the original pHellsgate12 resulted in unstable inserts, though at least 3 candidate gene silencing sequences were cloned into it, and infiltrated into plants. However, due to the difficulty in obtaining the double integration events using this vector, we have recently acquired new destination vectors, pK7GWIWG2(II) and pK7GWIWG2D(II). Currently, cloning is being done in parallel using the three vector systems. Agrobacterium infiltration using the final constructs is to begin in January 2012. A comparative proteomic study was completed to understand the pathogenic process of HLB in affected sweet orange leaves. Using the isobaric tags for relative and absolute quantification (iTRAQ) technique, we identified 686 unique proteins in the mature leaves of both mock-inoculated and diseased sweet orange plants. Microarray analysis showed that stress-related genes were significantly upregulated at the transcriptional level. Moreover, the transcriptional patterns of some of these upregulated proteins were examined at different stages of HLB disease development, providing information that may be used for early, presymptomatic detection of CLas infections. We have mined the BAC end-sequences from Dvorak’s lab and identified SNPs, that are being screened and verified to build a GoldenGate assay platform for hi-throughput genotyping of a large mapping family, to integrate the sweet orange genome sequence with genetic and physical linkage maps, thus improving the quality of the orange genome sequence assembly and its annotation.
Quarterly report for March 2011: Two full genome sequences have been assembled and annotated. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI and HudsonAlpha(US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. This genome was released through the Phytozome portal at JGI, as well as Tree Fruit GDR, at the International Plant and Animal Genome (PAG) Conference in January 2011. The current version, Citrus clementina 0.90, is based on ~6.4x coverage, and is a very preliminary product released to enable citrus research community access. BAC end sequences from the BAC library produced from the haploid have been included now, and a high-density genetic linkage map is being constructed by ICGC collaborators, to yield a chromosome-based assembly in the near future. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. This genome sequence is based on ~30x depth of sequence coverage and was assembled using Newbler software; it covers 319 Mb spread over 12,574 scaffolds. Half of the genome is accounted for by 236 scaffolds 251 kb or longer. The current gene set (orange1.1) integrates 3.8 million new ESTs (produced this year) with homology and ab initio-based gene predictions; 25,376 protein-coding loci have been predicted generating a total of 46,147 transcripts. The sweet orange genome also was presented at PAG in January 2011, and can be accessed through the portals indicated above. The 3.8 million sweet orange ESTs came from 17 different libraries that were produced and sequenced using the 454 platform. They represent various biotic/abiotic challenges including psyllid feeding on young seedlings, canker inoculation, and treatment with salicylic acid, among others. Substantial progress has also been made on the other objectives of this project. Studies comparing the time courses of gene expression in two sets of HLB-inoculated sweet orange and rough lemon plants, representing more susceptible and more tolerant types respectively, have been completed using Affymetrix and Agilent citrus chips (the latter developed by us at UF); some differentially expressed genes have been confirmed by RT-PCR. In addition, comparisons of carbohydrate metabolism and anatomical changes associated with gene expression differences in these same plants have been completed. For example, 4-fold induction of cell-wall-bound invertase activity was detected in symptomatic and asymptomatic leaves on diseased plants. Additionally,the expression profiles of starch breakdown genes indicated that the transcription of DPE2 and MEX1 was downregulated. Together with the reduction of maltose accumulation, it is suggested that the impairment of starch breakdown contributes to the starch accumulation in infected leaves. Our collaborators at UCR have updated the HarvEST-Citrus database, including sequences from Brazil and Spain, to provide an improved database for gene expression studies containing more than 465,000 publicly available ESTs. A preliminary list of candidate genes for silencing has been sent to our collaborator in Spain, and constructs are being prepared there to initiate silencing experiments to provide proof of their specific involvement in development of HLB disease symptoms.
Quarterly report June 2011: Two full genome sequences have been assembled and annotated, and made available to the citrus research community.. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI and HudsonAlpha(US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. Both genome sequence assemblies along with annotation are available at the Phytozome portal at JGI, as well as Tree Fruit GDR (citrusgenomedb.org). BAC end sequences from the BAC library produced from the haploid have been included now, and a high-density genetic linkage map has been constructed by ICGC collaborators; the map consists of 9 linkage groups, corresponding to the basic chromosome number for citrus, and it contains 952 sequence-derived markers (SNPs from Clementine BES and EST-SSRs), covering 1112cM. This map is strongly anchored on a large diploid Clementine BAC library resource, as well, and it supports the alignment of the haploid Clementine whole genome sequence in the framework of the ICGC collaborative project. The map, BAC end sequences, and assembled sequence scaffolds will be integrated to yield a chromosome-based assembly. Work has proceeded on the other objectives of this project. Our collaborators at UCR have updated the HarvEST-Citrus database, including sequences from Brazil and Spain, to provide an improved database for gene expression studies containing more than 465,000 publicly available ESTs. A preliminary list of candidate genes for silencing was sent to our collaborator in Spain, and constructs were prepared to initiate silencing experiments to provide proof of the gene’s specific involvement in development of HLB disease symptoms. However, in assessing the constructs it was found that use of the original pHellsgate12 resulted in unstable inserts, though at least 3 candidate gene silencing sequences were cloned into it, and infiltrated into plants. We are seeking a better vector to use for these experiments. The PI Gmitter will travel to meet the collaborator in Argentina soon, to coordinate efforts and to resolve technical issues. The collaborator in Spain reannotated the previously UF-developed Agilent microarray, making analysis and interpretation of our time course experiments in sensitive and tolerant host citrus plants easier and more meaningful. We have initiated collaboration with Jan Dvorak’s group from UC-Davis, to utilize a BAC-based physical map of sweet orange and his BAC end-sequences in an effort to integrate the sweet orange genome sequence with genetic and physical linkage maps to improve the quality of the genome sequence assembly and its annotation.
Funds for this project have now been received. The antibody service provider has provided a quote for anti-NodT antibody production. Antibody production and screening will be initiated soon and is expected to take approximately 3 months.
Results from four sweet orange rootstock field trials exposed to HLB were summarized and submitted for publication. The studies identified rootstock differences in tolerance to HLB that were discussed in the scientific publication. I will present this information to growers in an appropriate upcoming forum to use in making management decisions. Fruit quality, yield, and tree size data were collected from eight early season rootstock field trials. Detailed fruit quality data were collected from a large grapefruit rootstock trial at multiple harvest times to assess rootstock influence on grapefruit quality early, midseason, and late in the season. Two replicated field trials with 35 new supersour selections were planted in Lake County and Orange County. Source trees of 150 new supersour hybrids were selected and first stage propagations made to increase trees for specialized disease, abiotic, and field testing. Cuttings were made of 100 advanced supersour selections in preparation for cooperative field trials. Another supersour rootstock trial with 800 trees was prepared for field planting. Cooperative work was continued with a commercial nursery to multiply advanced supersour selections for placement of trees into cooperative field trials with growers. Work continued to assess supersour tolerance of CTV and calcareous soils (high pH). Studies continue to assess citrus germplasm tolerance to Liberibacter – Huanglongbing (HLB) and Phytophthora/Diaprepes in the greenhouse and under field conditions. Some trifoliate hybrid rootstocks, including US-802, US-812, US-897, and US-942 exhibit tolerance to HLB as seedling trees. Studies to compare the different components of tolerance in several rootstock selections were completed and a scientific publication is being prepared. Another study is underway to define the interaction of rootstock tolerance/susceptibility with scion tolerance/susceptibility. Collaborative work continues to study gene expression and metabolic changes associated with susceptible and tolerant plant responses to HLB, and to define genetic characteristics needed to prevent infection or avoid the damaging effects of the disease. Greenhouse and field studies are continuing to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. Selected anti-microbial and citrus plant resistance genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with increased resistance to HLB. Research is continuing to use HLB responsive citrus genes and promoters identified in the gene expression study published last year for inducing or engineering resistance in citrus. Thirty transgenic rootstocks with selected antimicrobial genes were propagated and entered into controlled greenhouse tests to assess tolerance to HLB. Data was collected from a field trial with selected transgenic rootstocks. Seed of the new rootstocks US-942, US-897, and US-802 was provided to the Florida Citrus Nursery Association for managed distribution to commercial nurseries.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, we have generated transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene. The transgenic plants are currently under canker resistance test. We will propagate these plants for citrus greening test. We have shown that overexpressing the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker, suggesting that the salicylic acid (SA) signaling pathway plays an important role in citrus disease resistance. We recently established an Arabidopsis-Xanthomonas citri subsp. citri (Xcc) pathosystem with the support of a USDA special grant. Using the Arabidopsis-Xcc pathosystem, we found that mutants of the SA signaling pathway are more susceptible to Xcc. A manuscript about these results has been accepted by PLoS ONE. We are trying to generate citrus transgenic plants that accumulate high levels of SA. For objective 2, we are continuing the screen with gamma ray-irradiated Ray Ruby grapefruit seeds. Two quarts of seeds treated with gamma-ray irradiation at 50 Gy have been plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots formed on the seeds previously plated were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Some shoots formed on these gamma irradiated seeds have been screened again on the selective medium. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test. We are also testing whether a direct genetic screen would work for identifying citrus greening-resistant varieties. We germinated gamma ray-irradiated Ray Ruby grapefruit seeds in soil and inoculated the seedlings with psyllids carrying greening bacteria. We are watching the development of greening symptoms on the seedlings.
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