The first objective of the project was to hire a Florida-based faculty scientist that could be trained under Dr. Leandro Pena in Spain, for the purpose of learning the mature tissue transformation technique and transferring the technology to Florida. The scientist (Dr. Cecilia Zapata) was hired, at the end of the first year of the three year project, and traveled to Dr. Pena’s lab at the IVIA, Spain, where she was trained in all tissue culture techniques associated with citrus mature transformation, starting with preparation of the source of material at the greenhouse and ending with the acclimatization of transformants in the greenhouse. It was emphasized that the preparation of plant material needed for mature transformation is the key to successfully and consistently obtaining mature transformants, and this can only be achieved by producing budsticks in a highly controlled and clean environment. The second objective of the project was to build a greenhouse at the Citrus Research and Education Center in Florida for the purpose of creating and growing citrus for mature transformation and to establish a Mature Transformation Laboratory. Unfortunately, the greenhouse was unable to be constructed due to miscalculations in the planning budget in the original proposal. Instead a growth room was constructed adding a clean head house structure that could be used as support of the growing area. It took approximately 7 months to construct the growth room. Up to date, almost a year after finishing the construction, we are still correcting a few problems with humidity, computer sensors and the water filtration system. Also, a generator needs to be purchased; without it, any prolonged electricity failure could jeopardize the whole project. The laboratory is fully operational. The third objective of the project was to obtain mature transgenic plants from the most important Florida citrus cultivars. We started using the growth room and plant the rootstocks at the beginning of April 2011. At the same time three (3) sweet orange varieties were indexed in vitro and micrografted; the cultivars introduced were Hamlin 1-4-1, Valencia SPB 1-14-19 and Pineapple F-60-3. A calendar was established in October 2011 and firsts mature transformation experiments were performed in November 2011, all the protocols developed at the IVIA are adjusting to our specific environmental conditions and clone specificities. In our conditions, mature Valencia was very responsive to organogenic regeneration. We obtained positive plants. Hamlin was also transformed but was less responsive to organogenic regeneration, we have a few positive plants, but we are still adjusting the protocol to improve the efficiency. Our control cultivar, Pineapple, has some quality problems with the starting material and new introductions are needed in the future, however since we don’t have too much space in the growth room, we are introducing another important cultivar for Florida. The Valencia positive plants are already growing in the growth room to be tested by PCR and Southern Blot.
We have achieved significant results in each area of focus of this project during the previous funding period. Substantial progress in developing and releasing new sweet orange and grapefruit cultivars, has resulted in the release of new sweet orange cultivars with improved juice quality, including Valquarius, a January-maturing Valencia, and a grapefruit hybrid, UF914 with potential to overcome the grapefruit juice effect. We have contributed substantially through international genetic mapping efforts in support of the International Citrus Genome Consortium project, to develop and make available new genome resources to the international research community in support of HLB research. We have developed base information on the nature of host-pathogen interactions to both HLB and citrus canker providing guidance for future research leading to development of resistant varieties. For example, through time course studies comparing tolerant rough lemon to susceptible sweet orange we have determined the underlying genetic mechanisms of tolerance and susceptibility. Proteomic analysis of infected orange revealed greater abundance of several stress related proteins, and microarray data indicated that their underlying genes were also upregulated. These studies together provide additional information for creating resistant citrus using native citrus genes, and developing platforms for disease detection at very early stages. We have planted out over 1200 plants containing various combinations of natural or synthetic genes, and various promoters, located at two permitted field trial locations, and many more are currently in greenhouse tests of disease resistance potential. Several canker tolerant transgenic grapefruit trees were identified. New transgenic plants were propagated for hot psyllid greenhouse tests and field planting. New candidate genes were identified from citrus and other plants for HLB and canker resistance; vectors were constructed for transgenic plant production. Citrus-specific promoters, transcription factors, and other elements were identified and incorporated into new constructs to produce consumer-friendly transgenic plants, by limiting foreign genes or controlling their expression in specific tissues. The effort to genetically engineer resistance to both HLB and citrus canker has been one of the highest priorities for our team. We have begun evaluations of existing plant materials in the field, exposed now for several years to endemic citrus canker and HLB, and have identified sources of tolerance or resistance to HLB and canker. Some of these hold promise as new varieties directly, while others provide breeding parents for further progress in developing disease resistance, and can serve as experimental plants to further dissect host pathogen interactions, and to highlight novel approaches toward disease resistance targets. Several new rootstock trials with more than 15,000 trees on a few hundred advanced selections have been planted throughout Florida, to assess their adaptation to evolving advanced citrus production systems, and most importantly the impact they will have on disease incidence and severity. Some of these have shown repression of HLB in greenhouse tests. Previous work to develop rootstocks with resistance or tolerance against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) has continued, and is particularly relevant as information accumulates on the confounding effects of biotic and abiotic stresses on HLB severity and tree decline. We have held two field days, and several impromptu events, where we have highlighted for the industry our new oranges and several new rootstock candidates that appear to support reasonably normal tree growth and good yields of high-quality fruit, even in the presence of HLB infection. We have continued all processes that are integral to further development of genetically superior new rootstock and scion varieties that can enable the Florida industry to survive the disease threats that currently threaten the future. These activities include basic genetics, gene discovery, development and testing of transgenics showing resistance to HLB in the field, selection and release of new varieties, extensive field trials and industry demonstration blocks, and identification of new rootstocks supporting tree growth and production in spite of HLB.
Preliminary examination of limb, scaffold and trunk phloem of healthy and HLB infected trees indicated that in diseased trees development of callose plugging was evident at all levels and started behind the newer phloem, was present in low amounts in healthy plants, could be seen in early infection stages and was readily seen by light microscopy with aniline blue or by TEM with normal fixation. Further evaluations by phloem age are underway. Huanglongbing (HLB) or citrus greening disease, caused by Candidatus Liberibacter asiaticus, is a phloem-limited fastidious pathogen transmitted by the Asian citrus psyllid, Diaphorina citri, and appears to be an intracellular pathogen that maintains an intimate association with the psyllid or the plant throughout its life cycle. The molecular basis of the interaction of this pathogen with its hosts is not well understood. We hypothesized that during infection, Ca. L. asiaticus differentially expresses the genes critical for its survival and pathogenicity in either host. To test this hypothesis, quantitative reverse transcription PCR was utilized to compare the gene expression of Ca. L. asiaticus in planta and in psyllid. Overall, 362 genes were analyzed for their gene expression in planta and in psyllid. Among them, 263 genes were up-regulated in planta compared with in psyllid, 18 genes were overexpressed in the psyllid, and 81 genes showed similar levels of expression in both plant and psyllid. Our study indicates that Ca. L. asiaticus adjusts its expression of genes involved in transport systems, secretion system, flagella, LPS, heme biosynthesis, stress resistance, hemolysin and serralysin in a host specific manner to adapt to the distinct environment of plant and insect. To our knowledge, this is the first large-scale study to evaluate the differential expression of Ca. L. asiaticus genes in a plant host and its insect vector. Efforts to propagate transgenic plants with the beta-glucanase gene continued, resulting in more than 150 Duncan and/or Valencia plants with this transgene, controlled by either or 35S promoter, or the phloem-specific Suc2 promoter. PCR analysis on a subset of these revealed that 90% are showing the specific band for the BG gene. Additional transgenic plants are being regenerated from in vitro cultures developed by Dr. Abdullah ‘ about 23 transgenic lines with the BG gene in either OLL#20 or Jin Cheng sweet oranges. These plantlets will be micrografted to rootstocks in 2012. Transgenic plants developed earlier are being moved to the Southern Gardens ‘hot psyllid’ greenhouse for inoculation.
Continued efforts to improve transformation efficiency: ‘ Evaluation of transgene expression of transgenic citrus plants with different phloem specific promoters. Several transgenic ‘Mexican lime’ lines containing the d35s promoter and the 4 phloem specific promoters were evaluated for transgene activity. Transgene analysis was carried out using PCR, RT-PCR, q-PCR and Southern Blot analyses. Publicataion: Dutt M., Ananthakrishnan G, Jaromin MK, Brlansky RH, & Grosser JW (2012) Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants. Tree Physiology 32(1):83-93. ‘ q-PCR approach to evaluate copy number and gene expression levels. Copy number of several transgenic lines has being evaluated using gene specific TaQMAN probes. Most transgenic lines had 1 ‘ 4 copies of the transgene stably incorporated into the genome. In addition a qPR-PCR approach is being used to evaluate gene expression levels in all transgenic lines. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Continued to grow selected precocious rootstock seedlings for subsequent budding with transgenic precocious sweet oranges (Vernia and OLL series). Several transgenic lines of our precious sweet orange and mandarin transgenic lines (B4-79, W. Murcott, B10-68 and OLL8) have been grafted onto precocious rootstock including Amblycarpa + Benton and Changsha + Benton. Grafted trees have been transplanted into airpots in a heated greenhouse for evaluation (Fig 1.), with plans to grow the trees in a RES (Rapid Evaluation System horticultural manipulation) type system in the greenhouse. We are now planning to establish a transgenic site on CREC property, and hope to be able to include a small structure to apply the RES technology (horticultural manipulation to reduce the time of juvenility) to actual transgenic plants. Transformation with early-flowering genes: ‘ We have regenerated many transgenic plants with the poplar FT behind either the 35S or heat shock promoter. Some of them are quite large now and the ones with HS promoter were maintained in a growth chamber at high temperature for several months, but none have bloomed yet. We are growing T1 tobacco with all 3 citrus FTs in order to determine phenotypes. We developed a co-transformation strategy to transform Carrizo citrange with two cassettes, one containing 35S-cft1 and the other containing AtSUC2 ‘ gus. We generated 122 transgenic Carrizo plants using the two vectors. PCR analysis revealed that 16 lines contained both cassettes. Plants have not flowered 12 months after transformation. Plants are currently being evaluated in an unheated greenhouse for cold stress in order to initiate flowering in spring 2012. Numerous transgenic plantlets of Hamlin and Carrizo were regenerated containing P27, P28, P29, PATFT and pPTFT.
Analysis of transgenic citrus lines: We confirmed the presence of transgenes in transformed citrus plants using standard PCR techniques. In one line, SUC2-snc1 mutant (20-7), it was unusually difficult to establish that the desired construct was present. Serial dilutions of genomic templates were employed to reduce interference of inhibitory substances present in plant genomic DNA extracts. This interference seemed to be correlated with this specific construct. It is possible, therefore, that constitutively expressed snc1 mutant may affect the production of phenolic or other interfering compounds. In order to evaluate the survival of Liberibacter asiaticus (Las) in our transformed citrus lines, we first focused on the development of an assay to detect the presence of the bacteria in heavily infected, symptomatic citrus leaves. Infected leaves were obtained from the UF Lake Alfred laboratory of Dr. William Dawson. These were sectioned into midveins and blades in order to determine the distribution of the infecting pathogen. Original quantities of tested materials (leaf samples) were in the range of 40 mg. PCR primers for detection of the Liberibacter asiaticus were based on 16S ribosomal DNA, and as plant controls, the cytochrome oxidase COX gene was used (Pelz-Stelinski et al., 2010, J. Econ. Entomol. 103, 1531-1541). We were able to detect Las and Cox amplicons in genomic DNA isolates from these relatively small quantities of transgenic citrus leaf material: either in green (asymptomatic) or yellow symptomatic Las-infected leaves. Setting up a calibrated curve for real time quantitative PCR: Next, we generated Las and Cox PCR amplicons to be used in standard curves in real-time PCR reactions for copy number determinations. The Wingless (Wg) gene that serves as the psyllid control was obtained from the genomic DNA isolated from 10 uninfected psyllids. Real-time PCR reactions required testing multiple variables in order to fine-tune the Las-detection assay, some being the primer and amplicon concentrations. We tested a range of amplicon concentrations from 10 ng to 1 pg (=12,190,283 copies), and in later experiments, down to 12 copies of Las and 14 copies of Cox and Wg. As a starting point, we tested the expression of AtPAD4-GUSplus transgenic plants responsive to wounding to correlate the wounding event itself with the actual psyllid feeding. Preliminary citrus wounding experiments by slit-cutting, or needle-puncturing determined that the AtPAD4 promoter was very specifically induced by wounding. We performed numerous histochemical studies, including aniline blue, acid fuchsin, toluidine blue, Evans blue as individual and with combined staining, and fluorescence antibody labeling techniques to detect psyllid stylet sheaths. These were performed on cross-sections identified by GUS staining spots generated in response to psyllid-feeding (=wounding). After numerous attempts we were unable to establish this technology as a useful tool to meet our early Liberibacter detection requirements in citrus plants.
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.
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