MANAGEMENT AND CHARACTERIZATION OF CITRUS AND DATE GENETIC RESOURCES AND ASSOCIATED INFORMATION

The mission of the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD), Riverside, CA is to access, maintain, preserve, evaluate, and distribute germplasm of Citrus and date palm (Phoenix dactylifera) and their wild relatives. In addition, research is conducted to enhance the viability and impact of the collections. A major focus of research is to reduce vulnerability of citrus and date palm genetic resources to natural disasters and virulent pathogens by devising cryopreservation methods for in vitro preservation and by developing more effective and efficient disease diagnostic procedures. This project�s comprehensive and coordinated genetic resource management efforts will result in ready access to pathogen-tested/pathogen-free propagative and breeding materials for citrus and date palm breeders, research scientists, and producers. Associated information will be made available to customers through the Genetic Resources Information Network, stakeholder meetings, and publications. The specific objectives are: 1) Develop and apply new methods for conserving and propagating citrus and date genetic resources which improve genebank operating efficiency and effectiveness and which enable pathogen-tested materials and associated information to be distributed worldwide. 1A) Efficiently and effectively maintain the National Plant Germplasm System collections of citrus and date palm genetic resources and associated information. Distribute accessions upon request to researchers and breeders world-wide. 1B) Develop and apply cryopreservation, in cooperative research with the National Center for Genetic Resources Preservation, Ft. Collins, as an alternate and efficient means of conserving and distributing citrus, citrus wild relatives, and date palm. 1C) Develop, validate, and apply alternative means, such as cryotherapy, to free citrus germplasm from graft transmissible pathogens. 2) Elucidate patterns of genetic divergence and diversity in citrus, dates, and related taxa, and apply that knowledge to strategically expand the collections and other facets of genetic resource management. Evaluate citrus and date genetic resources for priority genetic and horticultural traits. 2A) Characterize the genetic variability in citrus and citrus wild relatives by DNA sequencing to better classify accessions taxonomically. 2B) Evaluate horticultural quality and other important traits for priority accessions of date palm, citrus and citrus relatives. Incorporate the evaluation and characterization data in GRIN-Global and/ or other databases. 2C) Rescue unique and valuable citrus germplasm exposed to Huanglongbing (HLB) and citrus canker for cleanup from known pathogens and incorporation into the collection. Sub-objective 2D. Evaluate citrus and citrus relatives for tolerance or resistance to psyllids and/or HLB. 3) Develop more accurate and high through-put diagnostic methods for priority graft-transmissible pathogens of citrus and for insect-vectored pathogens of dates, to enable the exchange of pathogen-tested accessions, and to more efficiently evaluate accessions for host-plant resistance. New accessions will be acquired through exchange with university breeders, foreign country national programs, botanical gardens, or by plant exploration. New citrus accessions are released from quarantine following comprehensive testing on indicator plants and laboratory tests as required by CDFA and USDA APHIS, to ensure that graft-transmissible pathogens are no longer present. While citrus and date accessions have traditionally been maintained as living plants, methods for cryopreservation will be developed and applied to the 450 accessions held in the citrus protected collection which is used for distribution of budwood. For release from quarantine and before citrus accessions may be distributed, the accessions must be cleaned of graft transmissible pathogens. While the clean-up has traditionally been done by heat treatment or shoot tip grafting, recent research has shown that cryotherapy may be a more efficient method of eliminating graft transmissible pathogens from citrus. The protocols for cryotherapy will be further developed, standardized, and the efficiency of cleaning graft transmissible pathogens by shoot tip grafting and cryotherapy will be compared. In previous studies, the single nucleotide polymorphism of a specific nuclear gene, malate dehydrogenase, has been identified as informative to determine phylogenetic relationships among citrus accessions. This information will be utilized to develop a DNA barcode, based on parsimony-informative characters, to help identify citrus and related genera from the sub-family Aurantioideae. Huanglongbing (HLB) and other invasive diseases threaten germplasm held under unprotected conditions. In Florida where HLB is now endemic and in California where HLB threatens to become established, and other citrus producing states as needed; rare germplasm which is not already backed up elsewhere will be recovered, cleaned, and added to the protected collection following release from quarantine status for distribution upon request. Threatened germplasm will be identified by collaboration with stakeholders. Diverse citrus and citrus relative germplasm will be planted into the field in Florida for screening for tolerance/resistance to HLB and the psyllid vector of HLB. Half of the trees planted in the randomized complete block trial will be inoculated with HLB prior to planting in the field. The plants will be evaluated for HLB, citrus canker, incidence of Diaphorina citri, the psyllid vector of HLB, and other diseases at six month intervals over four years. High through-put methods of nucleic acid extraction will be coupled with high through-put (384-well) qPCR assays to lower the cost per test and to be able to test for multiple pathogens (both RNA and DNA genome pathogens) from one collection/sample using the Taqman� probes. Taqman� probes will be developed for the RNA transcripts of the housekeeping gene NADH dehydrogenase (NAD5) gene and for the DNA gene of NAD5 for use as an internal control to monitor the quality of RNA and DNA extractions, respectively. This is the final report for this project which completed its five-year term and been replaced by 2036-21000-011-00D, �Conservation, Management and Distribution of Citrus and Date Genetic Resources and Associated Information�. Please see the report for the new project for additional information. Objective 1: Significant progress was made in the development and application of new methods for conserving and propagating citrus and date palm genetic resources; Sub-objective 1B has been completed. In cooperation with the National Center for Genetic Resources Preservation (NCGRP), cryopreservation protocols were developed for citrus accessions. Citrus budwood of 500 apparently pathogen-free accessions was processed and is now stored for long term preservation in liquid nitrogen in Fort Collins, Colorado, at the NCGRP. The protocols were optimized and have become standard operating procedures for all citrus germplasm as it is released from quarantine. The development of similar methods was initiated for date palm accessions in tissue culture and will be optimized in the new plan. These methods improve the security of critical germplasm. Sub-objective 1C: Using cryopreservation methods to eliminate citrus pathogens from budwood was successful at a rate of only 80 to 85 percent. This rate is not acceptable by regulatory officials for the release of citrus accessions from quarantine currently. Therefore, the repository continues to use the standard and accepted methods of thermotherapy and shoot-tip grafting to qualify accessions as pathogen-free. The National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) currently maintains 1,617 accessions of Citrus and citrus relatives, 147 accessions of Phoenix (palms), and 102 isolates of citrus pathogens (used as positive controls for pathogen testing). Plant inventory is maintained in replicates; in a field planting, within protected structures, and in liquid nitrogen. As new accession data are obtained, the information is revised and/or added in the Germplasm Resources Information Network (GRIN) Global. During the term of this project, the repository distributed 2, 478 items to 488 requestors for developing cryopreservation techniques, genetic elucidation, taxonomic questions, various research programs, and for domestic and international certification programs. During August 2017, a federal and state Huanglongbing (HLB or citrus greening) quarantine was implemented in the city of Riverside including the University of California, Riverside (UCR) and the ARS Repository. A Hold Order prevented the distribution of citrus germplasm until required testing of the protected collection was conducted and a new compliance agreement executed. The distribution of citrus germplasm resumed in March 2018. Seeds of HLB-tolerant rootstocks were obtained from ARS citrus breeders in Fort Pierce, Florida. Re-propagation of the field variety collection onto these rootstocks will continue into the new project as an additional preventive measure against this disease. Sub-objective 2A: In cooperation with scientists at the UCR, genetic analysis of germplasm was conducted using 47 citron accessions; 30 samples from Yunnan, China, where citron is presumed to have originated, and 17 accessions from the Citrus Variety Collection (Mediterranean region, South America or unknown origin). Molecular markers (23 simple sequence repeat (SSRs) markers, single nucleotide polymorphisms (SNPs) for a nuclear gene and a chloroplast gene) were used to generate information that was analyzed to deduce population structure, identify genetic diversity and understand relationships. As a result, three distinct clusters were identified in the citrons included in this study. Further, the citrus genebank was analyzed using approximately 58,000 SNPs and the phylogenetic and admixture (method of inferring geographical origins based on an analysis of genetic ancestry) analyses are in progress. Results will be published as part of a doctoral thesis by the end of 2018. An assay system was developed for conducting digital droplet Polymerase Chain Reaction (PCR) and quantitative PCR tests using a single copy malate dehydrogenase (MDH) gene target. Since the currently used mitochondrial gene, cytochrome oxidase, is not suitable as an internal control gene for many assays, the newly described malate dehydrogenase assay was incorporated into the repository�s diagnostic procedures where applicable. ARS scientists in Riverside, California, contributed to genomic studies of date palms conducted by Weill Medical Center in Qatar, New York University Abu Dhabi, University of Georgia, and the French National Institute for Agricultural Research. Using germplasm from the date palm genebank as well as international sources, these studies showed that the domestication of date palms occurred as two separate events in different geographic regions. Additionally, a specific region of the genome was identified that determines the sex of the individual palm tree. This has provided a greater understanding of date genetics overall and specifically of sexual differentiation, which is vital towards the advancement of date breeding. These studies have resulted in four publications with additional reports in preparation. Sub-objective 2B: Date production suffers from a problem involving skin separation, called �puffy skin�. This is a fruit quality defect in which excessive portions of the skin of the date pull away from the flesh, causing a blistered or puffed appearance; thereby decreasing the market value of the date. This problem appears to be influenced by temperature and humidity during the khalal period of date fruit maturation. Khalal refers to the second of four recognized stages in the ripening of a date in which it reaches its full size and changes from green to red or yellow or a combination of the two colors. Research was conducted through a cooperative project with the University of California Riverside (UCR) to investigate bunch and irrigation management as tools to mitigate this problem. Water uptake measuring devices have been installed in production fields and preliminary results indicate overwatering may be the cause of this condition. Scant information has been published regarding the nutritional requirements of palms, for either date or ornamental varieties. A study was conducted to ascertain whether date palm cultivars growing under the same cultural conditions and environment exhibit differences in nutrient levels as measured in the leaves. The date palm genebank provided the opportunity to compare both date and ornamental palms within the genus Phoenix growing under the same cultural and climatic conditions. Some differences between date cultivars appear to exist. In general, nutrient levels of the ornamental species are lower than for date palm cultivars. Sub-objective 2C: In collaboration with scientists at the Universities of California and Florida and ARS scientists in Fort Pierce, Florida, funding was obtained through the USDA Animal Plant Health Inspection Service (APHIS) Multi-Agency Cooperative Program. Citrus accessions developed in Florida for tolerance to HLB are being sent to Riverside for evaluation. All federal and state permits were executed. Germplasm is in the process of therapy and testing to satisfy quarantine requirements for its release and planting in field evaluation plots. As the accessions are released from quarantine, the ARS Riverside team will propagate trees for planting. The Florida scientists will continue to evaluate this material for its potential resistance or tolerance to the disease and psyllid vectors in Florida where insect and disease pressure is high. In California where the insect and disease pressure are very low, material will be evaluated for horticultural traits and rootstock/scion compatibility under California growing conditions. Growers and nurserymen will be able to visit the evaluation plot to obtain information to make future farming decisions. A National Institute of Food and Agriculture (NIFA) multi-million-dollar grant was awarded to a collaborative team of scientists from Clemson University, University of Florida, and ARS scientists in Fort Pierce, Florida, and Riverside, California, to evaluate naturally occurring trees exhibiting tolerance to HLB within existing commercial groves in Florida. The genotypes of the �tolerant� trees were compared to trees of the same cultivar that are susceptible to the pathogen to identify target gene sequences. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas 9) technology was used to transfer the tolerant gene target to multiple commercially important cultivars. The resulting trees will be planted in field trials in Florida, Texas, and California to evaluate for tolerance to HLB and horticultural traits. Sub-objective 2D: In cooperation with scientists at UCR and ARS scientists in Fort Pierce, Florida, the citrus genebank was screened for potential resistance/tolerance/susceptibility to the disease HLB or citrus greening disease. Seeds from the genebank were sent to Florida and over 1000 trees were planted in the field to monitor accessions under naturally occurring high disease and insect pressure. Potential resistance traits have been identified in some citrus relatives including Australian limes, which are sexually compatible with the genus Citrus. Breeders are using these relatives to transfer resistance traits into commercial varieties through conventional breeding techniques. Objective 3: ARS scientists in Riverside, California, and Fort Pierce, Florida, collaborated with colleagues at the UCR and University of Hawaii, to develop a field detection kit using �loop-mediated amplification� technology to rapidly test psyllids for HLB-associated bacteria. The reagent kit and SmartDart� were validated and results were published. Accomplishments 01 Cryopreservation backup. There is an ongoing effort to backup material in the National Plant Germplasm System using cryopreservation technology and storing the germplasm for the long term in the ARS National Laboratory for Genetic Resource Preservation, Fort Collins, Colorado. ARS researchers optimized Citrus cryopreservation methods which are now standard operating procedures. Due to the increased threat to the citrus collection by the deadly bacteria causing huanglongbing (HLB) or citrus greening disease, additional resources were allocated to expedite the cryopreservation of all sanitized genotypes maintained at the ARS repository in Riverside, California. Currently, 550 accessions are in long term storage. ARS scientists in Riverside, California, processed 17 accessions to send to Ft. Collins for long-term preservation. 02 Collection development. ARS scientists in Riverside, California, acquired nine new citrus accessions. Repository germplasm holdings currently include 1866 total accessions; 1617 accessions are Aurantioideae (Citrus and citrus relatives), 147 are date palms, and 102 are citrus pathogens maintained in planta for positive controls used for diagnostics. Sanitized and pathogen- tested citrus accessions are maintained in a USDA Animal and Plant Health Inspection Service (APHIS) approved protective structure and number 550 accessions. Eight accessions were sanitized using shoot tip grafting methods and three accessions were sanitized using thermotherapy. Germplasm distributions were limited due to the implementation of the Huanglongbing Quarantine and Hold Order. Despite this setback, 161 accessions were distributed to 29 requestors. 03 Quarantine processing and release. ARS researchers in Riverside, California, extracted nucleic acids from 629 citrus inventory items held under quarantine and conducted 9,090 quantitative polymerase chain reaction (PCR) assays to determine pathogen status and which therapy method was necessary to sanitize each accession. Plants having gone through shoot tip grafting and thermotherapy were tested to determine pathogen status: 864 PCR assays were performed on 48 citrus inventory items. Of these, 222 accessions moved forward for further testing for viroid pathogens. Seventeen genotypes that tested negative in all lab tests were selected for the biological (release) index. Sixteen accessions were released from quarantine status. 04 Collection monitoring. ARS scientists in Riverside, California, conducted pathogen diagnostics on 448 citrus trees. Field 12B of the Citrus Variety Collection was surveyed for Huanglongbing (HLB)- associated pathogens. Twenty-two DNA extractions were made from each tree, and nearly 10,000 polymerase chain reaction (PCR) assays were conducted. All trees tested were negative for HLB-associated pathogens. 05 Diagnostic improvement. While conducting the Huanglongbing (HLB) survey in the citrus field planting, ARS researchers in Riverside, California, developed an in-house DNA extraction method which avoids the use of hazardous materials, including reagents and waste. The in- house extraction method was as sensitive as the commercial kits, however manual labor time increased by about 25 percent. A cost analysis was performed to compare the commercial kits with the in-house prepared reagents. The in-house kits generated a cost savings of approximately 65 percent as compared to the commercial kits. 06 Genomic analysis of citron germplasm. ARS scientists in Riverside, California, in cooperation with scientists at the University of California, Riverside, conducted genetic analysis of germplasm using 47 citron accessions. Molecular markers (23 simple sequence repeat markers, single nucleotide polymorphisms for a nuclear gene and a chloroplast gene) were used to generate the information necessary for genomic analyses. Resulting data was used to deduce population structure, identify genetic diversity, and understand genetic inter-relationships. As a result, three distinct clusters were identified in the citrons included in this study.