Progress 04/26/21 to 09/30/21
Outputs
Target Audience:Extension and outreach to the citrus grower community, who will benefit from applications of the research information we are generating. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three post-doctoral research associates and one graduate student have had the opportunity to receive advanced training in new skill sets through mentoring on the project. How have the results been disseminated to communities of interest?As goals and objectives are achieved, we are making genomic resources available to the broad research community through various public portals, including The Citrus Genome Database, Phytozome, and NCBI. Researchers can access these resources and apply them to gene editing-based solutions to HLB. Further, citrus growers, the ultimate beneficiaries of the work conducted, have been kept abreast of progress and developments through the course of the work. What do you plan to do during the next reporting period to accomplish the goals?We will proceed to follow the approaches described in the project document.
Impacts
What was accomplished under these goals?
HLB, also known as citrus greening, is the most destructive, devastating disease of citrus in the world. It was found in Florida in August 2005, and it poses an existential threat to the Florida citrus industry. Citrus is the most important agricultural industry in Florida, formerly generating a $9.3 billion annual economic impact on the state and providing as many as 80,000 jobs. In Florida, HLB is specifically associated with the bacterium 'Candidatus Liberibacter asiaticus' (CaLas) and is vectored by the Asian citrus psyllid (ACP, Diaphorina citri). HLB and ACP have already spread to Georgia, South Carolina, Louisiana, Texas, and California. Thus, HLB is truly posing a great threat to the entire U.S. citrus industry. HLB affects all citrus species, and commercial cultivars such as sweet oranges, grapefruit, and many mandarins, are most susceptible. The best long-term strategy, recommended by the National Academy of Sciences report on HLB, is to develop citrus cultivars that can tolerate or resist HLB. While there are no HLB-resistant cultivars, there are some selections and cultivars that are considered tolerant. Traditional breeding approaches to incorporate tolerance or resistance cannot preserve the unique quality attributes of most citrus fruit. Therefore, genetic engineering has been considered to develop tolerant or resistant cultivars, and any GMO projects have been pursued in different labs to achieve this goal. The unwillingness of consumers to accept GMO foods, however, has led to strong reluctance of the major beverage and food companies to embrace GMO citrus as a solution to HLB; in fact, all the major OJ products in the US carry the non-GMO label. An alternative approach, gene editing using CRISPR technology, has potential to address the concern over GMO citrus. If properly done no foreign DNA will remain in the new lines. The CRISPR-Cas9 system has been used successfully to modify citrus genes, and it is not currently regulated by USDA's Animal and Plant Health Inspection Service. To achieve effective and precise CRISPR gene editing, however, very high-quality genome sequence assemblies are required. The overarching goal of this project is to produce the highest quality genome assemblies possible of commercially important citrus cultivars, to enable efficient and effective gene editing, to produce HLB resistant plants that can allow the US industry to overcome the threat. Objective 1. Previously, we generated raw sequence data for Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T) and preliminary assemblies and analyses were carried out. Because of reduced sequencing costs, we were able to enter additional important genomes into the pipeline beyond those originally proposed, including Carrizo citrange, sour orange, and Shekwasha (an important breeding parent for HLB tolerance); these also have now been sequenced and assembled. Now, we have completed Hi-C sequencing of two genomes, and by incorporating these data with PacBio sequence assembly from two of our target genomes we have produced improved chromosome scale assemblies. Minor assembly errors in repetitive DNA regions have been repaired, by genome alignment and comparison to the Poncirus assembly we recently published, resulting in polished assemblies of these two accessions. Initial preliminary characterizations of the presence/absence variations among the four phased haploid genomes have been made. Notable variations among these four involve transposable elements including MITES with characteristic sizes. A genome graph-based approach is being pursued, with the goal of building a genome graph containing the different types of structural variations that are now being identified. The availability of high-quality assemblies for the 3 basic species (C. medica, reticulata, and maxima) will allow thorough and complete characterization of large-scale structural variations (SVs: deletions, insertions, etc.) in our target genomes of commercial interest. SVs are a driving force for phenotypic diversity especially among somatic mutants (e.g., different oranges, grapefruits, etc.), and this type of information will become more important as we test different sweet orange mutants exhibiting enhanced tolerance of HLB and attempt to determine the underlying causes of such tolerance. In a related effort, our team recently compared 69 new east Asian genomes and other mainland Asian citrus to reveal a previously unrecognized wild sexual species native to Japan's Ryukyu Islands: C. ryukyuensis, which hybridized with an ancient east Asian mandarin to produce Shekwasha (shiikuwasha) mandarin, a powerful source of HLB tolerance in rootstock breeding. Further, by studying the genomes of C. ryukyuensis-derived hybrids and other citrus. we traced the origin and spread of apomixis (nucellar embryony, a trait that is required for seed propagation of citrus rootstocks) from Mangshanyeju wild mandarins in China a few million years ago through most of the commonly known contemporary citrus types (orange, grapefruit, lemon, etc.). This work resulted in deeper understanding and new genome-based tools that can be exploited for two critically important traits in citrus genetic improvement, nucellar embryony and most importantly HLB tolerance; the research was published in Nature Communications in July 2021 (see https://doi.org/10.1038/s41467-021-24653-0). Objective 2. We now have some transcriptome data for two of our target genomes, using the Illumina sequencing platform, and genome annotation (i.e., identify all the genes within the genome) is in progress. Samples have been prepared and collected, and plans are in place to proceed with Hi-C sequencing of the target genomes not yet completed and polished, as well as to generate the transcriptome data required for genome annotation, and further characterization of large-scale structural variations within and among the genomes upon which we are focused. We used the PacBio Sequel platform to sequence full-length gene transcripts in the leaf tissues of sweet orange and trifoliate orange and reconstructed their leaf transcriptomes. We identified novel full-length transcripts that were not present in the published reference transcriptomes. Objective 3. We have phased the two parental chromosomes of the target genomes mentioned above using Illumina short reads from citrons, pummelos and mandarins. Objective 4. We found in the sweet orange and trifoliate orange full-length leaf transcriptomes that some NBS-encoding genes (nucleotide binding site genes, one typical class of disease resistance genes in plants) underwent alternative splicing. One alternatively spliced NBS transcript expressed in HLB symptomatic leaf and fruit of sweet orange, and another alternatively spliced NBS transcript was differentially expressed in CLas-infected trifoliate orange samples, suggesting that isoforms of some NBS-encoding genes may play an important role in HLB tolerance of trifoliate orange, or alternatively HLB susceptibility in sweet orange. The new transcriptomes will be useful to identify candidate genes for disease resistance that have been missed in the published citrus genomes and transcriptomes. Finally, we have collaborated with the USDA Germplasm Repository for Citrus and prepared genomic DNA from more than 120 citrus species, varieties, and relatives for enrichment and sequencing of specifically targeted genes. Sequencing libraries have been prepared and are in que for processing. Results from this effort will provide important insights for the evolution and domestication of select genes that are important for citrus resistance or susceptibility to Huanglongbing and other diseases. The results will also provide template gene sequences for genome editing for Huanglongbing resistance.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Wu, G.A., Sugimoto, C., Kinjo, H. Azama, C., Mitsube, F., Talon, M., Gmitter Jr.,F.G., and Rokhsar, D.S. Diversification of mandarin citrus by hybrid speciation and apomixis. Nat Commun 12, 4377 (2021). https://doi.org/10.1038/s41467-021-24653-0
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