Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Report Date: 12/03/2021
Project: 18-010   Year: 2021
Category: Plant Improvement
Author: Fred Gmitter
Sponsor: Citrus Research and Development Foundation

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 preliminarily assembled. The transcriptome data for two of our target genomes, produced previously by a commercial vendor on their PacBio platform was found to be inadequate. So, we are in the process of finding another vendor to redo the long-read sequencing on the most recent PacBio platform. Hi-C sequencing of the 7 remaining target genomes is underway; two were completed. These Hi-C results will be combined with the PacBio assemblies using Hi-Rise software, to produce improved chromosome scale assemblies. RNA samples of these 7 additional genomes have been prepared from young and old leaf tissue 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. However, we are attempting to increase the types and numbers of tissues sampled to have a fuller representation of the gene content of each genome.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.   


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