Goal and Objectives: The project goal is to create the highest quality genome sequence assembly resources for five commercially and biologically important citrus varieties using breakthrough technologies in sequencing and assembly, to provide the research community with the best possible tools to support development of HLB-resistant cultivars by genome editing or other techniques. The specific objectives are:1. Sequence sweet orange, grapefruit, Clementine (all sensitive), lemon and LB8-9 Sugar Bell® (both tolerant) genomes using PacBio and develop chromosome scale assemblies using proximity ligation technology. Sequence RNA transcript libraries using Oxford Nanopore technology, yielding full-length sequences, to aid annotation, assembly, etc.2. Annotate genomes, phase haplotypes, compare structural and relational genomics among the five genomes to develop a package of tools for researchers to effectively utilize sequence resources for gene discovery, gene sequence retrieval, genome editing, and ultimately the creation of HLB-tolerant or -resistant citrus cultivars.3. Make these resources freely available to the research community through USDOE-JGI’s Phytozome portal, and other genome sequence platforms.4. Extract full length, putative HLB-responsive gene sequences from all genomes, make comparisons of sequences, transcripts and protein structures as they may relate to host-pathogen interactions and disease development or its suppression, and predict candidate sites for guide RNA designing and gene editing.Timeline Year 1: Objective 1: Produce suitable plant materials for PacBio sequencing technical requirements, isolate high-quality genomic DNA samples for sequencing, generate PacBio genome sequence data, and assemble PacBio sequence reads as data are received. Objective 2: Prepare RNA libraries for transcriptome sequencing, generate full-length transcript sequence data on Nanopore sequencers.Results 1. We have produced the plant materials described, for Valencia orange (S, for sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, for tolerant), and Lisbon lemon (T).2. We isolated high-quality DNA (high molecular weight DNA, or HMWDNA) as required for sequencing on the PacBio Sequel II platform. We modified previously used procedures to meet the stringent quality standards required for this platform, in collaboration with Dr. Shana McDevitt, Director of the UC Berkeley Vincent J. Coates Genomics Sequencing Laboratory, This involved several iterations back and forth, with QC provided by Dr. McDevitt’s group, and modifications to the protocol by Gmitter’s lab, until stringent QC standards were achieved.3. We generated the PacBio raw sequence data for the 5 genomes listed above. The first genomes were finished running in late October, and preliminary output from the first genome run showed that >250 Gbp of sequence were produced, yielding potentially > 83X coverage of the genome, very near our target of 85X coverage. The other four have been completed, as well.4. Preliminary analyses and assemblies have been carried out. For four of the five genomes, the results have exceeded the quality of any other publicly available citrus reference genomes, and this is before the further steps using proximity ligation technology to finalize at the chromosome level. However, the quantity of grapefruit sequence is insufficient, so we will sequence further.5. We have prepared materials from two genomes for the Dovetail Omni-C proximity ligation sequencing, the next step toward assembling to yield a chromosome scale full length assembly. They were sent to Dovetail Genomics on 18 February 2020.6. We have begun preparing the samples for transcriptome sequencing, which will be used to annotate the genomes (i.e. to define and identify all the genes within the assembly).Conclusions1. We completed all genome sequencing work under Year 1, Objective 1; the exception is that we need to resample and generate more sequence data for the Ruby Red grapefruit.2. We have not yet generated the full-length transcript sequence data, as proposed for Year 1, Objective 2, though we have begun the process. This goal was compromised because budgetary uncertainty beyond year 1 precluded hiring a post-doc to carry out the work.3. Omni-C sequencing for ligation proximity and chromosome scale assembly is underway for 2 genomes.4. Once funding is assured, we will move quickly to meet the benchmarks in the Project Timeline. A post-doctoral candidate has been recruited to begin working on the project in late April. A no-cost extension was approved for year 1, to end in July 2020, and all tasks for year 1 (and some for year 2) will be completed by that time.