The project has been granted a no cost extension into July 2020; year 1 activities, and some of year 2 objectives, have been nearly completed. A comprehensive overview of progress since project inception through April 2020 follows below. 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 HMW DNA) 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 met.3. We generated the PacBio raw sequence data for the 5 genomes listed above. The first genomes were finished running in late October 2019, 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 genomes now 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 assembly at the chromosome level. However, the quantity of grapefruit sequence is insufficient, so we will sequence further. WE now have prepared the Ruby Red grapefruit HMW DNA for additional sequencing, but we will have to store it until the UCB Genome Sequencing Laboratory reopens for normal business; currently their resources are devoted entirely to COVID-19 screening. 5. We have prepared materials from two genomes for the Dovetail Omni-C proximity ligation sequencing, the next step toward producing a chromosome scale full length assembly. They were sent to Dovetail Genomics on 18 February 2020. They have completed their work on two genomes, and their data files have been transferred to the collaborators at UCB and JGI. These results are being integrated with the PacBio sequence data to complete assembly at the chromosome level. Further work on the proximity ligation of the other genomes in on hold until we can work at the UF-CREC labs, to prepare the next round of samples to submit to Dovetail Genomics.6. We had 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). However, this activity is currently on hold, pending reopening of the UF-CREC labs.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. This has been initiated, with our resampling of HMW DNA.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. Now this also is temporarily on hold, pending the reopening of the UF-CREC labs.3. Omni-C sequencing for proximity ligation has been completed for two genomes, and chromosome scale assembly is underway. 4. A post-doctoral candidate was recruited to begin working on the project in late April; however, his hiring has been put on hold because of COVID-19 travel restrictions. A no-cost extension was approved for year 1, to end in July 2020, and most tasks for year 1 (and some for year 2) will be completed by that time.