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 July 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 We have produced the plant materials and isolated the HMW-DNA from Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T). We generated raw sequence data for all 5 genomes on the PacBio Sequel II. Preliminary assemblies and analyses were carried out. For four of the five genomes, the results exceeded the quality of any other publicly available citrus reference genomes, even before Dovetail Hi-C proximity ligation sequencing to finalize assembly at the chromosome level. However, the quantity of grapefruit sequence is insufficient, so we have prepared the Ruby Red grapefruit HMW-DNA and additional sequencing is in progress. We prepared materials from two genomes for the Dovetail Hi-C sequencing. Incorporating these data with PacBio sequence of one of our target genomes has resulted in an improved chromosome scale assembly. The two parental chromosomes of the target genome have been phased/separated using Illumina short reads from citrons, pummelos and mandarins. By genome alignment and comparison to the Poncirus assembly (see below), minor assembly errors in repetitive regions have been fixed, resulting in a polished assembly. The availability of high-quality assemblies for the 3 basic species (C. medica, reticulata, and maxima) will allow a more thorough and complete characterization of large-scale structural variation (SVs: deletions, insertions, etc.) in genomes of commercial interest. These SVs are the driving force for phenotypic diversity especially among somatic mutants (e.g. different oranges, grapefruits).A previously funded CRDF project supported the initiation of a project producing the first ever high-quality reference genome of Poncirus trifoliata, and under this current project we have completed the task; a manuscript is under review in The Plant Journal, and the sequence will be released to the global citrus research community upon publication. By mining this new genome, we identified candidate genes within previously identified chromosomal regions for HLB tolerance, including a transcription factor gene and one disease resistance-like gene that are up-regulated by CLas and positively selected in trifoliate orange. These genes are promising candidate genes for further research.Conclusions1. We completed all genome sequencing work under Year 1, Objective 1, except for generating more sequence data for the Ruby Red grapefruit, currently underway.2. We have not yet generated the full-length transcript sequence data, as proposed for Year 1, Objective 2. This goal was compromised for several reasons beyond our control but should progress in year 2.3. Hi-C sequencing for proximity ligation was completed for two genomes, and along with Illumina short reads resulted in a phased chromosome scale assembly of one target genome. 4. We have produced a very high-quality genome assembly of Poncirus, an important source of resistance to CLas and HLB. This genome assembly was used to repair minor assembly errors in repetitive regions, and by mining the sequence we have identified genes to be targeted for HLB resistance.