Plant whole genome sequencing provides detailed information on gene content, genome organization, and evolutionary relationships as well as supports biotechnological applications such as gene editing. The first 3X draft genome sequence of papaya based on whole genome shotgun reads from the transgenic ‘SunUp’ papaya cultivar was published in 2008. Since then, advancements in sequencing and whole genome assembly enabled a near complete sequence of ‘SunUp’ and a detailed picture of events resulting from particle gun-mediated transformation. With current technology, the 372 Mb genome size of papaya makes it tractable for routine whole genome sequencing to characterize different cultivars and molecular events. In this study, we improved ease and speed of preparation, efficiency of recovery, and DNA quality through a combination of classical and contemporary plant nuclei or high molecular weight DNA isolation methods. Leveraging Hi-Fi sequencing and Hi-C technology, we achieved rapid chromosome-level sequence assembly of two local Hawaiian cultivars, Kapoho and Waimanalo. The assembled genomes of Kapoho and Waimanalo spanned 341.6 Mb and 337.4 Mb, respectively, with a total of 20,343 and 20,165 annotated protein-coding genes.
