Chromosomal length reference assembly for Diaphorina citri using single-molecule sequencing and Hi-C proximity ligation with manually curated genes in developmental, structural and immune pathways

Hemipterans include some of the most important insect pests of agricultural systems and vectors of plant pathogens. The vector, Diaphorina citri (Asian citrus psyllid) belonging to the Psylloidae superfamily, is the primary target of approaches to stop the spread of the pathogen Ca. Liberibacter asiaticus that causes Huanglongbing or citrus greening disease. High quality genomic resources enable rapid functional discovery that can target disease transmission and control. The previous psyllid genome (Diaci v1.1) available in NCBI is missing 25% of the single copy markers conserved in other Hemipterans. Manual genome curation helped to identify a significant number of genome anomalies including misassemblies and missing genes. We present an improved and highly contiguous de novo assembly based on PacBio long reads followed by Dovetail Chicago and Hi-C based scaffolding. The current assembly (Diaci v3) has 13 chromosomal length scaffolds with a genome size of 475 Mb. This is the first report of a chromosomal length assembly in the Hemiptera order according to our knowledge. Full-length cDNA transcripts were sequenced with PacBio Iso-Seq technology from diseased and healthy tissue at multiple life stages. Iso-Seq along with diverse Illumina RNAseq expression data were used to predict 19,049 protein-coding genes in psyllid using MAKER annotation pipeline. We also generated a genome independent transcriptome with a comprehensive catalog of all genes in the psyllid. Gene-targeting technologies like RNAi, antisense oligos and CRISPR require accurate annotation of genes. Lack of closely related and well characterized model organisms coupled with the diversity of insect genomes impacts the quality of predicted gene models. We used the improved genomic resources to create a high-quality manually curated gene set for developmental, structural and immune pathways. All resources are available on https://citrusgreening.org/, a portal for all omics resources for the citrus greening disease research community. The high quality ACP genome assembly, annotation based on transcriptomics evidence, manual curation of critical pathways and a genome independent de novo transcriptome will provide a foundation for comparative analysis among genomes of agricultural pests and vectors in the Hemiptera.