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Lasiodiplodia theobromae is a fungal pathogen associated with perennial tropical fruit plants worldwide. In citrus, L. theobromae causes stem-end rot (Diplodia stem-end rot), a damaging postharvest disease that is aggravated when trees are also infected with the citrus greening bacteria ‘Candidatus Liberibacter asiaticus’. Due to the latent infection of L. theobromae during the preharvest stage, it becomes difficult to control the disease by chemical or physical treatment. In the current study, we sequenced and assembled strain CITRA15, the first genome of L. theobromae obtained from diseased Citrus paradise ‘Flame’ grapefruit in Florida, and thereby provided a genomic resource for future research on diagnostics, and postharvest and preharvest disease management of citrus and other fruit crops.

Detailed annotation and comparative analysis were performed on the Asian citrus psyllid (ACP), Diaphorina citri, vacuolar-type ATP synthase (V-ATPase) to support the biological understanding and development of novel therapeutics to manage psyllid vectors. D. citri is a hemipteran insect that vectors the causative agent, the bacteria Candidatus Liberibacter asiaticus (CLas), of the citrus greening disease, Huanglongbing (HLB). Millions of citrus trees have been destroyed by citrus greening and every grove in Florida has been directly impacted. In eukaryotic organisms, V-ATPase is an abundant heterodimeric enzyme that serves the cell with essential compartment acidification through the active processes that transport protons across the membrane. Manual curation was completed on 15 putative V-ATPase genes in the D. citri genome. Comparative genomic analysis reveals that the D. citri V-ATPase subunits share domains and motifs with other insects, including the V-ATPase-A superfamily domain from the V-ATPase catalytic subunit A, which shares a 92% identity with Acyrthosiphon pisum. Phylogenetic analysis separates D. citri V-ATPase subunits into expected clades with orthologous sequences. Based on the results of annotation and comparative genomic analysis, RNAi therapies targeting D. citri V-ATPase genes, which have been successfully utilized in related hemipterans, are being pursued. Annotation of the D. citri genome is a critical step towards the development of directed-pest management that will lead to the reduced spread of the pathogens causing HLB throughout the citrus industry.

Ubiquitination is an ATP-dependent process that targets proteins for degradation by the proteasome. In this study, we annotated 15 genes from the ubiquitin-proteasome pathway in the Asian citrus psyllid, Diaphorina citri. This psyllid vector has come to prominence in the last decade due to its role in the transmission of the devastating bacterial pathogen, Candidatus Liberibacter asiaticus (CLas). Infection of citrus crops by this pathogen causes Huanglongbing (HLB or citrus greening disease) and results in the eventual death of citrus trees. The identification and correct annotation of these genes in D. citri will be useful for functional genomic studies that aid in the development of RNAi-based management strategies aimed at reducing the spread of HLB. Investigating the effects of CLas infection on the expression of ubiquitin-proteasome pathway genes may provide new information regarding the role that these genes play in the acquisition and transmission of CLas by D. citri.

Citrus greening disease is caused by the pathogenCandidatusLiberibacter asiaticus, which is transmitted by the Asian citrus psyllid,Diaphorina citri. There is no curative treatment or significant prevention mechanism for this detrimental disease that causes continued economic losses from reduced citrus production. A high quality genome ofD. citriis being manually annotated to provide accurate gene models required to identify novel control targets and increase understanding of this pest. Here, we annotated genes involved in glycolysis, gluconeogenesis, and trehaloneogenesis in theD. citrigenome, as these are core metabolic pathways and suppression could reduce this pest. Specifically, twenty-five genes were identified and annotated in the glycolysis and gluconeogenesis pathways and seven genes for the trehaloneogenesis pathway. Comparative analysis showed that the glycolysis genes inD. citriare highly conserved compared to orthologs in other insect systems, but copy numbers vary inD. citri.Expression levels of the annotated gene models were analyzed and several enzymes in the glycolysis pathway showed high expression in the thorax. This is consistent with the primary use of glucose by flight muscles located in the thorax. A few of the genes annotated inD. citrihave been targeted for gene knockdown as a proof of concept, for RNAi therapeutics. Thus, manual annotation of these core metabolic pathways provides accurate genomic foundations for developing gene-targeting therapeutics to reduceD. citri.

The circadian rhythm is a process involving multiple genes that generates an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the earth’s rotation on its axis. This report presents the results of the manual curation of twenty-seven genes likely associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect acts as the vector of the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease (Huanglongbing). This disease is the most severe detriment to citrus industries and has drastically decreased crop yields worldwide. Based on the genes identified in the psyllid genome, namely cry1 and cry2, D. citri likely possesses a circadian model similar to that of the lepidopteran butterfly, Danaus plexippus. Manual annotation of these genes will allow future molecular therapeutics to be developed that can disrupt the psyllid biology.

Insects have a segmented body plan that is established during embryogenesis when the anterior-posterior (A-P) axis is divided into repeated units by a cascade of gene expression. The cascade is initiated by protein gradients created by translation of maternally provided mRNAs, localized at the anterior and posterior poles of the embryo. Particular combinations of these proteins activate specific gap genes to divide the embryo into distinct regions along the A-P axis. Gap genes then activate pair-rule genes, which are usually expressed in part of every other segment. The pair-rule genes, in turn, activate expression of segment polarity genes in a portion of each segment. The segmentation genes are generally conserved among insects, although there is considerable variation in how they are deployed. We annotated 24 segmentation gene homologs in the Asian citrus psyllid, Diaphorina citri. We identified most of the genes that were expected to be present based on known phylogenetic distribution. Two exceptions were eagle and invected, which are present in at least some hemipterans, but were not identified in D. citri. Many of these genes are likely to be essential for D. citri development and thus may be useful targets for pest control methods.

Chitin deacetylases (CDAs) are one of the least understood components of insect chitin metabolism. The partial deacetylation of chitin polymers appears to be important for the proper formation of higher order chitin structures, such as long fibers and bundles, that contribute to the integrity of the insect exoskeleton and other structures. Some CDAs may also play a role in bacterial defense. Here we report the characterization of four CDA genes from the Asian citrus psyllid, Diaphorina citri, laying the groundwork for future study of these genes. D. citri is the vector for Candidatus Liberibacter asiaticus (CLas), which is responsible for the global outbreak of Huanglongbing (citrus greening) disease. The manual annotation was done as part of a collaborative community annotation project (https://citrusgreening.org/annotation/index).

Huanglongbing (HLB), also known as citrus greening disease, is caused by the bacterium Candidatus Liberibacter asiaticus (CLas) and represents a serious threat to global citrus production. This bacteria is transmitted by the Asian citrus psyllid, Diaphorina citri (Hemiptera) and there are no effective in-planta treatments for CLas. Therefore, one strategy is to manage the psyllid population. Manual annotation of the D. citri genome can identify and characterize gene families that could serve as novel targets for psyllid control. The yellow gene family represents an excellent target as yellow genes are linked to development and immunity due to their roles in melanization. Combined analysis of the genome with RNA-seq datasets, sequence homology, and phylogenetic trees were used to identify and annotate nine yellow genes for the D. citri genome. Phylogenetic analysis shows a unique duplication of yellow-y in D. citri, with life stage specific expression for these two genes. Genomic analysis also indicated the loss of a gene vital to the process of melanization, yellow-f, and the gain of a gene which seems to be unique to hemipterans, yellow 9. We suggest that yellow 9 or the gene yellow 8 (c), which consistently groups closely to yellow-f, may take on this role. Manual curation of genes in D. citri has provided an in-depth analysis of the yellow family among hemipteran insects and provides new targets for molecular control of this psyllid pest. Manual annotation was done as part of a collaborative community annotation project (https://citrusgreening.org/annotation/index).

The polysaccharide chitin is critical for the formation of many insect structures, including the exoskeleton, and is required for normal development. Here we report the annotation of three genes from the chitin synthesis pathway in the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), the vector of Huanglongbing (citrus greening disease). Most insects have two chitin synthase (CHS) genes but, like other hemipterans, D. citri has only one. In contrast, D. citri is unusual among insects in having two UDP-N-acetylglucosamine pyrophosphorylase (UAP) genes. One of the D. citri UAP genes is broadly expressed, while the other is expressed predominantly in males. Our work helps pave the way for potential utilization of these genes as pest control targets to reduce the spread of Huanglongbing.

The Asian citrus psyllid, Diaphorina citri, is an insect vector that transmits Candidatus Liberibacter asiaticus, the causal agent of the Huanglongbing (HLB) or citrus greening disease. This disease has devastated Florida’s citrus industry and threatens California’s industry as well as other citrus producing regions around the world. To find novel solutions to the disease, a better understanding of the vector is needed. The D. citri genome has been used to identify and characterize genes involved in Wnt signaling pathways. Wnt signaling is utilized for many important biological processes in metazoans, such as patterning and tissue generation. Curation based on RNA sequencing data and sequence homology confirm twenty four Wnt signaling genes within the D. citri genome, including homologs for beta-catenin, Frizzled receptors, and seven Wnt-ligands. Through phylogenetic analysis, we classify D. citri Wnt-ligands as Wg/Wnt1, Wnt5, Wnt6, Wnt7, Wnt10, Wnt11, and WntA. The D. citri version 3.0 genome with chromosomal length scaffolds reveals a conserved Wnt1-Wnt6-Wnt10 gene cluster with gene configuration similar to that in Drosophila melanogaster. These findings provide a greater insight into the evolutionary history of D. citri and Wnt signaling in this important hemipteran vector. Manual annotation was essential for identifying high quality gene models. These gene models can further be used to develop molecular systems, such as CRISPR and RNAi, that target and control D. citri populations, to manage the spread of HLB. Manual annotation of Wnt signaling pathways was done as part of a collaborative community annotation project (https://citrusgreening.org/annotation/index).

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 RNA-Seq 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.

We have created an open access web portal with pathosystem-wide resources and bioinformatics tools for the host citrus, the vector Asian citrus psyllid (ACP) and multiple pathogens including Ca. Liberibacter asiaticus. To the best of our knowledge, this is the first example of a database to use the pathosystem as a holistic framework to understand an insect transmitted plant disease. This endeavor integrates and enables the analysis of data sets generated by the community to study the citrus greening disease complex. Users can submit relevant data sets to enable sharing and allow the community to better analyze their data within an integrated system. The portal contains a variety of tools for omics data. Metabolic pathway databases, CitrusCyc and DiaphorinaCyc provide organism specific pathways that can be used to mine metabolomics, transcriptomics and proteomics results to identify pathways and regulatory mechanism involved in disease response. Psyllid Expression Network (PEN) contains expression profiles of ACP genes from multiple life stages, tissues, conditions and hosts. Citrus Expression Network (CEN) contains public expression data from multiple tissues and conditions for various citrus hosts. All tools like Apollo/JBrowse, Biocyc, Blast, CEN and PEN connect to a central database containing gene models for citrus, ACP and multiple Liberibacter pathogens. The portal also includes electrical penetration graph (EPG) recordings of ACP feeding on citrus, information about citrus rootstock trials and metabolomics data in addition to traditional omics data types with a goal of combining and mining all information related to a pathosystem. The portal includes user-friendly manual curation tools to allow the research community to continuously improve this knowledge base as more experimental research is published. Bulk downloads are available for all genome and annotation datasets from the FTP site (ftp://ftp.citrusgreening.org). The portal can be accessed at https://citrusgreening.org/.

The original Heinz 1706 reference genome was produced by a large team of scientists from across the globe from a variety of input sources that included 454 sequences in addition to full-length BACs, BAC and fosmid ends sequenced with Sanger technology. We present here the latest tomato reference genome (SL4.0) assembledde novofrom PacBio long reads and scaffolded using Hi-C contact maps. The assembly was validated using Bionano optical maps and 10X linked-read sequences. This assembly is highly contiguous with fewer gaps compared to previous genome builds and almost all scaffolds have been anchored and oriented to the 12 tomato chromosomes. We have found more repeats compared to the previous versions and one of the largest repeat classes identified are the LTR retrotransposons. We also describe updates to the reference genome and annotation since the last publication. The corresponding ITAG4.0 annotation has 4,794 novel genes along with 29,281 genes preserved from ITAG2.4. Most of the updated genes have extensions in the 5’ and 3’ UTRs resulting in doubling of annotated UTRs per gene. The genome and annotation can be accessed using SGN through BLAST database, Pathway database (SolCyc), Apollo, JBrowse genome browser and FTP available athttps://solgenomics.net.

High quality gene models are necessary to expand the molecular and genetic tools available for a target organism, but these are available for only a handful of model organisms that have undergone extensive curation and experimental validation over the course of many years. The majority of gene models present in biological databases today have been identified in draft genome assemblies using automated annotation pipelines that are frequently based on orthologs from distantly related model organisms and usually have minor or major errors. Manual curation is time consuming and often requires substantial expertise, but is instrumental in improving gene model structure and identification. Manual annotation may seem to be a daunting and cost-prohibitive task for small research communities but involving undergraduates in community genome annotation consortiums can be mutually beneficial for both education and improved genomic resources. We outline a workflow for efficient manual annotation driven by a team of primarily undergraduate annotators. This model can be scaled to large teams and includes quality control processes through incremental evaluation. Moreover, it gives students an opportunity to increase their understanding of genome biology and to participate in scientific research in collaboration with peers and senior researchers at multiple institutions.

The Asian citrus psyllid, Diaphorina citri, is an insect vector that transmits Candidatus Liberibacter asiaticus, the causal agent of the Huanglongbing (HLB), or citrus greening disease. This disease has devastated Florida’s citrus industry, and threatens California’s industry as well as other citrus producing regions around the world. To find novel solutions to the disease, a better understanding of the vector is needed. The D. citri genome has been used to identify and characterize genes involved in Wnt signaling pathways. Wnt signaling is utilized for many important biological processes in metazoans, such as patterning and tissue generation. Curation based on RNA sequencing data and sequence homology confirms 24 Wnt signaling genes within the D. citri genome, including homologs for beta-catenin, Frizzled receptors, and seven Wnt-ligands. Through phylogenetic analysis, we classify D. citri Wnt ligands as Wg/Wnt1, Wnt5, Wnt6, Wnt7, Wnt10, Wnt11, and WntA. The D. citri version 3.0 genome with chromosomal length scaffolds reveals a conserved Wnt1-Wnt6-Wnt10 gene cluster with a gene configuration like that in Drosophila melanogaster. These findings provide greater insight into the evolutionary history of D. citri and Wnt signaling in this important hemipteran vector. Manual annotation was essential for identifying high quality gene models. These gene models can be used to develop molecular systems, such as CRISPR and RNAi, which target and control psyllid populations to manage the spread of HLB. Manual annotation of Wnt signaling pathways was done as part of a collaborative community annotation project.

Insects have a segmented body plan that is established during embryogenesis when the anterior–posterior (A–P) axis is divided into repeated units by a cascade of gene expression. The cascade is initiated by protein gradients created by translation of maternally provided mRNAs, localized at the anterior and posterior poles of the embryo. Combinations of these proteins activate specific gap genes to divide the embryo into distinct regions along the anterior–posterior axis. Gap genes then activate pair-rule genes, which are usually expressed in parts of every other segment. The pair-rule genes, in turn, activate expression of segment polarity genes in a portion of each segment. The segmentation genes are generally conserved among insects, although there is considerable variation in how they are deployed. We annotated 25 segmentation gene homologs in the Asian citrus psyllid, Diaphorina citri. Most of the genes expected to be present in D. citri based on their phylogenetic distribution in other insects were identified and annotated. Two exceptions were eagle and invected, which are present in at least some hemipterans, but were not found in D. citri. Many of the segmentation pathway genes are likely to be essential for D. citri development, and thus they may be useful targets for gene-based pest control methods.

Huanglongbing (HLB), also known as citrus greening disease, is caused by the bacterium Candidatus Liberibacter asiaticus (CLas). It is a serious threat to global citrus production. This bacterium is transmitted by the Asian citrus psyllid, Diaphorina citri (Hemiptera). There are no effective in planta treatments for CLas. Therefore, one strategy is to manage the psyllid population. Manual annotation of the D. citri genome can identify and characterize gene families that could be novel targets for psyllid control. The yellow gene family is an excellent target because yellow genes, which have roles in melanization, are linked to development and immunity. Combined analysis of the genome with RNA-seq datasets, sequence homology, and phylogenetic trees were used to identify and annotate nine yellow genes in the D. citri genome. Manual curation of genes in D. citri provided in-depth analysis of the yellow family among hemipteran insects and provides new targets for molecular control of this psyllid pest. Manual annotation was done as part of a collaborative Citrus Greening community annotation project.

Chitin deacetylases (CDAs) are one of the least understood components of insect chitin metabolism. The partial deacetylation of chitin polymers appears to be important for the proper formation of higher order chitin structures, such as long fibers and bundles, which contribute to the integrity of the insect exoskeleton and other structures. Some CDAs may also be involved in bacterial defense. Here, we report the manual annotation of four CDA genes from the Asian citrus psyllid, Diaphorina citri, laying the groundwork for future study of these genes.

The polysaccharide chitin is critical for the formation of many insect structures, including the exoskeleton, and is required for normal development. Here we report the annotation of three genes from the chitin synthesis pathway in the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), the vector of Huanglongbing (citrus greening disease). Most insects have two chitin synthase (CHS) genes but, like other hemipterans, D. citri has only one. In contrast, D. citri is unusual among insects in having two UDP-N-acetylglucosamine pyrophosphorylase (UAP) genes. One of the D. citri UAP genes is broadly expressed, while the other is expressed predominantly in males. Our work helps pave the way for potential utilization of these genes as pest control targets to reduce the spread of Huanglongbing.