Improved chromosome-level genome assembly of the Glanville fritillary butterfly (Melitaea cinxia) integrating Pacific Biosciences long reads and a high-density linkage map

Background The Glanville fritillary (Melitaea cinxia) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome-level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. Results The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO indicates that the genome contains 92–94% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models. Conclusions The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome, and genetics studies on this species.

Identification of Candidate Genes and Genomic Regions Associated with Adult Plant Resistance to Stripe Rust in Spring Wheat

Wheat stripe rust (caused by Puccinia striiformis f. sp. tritici) is a major disease that damages wheat plants and affects wheat yield all over the world. In recent years, stripe rust became a major problem that affects wheat yield in Egypt. New races appeared and caused breakdowns in the resistant genotypes. To improve resistance in the Egyptian genotypes, new sources of resistance are urgently needed. In the recent research, a set of 95 wheat genotypes collected from 19 countries, including Egypt, were evaluated for their resistance against the Egyptian race(s) of stripe rust under field conditions in the two growing seasons 2018/2019 and 2019/2020. A high genetic variation was found among the tested genotypes. Single marker analysis was conducted using a subset of 71 genotypes and 424 diversity array technology (DArT) markers, well distributed across the genome. Out of the tested markers, 13 stable markers were identified that were significantly associated with resistance in both years (p-value ≤ 0.05). By using the sequence of the DArT markers, the chromosomal position of the significant DArT markers was detected, and nearby gene models were identified. Two markers on chromosomes 5A and 5B were found to be located within gene models functionally annotated with disease resistance in plants. These two markers could be used in marker-assisted selection for stripe rust resistance under Egyptian conditions. Two German genotypes were carrying the targeted allele of all the significant DArT markers associated with stripe rust resistance and could be used to improve resistance under Egyptian conditions.

Unravelling consensus genomic regions associated with quality traits in wheat (Triticum aestivum L.) using meta-analysis of quantitative trait loci

A meta-analysis of quantitative trait loci (QTLs) associated with following six major quality traits (i) arabinoxylan, (ii) dough rheology properties, (iii) nutritional traits, (iv) polyphenol content, (v) processing quality traits, and (vi) sedimentation volume was conducted in wheat. For this purpose, as many as 2458 QTLs were collected from the 50 mapping studies published during 2013-20. Of the total QTLs, 1126 QTLs were projected on to the consensus map saturated with 2,50,077 markers resulting into the identification of 110 meta-QTLs (MQTLs) with average confidence interval (CI) of 5.6 cM. These MQTLs had 18.84 times reduced CI compared to CI of initial QTLs. Fifty-one (51) MQTLs were also verified with the marker-trait associations (MTAs) detected in earlier genome-wide association studies (GWAS). Physical region occupied by a single MQTL ranged from 0.12 to 749.71 Mb with an average of 130.25 Mb. Candidate gene mining allowed the identification of 2533 unique gene models from the MQTL regions. In-silico expression analysis discovered 439 differentially expressed gene models with >2 transcripts per million (TPM) expression in grains and related tissues which also included 44 high-confidence candidate genes known to be involved in the various cellular and biochemical processes related to quality traits. Further, nine functionally characterized wheat genes associated with grain protein content, high molecular weight glutenin and starch synthase enzymes were also found to be co-localized with some of the MQTLs. In addition, synteny analysis between wheat and rice MQTL regions identified 23 wheat MQTLs syntenic to 16 rice MQTLs. Furthermore, 64 wheat orthologues of 30 known rice genes were detected in 44 MQTL regions. These genes encoded proteins mainly belonging to the following families: starch synthase, glycosyl transferase, aldehyde dehydrogenase, SWEET sugar transporter, alpha amylase, glycoside hydrolase, glycogen debranching enzyme, protein kinase, peptidase, legumain and seed storage protein enzyme.

Numerous expansions in TRP ion channel diversity highlight widespread evolution of molecular sensors in animal diversification.

Transient Potential Receptor (TRP) ion channels are a diverse superfamily of multimodal molecular sensors that respond to a wide variety of stimuli, including mechanical, chemical, and thermal. TRP channels are present in most eukaryotes but best understood in mammalian, worm, and fly genetic models, where they are expressed in diverse cell-types and commonly associated with the nervous system. Here, we characterized TRP superfamily gene and genome evolution to better understand origins and evolution of molecular sensors, brains, and behavior in animals and help advance development of novel genetic technologies, like sonogenetics. We developed a flexible push-button bioinformatic and phylogenomic pipeline, GIGANTIC, that generated genome-based gene and species trees and enabled phylogenetic characterization of challenging remote homologs and distantly-related organisms deep in evolution. We identified complete sets of TRP superfamily ion channels, with over 3000 genes in 22 animal phyla and 70 species having publicly-available sequenced genomes, including 3 unicellular outgroups. We then identified clusters of TRP family members in genomes, evaluated gene models per cluster, and repaired split gene models. We also produced whole-organism PacBio transcriptomes for five species to independently validate our gene model assessment and model repairs. We find that many TRP families exhibited numerous and often extensive expansions in different phyla. Some expansions represent local clusters on respective genomes, a trend that is likely undercounted due to varied quality in genome assemblies and annotations of non-model organisms. Our work expands known TRP diversity across animals, including addition of previously uncharacterized phyla and identification of unrecognized homologs in previously characterized species.

Annotation of glycolysis, gluconeogenesis, and trehaloneogenesis pathways provide insight into carbohydrate metabolism in the Asian citrus psyllid

Citrus greening disease is caused by the pathogen Candidatus Liberibacter 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 of D. citri is 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 the D. citri genome, 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 in D. citri are highly conserved compared to orthologs in other insect systems, but copy numbers vary in D. 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 in D. citri have 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 reduce D. citri.

Single cell Iso-Sequencing enables rapid genome annotation for scRNAseq analysis

ABSTRACTSingle cell RNA sequencing (scRNAseq) is a powerful technique that continues to expand across various biological applications. However, incomplete 3′ UTR annotations in less developed or non-model systems can impede single cell analysis resulting in genes that are partially or completely uncounted. Performing scRNAseq with incomplete 3′ UTR annotations can impede the identification of cell identities and gene expression patterns and lead to erroneous biological inferences. We demonstrate that performing single cell isoform sequencing (ScISOr-Seq) in tandem with scRNAseq can rapidly improve 3′ UTR annotations. Using threespine stickleback fish (Gasterosteus aculeatus), we show that gene models resulting from a minimal embryonic ScISOr-Seq dataset retained 26.1% greater scRNAseq reads than gene models from Ensembl alone. Furthermore, pooling our ScISOr-Seq isoforms with a previously published adult bulk Iso-Seq dataset from stickleback, and merging the annotation with the Ensembl gene models, resulted in a marginal improvement (+0.8%) over the ScISOr-Seq only dataset. In addition, isoforms identified by ScISOr-Seq included thousands of new splicing variants. The improved gene models obtained using ScISOr-Seq lead to successful identification of cell types and increased the reads identified of many genes in our scRNAseq stickleback dataset. Our work illuminates ScISOr-Seq as a cost-effective and efficient mechanism to rapidly annotate genomes for scRNAseq.

Gene Validation and Remodelling Using Proteogenomics of Phytophthora cinnamomi, the Causal Agent of Dieback

Phytophthora cinnamomi is a pathogenic oomycete that causes plant dieback disease across a range of natural ecosystems and in many agriculturally important crops on a global scale. An annotated draught genome sequence is publicly available (JGI Mycocosm) and suggests 26,131 gene models. In this study, soluble mycelial, extracellular (secretome), and zoospore proteins of P. cinnamomi were exploited to refine the genome by correcting gene annotations and discovering novel genes. By implementing the diverse set of sub-proteomes into a generated proteogenomics pipeline, we were able to improve the P. cinnamomi genome annotation. Liquid chromatography mass spectrometry was used to obtain high confidence peptides with spectral matching to both the annotated genome and a generated 6-frame translation. Two thousand seven hundred sixty-four annotations from the draught genome were confirmed by spectral matching. Using a proteogenomic pipeline, mass spectra were used to edit the P. cinnamomi genome and allowed identification of 23 new gene models and 60 edited gene features using high confidence peptides obtained by mass spectrometry, suggesting a rate of incorrect annotations of 3% of the detectable proteome. The novel features were further validated by total peptide support, alongside functional analysis including the use of Gene Ontology and functional domain identification. We demonstrated the use of spectral data in combination with our proteogenomics pipeline can be used to improve the genome annotation of important plant diseases and identify missed genes. This study presents the first use of spectral data to edit and manually annotate an oomycete pathogen.

Utilizing a chromosomal-length genome assembly to annotate the Wnt signaling pathway in the Asian citrus psyllid, Diaphorina citri

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.

Deciphering the Monilinia fructicola Genome to Discover Effector Genes Possibly Involved in Virulence

Brown rot is the most economically important fungal disease of stone fruits and is primarily caused by Monilinia laxa and Monlinia fructicola. Both species co-occur in European orchards although M. fructicola is considered to cause the most severe yield losses in stone fruit. This study aimed to generate a high-quality genome of M. fructicola and to exploit it to identify genes that may contribute to pathogen virulence. PacBio sequencing technology was used to assemble the genome of M. fructicola. Manual structural curation of gene models, supported by RNA-Seq, and functional annotation of the proteome yielded 10,086 trustworthy gene models. The genome was examined for the presence of genes that encode secreted proteins and more specifically effector proteins. A set of 134 putative effectors was defined. Several effector genes were cloned into Agrobacterium tumefaciens for transient expression in Nicotiana benthamiana plants, and some of them triggered necrotic lesions. Studying effectors and their biological properties will help to better understand the interaction between M. fructicola and its stone fruit host plants.

Chromosomal-level genome assembly of the painted sea urchin Lytechinus pictus, a genetically enabled model system for cell biology and embryonic development

The painted urchin Lytechinus pictus is a sea urchin in the family Toxopneustidae and one of several sea urchin species that are routinely used as an experimental research organism. Recently, L. pictus has emerged as a tractable model system for establishing transgenic sea urchin lines due to its amenability to long term laboratory culture. We present the first published genome of L. pictus. This chromosomal-level assembly was generated using Illumina sequencing in conjunction with Oxford Nanopore Technologies long read sequencing and HiC chromatin conformation capture sequencing. The 998.9 Mb assembly exhibits high contiguity and has a scaffold length N50 of 46.0 Mb with 97% of the sequence assembled into 19 chromosomal-length scaffolds. These 19 scaffolds exhibit a high degree of synteny compared to the 19 chromosomes of a related species Lytechinus variegatus. Ab initio and transcript evidence gene modeling, combined with sequence homology, identified 28,631 gene models that capture 92% of BUSCO orthologs. This annotation strategy was validated by manual curation of gene models for the ABC transporter superfamily, which confirmed the completeness and accuracy of the annotations. Thus, this genome assembly, in conjunction with recent high contiguity assemblies of related species, positions Lytechinus pictus as an exceptional model system for comparative functional genomics and it will be a key resource for the developmental, toxicological, and ecological biology scientific communities.