Organellar (plastid and mitochondrial) genomes play an important role in resolving phylogenetic relationships, and next-generation sequencing technologies have led to a burst in their availability. The ongoing massive sequencing efforts require software tools for routine assembly and annotation of organellar genomes as well as their display as physical maps. OrganellarGenomeDRAW (OGDRAW) has become the standard tool to draw graphical maps of plastid and mitochondrial genomes. Here, we present a new version of OGDRAW equipped with a new front end. Besides several new features, OGDRAW now has access to a local copy of the organelle genome database of the NCBI RefSeq project. Together with batch processing of (multi-)GenBank files, this enables the user to easily visualize large sets of organellar genomes spanning entire taxonomic clades. The new OGDRAW server can be accessed at https://chlorobox.mpimp-golm.mpg.de/OGDraw.html.

OrganellarGenomeDRAW (OGDRAW) version 1.3.1: expanded toolkit for the graphical visualization of organellar genomes.

Plastome (plastid genome) sequences provide valuable information for understanding the phylogenetic relationships and evolutionary history of plants. Although the rapid development of high-throughput sequencing technology has led to an explosion of plastome sequences, annotation remains a significant bottleneck for plastomes. User-friendly batch annotation of multiple plastomes is an urgent need. We introduce Plastid Genome Annotator (PGA), a standalone command line tool that can perform rapid, accurate, and flexible batch annotation of newly generated target plastomes based on well-annotated reference plastomes. In contrast to current existing tools, PGA uses reference plastomes as the query and unannotated target plastomes as the subject to locate genes, which we refer to as the reverse query-subject BLAST search approach. PGA accurately identifies gene and intron boundaries as well as intron loss. The program outputs GenBank-formatted files as well as a log file to assist users in verifying annotations. Comparisons against other available plastome annotation tools demonstrated the high annotation accuracy of PGA, with little or no post-annotation verification necessary. Likewise, we demonstrated the flexibility of reference plastomes within PGA by annotating the plastome of Rosa roxburghii using that of Amborella trichopoda as a reference. The program, user manual and example data sets are freely available at https://github.com/quxiaojian/PGA
 . PGA facilitates rapid, accurate, and flexible batch annotation of plastomes across plants. For projects in which multiple plastomes are generated, the time savings for high-quality plastome annotation are especially significant.

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PGA: a software package for rapid, accurate, and flexible batch annotation of plastomes

The complete sequences of chloroplast genomes provide wealthy information regarding the evolutionary history of species. With the advance of next-generation sequencing technology, the number of completely sequenced chloroplast genomes is expected to increase exponentially, powerful computational tools annotating the genome sequences are in urgent need. We have developed a web server CPGAVAS. The server accepts a complete chloroplast genome sequence as input. First, it predicts protein-coding and rRNA genes based on the identification and mapping of the most similar, full-length protein, cDNA and rRNA sequences by integrating results from Blastx, Blastn, protein2genome and est2genome programs. Second, tRNA genes and inverted repeats (IR) are identified using tRNAscan, ARAGORN and vmatch respectively. Third, it calculates the summary statistics for the annotated genome. Fourth, it generates a circular map ready for publication. Fifth, it can create a Sequin file for GenBank submission. Last, it allows the extractions of protein and mRNA sequences for given list of genes and species. The annotation results in GFF3 format can be edited using any compatible annotation editing tools. The edited annotations can then be uploaded to CPGAVAS for update and re-analyses repeatedly. Using known chloroplast genome sequences as test set, we show that CPGAVAS performs comparably to another application DOGMA, while having several superior functionalities. CPGAVAS allows the semi-automatic and complete annotation of a chloroplast genome sequence, and the visualization, editing and analysis of the annotation results. It will become an indispensible tool for researchers studying chloroplast genomes. The software is freely accessible from
 http://www.herbalgenomics.org/cpgavas
 .

/pdf/cpgavas-an-integrated-web-server-for-the-annotation-cim391ivj8.pdf

CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences.

We previously developed a web server CPGAVAS for annotation, visualization and GenBank submission of plastome sequences. Here, we upgrade the server into CPGAVAS2 to address the following challenges: (i) inaccurate annotation in the reference sequence likely causing the propagation of errors; (ii) difficulty in the annotation of small exons of genes petB, petD and rps16 and trans-splicing gene rps12; (iii) lack of annotation for other genome features and their visualization, such as repeat elements; and (iv) lack of modules for diversity analysis of plastomes. In particular, CPGAVAS2 provides two reference datasets for plastome annotation. The first dataset contains 43 plastomes whose annotation have been validated or corrected by RNA-seq data. The second one contains 2544 plastomes curated with sequence alignment. Two new algorithms are also implemented to correctly annotate small exons and trans-splicing genes. Tandem and dispersed repeats are identified, whose results are displayed on a circular map together with the annotated genes. DNA-seq and RNA-seq data can be uploaded for identification of single-nucleotide polymorphism sites and RNA-editing sites. The results of two case studies show that CPGAVAS2 annotates better than several other servers. CPGAVAS2 will likely become an indispensible tool for plastome research and can be accessed from http://www.herbalgenomics.org/cpgavas2.

/pdf/cpgavas2-an-integrated-plastome-sequence-annotator-and-1ovrtqcarg.pdf

CPGAVAS2, an integrated plastome sequence annotator and analyzer

The chloroplast genome encodes proteins required for photosynthesis, gene expression, and other essential organellar functions. Derived from a cyanobacterial ancestor, the chloroplast combines prokaryotic and eukaryotic features of gene expression and is regulated by many nucleus-encoded proteins. This review covers four major chloroplast posttranscriptional processes: RNA processing, editing, splicing, and turnover. RNA processing includes the generation of transcript 5' and 3' termini, as well as the cleavage of polycistronic transcripts. Editing converts specific C residues to U and often changes the amino acid that is specified by the edited codon. Chloroplasts feature introns of groups I and II, which undergo protein-facilitated cis- or trans-splicing in vivo. Each of these RNA-based processes involves proteins of the pentatricopeptide motif-containing family, which does not occur in prokaryotes. Plant-specific RNA-binding proteins may underpin the adaptation of the chloroplast to the eukaryotic context.

Chloroplast RNA metabolism.

We have developed the web application GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) for the rapid and accurate annotation of organellar genome sequences, in particular chloroplast genomes. In contrast to existing tools, GeSeq combines batch processing with a fully customizable reference sequence selection of organellar genome records from NCBI and/or references uploaded by the user. For the annotation of chloroplast genomes, the application additionally provides an integrated database of manually curated reference sequences. GeSeq identifies genes or other feature-encoding regions by BLAT-based homology searches and additionally, by profile HMM searches for protein and rRNA coding genes and two de novo predictors for tRNA genes. These unique features enable the user to conveniently compare the annotations of different state-of-the-art methods, thus supporting high-quality annotations. The main output of GeSeq is a GenBank file that usually requires only little curation and is instantly visualized by OGDRAW. GeSeq also offers a variety of optional additional outputs that facilitate downstream analyzes, for example comparative genomic or phylogenetic studies.

GeSeq - versatile and accurate annotation of organelle genomes.

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.

/pdf/the-evolution-of-chloroplast-rna-editing-3ews7z85qf.pdf

The evolution of chloroplast RNA editing.

Chloroplast ribonucleoproteins (cpRNPs) are nuclear-encoded, highly abundant, and light-regulated RNA binding proteins. They have been shown to be involved in chloroplast RNA processing and stabilization in vitro and are phylogenetically related to the well-described heterogeneous nuclear ribonucleoproteins (hnRNPs). cpRNPs have been found associated with mRNAs present in chloroplasts and have been regarded as nonspecific stabilizers of chloroplast transcripts. Here, we demonstrate that null mutants of the cpRNP family member CP31A exhibit highly specific and diverse defects in chloroplast RNA metabolism. First, analysis of cp31a and cp31a/cp31b double mutants uncovers that these 2 paralogous genes participate nonredundantly in a combinatorial fashion in processing a subset of chloroplast editing sites in vivo. Second, a genome-wide analysis of chloroplast transcript accumulation in cp31a mutants detected a virtually complete loss of the chloroplast ndhF mRNA and lesser reductions for specific other mRNAs. Fluorescence analyses show that the activity of the NADH dehydrogenase complex, which also includes the NdhF subunit, is defective in cp31a mutants. This indicates that cpRNPs are important in vivo for calibrating the expression levels of specific chloroplast mRNAs and impact chloroplast physiology. Taken together, the specificity and combinatorial aspects of cpRNP functions uncovered suggest that these chloroplast proteins are functional equivalents of nucleocytosolic hnRNPs.

https://www.pnas.org/content/pnas/106/14/6002.full.pdf

Pascal Lehwark

Papers

GeSeq - versatile and accurate annotation of organelle genomes.

OrganellarGenomeDRAW (OGDRAW) version 1.3.1: expanded toolkit for the graphical visualization of organellar genomes.

The evolution of chloroplast RNA editing.

Chloroplast ribonucleoprotein CP31A is required for editing and stability of specific chloroplast mRNAs

GB2sequin - A file converter preparing custom GenBank files for database submission.