Pervasive transcription of plant organelle genomes: functional noncoding transcriptomes?

Plant mitochondrial and plastid genomes typically show pervasive, genome-wide transcription. Little is known, however, about the utility of organelle noncoding RNAs, which often make up most of the transcriptome. Here, we suggest that long-read sequencing data combined with dedicated RNA databases could help identify putative functional organelle noncoding transcripts.

HSDatabase-a database of highly similar duplicate genes from plants, animals, and algae.

Gene duplication is an important evolutionary mechanism capable of providing new genetic material, which in some instances can help organisms adapt to various environmental conditions. Recent studies, for example, have indicated that highly similar duplicate genes (HSDs) are aiding adaptation to extreme conditions via gene dosage. However, for most eukaryotic genomes HSDs remain uncharacterized, partly because they can be hard to identify and categorize efficiently and effectively. Here, we collected and curated HSDs in nuclear genomes from various model animals, land plants and algae and indexed them in an online, open-access sequence repository called HSDatabase. Currently, this database contains 117 864 curated HSDs from 40 distinct genomes; it includes statistics on the total number of HSDs per genome as well as individual HSD copy numbers/lengths and provides sequence alignments of the duplicate gene copies. HSDatabase also allows users to download sequences of gene copies, access genome browsers, and link out to other databases, such as Pfam and Kyoto Encyclopedia of Genes and Genomes. What is more, a built-in Basic Local Alignment Search Tool option is available to conveniently explore potential homologous sequences of interest within and across species. HSDatabase has a user-friendly interface and provides easy access to the source data. It can be used on its own for comparative analyses of gene duplicates or in conjunction with HSDFinder, a newly developed bioinformatics tool for identifying, annotating, categorizing and visualizing HSDs. Database URL: http://hsdfinder.com/database/.

An overview of online resources for intra-species detection of gene duplications.

Gene duplication plays an important role in evolutionary mechanism, which can act as a new source of genetic material in genome evolution. However, detecting duplicate genes from genomic data can be challenging. Various bioinformatics resources have been developed to identify duplicate genes from single and/or multiple species. Here, we summarize the metrics used to measure sequence identity among gene duplicates within species, compare several computational approaches that have been used to predict gene duplicates, and review recent advancements of a Basic Local Alignment Search Tool (BLAST)-based web tool and database, allowing future researchers to easily identify intra-species gene duplications. This article is a quick reference guide for research tools used for detecting gene duplicates.

The evolution of extremely diverged plastomes in Selaginellaceae (lycophyte) is driven by repeat patterns and the underlying DNA maintenance machinery.

Two factors are proposed to account for the unusual features of organellar genomes: the disruptions of organelle-targeted DNA replication, repair, and recombination (DNA-RRR) systems in the nuclear genome and repetitive elements in organellar genomes. Little is known about how these factors affect organellar genome evolution. The deep-branching vascular plant family Selaginellaceae is known to have a deficient DNA-RRR system and convergently evolved organellar genomes. However, we found that the plastid genome (plastome) of Selaginella sinensis has extremely accelerated substitution rates, a low GC content, pervasive repeat elements, a dynamic network structure, and it lacks direct or inverted repeats. Unexpectedly, its organelle DNA-RRR system is short of a plastid-targeted Recombinase A1 (RecA1) and a mitochondrion-targeted RecA3, in line with other explored Selaginella species. The plastome contains a large collection of short- and medium-sized repeats. Given the absence of RecA1 surveillance, we propose that these repeats trigger illegitimate recombination, accelerated mutation rates, and structural instability. The correlations between repeat quantity and architectural complexity in the Selaginella plastomes support these conclusions. We, therefore, hypothesize that the interplay of the deficient DNA-RRR system and the high repeat content has led to the extraordinary divergence of the S. sinensis plastome. Our study not only sheds new light on the mechanism of plastome divergence by emphasizing the power of cytonuclear integration, but it also reconciles the longstanding contradiction on the effects of DNA-RRR system disruption on genome structure evolution.

Genome evolution: Minicircular mtDNA and unusual heteroplasmy in a parasitic plant.

Minicircular organelle genomes exist in diverse species but have never been observed in plants - that is, until now. The mitochondrial genome of the holoparasite Rhopalocnemis phalloides comprises 21 minicircles, which are extremely heteroplasmic, providing an exceptional example of convergent organelle evolution across disparate lineages.

Simple Matching Using QIIME 2 and RDP Reveals Misidentified Sequences and an Underrepresentation of Fungi in Reference Datasets.

Simple nucleotide matching identification methods are not as accurate as once thought at identifying environmental fungal sequences. This is largely because of incorrect naming and the underrepresentation of various fungal groups in reference datasets. Here, we explore these issues by examining an environmental metabarcoding dataset of partial large subunit rRNA sequences of Basidiomycota and basal fungi. We employed the simple matching method using the QIIME 2 classifier and the RDP Classifier in conjunction with the latest releases of the SILVA (138.1, 2020) and RDP (11, 2014) reference datasets and then compared the results with a manual phylogenetic binning approach. Of the 71 query sequences tested, 21 and 42% were misidentified using QIIME 2 and the RDP Classifier, respectively. Of these simple matching misidentifications, more than half resulted from the underrepresentation of various groups of fungi in the SILVA and RDP reference datasets. More comprehensive reference datasets with fewer misidentified sequences will increase the accuracy of simple matching identifications. However, we argue that the phylogenetic binning approach is a better alternative to simple matching since, in addition to better accuracy, it provides evolutionary information about query sequences.

Mutational Effects of Mobile Introns on the Mitochondrial Genomes of Metschnikowia Yeasts.

It has been argued that DNA repair by homologous recombination in the context of endonuclease-mediated cleavage can cause mutations. To better understand this phenomenon, we examined homologous recombination following endonuclease cleavage in a native genomic context: the movement of self-splicing introns in the mitochondrial genomes of Metschnikowia yeasts. Self-splicing mitochondrial introns are mobile elements, which can copy and paste themselves at specific insertion sites in mitochondrial DNA using a homing endonuclease in conjunction with homologous recombination. Here, we explore the mutational effects of self-splicing introns by comparing sequence variation within the intron-rich cox1 and cob genes from 71 strains (belonging to 40 species) from the yeast genus Metschnikowia. We observed a higher density of single nucleotide polymorphisms around self-splicing-intron insertion sites. Given what is currently known about the movement of organelle introns, it is likely that their mutational effects result from the high binding affinity of endonucleases and their interference with repair machinery during homologous recombination (or, alternatively, via gene conversion occurring during the intron insertion process). These findings suggest that there are fitness costs to harbouring self-splicing, mobile introns and will help us better understand the risks associated with modern biotechnologies that use endonuclease-mediated homologous recombination, such as CRISPR-Cas9 gene editing.

Protocol for using NoBadWordsCombiner to merge and minimize "bad words" from BLAST hits against multiple eukaryotic gene annotation databases.

Annotating protein-coding genes can be challenging, especially when searching for the best hits against multiple functional databases. This is partly because of "bad words" appearing as top hits, such as hypothetical or uncharacterized proteins. To help alleviate some of these issues, we designed a bioinformatics tool called NoBadWordsCombiner, which efficiently merges the hits from various databases, strengthening gene definitions by minimizing functional descriptions containing "bad words." Unlike other available tools, NoBadWordsCombiner is user friendly, but it does require users to have some general bioinformatics skills, including a basic understanding of the BLAST package and dash shell in Linux/Unix environments. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021a).

Protocol for HSDFinder: Identifying, annotating, categorizing, and visualizing duplicated genes in eukaryotic genomes.

Although gene duplications have been documented in many species, the precise numbers of highly similar duplicated genes (HSDs) in eukaryotic nuclear genomes remain largely unknown and can be time-consuming to explore. We developed HSDFinder to identify, categorize, and visualize HSDs in eukaryotic nuclear genomes using protein family domains and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In contrast to existing tools, HSDFinder allows users to compare HSDs among different species and visualize results in different KEGG pathway functional categories via heatmap plotting. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Draft genome sequence of the Antarctic green alga Chlamydomonas sp. UWO241.

Antarctica is home to an assortment of psychrophilic algae, which have evolved various survival strategies for coping with their frigid environments. Here, we explore Antarctic psychrophily by examining the ∼212 Mb draft nuclear genome of the green alga Chlamydomonas sp. UWO241, which resides within the water column of a perennially ice-covered, hypersaline lake. Like certain other Antarctic algae, UWO241 encodes a large number (≥37) of ice-binding proteins, putatively originating from horizontal gene transfer. Even more striking, UWO241 harbors hundreds of highly similar duplicated genes involved in diverse cellular processes, some of which we argue are aiding its survival in the Antarctic via gene dosage. Gene and partial gene duplication appear to be an ongoing phenomenon within UWO241, one which might be mediated by retrotransposons. Ultimately, we consider how such a process could be associated with adaptation to extreme environments but explore potential non-adaptive hypotheses as well.

HSDFinder: A BLAST-Based Strategy for Identifying Highly Similar Duplicated Genes in Eukaryotic Genomes.

Gene duplication is an important evolutionary mechanism capable of providing new genetic material for adaptive and nonadaptive evolution. However, bioinformatics tools for identifying duplicate genes are often limited to the detection of paralogs in multiple species or to specific types of gene duplicates, such as retrocopies. Here, we present a user-friendly, BLAST-based web tool, called HSDFinder, which can identify, annotate, categorize, and visualize highly similar duplicate genes (HSDs) in eukaryotic nuclear genomes. HSDFinder includes an online heatmap plotting option, allowing users to compare HSDs among different species and visualize the results in different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional categories. The external software requirements are BLAST, InterProScan, and KEGG. The utility of HSDFinder was tested on various model eukaryotic species, including Chlamydomonas reinhardtii, Arabidopsis thaliana, Oryza sativa, and Zea mays as well as the psychrophilic green alga Chlamydomonas sp. UWO241, and was proven to be a practical and accurate tool for gene duplication analyses. The web tool is free to use at http://hsdfinder.com. Documentation and tutorials can be found via the GitHub: https://github.com/zx0223winner/HSDFinder.

Depositing annotated sequences in GenBank: there needs to be a better way.

Submitting sequences to the National Center for Biotechnology Information (NCBI) is an integral part of research and the publication process for many disciplines within the life sciences, and it will only become more important as sequencing technologies continue to improve. Here, I argue that the available infrastructure and resources for uploading data to NCBI-especially the associated annotations of eukaryotic genomes-are inefficient, hard to use and sometimes just plain bad. This, in turn, is causing some researchers to forgo annotations entirely in their submissions. The time is overdue for the development of sophisticated, user-friendly software for depositing annotated sequences in GenBank.

The strange mitochondrial genomes of Metschnikowia yeasts.

While sequencing and characterizing the mitochondrial genomes of 71 strains from the yeast genus Metschnikowia [1] (close cousin to the model species Candida albicans), we uncovered one of the most extreme examples of mitochondrial genome architectural diversity observed to date. These Metschnikowia mitochondrial DNAs (mtDNAs) capture nearly the entire known gene-size and intron-content range for cox1 and cob across all eukaryotic life and show remarkable differences in structure and noncoding content. This genomic variation can be seen both among species and between strains of the same species, raising the question: why are Metschnikowia mitogenomes so malleable?

Common Repeat Elements in the Mitochondrial and Plastid Genomes of Green Algae.

Despite both originating from endosymbiotic bacteria, one does not typically expect mitochondrial DNA (mtDNA) to show strong sequence identity to plastid DNA (ptDNA). Nevertheless, a recent analysis of Haematococcus lacustris revealed exactly that. A common repeat element has proliferated throughout the mtDNA and ptDNA of this chlamydomonadalean green alga, resulting in the unprecedented situation whereby these two distinct organelle genomes are largely made up of nearly identical sequences. In this short update to the work on H. lacustris, I highlight another chlamydomonadalean species (Stephanosphaera pluvialis) for which matching repeats have spread throughout its organelle genomes (but to a lesser degree than in H. lacustris). What's more, the organelle repeats from S. pluvialis are similar to those from H. lacustris, suggesting that they have a shared origin, and perhaps existed in the mtDNA and ptDNA of the most recent common ancestor of these two species. However, my examination of organelle genomes from other close relatives of H. lacustris and S. pluvialis did not uncover further compelling examples of common organelle repeat elements, meaning that the evolutionary history of these repeats might be more complicated than initially thought.

Revisiting Ceriantharian (Anthozoa) Mitochondrial Genomes: Casting Doubts about Their Structure and Size.

Recently, Stampar et al. (2019. Linear mitochondrial genome in Anthozoa (Cnidaria): a case study in. Sci Rep. 9(1):6094.) uncovered highly atypical mitochondrial genome structures in the cnidarian species Pachycerianthus magnus and Isarachnanthus nocturnus (Anthozoa, Ceriantharia). These two mitochondrial DNAs assembled as linear fragmented genomes, comprising eight and five chromosomes, respectively-architectures unlike any other anthozoan mitogenome described to date. What's more, they have cumulative lengths of 77.8 (P. magnus) and 80.9 kb (I. nocturnus), making them the largest animal mitochondrial DNAs on record, a finding which garnered significant attention by various news media. Here, I take a closer look at the work of Stampar et al. and question their key results. I provide evidence that the currently available mitogenome sequences for I. nocturnus and P. magnus, including their structures, sizes, and chromosome numbers, should be treated with caution. More work must be done on these genomes before one can say with any certainty that they are linear, fragmented, or the largest animal mitogenomes observed to date.

Can Green Algal Plastid Genome Size Be Explained by DNA Repair Mechanisms?

A major finding in organelle biology over the past decade is that land plant mitochondrial genomes, which are the largest among eukaryotes, can have a "Jekyll and Hyde" mutational pattern: low for synonymous sites, high for intergenic ones. This has led to the theory that double-strand breaks (DSBs) in the intergenic DNA of plant mitogenomes are repaired by inaccurate mechanisms, such as break-induced replication, which can result in large insertions and, thus, could explain why these genomes are so prone to expansion. But how universal is this theory? Can it apply to other giant organelle DNAs, such as the massive plastid DNAs (ptDNAs) of chlamydomonadalean green algae? Indeed, it can. Analysis of the expanded plastomes from two distinct isolates of the unicellular chlamydomonadalean Chlorosarcinopsis eremi uncovered exceptionally low rates of synonymous substitution in the coding regions but high substitution rates, including frequent indels, in the noncoding ptDNA, mirroring the trend from land plant mitogenomes. Remarkably, nearly all of the substitutions and indels identified in the noncoding ptDNA of C. eremi occur adjacent to or within short inverted palindromic repeats, suggesting that these elements are mutational hotspots. Building upon earlier studies, I propose that these palindromic repeats are predisposed to DSBs and that error-prone repair of these breaks is contributing to genomic expansion. Short palindromic repeats are a common theme among bloated plastomes, including the largest one on record, meaning that these data could have wide-reaching implications for our understanding of ptDNA expansion.

Presence and absence of light-independent chlorophyll biosynthesis among Chlamydomonas green algae in an ice-covered Antarctic lake.

The cold, permanently ice-covered waters of Lake Bonney, Antarctica, may seem like an uninviting place for an alga, but they are home to a diversity of photosynthetic life, including Chlamydomonas sp. UWO241, a psychrophile residing in the deep photic zone. Recently, we found that UWO241 has lost the genes responsible for light-independent chlorophyll biosynthesis, which is surprising given that this green alga comes from a light-limited environment and experiences extended periods of darkness during the Antarctic winter. Why discard such a process? We argued that it might be linked to the very high dissolved oxygen concentration of Lake Bonney at the depth at which UWO241 is found. Oxygen is the Achilles' heel of the key enzyme involved in light-independent chlorophyll biosynthesis: DPOR. If this hypothesis is true, then other algae in Lake Bonney should also be susceptible to losing DPOR, such as Chlamydomonas sp. ICE-MDV, which predominantly resides in the chemocline, a depth with an even higher oxygen concentration than that where UWO241 exists. Here, we report that, contrary to our earlier prediction, ICE-MDV has maintained the genes encoding DPOR. We briefly discuss the implications of this finding in relation to the loss of light-independent chlorophyll synthesis in UWO241.

The mitochondrial and chloroplast genomes of the green alga Haematococcus are made up of nearly identical repetitive sequences.

The chlamydomonadalean green alga Haematococcus lacustris (strain UTEX 2505) has the largest chloroplast genome on record: 1352 kb with ∼90% non-coding DNA [1,2]. But what of the mitochondrial genome? Here we present sequencing, assembly, and analysis of the mitogenome that shows that it, too, is extremely expanded. What's more, the same repetitive elements have spread throughout the mitochondrial and chloroplast (or plastid) DNA (mtDNA and ptDNA, respectively), resulting in the situation whereby these two distinct organelle genomes are made up of nearly identical sequences.