OTP970 Is Required for RNA Editing of Chloroplast ndhB Transcripts in Arabidopsis thaliana

RNA editing is essential for compensating for defects or mutations in haploid organelle genomes and is regulated by numerous trans-factors. Pentatricopeptide repeat (PPR) proteins are the prime factors that are involved in RNA editing; however, many have not yet been identified. Here, we screened the plastid-targeted PLS-DYW subfamily of PPR proteins belonging to Arabidopsis thaliana and identified ORGANELLE TRANSCRIPT PROCESSING 970 (OTP970) as a key player in RNA editing in plastids. A loss-of-function otp970 mutant was impaired in RNA editing of ndhB transcripts at site 149 (ndhB-C149). RNA-immunoprecipitation analysis indicated that OTP970 was associated with the ndhB-C149 site. The complementation of the otp970 mutant with OTP970 lacking the DYW domain (OTP970∆DYW) failed to restore the RNA editing of ndhB-C149. ndhB gene encodes the B subunit of the NADH dehydrogenase-like (NDH) complex; however, neither NDH activity and stability nor NDH-PSI supercomplex formation were affected in otp970 mutant compared to the wild type, indicating that alteration in amino acid sequence is not necessary for NdhB function. Together, these results suggest that OTP970 is involved in the RNA editing of ndhB-C149 and that the DYW domain is essential for its function.

ReGSP: a visualized application for homology-based gene searching and plotting using multiple reference sequences

The massively parallel nature of next-generation sequencing technologies has contributed to the generation of massive sequence data in the last two decades. Deciphering the meaning of each generated sequence requires multiple analysis tools, at all stages of analysis, from the reads stage all the way up to the whole-genome level. Homology-based approaches based on related reference sequences are usually the preferred option for gene and transcript prediction in newly sequenced genomes, resulting in the popularity of a variety of BLAST and BLAST-based tools. For organelle genomes, a single-reference–based gene finding tool that uses grouping parameters for BLAST results has been implemented in the Genome Search Plotter (GSP). However, this tool does not accept multiple and user-customized reference sequences required for a broad homology search. Here, we present multiple Reference–based Gene Search and Plot (ReGSP), a simple and convenient web tool that accepts multiple reference sequences for homology-based gene search. The tool incorporates cPlot, a novel dot plot tool, for illustrating nucleotide sequence similarity between the query and the reference sequences. ReGSP has an easy-to-use web interface and is freely accessible at https://ds.mju.ac.kr/regsp.

Organelle Comparative Genome Analysis Reveals Novel Alloplasmic Male Sterility with orf112 in Brassica oleracea L.

B. oleracea Ogura CMS is an alloplasmic male-sterile line introduced from radish by interspecific hybridization and protoplast fusion. The introduction of alien cytoplasm resulted in many undesirable traits, which affected the yield of hybrids. Therefore, it is necessary to identify the composition and reduce the content of alien cytoplasm in B. oleracea Ogura CMS. In the present study, we sequenced, assembled, and compared the organelle genomes of Ogura CMS cabbage and its maintainer line. The chloroplast genome of Ogura-type cabbage was completely derived from normal-type cabbage, whereas the mitochondrial genome was recombined from normal-type cabbage and Ogura-type radish. Nine unique regions derived from radish were identified in the mitochondrial genome of Ogura-type cabbage, and the total length of these nine regions was 35,618 bp, accounting for 13.84% of the mitochondrial genome. Using 32 alloplasmic markers designed according to the sequences of these nine regions, one novel sterile source with less alien cytoplasm was discovered among 305 materials and named Bel CMS. The size of the alien cytoplasm in Bel CMS was 21,587 bp, accounting for 8.93% of its mtDNA, which was much less than that in Ogura CMS. Most importantly, the sterility gene orf138 was replaced by orf112, which had a 78-bp deletion, in Bel CMS. Interestingly, Bel CMS cabbage also maintained 100% sterility, although orf112 had 26 fewer amino acids than orf138. Field phenotypic observation showed that Bel CMS was an excellent sterile source with stable 100% sterility and no withered buds at the early flowering stage, which could replace Ogura CMS in cabbage heterosis utilization.

Structure and Phylogeny of the Curly Birch Chloroplast Genome

Curly birch [Betula pendula var. carelica (Merckl.) Hämet-Ahti] is a relatively rare variety of silver birch (B. pendula Roth) that occurs mainly in Northern Europe and northwest part of Russia (Karelia). It is famous for the beautiful decorative texture of wood. Abnormal xylogenesis underlying this trait is heritable, but its genetic mechanism has not yet been fully understood. The high number of potentially informative genetic markers can be identified through sequencing nuclear and organelle genomes. Here, the de novo assembly, complete nucleotide sequence, and annotation of the chloroplast genome (plastome) of curly birch are presented for the first time. The complete plastome length is 160,523 bp. It contains 82 genes encoding structural and enzymatic proteins, 37 transfer RNAs (tRNAs), and eight ribosomal RNAs (rRNAs). The chloroplast DNA (cpDNA) is AT-rich containing 31.5% of A and 32.5% of T nucleotides. The GC-rich regions represent inverted repeats IR1 and IR2 containing genes of rRNAs (5S, 4.5S, 23S, and 16S) and tRNAs (trnV, trnI, and trnA). A high content of GC was found in rRNA (55.2%) and tRNA (53.2%) genes, but only 37.0% in protein-coding genes. In total, 384 microsatellite or simple sequence repeat (SSR) loci were found, mostly with mononucleotide motifs (92% of all loci) and predominantly A or T motifs (94% of all mononucleotide motifs). Comparative analysis of cpDNA in different plant species revealed high structural and functional conservatism in organization of the angiosperm plastomes, while the level of differences depends on the phylogenetic relationship. The structural and functional organization of plastome in curly birch was similar to cpDNA in other species of woody plants. Finally, the identified cpDNA sequence variation will allow to develop useful genetic markers.

Organelle Genome Variation in the Red Algal Genus Ahnfeltia (Florideophyceae)

The agarophyte Ahnfeltia (Ahnfeltiales, Rhodophyta) is a globally widespread genus with 11 accepted species names. Two of the most widespread species in this genus, A. plicata and A. fastigiata, may have diverged genetically due to past geographic changes and subsequent geographic isolation. To investigate this genomic and genetic diversity, we generated new plastid (ptDNAs) and mitochondrial genomes (mtDNAs) of these Ahnfeltia species from four different regions (A. plicata - Chile and UK and A. fastigiata - Korea and Oregon). Two architecture variations were found in the Ahnfeltia genomes: in ptDNA of A. fastigiata Oregon, the hypothetical pseudogene region was translocated, likely due to recombination with palindromic repeats or a gene transfer from a red algal plasmid. In mtDNA of A. fastigiata Korea, the composition of the group II intronic ORFs was distinct from others suggesting different scenarios of gain and loss of group II intronic ORFs. These features resulted in genome size differences between the two species. Overall gene contents of organelle genomes of Ahnfeltia were conserved. Phylogenetic analysis using concatenated genes from ptDNAs and mtDNAs supported the monophyly of the Ahnfeltiophycidae. The most probable individual gene trees showed that the Ahnfeltia populations were genetically diversified. These trees, the cox1 haplotype network, and a dN/dS analysis all supported the theory that these Ahnfeltia populations have diversified genetically in accordance with geographic distribution.

Evaluation of Four Methods to Identify the Homozygotic Sex Chromosome in Small Populations

BackgroundWhole genomes are commonly assembled into a collection of scaffolds and often lack annotations of autosomes, sex chromosomes, and organelle genomes (i.e., mitochondrial and chloroplast). As these chromosome types differ in effective population size and can have highly disparate evolutionary histories, it is imperative to take this information into account when analysing genomic variation. Here we assessed the accuracy of four methods for identifying the homogametic sex chromosome in a small population using two whole genome sequences (WGS) and 133 RAD sequences of white-tailed eagles (Haliaeetus albicilla): i) difference in read depth per scaffold in a male and a female, ii) heterozygosity per scaffold in a male and a female, iii) mapping to a reference genome of a related species (chicken) with identified sex chromosomes, and iv) analysis of SNP-loadings from a principal components analysis (PCA), based on the low-depth RADseq data. ResultsThe best performing approach was the reference mapping (method iii), which identified 98.12% of the expected homogametic sex chromosome (Z). The read depth per scaffold (method i) identified 86.41% of the homogametic sex chromosome with few false positives. The SNP-loading scores (method iv) found 78.6% of the Z-chromosome and had a false positive discovery rate of more than 10%. The heterozygosity per scaffold (method ii) did not provide clear results due to a lack of diversity in both the Z and autosomal chromosomes, and potential interference from the heterogametic sex chromosome (W). The evaluation of these methods also revealed 10 Mb of likely PAR and gametologous regions.ConclusionIdentification of the homogametic sex chromosome in a small population is best accomplished by reference mapping or examining read depth differences between sexes.

Organelle Genomes and Transcriptomes of Nymphaea Reveal the Interplay between Intron Splicing and RNA Editing

Posttranscriptional modifications, including intron splicing and RNA editing, are common processes during regulation of gene expression in plant organelle genomes. However, the intermediate products of intron-splicing, and the interplay between intron-splicing and RNA-editing were not well studied. Most organelle transcriptome analyses were based on the Illumina short reads which were unable to capture the full spectrum of transcript intermediates within an organelle. To fully investigate the intermediates during intron splicing and the underlying relationships with RNA editing, we used PacBio DNA-seq and Iso-seq, together with Illumina short reads genome and transcriptome sequencing data to assemble the chloroplast and mitochondrial genomes of Nymphaea ‘Joey Tomocik’ and analyze their posttranscriptional features. With the direct evidence from Iso-seq, multiple intermediates partially or fully intron-spliced were observed, and we also found that both cis- and trans-splicing introns were spliced randomly. Moreover, by using rRNA-depleted and non-Oligo(dT)-enrichment strand-specific RNA-seq data and combining direct SNP-calling and transcript-mapping methods, we identified 98 and 865 RNA-editing sites in the plastome and mitogenome of N. ‘Joey Tomocik’, respectively. The target codon preference, the tendency of increasing protein hydrophobicity, and the bias distribution of editing sites are similar in both organelles, suggesting their common evolutionary origin and shared editing machinery. The distribution of RNA editing sites also implies that the RNA editing sites in the intron and exon regions may splice synchronously, except those exonic sites adjacent to intron which could only be edited after being intron-spliced. Our study provides solid evidence for the multiple intermediates co-existing during intron-splicing and their interplay with RNA editing in organelle genomes of a basal angiosperm.

Identification of the homozygotic sex chromosome of non-model organisms

Whole genomes are commonly assembled into a collection of scaffolds and often lack annotations of autosomes, sex chromosomes and, and organelle genomes (i.e., mitochondrial and chloroplast). As these chromosome types can have highly disparate evolutionary histories, it is imperative to take this information into account when analyzing genomic variation. Here we assessed the accuracy of four methods for identifying the homogametic sex chromosome using two whole genome sequenced (WGS) and 133 RAD sequenced white-tailed eagles (Haliaeetus albicilla): i) difference in read depth per scaffold, ii) heterozygosity per scaffold in a male and female bird, iii) mapping to a reference genome of a related species (chicken) with identified sex chromosomes, and iv) an analysis of SNP-loadings from a principal components analysis (PCA), based on low-depth RADseq data from 133 individuals. In i and ii, the WGS were mapped to a reference genome consisting of 1142 assembled scaffolds from the golden eagle (Aquila chrysaetos) with no identified chromosomes. The read depth per scaffold identified 86.41% of the homogametic sex chromosome (Z) with few false positives. The SNP-loading scores found 78.6% of the Z-chromosome but had a false positive discovery rate of more than 10%. The heterozygosity per scaffold did not provide clear results due to a lack of diversity in both the Z and autosomal chromosomes, and potential interference from the heterogametic sex chromosome (W).

Ultra-deep sequencing reveals dramatic alteration of organellar genomes in Physcomitrella patens due to biased asymmetric recombination

Destabilization of organelle genomes causes organelle dysfunction that appears as abnormal growth in plants and diseases in human. In plants, loss of the bacterial-type homologous recombination repair (HRR) factors RECA and RECG induces organelle genome instability. In this study, we show the landscape of organelle genome instability in Physcomitrella patens HRR knockout mutants by deep sequencing in combination with informatics approaches. Genome-wide maps of rearrangement positions in the organelle genomes, which exhibited prominent mutant-specific patterns, were highly biased in terms of direction and location and often associated with dramatic variation in read depth. The rearrangements were location-dependent and mostly derived from the asymmetric products of microhomology-mediated recombination. Our results provide an overall picture of organelle-specific gross genomic rearrangements in the HRR mutants, and suggest that chloroplasts and mitochondria share common mechanisms for replication-related rearrangements.

Horizontal Gene Transfer Involving Chloroplasts

Horizontal gene transfer (HGT)- is defined as the acquisition of genetic material from another organism. However, recent findings indicate a possible role of HGT in the acquisition of traits with adaptive significance, suggesting that HGT is an important driving force in the evolution of eukaryotes as well as prokaryotes. It has been noted that, in eukaryotes, HGT is more prevalent than originally thought. Mitochondria and chloroplasts lost a large number of genes after their respective endosymbiotic events occurred. Even after this major content loss, organelle genomes still continue to lose their own genes. Many of these are subsequently acquired by intracellular gene transfer from the original plastid. The aim of our review was to elucidate the role of chloroplasts in the transfer of genes. This review also explores gene transfer involving mitochondrial and nuclear genomes, though recent studies indicate that chloroplast genomes are far more active in HGT as compared to these other two DNA-containing cellular compartments.