Comparative Analysis of Complete Chloroplast Genomes of 13 Species in Epilobium, Circaea, and Chamaenerion and Insights Into Phylogenetic Relationships of Onagraceae

The evening primrose family, Onagraceae, is a well defined family of the order Myrtales, comprising 22 genera widely distributed from boreal to tropical areas. In this study, we report and characterize the complete chloroplast genome sequences of 13 species in Circaea, Chamaenerion, and Epilobium using a next-generation sequencing method. We also retrieved chloroplast sequences from two other Onagraceae genera to characterize the chloroplast genome of the family. The complete chloroplast genomes of Onagraceae encoded an identical set of 112 genes (with exclusion of duplication), including 78 protein-coding genes, 30 transfer RNAs, and four ribosomal RNAs. The chloroplast genomes are basically conserved in gene arrangement across the family. However, a large segment of inversion was detected in the large single copy region of all the samples of Oenothera subsect. Oenothera. Two kinds of inverted repeat (IR) region expansion were found in Oenothera, Chamaenerion, and Epilobium samples. We also compared chloroplast genomes across the Onagraceae samples in some features, including nucleotide content, codon usage, RNA editing sites, and simple sequence repeats (SSRs). Phylogeny was inferred by the chloroplast genome data using maximum-likelihood (ML) and Bayesian inference methods. The generic relationship of Onagraceae was well resolved by the complete chloroplast genome sequences, showing potential value in inferring phylogeny within the family. Phylogenetic relationship in Oenothera was better resolved than other densely sampled genera, such as Circaea and Epilobium. Chloroplast genomes of Oenothera subsect. Oenothera, which are biparental inheritated, share a syndrome of characteristics that deviate from primitive pattern of the family, including slightly expanded inverted repeat region, intron loss in clpP, and presence of the inversion.

In planta dynamics, transport-biases and endogenous functions of mobile siRNAs in Arabidopsis

In RNA interference (RNAi), small-interfering (si)RNAs processed from double-stranded RNA guide ARGONAUTE(AGO) proteins to silence sequence-complementary RNA/DNA. Plant RNAi can propagate locally and systemically, but despite recent mechanistic advances, basic questions/hurdles remain unaddressed. For instance, RNAi is inferred to diffuse through plasmodesmata, yet how its dynamics in planta compares with that of established symplastic-diffusion markers remains unknown. Also unknown is why select siRNA species, or size-classes thereof, are recovered in RNAi-recipient tissues, yet only under some experimental settings. Finally, RNAi shootward movement in micro-grafted Arabidopsis -necessary to study its presumptive transgenerational effects- has not been achieved thus far and endogenous functions of mobile RNAi remain scarcely documented. Focusing on non-amplified RNAi in Arabidopsis, we show here that (i) transgenic RNAi-movement, although symplasmic, only partially recapitulates the diffusion pattern of free GFP in planta, (ii) the presence/absence of specific AGOs in incipient/traversed/recipient tissues likely explains the apparent siRNA-selectivity observed during vascular movement, (iii) stress application allows endo-siRNA translocation against the shoot-to-root phloem flow, and (iv) mobile endo-siRNAs generated from a single inverted-repeat(IR) locus, have the potential to regulate hundreds of transcripts. Our results close important knowledge-gaps, rationalize previously-noted inconsistencies between mobile RNAi settings, and provide a framework for functional endo-siRNA studies.

Temporal Changes in Transcripts of MITE Transposable Elements during Rice Endosperm Development

The repression of transcription from transposable elements (TEs) by DNA methylation is necessary to maintain genome integrity and prevent harmful mutations. However, under certain circumstances, TEs are thought to escape from the host defense system and reactivate their transcription. In Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), DNA demethylases target the sequences derived from TEs in the central cell, the progenitor cell for the endosperm in the female gametophyte. This genome-wide DNA demethylation is also observed in the endosperm after fertilization. In this study, we used a custom microarray to survey the transcripts generated from TEs during the rice endosperm development and at selected timepoints in the embryo as a control. The expression patterns of TE transcripts are dynamically up- and downregulated during endosperm development, especially for miniature inverted-repeat transposable elements (MITEs). Surprisingly, some TE transcripts were directionally controlled, while the other DNA transposons and retrotransposons were not. We also discovered the NF-Y binding motif, CCAAT, in the region near the 5′ terminal inverted repeat of Youren, one of the transcribed MITEs in the endosperm. Our results uncover dynamic changes in TE activity during endosperm development in rice.

Optimized RNA-Silencing Strategies for Rice Ragged Stunt Virus Resistance in Rice

Rice ragged stunt virus (RRSV) is one of the most damaging viruses of the rice culture area in south and far-eastern Asia. To date, no genetic resistance has been identified and only expensive and non-environmentally friendly chemical treatments are deployed to fight this important disease. Non-chemical approaches based on RNA-silencing have been developed as resistance strategies against viruses. Here, we optimized classical miRNA and siRNA-based strategies to obtain efficient and durable resistance to RRSV. miRNA-based strategies are involved in producing artificial miRNA (amiR) targeting viral genomes in plants. Classically, only one amiR is produced from a single construct. We demonstrated for the first time that two amiRs targeting conserved regions of RRSV genomes could be transgenically produced in Nicotiana benthamiana and in rice for a single precursor. Transgenic rice plants producing either one or two amiR were produced. Despite efficient amiR accumulations, miRNA-based strategies with single or double amiRs failed to achieve efficient RRSV resistance in transformed rice plants. This suggests that this strategy may not be adapted to RRSV, which could rapidly evolve to counteract them. Another RNA-silencing-based method for viral resistance concerns producing several viral siRNAs targeting a viral fragment. These viral siRNAs are produced from an inverted repeat construct carrying the targeted viral fragment. Here, we optimized the inverted repeat construct using a chimeric fragment carrying conserved sequences of three different RRSV genes instead of one. Of the three selected homozygous transgenic plants, one failed to accumulate the expected siRNA. The two other ones accumulated siRNAs from either one or three fragments. A strong reduction of RRSV symptoms was observed only in transgenic plants expressing siRNAs. We consequently demonstrated, for the first time, an efficient and environmentally friendly resistance to RRSV in rice based on the siRNA-mediated strategy.

Recent amplification of microsatellite-associated miniature inverted-repeat transposable elements in the pineapple genome

Background Miniature inverted-repeat transposable elements (MITEs) are non-autonomous DNA transposable elements that play important roles in genome organization and evolution. Genome-wide identification and characterization of MITEs provide essential information for understanding genome structure and evolution. Results We performed genome-wide identification and characterization of MITEs in the pineapple genome. The top two MITE families, accounting for 29.39% of the total MITEs and 3.86% of the pineapple genome, have insertion preference in (TA) n dinucleotide microsatellite regions. We therefore named these MITEs A. comosus microsatellite-associated MITEs (Ac-mMITEs). The two Ac-mMITE families, Ac-mMITE-1 and Ac-mMITE-2, shared sequence similarity in the terminal inverted repeat (TIR) regions, suggesting that these two Ac-mMITE families might be derived from a common or closely related autonomous elements. The Ac-mMITEs are frequently clustered via adjacent insertions. Among the 21,994 full-length Ac-mMITEs, 46.1% of them were present in clusters. By analyzing the Ac-mMITEs without (TA) n microsatellite flanking sequences, we found that Ac-mMITEs were likely derived from Mutator-like DNA transposon. Ac-MITEs showed highly polymorphic insertion sites between cultivated pineapples and their wild relatives. To better understand the evolutionary history of Ac-mMITEs, we filtered and performed comparative analysis on the two distinct groups of Ac-mMITEs, microsatellite-targeting MITEs (mt-MITEs) that are flanked by dinucleotide microsatellites on both sides and mutator-like MITEs (ml-MITEs) that contain 9/10 bp TSDs. Epigenetic analysis revealed a lower level of host-induced silencing on the mt-MITEs in comparison to the ml-MITEs, which partially explained the significantly higher abundance of mt-MITEs in pineapple genome. The mt-MITEs and ml-MITEs exhibited differential insertion preference to gene-related regions and RNA-seq analysis revealed their differential influences on expression regulation of nearby genes. Conclusions Ac-mMITEs are the most abundant MITEs in the pineapple genome and they were likely derived from Mutator-like DNA transposon. Preferential insertion in (TA) n microsatellite regions of Ac-mMITEs occurred recently and is likely the result of damage-limiting strategy adapted by Ac-mMITEs during co-evolution with their host. Insertion in (TA) n microsatellite regions might also have promoted the amplification of mt-MITEs. In addition, mt-MITEs showed no or negligible impact on nearby gene expression, which may help them escape genome control and lead to their amplification.

Proposal to recognise the tribes Adinobotryeae and Glycyrrhizeae (Leguminosae subfamily Papilionoideae) based on chloroplast phylogenomic evidence

Within the legume family, the taxonomic status of subtribe Glycyrrhizinae of tribe Galegeae and of the genus Adinobotrys has been re-assessed. Based on genome skimming data, we conducted phylogenomic analyses of the inverted repeat-lacking clade within subfamily Papilionoideae. The results support the sister relationship between Glycyrrhizeae and Adinobotrys. Glycyrrhizeae is resurrected based on Glycyrrhiza and Glycyrrhizopsis, and a new tribe, Adinobotryeae, is proposed to accommodate Adinobotrys.

Complete Chloroplast Genomes of Fagus sylvatica L. Reveal Sequence Conservation in the Inverted Repeat and the Presence of Allelic Variation in NUPTs

Growing amounts of genomic data and more efficient assembly tools advance organelle genomics at an unprecedented scale. Genomic resources are increasingly used for phylogenetic analyses of many plant species, but are less frequently used to investigate within-species variability and phylogeography. In this study, we investigated genetic diversity of Fagus sylvatica, an important broadleaved tree species of European forests, based on complete chloroplast genomes of 18 individuals sampled widely across the species distribution. Our results confirm the hypothesis of a low cpDNA diversity in European beech. The chloroplast genome size was remarkably stable (158,428 ± 37 bp). The polymorphic markers, 12 microsatellites (SSR), four SNPs and one indel, were found only in the single copy regions, while inverted repeat regions were monomorphic both in terms of length and sequence, suggesting highly efficient suppression of mutation. The within-individual analysis of polymorphisms showed >9k of markers which were proportionally present in gene and non-gene areas. However, an investigation of the frequency of alternate alleles revealed that the source of this diversity originated likely from nuclear-encoded plastome remnants (NUPTs). Phylogeographic and Mantel correlation analysis based on the complete chloroplast genomes exhibited clustering of individuals according to geographic distance in the first distance class, suggesting that the novel markers and in particular the cpSSRs could provide a more detailed picture of beech population structure in Central Europe.

Complete Chloroplast Genome Sequence and Comparative and Phylogenetic Analyses of the Cultivated Cyperus esculentus

Cyperus esculentus produces large amounts of oil as one of the main oil storage reserves in underground tubers, making this crop species not only a promising resource for edible oil and biofuel in food and chemical industry, but also a model system for studying oil accumulation in non-seed tissues. In this study, we determined the chloroplast genome sequence of the cultivated C. esculentus (var. sativus Boeckeler). The results showed that the complete chloroplast genome of C. esculentus was 186,255 bp in size, and possessed a typical quadripartite structure containing one large single copy (100,940 bp) region, one small single copy (10,439 bp) region, and a pair of inverted repeat regions of 37,438 bp in size. Sequence analyses indicated that the chloroplast genome encodes 141 genes, including 93 protein-coding genes, 40 transfer RNA genes, and 8 ribosomal RNA genes. We also identified 396 simple-sequence repeats and 49 long repeats, including 15 forward repeats and 34 palindromes within the chloroplast genome of C. esculentus. Most of these repeats were distributed in the noncoding regions. Whole chloroplast genome comparison with those of the other four Cyperus species indicated that both the large single copy and inverted repeat regions were more divergent than the small single copy region, with the highest variation found in the inverted repeat regions. In the phylogenetic trees based on the complete chloroplast genomes of 13 species, all five Cyperus species within the Cyperaceae formed a clade, and C. esculentus was evolutionarily more related to C. rotundus than to the other three Cyperus species. In summary, the chloroplast genome sequence of the cultivated C. esculentus provides a valuable genomic resource for species identification, evolution, and comparative genomic research on this crop species and other Cyperus species in the Cyperaceae family.

airpg: automatically accessing the inverted repeats of archived plastid genomes

Background In most flowering plants, the plastid genome exhibits a quadripartite genome structure, comprising a large and a small single copy as well as two inverted repeat regions. Thousands of plastid genomes have been sequenced and submitted to public sequence repositories in recent years. The quality of sequence annotations in many of these submissions is known to be problematic, especially regarding annotations that specify the length and location of the inverted repeats: such annotations are either missing or portray the length or location of the repeats incorrectly. However, many biological investigations employ publicly available plastid genomes at face value and implicitly assume the correctness of their sequence annotations. Results We introduce , a Python package that automatically assesses the frequency of incomplete or incorrect annotations of the inverted repeats among publicly available plastid genomes. Specifically, the tool automatically retrieves plastid genomes from NCBI Nucleotide under variable search parameters, surveys them for length and location specifications of inverted repeats, and confirms any inverted repeat annotations through self-comparisons of the genome sequences. The package also includes functionality for automatic identification and removal of duplicate genome records and accounts for taxa that genuinely lack inverted repeats. A survey of the presence of inverted repeat annotations among all plastid genomes of flowering plants submitted to NCBI Nucleotide until the end of 2020 using , followed by a statistical analysis of potential associations with record metadata, highlights that release year and publication status of the genome records have a significant effect on the frequency of complete and equal-length inverted repeat annotations. Conclusion The number of plastid genomes on NCBI Nucleotide has increased dramatically in recent years, and many more genomes will likely be submitted over the next decade. enables researchers to automatically access and evaluate the inverted repeats of these plastid genomes as well as their sequence annotations and, thus, contributes to increasing the reliability of publicly available plastid genomes. The software is freely available via the Python package index at http://pypi.python.org/pypi/airpg.