Phylogenomic discordance suggests polytomies along the backbone of the large genus Solanum.

PREMISE: Evolutionary studies require solid phylogenetic frameworks, but increased volumes of phylogenomic data have revealed incongruent topologies among gene trees in many organisms both between and within genomes. Some of these incongruences indicate polytomies that may remain impossible to resolve. Here we investigate the degree of gene-tree discordance in Solanum, one of the largest flowering plant genera that includes the cultivated potato, tomato, and eggplant, as well as 24 minor crop plants. METHODS: A densely sampled species-level phylogeny of Solanum is built using unpublished and publicly available Sanger sequences comprising 60% of all accepted species (742 spp.) and nine regions (ITS, waxy, and seven plastid markers). The robustness of this topology is tested by examining a full plastome dataset with 140 species and a nuclear target-capture dataset with 39 species of Solanum (Angiosperms353 probe set). RESULTS: While the taxonomic framework of Solanum remained stable, gene tree conflicts and discordance between phylogenetic trees generated from the target-capture and plastome datasets were observed. The latter correspond to regions with short internodal branches, and network analysis and polytomy tests suggest the backbone is composed of three polytomies found at different evolutionary depths. The strongest area of discordance, near the crown node of Solanum, could potentially represent a hard polytomy. CONCLUSIONS: We argue that incomplete lineage sorting due to rapid diversification is the most likely cause for these polytomies, and that embracing the uncertainty that underlies them is crucial to understand the evolution of large and rapidly radiating lineages.

Phylogenomic proof of Recurrent Demipolyploidization and Evolutionary Stalling of the "Triploid Bridge" in Arundo (Poaceae).

Polyploidization is a frequent phenomenon in plants, which entails the increase from one generation to the next by multiples of the haploid number of chromosomes. While tetraploidization is arguably the most common and stable outcome of polyploidization, over evolutionary time triploids often constitute only a transient phase, or a "triploid bridge", between diploid and tetraploid levels. In this study, we reconstructed in a robust phylogenomic and statistical framework the evolutionary history of polyploidization in Arundo, a small genus from the Poaceae family with promising biomass, bioenergy and phytoremediation species. Through the obtainment of 10 novel leaf transcriptomes for Arundo and outgroup species, our results prove that recurrent demiduplication has likely been a major driver of evolution in this species-poor genus. Molecular dating further demonstrates that the species originating by demiduplication stalled in the "triploid bridge" for evolutionary times in the order of millions of years without undergoing tetratploidization. Nevertheless, we found signatures of molecular evolution highlighting some of the processes that accompanied the genus radiation. Our results clarify the complex nature of Arundo evolution and are valuable for future gene functional validation as well as reverse and comparative genomics efforts in the Arundo genus and other Arundinoideae.

Exploring the phylogeny of the marattialean ferns.

The eusporangiate marattialean ferns represent an ancient radiation with a rich fossil record but limited modern diversity in the tropics. The long evolutionary history without close extant relatives has confounded studies of the phylogenetic origin, rooting and timing of marattialean ferns. Here we present new complete plastid genomes of six marattialean species and compiled a plastid genome dataset representing all of the currently accepted marattialean genera. We further supplemented this dataset by compiling a large dataset of mitochondrial genes and a phenotypic data matrix covering both extant and extinct representatives of the lineage. Our phylogenomic and total-evidence analyses corroborated the postulated position of marattialean ferns as the sister to leptosporangiate ferns, and the position of Danaea as the sister to the remaining extant marattialean genera. However, our results provide new evidence that Christensenia is sister to Marattia and that M. cicutifolia actually belongs to Eupodium. The apparently highly reduced rate of molecular evolution in marattialean ferns provides a challenge for dating the key phylogenetic events with molecular clock approaches. We instead applied a parsimony-based total-evidence dating approach, which suggested a Triassic age for the extant crown group. The modern distribution can best be explained as mainly resulting from vicariance following the breakup of Pangaea and Gondwana. We resolved the fossil genera Marattiopsis, Danaeopsis and Qasimia as members of the monophyletic family Marattiaceae, and the Carboniferous genera Sydneia and Radstockia as the monophyletic sister of all other marattialean ferns.

Protocols for miRNA Target Prediction in Plants.

Next-generation sequencing has opened up new avenues for the identification of microRNAs (miRNAs) and their corresponding roles in abiotic and biotic stress responses. Recently, a plethora of evidence suggests a canonical action of miRNA-mRNA interactions to regulate plant systems biology at the posttranscriptional level, thus leading to the gain or loss of genetic adaptation in plants. In this chapter, we present a detailed protocol for the identification of miRNA targets using six different prediction tools.

NAMS: Noncoding Assessment of long RNAs in Magnoliophyta Species.

Regulation of plant transcriptional machinery has been recently demonstrated to be widely regulated by a class of long noncoding RNAs (lncRNAs) with size larger than 200 nt. The lncRNAs have been demonstrated to play key roles in abiotic stress. Taking into account the rapid pace in the development of the sequencing technologies, accelerated identification of lncRNAs with potential involvement in regulating the gene expression has been witnessed. Although progress has been made to identify the lncRNAs, however, accurate classification of the lncRNAs particularly in the case of plants is still challenging. In this protocol chapter, we present NAMS, which provides large-scale noncoding assessment of the lncRNAs specifically designed for Magnoliophyta species. We describe the approach and the usage of NAMS with potential applications for the lncRNA discovery.

Sequencing the Plastid Genome of Giant Ragweed (Ambrosia trifida, Asteraceae) From a Herbarium Specimen.

We report the first plastome sequence of giant ragweed (Ambrosia trifida); with this new genome information, we assessed the phylogeny of Asteraceae and the transcriptional profiling against glyphosate resistance in giant ragweed. Assembly and genic features show a normal angiosperm quadripartite plastome structure with no signatures of deviation in gene directionality. Comparative analysis revealed large inversions across the plastome of giant ragweed and the previously sequenced members of the plant family. Asteraceae plastid genomes contain two inversions of 22.8 and 3.3 kb; the former is located between trnS-GCU and trnG-UCC genes, and the latter between trnE-UUC and trnT-GGU genes. The plastid genome sequences of A. trifida and the related species, Ambrosia artemisiifolia, are identical in gene content and arrangement, but they differ in length. The phylogeny is well-resolved and congruent with previous hypotheses about the phylogenetic relationship of Asteraceae. Transcriptomic analysis revealed divergence in the relative expressions at the exonic and intronic levels, providing hints toward the ecological adaptation of the genus. Giant ragweed shows various levels of glyphosate resistance, with introns displaying higher expression patterns at resistant time points after the assumed herbicide treatment.

Diff isomiRs: Large-scale detection of differential isomiRs for understanding non-coding regulated stress omics in plants.

Plants have an amazing ability to cope with wide variety of stresses by regulating the expression of genes and thus by altering the physiological status. In the past few years, canonical microRNA variants (isomiRs) have been shown to play pivotal roles by acting as regulators of the transcriptional machinery. In the present research, we present Diff isomiRs, a web-based exploratory repository of differential isomiRs across 16 sequenced plant species representing a total of 433 datasets across 21 different stresses and 158 experimental states. Diff isomiRs provides the high-throughput detection of differential isomiRs using mapping-based and model-based differential analysis revealing a total of 16,157 and 2,028 differential isomiRs, respectively. Easy-to-use and web-based exploration of differential isomiRs provides several features such as browsing of the differential isomiRs according to stress or species, as well as association of the differential isomiRs to targets and plant endogenous target mimics (PeTMs). Diff isomiRs also provides the relationship between the canonical miRNAs, isomiRs and the miRNA-target interactions. This is the first web-based large-scale repository for browsing differential isomiRs and will facilitate better understanding of the regulatory role of the isomiRs with respect to the canonical microRNAs. Diff isomiRs can be accessed at: www.mcr.org.in/diffisomirs .

AtCircDB: a tissue-specific database for Arabidopsis circular RNAs.

Circular RNAs are widely existing in eukaryotes. However, there is as yet no tissue-specific Arabidopsis circular RNA database, which hinders the study of circular RNA in plants. Here, we used 622 Arabidopsis RNA sequencing data sets from 87 independent studies hosted at NCBI SRA and developed AtCircDB to systematically identify, store and retrieve circular RNAs. By analyzing back-splicing sites, we characterized 84 685 circular RNAs, 30 648 tissue-specific circular RNAs and 3486 microRNA-circular RNA interactions. In addition, we used a metric (detection score) to measure the detection ability of the circular RNAs using a big-data approach. By experimental validation, we demonstrate that this metric improves the accuracy of the detection algorithm. We also defined the regions hosting enriched circular RNAs as super circular RNA regions. The results suggest that these regions are highly related to alternative splicing and chloroplast. Finally, we developed a comprehensive tissue-specific database (AtCircDB) to help the community store, retrieve, visualize and download Arabidopsis circular RNAs. This database will greatly expand our understanding of circular RNAs and their related regulatory networks. AtCircDB is freely available at http://genome.sdau.edu.cn/circRNA.

In silico identification and characterization of a diverse subset of conserved microRNAs in bioenergy crop Arundo donax L.

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the post-transcriptional regulation of gene expression in plants. Arundo donax L. is a perennial C3 grass considered one of the most promising bioenergy crops. Despite its relevance, many fundamental aspects of its biology still remain to be elucidated. In the present study we carried out the first in silico mining and tissue-specific characterization of microRNAs and their putative targets in A. donax. We identified a total of 141 miRNAs belonging to 14 families along with the corresponding primary miRNAs, precursor miRNAs and a total of 462 high-confidence predicted targets and novel target sites were validated by 5'-race. Gene Ontology functional annotation showed that miRNA targets are constituted mainly by transcription factors, but three of the newly validated targets are enzymes involved in novel functions like RNA editing, acyl lipid metabolism and post-Golgi trafficking. Folding variability of pre-miRNA loops and phylogenetic analyses indicate variable selective pressure acting on the different miRNA families. The set of miRNAs identified in this study will pave the road to further miRNA research in Arundo donax and contribute towards a better understanding of miRNA-mediated gene regulatory processes in other bioenergy crops.

SeagrassDB: An open-source transcriptomics landscape for phylogenetically profiled seagrasses and aquatic plants.

Seagrasses and aquatic plants are important clades of higher plants, significant for carbon sequestration and marine ecological restoration. They are valuable in the sense that they allow us to understand how plants have developed traits to adapt to high salinity and photosynthetically challenged environments. Here, we present a large-scale phylogenetically profiled transcriptomics repository covering seagrasses and aquatic plants. SeagrassDB encompasses a total of 1,052,262 unigenes with a minimum and maximum contig length of 8,831 bp and 16,705 bp respectively. SeagrassDB provides access to 34,455 transcription factors, 470,568 PFAM domains, 382,528 prosite models and 482,121 InterPro domains across 9 species. SeagrassDB allows for the comparative gene mining using BLAST-based approaches and subsequent unigenes sequence retrieval with associated features such as expression (FPKM values), gene ontologies, functional assignments, family level classification, Interpro domains, KEGG orthology (KO), transcription factors and prosite information. SeagrassDB is available to the scientific community for exploring the functional genic landscape of seagrass and aquatic plants at: http://115.146.91.129/index.php .

Plant IsomiR Atlas: Large Scale Detection, Profiling, and Target Repertoire of IsomiRs in Plants.

microRNAs (miRNAs) play an important role as key regulators controlling the post-transcriptional events in plants across development, abiotic and biotic stress, tissue polarity and also in defining the evolutionary basis of the origin of the post-transcriptional machinery. Identifying patterns of regulated and co-regulated small RNAs, in particular miRNAs and their sequence variants with the availability of next generation sequencing approaches has widely demonstrated the role of miRNAs and their temporal regulation in maintaining plant development and their response to stress conditions. Although the role of canonical miRNAs has been widely explored and functional diversity is revealed, those works for isomiRs are still limited and urgent to be carried out across plants. This relative lack of information with respect to isomiRs might be attributed to the non-availability of large-scale detection of isomiRs across wide plant species. In the present research, we addressed this by developing Plant isomiR Atlas, which provides large-scale detection of isomiRs across 23 plant species utilizing 677 smallRNAs datasets and reveals a total of 98,374 templated and non-templated isomiRs from 6,167 precursors. Plant isomiR Atlas provides several visualization features such as species specific isomiRs, isomiRs and canonical miRNAs overlap, terminal modification classifications, target identification using psRNATarget and TargetFinder and also canonical miRNAs:target interactions. Plant isomiR Atlas will play a key role in understanding the regulatory nature of miRNAome and will accelerate to understand the functional role of isomiRs. Plant isomiR Atlas is available at www.mcr.org.in/isomir. One Sentence Summary  Plant isomiR Atlas will play a key role in understanding the regulatory nature of miRNAome and will accelerate the understanding and diversity of functional targets of plants isomiRs.

Comparative assessment of chloroplast transcriptional responses highlights conserved and unique patterns across Triticeae members under salt stress.

Chloroplast functional genomics, in particular understanding the chloroplast transcriptional response is of immense importance mainly due to its role in oxygenic photosynthesis. As a photosynthetic unit, its efficiency and transcriptional activity is directly regulated by reactive oxygen species during abiotic and biotic stress and subsequently affects carbon assimilation, and plant biomass. In crops, understanding photosynthesis is crucial for crop domestication by identifying the traits that could be exploited for crop improvement. Transcriptionally and translationally active chloroplast plays a key role by regulating the PSI and PSII photo-reaction centres, which ubiquitously affects the light harvesting. Using a comparative transcriptomics mapping approach, we identified differential regulation of key chloroplast genes during salt stress across Triticeae members with potential genes involved in photosynthesis and electron transport system such as CytB6f. Apart from differentially regulated genes involved in PSI and PSII, we found widespread evidence of intron splicing events, specifically uniquely spliced petB and petD in Triticum aestivum and high proportion of RNA editing in ndh genes across the Triticeae members during salt stress. We also highlight the role and differential regulation of ATP synthase as member of CF0CF1 and also revealed the effect of salt stress on the water-splitting complex under salt stress. It is worthwhile to mention that the observed conserved down-regulation of psbJ across the Triticeae is limiting the assembly of water-splitting complexes and thus making the BEP clade Triticeae members more vulnerable to high light during the salt stress. Comparative understanding of the chloroplast transcriptional dynamics and photosynthetic regulation will improve the approaches for improved crop domestication.

Estimate Codon Usage Bias Using Codon Usage Analyzer (CUA).

One amino acid is added to a growing peptide by a ribosome through reading triple nucleotides, i.e., a codon, each time. Twenty species of amino acids are often coded by 61 codons, so one amino acid can be coded by more than one codon and the codons coding the same amino acid are called synonymous. Intriguingly, synonymous codons' usage is often uneven: some are used more often than their alternatives in a genome. The unevenness of codon usage is termed codon usage bias (CUB). CUB is widespread, and its causes and consequences have been under intensive investigation. To facilitate the studying of CUB, in this chapter we present a protocol of estimating CUB by using the free software Codon Usage Analyzer, and apply it to Brachypodium distachyon as an example. To accomplish this protocol, the readers need some basic command-line skills. Briefly, the protocol comprises four major steps: downloading data and software, setting up computing environment, preparing data, and estimating CUB.

Method for the Large-Scale Identification of phasiRNAs in Brachypodium distachyon.

Postranscriptional regulation has been widely shown to be regulated by several classes of small non-coding RNAs; most abundantly, microRNAs, which have been shown to be the first dominant class and has been widely characterized as post-transcriptional regulators. In addition to microRNAs, triggered by miRNAs, transcripts called as PHAS (or TAS) generate abundant class of small RNAs in 21-nt manner, which is a pattern formed by DICER-LIKE 4 (DCL4) processing. Although PHAS can be identified by aligning transcripts to reported PHAS in other species, the most sensitive and accurate way to discovery them is by mapping of the smallRNAs taking into account the transcript coordinates. Here, we describe a workflow that can be used for the identification PHAS and corresponding phasiRNAs in Brachypodium distachyon using publically availabe smallRNAs datasets.

Evaluation of Genome-Wide Markers and Orthologous Markers in Brachypodium distachyon.

Molecular markers play an important role in identifying the species variation, characterizing the genic diversity, and also linking the identified markers to trait of interest. Genome- and transcriptome-derived molecular markers have been widely used to understand the geographical diversity and have also played a major role in the development of high-density linkage maps. In the present protocol, we present a detailed protocol on bioinformatics approaches towards the whole-genome and transcriptome-assisted simple sequence repeats (SSRs) marker mining in Brachypodium distachyon and identification of orthologus SSRs and their validation in Brachypodium ecotypes. We also present a protocol for the validation of the identified markers.

Protocol for Coexpression Network Construction and Stress-Responsive Expression Analysis in Brachypodium.

Identifying functionally coexpressed genes and modules has increasingly become important to understand the transcriptional flux and to understand large scale gene association. Application of the graph theory and combination of tools has allowed to understand the genic interaction and to understand the role of hub and non-hub proteins in plant development and its ability to cope with stress. Association genetics has also been coupled with network modules to map these key genes as e-QTLs. High throughput sequencing approaches has revolutionized the mining of the gene behavior and also the association of the genes over time-series. The present protocol chapter presents a unified workflow to understand the transcriptional modules in Brachypodium distachyon using weighted coexpressed gene network analysis approach.

Transcriptome analysis of Brachypodium during fungal pathogen infection reveals both shared and distinct defense responses with wheat.

Fusarium crown rot (FCR) of wheat and barley, predominantly caused by the fungal pathogen Fusarium pseudograminearum, is a disease of economic significance. The quantitative nature of FCR resistance within cultivated wheat germplasm has significantly limited breeding efforts to enhanced FCR resistance in wheat. In this study, we characterized the molecular responses of Brachypodium distachyon (Brachypodium hereafter) to F. pseudograminearum infection using RNA-seq to determine whether Brachypodium can be exploited as a model system towards better understanding of F. pseudograminearum-wheat interaction. The transcriptional response to infection in Brachypodium was strikingly similar to that previously reported in wheat, both in shared expression patterns of wheat homologs of Brachypodium genes and functional overlap revealed through comparative gene ontology analysis in both species. Metabolites produced by various biosynthetic pathways induced in both wheat and Brachypodium were quantified, revealing a high degree of overlap between these two species in metabolic response to infection but also showed Brachypodium does not produce certain defence-related metabolites found in wheat. Functional analyses of candidate genes identified in this study will improve our understanding of resistance mechanisms and may lead to the development of new strategies to protect cereal crops from pathogen infection.

Emerging Roles and Landscape of Translating mRNAs in Plants.

Plants use a wide range of mechanisms to adapt to different environmental stresses. One of the earliest responses displayed under stress is rapid alterations in stress responsive gene expression that has been extensively analyzed through expression profiling such as microarrays and RNA-sequencing. Recently, expression profiling has been complemented with proteome analyses to establish a link between transcriptional and the corresponding translational changes. However, proteome profiling approaches have their own technical limitations. More recently, ribosome-associated mRNA profiling has emerged as an alternative and a robust way of identifying translating mRNAs, which are a set of mRNAs associated with ribosomes and more likely to contribute to proteome abundance. In this article, we briefly review recent studies that examined the processes affecting the abundance of translating mRNAs, their regulation during plant development and tolerance to stress conditions and plant factors affecting the selection of translating mRNA pools. This review also highlights recent findings revealing differential roles of alternatively spliced mRNAs and their translational control during stress adaptation. Overall, better understanding of processes involved in the regulation of translating mRNAs has obvious implications for improvement of stress tolerance in plants.

tRNA Derived smallRNAs: smallRNAs Repertoire Has Yet to Be Decoded in Plants.

Among several smallRNAs classes, microRNAs play an important role in controlling the post-transcriptional events. Next generation sequencing has played a major role in extending the landscape of miRNAs and revealing their spatio-temporal roles in development and abiotic stress. Lateral evolution of these smallRNAs classes have widely been seen with the recently emerging knowledge on tRNA derived smallRNAs. In the present perspective, we discussed classification, identification and roles of tRNA derived smallRNAs across plants and their potential involvement in abiotic and biotic stresses.

Draft Genome Sequence of the Nitrogen-Fixing Rhizobium sullae Type Strain IS123T Focusing on the Key Genes for Symbiosis with its Host Hedysarum coronarium L.

The prominent feature of rhizobia is their molecular dialogue with plant hosts. Such interaction is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant. The genome of the Rhizobium sullae type strain IS123T nodulating the legume Hedysarum coronarium, was sequenced and resulted in 317 scaffolds for a total assembled size of 7,889,576 bp. Its features were compared with those of genomes from rhizobia representing an increasing gradient of taxonomical distance, from a conspecific isolate (Rhizobium sullae WSM1592), to two congeneric cases (Rhizobium leguminosarum bv. viciae and Rhizobium etli) and up to different genera within the legume-nodulating taxa. The host plant is of agricultural importance, but, unlike the majority of other domesticated plant species, it is able to survive quite well in the wild. Data showed that that the type strain of R. sullae, isolated from a wild host specimen, is endowed with a richer array of symbiotic genes in comparison to other strains, species or genera of rhizobia that were rescued from domesticated plant ecotypes. The analysis revealed that the bacterium by itself is incapable of surviving in the extreme conditions that its host plant can tolerate. When exposed to drought or alkaline condition, the bacterium depends on its host to survive. Data are consistent with the view of the plant phenotype as the primary factor enabling symbiotic nitrogen fixing bacteria to survive in otherwise limiting environments.