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SUMMARY: Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium‐mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant‐microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long‐read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium‐mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large‐scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants. Significance Statement: Agrobacterium‐mediated transformation (AMT) of plants is used extensively as a tool to create new genetic variants for research purposes or agricultural needs. In this work, we summarize the molecular mechanisms involved in AMT and the effects on plant genomes resulting from the integration of exogenous DNA through this method. [ABSTRACT FROM AUTHOR]

DNA methylation provides an important epigenetic mechanism that critically regulates gene expression, genome imprinting, and retrotransposon silencing. In plants, DNA methylation is prevalent not only in a CG dinucleotide context but also in non-CG contexts, namely CHG and CHH (H = C, T, or A) methylation. It has been established that plant non-CG DNA methylation is highly context dependent, with the +1- and +2-flanking sequences enriched with A/T nucleotides. How DNA sequence, conformation, and dynamics influence non-CG methylation remains elusive. Here, we report structural and biochemical characterizations of the intrinsic substrate preference of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a plant DNA methyltransferase responsible for establishing all cytosine methylation and maintaining CHH methylation. Among nine CHH motifs, the DRM2 methyltransferase (MTase) domain shows marked substrate preference toward CWW (W = A or T) motifs, correlating well with their relative abundance in planta. Furthermore, we report the crystal structure of DRM2 MTase in complex with a DNA duplex containing a flexible TpA base step at the +1/+2-flanking sites of the target nucleotide. Comparative structural analysis of the DRM2-DNA complexes provides a mechanism by which flanking nucleotide composition impacts DRM2-mediated DNA methylation. Furthermore, the flexibility of the TpA step gives rise to two alternative DNA conformations, resulting in different interactions with DRM2 and consequently temperature-dependent shift of the substrate preference of DRM2. Together, this study provides insights into how the interplay between the conformational dynamics of DNA and temperature as an environmental factor contributes to the context-dependent CHH methylation by DRM2. [ABSTRACT FROM AUTHOR]

A DNA barcode is a short piece of standard DNA sequence used for species determination and discrimination. Representation of DNA barcodes is essential for DNA barcodes' applications in the transportation and recognition of biological materials. Previously, we have compared different strategies for representing the DNA barcodes. In the present study, we have developed a compression algorithm based on binary coding or Huffman coding scheme, followed by converting the binary digits into Base64 digits. The combination of this compression algorithm and the QR representation leads to the dynamic DNA QR coding algorithm (DDQR). We tested the DDQR algorithm on simulated data and real DNA barcode sequences from the commonly used plant and animal DNA barcode markers: rbcL, matK, trnH-psbA, ITS2, and COI. We compared the compression efficiency of DDQR and another state-of-the-art DNA compression algorithm GeCo3 for sequences with various base compositions and lengths. We found that DDQR had a higher compression rate than GeCo3 for DNA sequences shorter than 800 bp, which is the typical size range for DNA barcodes. We also upgraded a web server (http://www.1kmpg.cn/ddqr) that provides three functions: retrieval of DNA barcode sequences, encoding DNA barcode sequences to DDQR codes, and decoding DDQR codes to DNA barcode sequences. The DDQR algorithm and the webserver will be invaluable to applying DNA barcode technology in the food and traditional medicine industries. [ABSTRACT FROM AUTHOR]

Accurate identification of plants remains a significant challenge for taxonomists and is the basis for plant diversity conservation. Although DNA barcoding methods are commonly used for plant identification, these are limited by the low amplification success and low discriminative power of selected genomic regions. In this study, we developed a k‐mer–based approach, the DNA signature sequence (DSS), to accurately identify plant taxon‐specific markers, especially at the species level. DSS is a constant‐length nucleotide sequence capable of identifying a taxon and distinguishing it from other taxa. In this study, we performed the first large‐scale study of DSS markers in plants. DSS candidates of 3899 angiosperm plant species were calculated based on a chloroplast data set with 4356 assemblies. Using Sanger sequencing of PCR amplicons and high‐throughput sequencing, DSSs were validated in four and 165 species, respectively. Based on this, the universality of the DSSs was over 79.38%. Several indicators influencing DSS marker identification and detection have also been evaluated, and common criteria for DSS application in plant identification have been proposed. [ABSTRACT FROM AUTHOR]

Pack-TYPE transposable elements (TEs) are a group of non-autonomous DNA transposons found in plants. These elements can efficiently capture and shuffle coding DNA across the host genome, accelerating the evolution of genes. Despite their relevance for plant genome plasticity, the detection and study of Pack-TYPE TEs are challenging due to the high similarity these elements have with genes. Here, we produced an automated annotation pipeline designed to study Pack-TYPE elements and used it to successfully annotate and analyse more than 10,000 new Pack-TYPE TEs in the rice and maize genomes. Our analysis indicates that Pack-TYPE TEs are an abundant and heterogeneous group of elements. We found that these elements are associated with all main superfamilies of Class II DNA transposons in plants and likely share a similar mechanism to capture new chromosomal DNA sequences. Furthermore, we report examples of the direct contribution of these TEs to coding genes, suggesting a generalised and extensive role of Pack-TYPE TEs in plant genome evolution. Author summary: Transposable Elements (TEs) are genetic DNA sequences able to move across the genome, and their transposition activity is associated with genome plasticity and gene evolution. However, most of these elements exhibit "selfish" behaviour, meaning that they mainly transpose their own DNA sequence and only exceptionally might rearrange the DNA of coding genes. Pack-TYPE TEs, found in plants, represent an important exception, and they can efficiently capture and shuffle DNA sequences captured from the genome, accelerating the evolution of genes. We provide here the first automatic pipeline designed explicitly for the annotation of Pack-TYPE TEs. We used our approach to systematically investigate Pack-TYPE TEs in the rice and maize reference genomes, and annotated thousands of new elements in these species. We demonstrate that Pack-TYPE elements are abundant in plants and we report several examples of coding genes originated as a consequence of the mobilization of these elements. [ABSTRACT FROM AUTHOR]

Objectives: Development of sequencing technology has opened up vast opportunities for tree genomic research in the tropics. One of the aforesaid technologies named ONT (Oxford Nanopore Technology) has attracted researchers in undertaking testings and experiments due to its affordability and accessibility. To the best of our knowledge, there has been no published reports on the use of ONT for genomic analysis of Indonesian tree species. This progress is promising for further improvement in order to acquire more genomic data for research purposes. Therefore, the present study was carried out to determine the effectiveness of ONT in generating long-read DNA sequences using DNA isolated from leaves and wood cores of Macassar ebony (Diospyros celebica Bakh.). Data description: Long-read sequences data of leaves and wood cores of Macassar ebony were generated by using the MinION device and MinKnow v3.6.5 (ONT). The obtained data, as the first long-read sequence dataset for Macassar ebony, is of great importance to conserve the genetic diversity, understanding the molecular mechanism, and sustainable use of plant genetic resources for downstream applications. [ABSTRACT FROM AUTHOR]

Thrixspermum (T.) is one of the genus in Orchidaceae that has small flowers. Among species in this genus has a high homology and also has many synonyms. Identification using morphological characters can be constrained since Thrixspermum flowering time occurs in a very short period. This study aimed to conduct molecular-based identification of Thrixspermum orchids using DNA barcoding. This method applied molecular-based species identification technique using DNA sequences from genomic fragments that are considered fast, accurate, and consistent. The molecular markers used were Internal Transcribed Spacer 2 (ITS2), while the samples used were T. centipeda, T. lucidum, and T. angustifolium. BLAST results show that T. centipeda has a close relationship with T. centipeda from Malaysia (KX679332) with 99.79% percent identity, T. lucidum has a high homology with T. linusii (KX679333; 97.30%), while T. angustifolium has a high homology with T. triangulare (KX679348; 99.38%). There is a unique sequence that only T. lucidum and T. angustifolium have that distinguishes the two from other species. ITS2 can be recommended as a molecular marker for determining the Thrixspermum orchid barcode. The benefit obtained from this research is the DNA barcode sequences (ITS2) of Thrixspermum orchids would be very useful to enrich the plant barcodes database for further molecular taxonomy and biodiversity of orchid. [ABSTRACT FROM AUTHOR]

DNA barcoding based on mitochondrial (mt) nucleotide sequences is an enigma. Neutral models of mt evolution predict DNA barcoding cannot work for recently diverged taxa, and yet, mt DNA barcoding accurately delimits species for many bilaterian animals. Meanwhile, mt DNA barcoding often fails for plants and fungi. I propose that because mt gene products must cofunction with nuclear gene products, the evolution of mt genomes is best understood with full consideration of the two environments that impose selective pressure on mt genes: the external environment and the internal genomic environment. Moreover, it is critical to fully consider the potential for adaptive evolution of not just protein products of mt genes but also of mt transfer RNAs and mt ribosomal RNAs. The tight linkage of genes on mt genomes that do not engage in recombination could facilitate selective sweeps whenever there is positive selection on any element in the mt genome, leading to the purging of mt genetic diversity within a population and to the rapid fixation of novel mt DNA sequences. Accordingly, the most important factor determining whether or not mt DNA sequences diagnose species boundaries may be the extent to which the mt chromosomes engage in recombination. [ABSTRACT FROM AUTHOR]

Berkeleyomyces rouxiae, a causal agent of black root rot of lettuce, is morphologically and physiologically indistinguishable from the closely related species B. basicola. PCR primers specific for these species were designed based on the nucleotide sequence of genes specific to the species. The primer pair specific to B. rouxiae did not amplify DNA of other plant pathogens that invade root or stem base of lettuce and was useful for diagnosis and quantitating the pathogen from lettuce root tissue. [ABSTRACT FROM AUTHOR]

The palm family (Arecaceae) is of high ecological and economic value, yet identification in the family remains a challenge for both taxonomists and horticulturalists. The family consists of approximately 2600 species across 181 genera and DNA barcoding may be a useful tool for species identification within the group. However, there have been few systematic evaluations of DNA barcodes for the palm family. In the present study, five DNA barcodes (rbcL, matK, trnH-psbA, ITS, ITS2) were evaluated for species identification ability across 669 samples representing 314 species and 100 genera in the Arecaceae, employing four analytical methods. The ITS gene region was found to not be a suitable barcode for the palm family, due in part, to low recovery rates and paralogous gene copies. Among the four analyses used, species resolution for ITS2 was much higher than that achieved with the plastid barcodes alone (rbcL, matK, trnH-psbA), and the barcode combination ITS2 + matK + rbcL gave the highest resolution among all single barcodes and their combinations, followed by ITS2 + matK. Among 669 palm samples analyzed, 110 samples (16.3%) were found to be misidentified. The 2992 DNA barcode sequences generated in this study greatly enriches the existing identification toolbox available to plant taxonomists that are interested in researching genetic relationships among palm taxa as well as for horticulturalists that need to confirm palm collections for botanical garden curation and horticultural applications. Our results indicate that the use of the ITS2 DNA barcode gene region provides a useful and cost-effective tool to confirm the identity of taxa in the Palm family. [ABSTRACT FROM AUTHOR]

Due to the sequence homology between the bacterial 16S rRNA gene and plant chloroplast and mitochondrial DNA, the taxonomic characterization of plant microbiome using amplicon-based high throughput sequencing often results in the overwhelming presence of plant-affiliated reads, preventing the thorough description of plant-associated microbial communities. In this work we developed a PCR blocking primer assay targeting the taxonomically informative V5-V6 region of the 16S rRNA gene in order to reduce plant DNA co-amplification, and increase diversity coverage of associated prokaryotic communities. Evaluation of our assay on the characterization of the prokaryotic endophytic communities of Zea mays , Pinus taeda and Spartina alternifora leaves led to significantly reducing the proportion of plant reads, yielded 20 times more prokaryotic reads and tripled the number of detected OTUs compared to a commonly used V5-V6 PCR protocol. To expand the application of our PCR-clamping assay across a wider taxonomic spectrum of plant hosts, we additionally provide an alignment of chloroplast and mitochondrial DNA sequences encompassing more than 200 terrestrial plant families as a supporting tool for customizing our blocking primers. [ABSTRACT FROM AUTHOR]

Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq). [ABSTRACT FROM AUTHOR]

Background and objective: A simple, rapid, efficient, and reproducible protocol for callus induction and regeneration of plantlets from callus of Jatropha curcas plant was established. Materials and methods: Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses were used to determine the genetic variation between the regenerated, micropropagated, and mother plants. Results: The highest callus induction percentage from leaf explant was recorded with MS medium containing 2.5 mg/l BA (6-benzylaminopurine) + 1.0 mg/l NAA (1-naphthaleneacetic acid). Leaf-derived callus was grown on medium containing 2.0 mg/l BA + 0.2 mg/l IBA (indole-3-butyric acid) for adventitious shoot regeneration. In addition, using five random RAPD primers with the tested samples produced 117 amplified products out of which 25 were polymorphic bands resulting in 21.37% polymorphism, whereas the five ISSR primers used yielded 116 scorable bands out of which 22 were polymorphic bands producing a polymorphism pecentage of 18.96. Conclusion: An optimized protocol for large-scale production of J. curcas plants using plant biotechnology tools was achieved. RAPD and ISSR techniques would introduce an alternative system for large-scale production and establishment of genetically stable plants. [ABSTRACT FROM AUTHOR]

Information about the positioning of individual loci in the nucleus and the status of epigenetic modifications at these loci in each cell contained in plant tissue increases our understanding of how cells in a tissue coordinate gene expression. To obtain such information, a less damaging method of visualizing DNA in tissue that can be used with immunohistochemistry is required. Recently, a less damaging DNA visualization method using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/associated caspase 9) system, named RNA-guided endonuclease– in situ labeling (RGEN-ISL), was reported. This system made it possible to visualize a target DNA locus in a nucleus fixed on a glass slide with a set of simple operations, but it could not be applied to cells in plant tissues. In this work, we have developed a modified RGEN-ISL method with decrosslinking that made it possible to simultaneously detect the DNA loci and immunohistochemistry signals, including histone modification, in various types of plant tissues and species. [ABSTRACT FROM AUTHOR]

DNA sequencing technologies continue to improve, and there has been a corresponding expansion of DNA-based applications in the forensic sciences. DNA recovered from dust and environmental debris can be used to identify the organisms associated with these sample types, including bacteria, plants, fungi, and insects. Such results can then be leveraged to discern sample origin or geolocation and investigate individual identification. Here, we take a critical look at the current DNA-based technologies using microbiome and environmental sample sources that are focused on the generation of some investigative tools for use in forensic science. We discuss the pitfalls and contentions associated with the use of these techniques and highlight some of the future research required to expand the utility of these methods in the forensic sciences. [ABSTRACT FROM AUTHOR]

Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of morphological information from herbarium specimens, especially for small plants with few leaves, and reduces the voucher value of specimens. Here, we developed a new, non-destructive DNA extraction protocol (Protocol 1) that only needs a small piece of leaf (< 25 mm2) to obtain DNA suitable for DNA sequencing from fragile herbarium specimens. The protocol was very simple and rapid; an extraction buffer was placed on the leaf surface of an intact specimen for 30 min at room temperature (20 °C). The quality of extracted DNA was checked by PCR amplification of two standard plant DNA barcode regions, the maturase K gene (matK, ca. 850 bp) and the ribulose-1,5-bisphosphatecarboxylase/oxygenase gene (rbcL, ca. 550 bp), for 14 vascular plant species encompassing various taxonomic groups. The protocol retrieved sequences from 80.0% of specimens for matK and 46.2% of specimens for rbcL. Placing of the extraction buffer onto specimens did not cause any tears or deformation, but caused discoloration in some plants. To improve DNA yield for specimens incompatible with Protocol 1, we developed an alternative protocol for DNA extraction with minimally invasive destruction of specimens (Protocol 2). In this protocol, a cut leaf was immersed in the extraction buffer for 30 min and stored subsequently in a fragment pocket on the specimen sheet. This alternative method retrieved matK sequences from 80.0% of specimens and rbcL sequences from 92.8% of specimens. The combination of Protocols 1 and 2 enabled us to obtain matK sequences from 90.0% of specimens and rbcL sequences form 92.8% of specimens. The new protocols facilitate the use of museum specimens for use of DNA of museum specimens while still preserving morphological information. [ABSTRACT FROM AUTHOR]

The CRISPR–Cas9 system is a powerful tool for editing genes of interest in specific plant genomes. Indeed, genome-editing systems have been used to enhance a variety of agricultural traits and to study gene functions in many plant species, and the plasmid-mediated delivery of Cas9 and single guide RNA (sgRNA) to plants has been reported to facilitate highly efficient editing. However, the random and stable integration of plasmid DNA sequences into plant genomes can cause insertional mutagenesis, and an additional step is required to remove such foreign sequences from edited plant genomes. Accordingly, the aim of the present study was to investigate the effectiveness of directly delivering purified CRISPR–Cas9 ribonucleoproteins (RNPs) to protoplasts from cabbage (Brassica oleracea var. capitata), an important cruciferous vegetable. The flowering-time regulator gene GIGANTEA (GI) was targeted, with the goal of delaying flowering time and prolonging vegetative growth. We investigated the targeted mutagenesis insertion and deletion rates using targeted deep sequencing. The mutation frequency achieved using one of the sgRNAs (sgRNA2) was 2% in the infected protoplast. The shoots were regenerated from 44% (46/103) of protoplast-derived calli. Consequently, three independent and completely transgene-free mutants were obtained, including one homogeneous biallelic line in which both GI alleles were successfully edited, thereby yielding a complete GI knockout line. These results suggest that the transgene-free CRISPR–Cas9 system is a promising tool for improving agricultural beneficial traits of cabbage. [ABSTRACT FROM AUTHOR]

Complete plastid genome (plastome) sequences and nuclear ribosomal DNA (nrDNA) regions have been proposed as candidates for the next generation of DNA barcodes for plant species discrimination. However, the efficacy of this approach still lacks comprehensive evaluation. We carried out a case study in the economically important but phylogenetically and taxonomically difficult genus Panax (Araliaceae). We generated a large data set of plastomes and nrDNA sequences from multiple accessions per species. Our data improved the phylogenetic resolution and levels of species discrimination in Panax, compared to any previous studies using standard DNA barcodes. This provides new insights into the speciation, lineage diversification and biogeography of the genus. However, both plastome and nrDNA failed to completely resolve the phylogenetic relationships in the Panax bipinnatifidus species complex, and only half of the species within it were recovered as monophyletic units. The results suggest that complete plastome and ribosomal DNA sequences can substantially increase species discriminatory power in plants, but they are not powerful enough to fully resolve phylogenetic relationships and discriminate all species, particularly in evolutionarily young and complex plant groups. To gain further resolving power for closely related species, the addition of substantial numbers of nuclear markers is likely to be required. [ABSTRACT FROM AUTHOR]

Background: The submission of DNA sequences to public sequence databases is an essential, but insufficiently automated step in the process of generating and disseminating novel DNA sequence data. Despite the centrality of database submissions to biological research, the range of available software tools that facilitate the preparation of sequence data for database submissions is low, especially for sequences generated via plant and fungal DNA barcoding. Current submission procedures can be complex and prohibitively time expensive for any but a small number of input sequences. A user-friendly software tool is needed that streamlines the file preparation for database submissions of DNA sequences that are commonly generated in plant and fungal DNA barcoding. Methods: A Python package was developed that converts DNA sequences from the common EMBL and GenBank flat file formats to submission-ready, tab-delimited spreadsheets (so-called ‘checklists’) for a subsequent upload to the annotated sequence section of the European Nucleotide Archive (ENA). The software tool, titled ‘EMBL2checklists’, automatically converts DNA sequences, their annotation features, and associated metadata into the idiosyncratic format of marker-specific ENA checklists and, thus, generates files that can be uploaded via the interactive Webin submission system of ENA. Results: EMBL2checklists provides a simple, platform-independent tool that automates the conversion of common DNA barcoding sequences into easily editable spreadsheets that require no further processing but their upload to ENA via the interactive Webin submission system. The software is equipped with an intuitive graphical as well as an efficient command-line interface for its operation. The utility of the software is illustrated by its application in four recent investigations, including plant phylogenetic and fungal metagenomic studies. Discussion: EMBL2checklists bridges the gap between common software suites for DNA sequence assembly and annotation and the interactive data submission process of ENA. It represents an easy-to-use solution for plant and fungal biologists without bioinformatics expertise to generate submission-ready checklists from common DNA sequence data. It allows the post-processing of checklists as well as work-sharing during the submission process and solves a critical bottleneck in the effort to increase participation in public data sharing. [ABSTRACT FROM AUTHOR]

Abstract Ancient hybridization leading to plastid capture is a likely cause of cytonuclear discordance, although discordance may also be attributed to other mechanisms. Cytonuclear discordance is ubiquitous in a clade of southern African Oxalis known as the Hirta clade, despite a notable lack of documented field hybrids for this genus. The aims of this study were to determine whether hybridization is possible between different Hirta clade species, what the fitness effects on seed set of possible hybridizations are, and to determine the mode of inheritance of plastid DNA in Oxalis. We tested these aims using manual interspecific and conspecific crosses, as well as self-pollination control crosses of species within this clade, conducted in a research collection context. Roughly 10% of interspecific crosses produced seed, most of which were confirmed as unambiguous hybrid plants using DNA sequencing of the Internal Transcribed Spacer (ITS). Low levels of self-compatibility were recorded in several species, while one accession showing very high levels of self-compatibility (accession MO1218). Interspecific crosses produced significantly less seed per capsule compared to conspecific crosses with respective parents, suggesting decreased fitness in the establishment of the F1 hybrids. Selfing seed set was significantly lower than interspecific and conspecific seed set upon removal of MO1218. Using plastid fragment length polymorphisms, Oxalis was found to have predominantly or exclusively maternally inherited plastid DNA. This study establishes the viability of the earliest stages of plastid capture in Oxalis , and provides context relevant to the lack of documented field hybrids in the southern African members of this genus. Future studies should focus on F1 survival and fertility and the maternal fitness effects of subsequent backcrosses to the maternal parent, as well as on hybridization rates under field conditions, to assess whether hybridization occurs at rates sufficient to affect Oxalis evolution in southern Africa. Highlights • Produced the first, unequivocal confirmed Oxalis F1 hybrids • Confirm some of the hybrids to be heteroploid • First unequivocal evidence of self-compatibility in SA Oxalis • Show that plastid DNA is maternally inherited in Oxalis [ABSTRACT FROM AUTHOR]

The possibility to predict the outcome of targeted DNA double-stranded break ( DSB) repair would be desirable for genome editing. Furthermore the consequences of mis-repair of potentially cell-lethal DSBs and the underlying pathways are not yet fully understood. Here we study the clustered regularly interspaced short palindromic repeats ( CRISPR)/Cas9-induced mutation spectra at three selected endogenous loci in Arabidopsis thaliana by deep sequencing of long amplicon libraries. Notably, we found sequence-dependent genomic features that affected the DNA repair outcome. Deletions of 1-bp to <1000-bp size and/or very short insertions, deletions >1 kbp (all due to NHEJ) and deletions combined with insertions between 5-bp to >100 bp [caused by a synthesis-dependent strand annealing ( SDSA)-like mechanism] occurred most frequently at all three loci. The appearance of single-stranded annealing events depends on the presence and distance between repeats flanking the DSB. The frequency and size of insertions is increased if a sequence with high similarity to the target site was available in cis. Most deletions were linked to pre-existing microhomology. Deletion and/or insertion mutations were blunt-end ligated or via de novo generated microhomology. While most mutation types and, to some degree, their predictability are comparable with animal systems, the broad range of deletion mutations seems to be a peculiar feature of the plant A. thaliana. [ABSTRACT FROM AUTHOR]

DNase I hypersensitive sites (DHSs) are accessible chromatin regions hypersensitive to cleavages by DNase I endonucleases. DHSs are indicative of cis -regulatory DNA elements (CREs), all of which play important roles in global gene expression regulation. It is helpful for discovering CREs by recognition of DHSs in genome. To accelerate the investigation, it is an important complement to develop cost-effective computational methods to identify DHSs. However, there is a lack of tools used for identifying DHSs in plant genome. Here we presented pDHS-SVM, a computational predictor to identify plant DHSs. To integrate the global sequence-order information and local DNA properties, reverse complement kmer and dinucleotide-based auto covariance of DNA sequences were applied to construct the feature space. In this work, fifteen physical-chemical properties of dinucleotides were used and Support Vector Machine (SVM) was employed. To further improve the performance of the predictor and extract an optimized subset of nucleotide physical-chemical properties positive for the DHSs, a heuristic nucleotide physical-chemical property selection algorithm was introduced. With the optimized subset of properties, experimental results of Arabidopsis thaliana and rice ( Oryza sativa ) showed that pDHS-SVM could achieve accuracies up to 87.00%, and 85.79%, respectively. The results indicated the effectiveness of proposed method for predicting DHSs. Furthermore, pDHS-SVM could provide a helpful complement for predicting CREs in plant genome. Our implementation of the novel proposed method pDHS-SVM is freely available as source code, at https://github.com/shanxinzhang/pDHS-SVM . [ABSTRACT FROM AUTHOR]

Parasenecio with c. 70 species is a major component of the eastern Asian Tussilagininae (Asteraceae, Senecioneae). Previous molecular phylogenetic studies, although with limited taxon sampling, have suggested that it is polyphyletic and forms a complex with ten other eastern Asian genera of Tussilagininae, i.e. the Ligularia-Cremanthodium- Parasenecio (LCP) complex. Here we sample Parasenecio and its relatives in the LCP complex densely and generate two phylogenetic hypotheses for the genus using four plastid DNA (trnCGCA-petN, trnGUCC-trnTGGU-psbD, trnTUGUtrnLUAA and rpl32-ndhF) and two nuclear ribosomal (nr)DNA [internal and external transcribed spacer (ITS and ETS)] regions, respectively. Analyses of the plastid DNA data produce a robust generic-level phylogenetic tree for Parasenecio and clearly demonstrate the polyphyletic pattern of the genus. Most notably, two sections of Parasenecio, P. section Delphiniifolii s.s. and P. section Taimingasa, are shown to be distantly related to core Parasenecio, being sister to Miricacalia and Syneilesis, respectively, with strong support. The small genus Sinacalia is also revealed to be polyphyletic, with S. caroli nested in core Parasenecio, and S. tangutica and S. davidii forming a clade sister to the core Parasenecio-S. caroli clade. The nrDNA tree is, however, relatively poorly resolved, only confirming the sister relationship between P. section Delphiniifolii s.s. and Miricacalia. Sinacalia s.s. is resolved as being deeply nested in core Parasenecio with nrDNA data, which is the only significant incongruence with the plastid DNA hypothesis relevant to the generic circumscription of Parasenecio. Statistical topology (approximately unbiased and Swofford- Olsen-Waddell-Hillis) tests suggest that, except for the position of Sinacalia s.s., the generic-level relationships of Parasenecio inferred from the plastid DNA data are not incongruent with those from the nrDNA data and, on the basis of current sequence data, the inferred molecular phylogenetic relationships should be preferred over the current classification. Based on these results and evidence from morphology and karyology, we propose a new circumscription of Parasenecio, involving transferring S. caroli to Parasenecio as P. caroli comb. nov. and segregating P. section Delphiniifolii s.s. and P. section Taimingasa as the new genera Japonicalia gen. nov. and Taimingasa gen. nov., respectively. Sinacalia s.s., with significant incongruence between the plastid DNA and nrDNA trees, is tentatively retained as an independent genus pending future studies. A revised key to Parasenecio and its close relatives is provided. [ABSTRACT FROM AUTHOR]

Prasinophytes form a paraphyletic assemblage of early diverging green algae, which have the potential to reveal the traits of the last common ancestor of the main two green lineages: (i) chlorophyte algae and (ii) streptophyte algae. Understanding the genetic composition of prasinophyte algae is fundamental to understanding the diversification and evolutionary processes that may have occurred in both green lineages. In this study, we sequenced the chloroplast genome of Pyramimonas parkeae NIES254 and compared it with that of P. parkeae CCMP726, the only other fully sequenced P. parkeae chloroplast genome. The results revealed that P. parkeae chloroplast genomes are surprisingly variable. The chloroplast genome of NIES254 was larger than that of CCMP726 by 3,204 bp, the NIES254 large single copy was 288 bp longer, the small single copy was 5,088 bp longer, and the IR was 1,086 bp shorter than that of CCMP726. Similarity values of the two strains were almost zero in four large hot spot regions. Finally, the strains differed in copy number for three protein-coding genes: ycf20, psaC, and ndhE. Phylogenetic analyses using 16S and 18S rDNA and rbcL sequences resolved a clade consisting of these two P. parkeae strains and a clade consisting of these plus other Pyramimonas isolates. These results are consistent with past studies indicating that prasinophyte chloroplast genomes display a higher level of variation than is commonly found among land plants. Consequently, prasinophyte chloroplast genomes may be less useful for inferring the early history of Viridiplantae than has been the case for land plant diversification. [ABSTRACT FROM AUTHOR]

Background: Strawberry vein banding virus (SVBV) is a double-stranded DNA plant virus, which has been found in North America, Australia, Brazil, Japan, Europe and several provinces of China. Infected strawberry plants exhibit mild vein-banding symptoms and chlorosis along the veins. It is one of the most economically important diseases in Asiatic, European and North American strawberry-growing areas. Findings: The complete genome of an SVBV Chinese isolate (SVBV-CN) was isolated and cloned from a naturally infected strawberry (Fragaria × ananassa cv. Sachinoka) sample found in Shenyang city of Liaoning province. Sequence analysis revealed a complete genome of 7864 nucleotides (nts) that indicated SVBV-CN was most closely related to SVBV from the United States (SVBV-US) with a sequence similarity of 85.8 %. Two major clades were identified based on phylogenetic analysis of the complete genome sequences of caulimoviruses. SVBV-CN clustered together with SVBV-US, whereas other caulimoviruses formed a separate branch. Agrobacterium-mediated inoculation of Fragaria vesca with an infectious clone of SVBV-CN results in systemic infection with distinct symptoms of yellowing bands along the main leaf veins. This suggests that the SVBV-CN infectious clone can recapitulate the symptoms observed in naturally infected strawberries, and therefore is likely the causal agent of the original disease observed in strawberries. Furthermore, strawberry plants inoculated with the infectious clone using vacuum infiltration developed symptoms with a very high infection rate of 86–100 % in 4-5 weeks post-inoculation. This compares to an infection rate of 20–40 % in 8–9 weeks post-inoculation using syringe-inoculation. Conclusions: The complete nucleotide sequence of SVBV from a naturally infected strawberry was determined. Agroinfiltration of strawberry plants using an infectious clone of SVBV-CN resulted in symptoms typically found in infected strawberries from Shenyang city of Liaoning province in China. This is the first report describing an infectious clone of SVBV-CN, and that vacuum infiltration can be potentially used as a new and highly efficient means for inoculation of strawberry plants. [ABSTRACT FROM AUTHOR]

Phylogenetic relationships were studied based on morphology and nuclear ribosomal DNA internal transcript spacer (nrDNA ITS) sequences in 49 populations of 36 Onobrychis taxa belonging to 5 sections (Dendobrychis, Lophobrychis, Onobrychis, Heliobrychis and Hymenobrychis) naturally grown in Turkey. The morphological data (46 quantitative characters) were standardized and submitted for PCA analysis before the performance of hierarchical clustering analysis. In addition, a 577- bp region of the nrDNA ITS sequences was compared to generate a dendrogram using the neighbor-joining method. According to the obtained results, the Onobrychis genus is clearly divided into the Onobrychis and Sisyrosema subgenera both molecularly and morphologically; however, in terms of molecular data, while the Onobrychis subgenus showed polyphyletic features, the Sisyrosema subgenus had monophyletic features. Additionally, long-wing taxa belonging to Dendobrychis and Onobrychis and O. crista-galli belonging to Lophobrychis located in the same clade in Onobrychis showed polyphyletic features. This finding was confirmed in terms of long-wing taxa as well as morphology and indicated that these three sections were entangled with each other under the Onobrychis subgenus. [ABSTRACT FROM AUTHOR]

Centromeres on eukaryotic chromosomes consist of large arrays of DNA repeats that undergo very rapid evolution. Nelumbo nucifera Gaertn. (sacred lotus) is a phylogenetic relict and an aquatic perennial basal eudicot. Studies concerning the centromeres of this basal eudicot species could provide ancient evolutionary perspectives. In this study, we characterized the centromeric marker protein NnCenH3 (sacred lotus centromere-specific histone H3 variant), and used a chromatin immunoprecipitation (Ch IP)-based technique to recover the NnCenH3 nucleosome-associated sequences of sacred lotus. The properties of the centromere-binding protein and DNA sequences revealed notable divergence between sacred lotus and other flowering plants, including the following factors: (i) an NnCenH3 alternative splicing variant comprising only a partial centromere-targeting domain, (ii) active genes with low transcription levels in the NnCenH3 nucleosomal regions, and (iii) the prevalence of the Ty1/ copia class of long terminal repeat ( LTR) retrotransposons in the centromeres of sacred lotus chromosomes. In addition, the dynamic natures of the centromeric region showed that some of the centromeric repeat DNA sequences originated from telomeric repeats, and a pair of centromeres on the dicentric chromosome 1 was inactive in the metaphase cells of sacred lotus. Our characterization of the properties of centromeric DNA structure within the sacred lotus genome describes a centromeric profile in ancient basal eudicots and might provide evidence of the origins and evolution of centromeres. Furthermore, the identification of centromeric DNA sequences is of great significance for the assembly of the sacred lotus genome. [ABSTRACT FROM AUTHOR]

Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders' needs. [ABSTRACT FROM AUTHOR]

Background: The continuing increase in size and quality of the "short reads" raw data is a significant help for the quality of the assembly obtained through various bioinformatics tools. However, building a reference genome sequence for most plant species remains a significant challenge due to the large number of repeated sequences which are problematic for a whole-genome quality de novo assembly. Furthermore, for most SNP identification approaches in plant genetics and breeding, only the "Gene-space" regions including the promoter, exon and intron sequences are considered. Results: We developed the iPea protocol to produce a de novo Gene-space assembly by reconstructing, in an iterative way, the non-coding sequence flanking the Unigene cDNA sequence through addition of next-generation DNA-seq data. The approach was elaborated with the large diploid genome of pea (Pisum sativum L.), rich in repetitive sequences. The final Gene-space assembly included 35,400 contigs (97 Mb), covering 88 % of the 40,227 contigs (53.1 Mb) of the PsCam_low-copy Unigen set. Its accuracy was validated by the results of the built GenoPea 13.2 K SNP Array. Conclusion: The iPEA protocol allows the reconstruction of a Gene-space based from RNA-Seq and DNA-seq data with limited computing resources. [ABSTRACT FROM AUTHOR]

Bouteloua (Poaceae: Chloridoideae: Cynodonteae; Boutelouiane) is an important genus of forage grasses containing 60 species found primarily in the Americas with a center of diversity in northern Mexico. A modern subgeneric classification is lacking. The goals of our study were to reconstruct the evolutionary history among the species of Bouteloua using molecular data with increased species sampling compared to previous studies. A phylogenetic analysis was conducted on 209 samples, of which 59 species (206 individuals) were in Bouteloua, using two plastid ( rpl32-trnL spacer and rps16-trnK spacer) and nuclear ITS 1&2 (ribosomal internal transcribed spacer) sequences to infer evolutionary relationships and produce a subgeneric classification. Overall, ITS and plastid phylogenies rendered similar patterns. However, the ITS phylogeny lacked backbone structure, recovering only four internal clades out of nine found in the plastid phylogeny. The ITS network shows a radiative evolutionary pattern and indicates a number of incompatible splits, suggesting past hybridization between species of different sections. The maximum-likelihood tree from the combined plastid and ITS regions is well resolved and depicts a strongly supported monophyletic Bouteloua that includes ten strongly supported clades and one moderately supported clade. The molecular results support the recognition of 10 sections and two subsections within Bouteloua s.l.; three sections are new: Barbata, Hirsuta, and Trifida; four sections are new combinations: Buchloe, Cyclostachya, Opizia, and Triplathera; and two subsections are new: Eriopoda and Hirsuta. Based on our molecular results and the possession of unique morphological characters we describe a new species from Nuevo León, Bouteloua herrera-arrietae. [ABSTRACT FROM AUTHOR]

During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase ( STS1 , STS10 ) genes of Vitis amurensis , the Actin2 gene of Arabidopsis thaliana , the internal transcribed spacer ( AtITS ), and tRNAPro of A. thaliana . The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs. [ABSTRACT FROM AUTHOR]

The article discusses a study that described the Anathallis roseopapillosa as the new species of Anathallis that thrive in the Brazilian Amazon. Topics covered include analysis of clade with data of nuclear deoxyribonucleic acid (DNA) sequences and evidence that the new species have phylogenetic position in the Pleurothallidinae subtribe. Results showed morphological relation of A. reseopapillosa to A. gracilenta but with shorter racemes and being tri-lobed with papillose apex in petals.

The article discusses a study that determined composition of Carolinense clade of solanum (Solanaceae) through the plastid and nuclear deoxyribonucleic acid (DNA) sequences. Topics covered included use of maximum parsimony for phylogenetic analyses, internal transcribed spacer (ITS) analysis and granule-bound starch synthase (GBSSI). Results showed 10 taxa in Carolinense clade with conflicting phylogenies of three species including Solanum perplexum indicating introgression or allopolyploidy.

Ramie (Boehmeria nivea L. Gaud) is a highly versatile herbaceous plant which is widely cropped in southern China. The success of this herbaceous plant relies on wide use in modern industry. Understanding the profiling of expressed genes in phloem and xylem of ramie is crucial for improving its industrial performance. Herein, we uncover the transcriptome profile in phloem and xylem in present study. Using Illumina paired-end sequencing technology, 57 million high quality reads were generated. De novo assembly yielded 87,144 unigenes with an average length of 635 bp. By sequence similarity searching for public databases, a total of 32,541 (41.77%) unigenes were annotated for their function. Among these genes, 57,873 (66.4%) and 28,678 (32.9%) unigenes were assigned to categories of Gene Ontology and Orthologous Groups database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 18,331 (21.0%) unigenes were mapped to 125 pathways. The metabolic pathways were assigned the most unigene (4,793, 26.2%). Furthermore, Pol II and Pol III subunits as well as the genes of Galactose metabolism pathway had higher expression in phloem compared to xylem. In addition, fatty acid metabolism pathway genes showed more abundant in xylem than phloem. These results suggest that high activities of RNA synthesis and Galactose metabolism pathway promises fiber synthesis in phloem. The present study is the initial exploration to uncover the fiber biosynthesis difference between phloem and xylem in ramie through the analysis of deep sequencing data. [ABSTRACT FROM AUTHOR]

The phylogeny of Astragalus L. section Anthylloidei DC. and its interrelationship with allies were examined. The study was conducted using nrDNA ITS and plastid rpl32-trnL(UAG) sequences. Astragalus sect. Anthylloidei is nonmonophyletic, and its members are scattered across the tree in 4 well-supported clades and intermixed with members of other spiny sections. All the multispecific informal groups of the section, with the exception of A. murinus Boiss. group, are not monophyletic. Morphological character evolution was mapped on the molecular tree. Our results suggest that morphology cannot elucidate infrageneric relationships in spiny Astragalus accurately; analyzed characters have evolved several times in sect. Anthylloidei and, thus, show high levels of homoplasy. Distribution of members of the section matches, more or less, certain geographic patterns, ranging from the Zagros mountains, Northwest Iran and Eastern Turkey, Central Iran to Northeast Iran, and Turkmenistan and Afghanistan. A new taxonomic system for this group of species is needed. The present study suggests that sect. Halicacabus Bunge, which has been merged with sect. Anthylloidei, should be resurrected since 8 species, including A. halicacabus Lam; A. wagneri Bartl. ex Bunge; A. distans Fisch.; A. raswendicus Hausskn. & Bornm.; A. veiskaramii Zarre, Podlech & T.Sabaii; A. submitis Boiss. & Hohen.; and A. chardinii Boiss. (sect. Anthylloidei) as well as A. semnanensis Bornm. & Rech. f. (sect. Semnanenses Podlech & Zarre), form a distinct clade. Sect. Eriostoma Bornm. is a distinct lineage from sect. Anthylloidei. [ABSTRACT FROM AUTHOR]

Eukaryotic genomes contain highly variable amounts of DNA with no apparent function. This so-called junk DNA is composed of two components: repeated and repeat-derived sequences (together referred to as the repeatome), and non-annotated sequences also known as genomic dark matter. Because of their high duplication rates as compared to other genomic features, transposable elements are predominant contributors to the repeatome and the products of their decay is thought to be a major source of genomic dark matter. Determining the origin and composition of junk DNA is thus important to help understanding genome evolution as well as host biology. In this study, we have used a combination of tools enabling to show that the repeatome from the small and reducing A. thaliana genome is significantly larger than previously thought. Furthermore, we present the concepts and results from a series of innovative approaches suggesting that a significant amount of the A. thaliana dark matter is of repetitive origin. As a tentative standard for the community, we propose a deep compendium annotation of the A. thaliana repeatome that may help addressing farther genome evolution as well as transcriptional and epigenetic regulation in this model plant. [ABSTRACT FROM AUTHOR]

Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions. [ABSTRACT FROM AUTHOR]

Trunk rot poses a substantial threat to Sanbu-sugi, one of the most economically important cultivars of Japanese cedar (Cryptomeria japonica). The etiology of this disease, inchtding its main agents, is incompletely known. This trunk rot was attributed to Fomitiporia (Phellinus) hartigii or F. (Phellinus) punctata. Here we phylogenetically analyzed DNA sequences of four markers from a set of strains isolated from trunk-rot symptoms and recovered a single, monophyletic clade, indicating that a single taxon is involved. This clade was identified as Fomitiporia torreyae, a species described from eastern China. This analysis also proved that trunk rot and/or dieback of other conifers and broadleaf trees, including Sawara cypress (Chamaecyparis pisifera), Japanese umbrella pine (Sciadopitys verticillata) and Japanese pear (Pyrus pyrifolia var. culta), were caused by the same species. The phylogenetic approach to Fomitiporia revealed that the F. torreyae clade was closely related to F. bannaensis but clearly distinct from F. punctata, which originally was thought to be the cause of trunk rot in Sanbu-sugi. Fomitiporia torreyae is redescribed on the basis of more than 40 specimens from multiple hosts from Japan and China. Fomitiporia juniperina comb. nov. is proposed. [ABSTRACT FROM AUTHOR]

Phytoplasma-associated diseases are reported for more than 1,000 plant species worldwide. Only a few genome sequences are available in contrast to the economical importance of these bacterial pathogens. A new strategy was used to retrieve phytoplasma strain-specific genome data. Multiple displacement amplification was performed on DNA obtained from <3 g of plant tissue from tobacco and parsley samples infected with 'stolbur' strains. Random hexamers and Phi29 polymerase were evaluated with and without supplementation by group-assigned oligonucleotides providing templates for Illumina's sequencing approach. Metagenomic drafts derived from individual and pooled strain-specific de novo assemblies were analyzed. Supplementation of the Phi29 reaction with the group-assigned oligonucleotides resulted in an about 2-fold enrichment of the percentage of phytoplasma-assigned reads and thereby improved assembly results. The obtained genomic drafts represent the largest datasets available from 'stolbur' phytoplasmas. Sequences of the two strains (558 kb, 448 proteins and 516 kb, 346 proteins, respectively) were annotated allowing the identification of prominent membrane proteins and reconstruction of core pathways. Analysis of a putative truncated sucrose phosphorylase provides hints on sugar degradation. Furthermore, it is shown that drafts obtained from repetitive-rich genomes allow only limited analysis on multicopy regions and genome completeness. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Highlights: [•] A phylogeny is provided for the genus Picea (spruces), including all 37 species. [•] It is based on chloroplast, nuclear, and mitochondrial sequences. [•] Fossil-calibrated DNA clocks indicate that today’s spruces date to max. 28Ma. [•] Previous molecular-biogeographic inferences are not supported. [•] North America has ten, not eight species, and Norway spruce appears to be polyphyletic. [ABSTRACT FROM AUTHOR]

Dianthus polylepis complex consists of two already known endemic species, Dianthus polylepis and D. binaludensis, in Khorassan-Kopetdagh floristic province. The taxonomic position of these species has long been debated. The aim of the present study is to shed light on the evolutionary relationships of the members of the complex using morphological and molecular data. In morphological study, firstly, 56 vegetative and floral characters were measured on 33 specimens of the both species. Multivariate analyses were performed on 25 (out of 56) significantly discriminating morphological traits. In molecular study, we sequenced alleles obtained from a region between 2nd and 6th exons of the gene coding for the enzyme dihydroflavonol 4-reductase copy1 (DFR1). Morphological results show that most of a priori identified accessions were not grouped in a posteriori classification. It is difficult to discriminate D. polylepis from D. binaludensis in morphological continuum among the accessions. Results obtained from the molecular data indicated no monophyly for the members of the D. polylepis complex. Consistency between the morphological and molecular results shows that D. polylepis and D. binaludensis were not morphologically and molecularly well differentiated. Therefore, we propose a new combination as D. polylepis subsp. binaludensis. [ABSTRACT FROM AUTHOR]

Almonds and peaches are among the most economically important fruit crops in subtropical and temperate regions. Questions regarding the origin and domestication of the crop species, interspecific relationships, and evolutionary patterns of fruits have remained unanswered due to the lack of phylogenetic data of Prunus subgenus Amygdalus, in which both almond and peach are classified. Twenty-two species of subg. Amygdalus were included in phylogenetic analyses of plastid ndhF- rpl32, rpL16, trnH- psbA, trnL- trnF, trnQ- 5′ rps16, and trnS- trnG and nuclear s6pdh genes to investigate the circumscription of subg. Amygdalus, phylogenetic relationships among the species, and implication for mesocarp and endocarp evolution. Prunus subg. Amygdalus is best circumscribed to include both almonds and peaches, comprising two sections, Amygdalus and Persica. Species traditionally placed close to almonds, such as P. tenella, P. petunnikowii, P. triloba, and P. pedunculata, should be excluded from subg. Amygdalus. Characteristics used to diagnose subg. Amygdalus, such as dry, splitting mesocarp and the ornamentation of endocarp evolved multiple times in Prunus; thus classification based solely on these morphological characters produces a taxonomic scheme that might not represent common ancestry. A dry, splitting mesocarp is likely to be ancestral for the common ancestor of subg. Amygdalus, and the fleshy, non-splitting mesocarp found in peaches was derived from the dry, splitting mesocarp, presumably by domestication and selection for a fleshy fruit in China. Very low genetic divergence among cultivated almond and its close relatives might be associated with gene flow and/or rapid speciation within the group. [ABSTRACT FROM AUTHOR]

The article discusses the development of next-generation technology for genetic sequencing. Sequencing projects emerged in laboratories around the world because of this technological development. Researchers are warned about some types of genomic DNA that cannot be sequenced by high-throughput methods. The application of the technology in plant sequencing is also explored.

Diverse and rapidly evolving pathogens cause plant diseases and epidemics that threaten crop yield and food security around the world. Research over the last 25 years has led to an increasingly clear conceptual understanding of the molecular components of the plant immune system. Combined with ever-cheaper DNA-sequencing technology and the rich diversity of germ plasm manipulated for over a century by plant breeders, we now have the means to begin development of durable (long-lasting) disease resistance beyond the limits imposed by conventional breeding and in a manner that will replace costly and unsustainable chemical controls. [ABSTRACT FROM AUTHOR]

We demonstrate quantitatively that, as predicted by evolutionary theory, sequences of homologous proteins from different species converge as we go further and further back in time. The converse, a non-evolutionary model can be expressed as probabilities, and the test works for chloroplast, nuclear and mitochondrial sequences, as well as for sequences that diverged at different time depths. Even on our conservative test, the probability that chance could produce the observed levels of ancestral convergence for just one of the eight datasets of 51 proteins is ≈1×10−19 and combined over 8 datasets is ≈1×10−132. By comparison, there are about 1080 protons in the universe, hence the probability that the sequences could have been produced by a process involving unrelated ancestral sequences is about 1050 lower than picking, among all protons, the same proton at random twice in a row. A non-evolutionary control model shows no convergence, and only a small number of parameters are required to account for the observations. It is time that that researchers insisted that doubters put up testable alternatives to evolution. [ABSTRACT FROM AUTHOR]

The article discusses the phylogenetic analyses that were performed based on four plastid deoxyribonucleic acid (DNA) sequences to determine if the Daturae and Datura are monophyletic and to understand their relationships and classification. It cites the division of species of Datura into sections Ceratocaulis, Datura, and Dutrab based upon the position and type of the fruit. It also notes the monophyly of the Daturae clade with two genera and the two distinct lineages of the Datura clade.

The article explores the phylogenetic relationships of Pogoyne (Lamiacea), a small genus of annual plants that occur almost entirely in vernal pool habitats. It discusses the use of both parsimony and Bayesian phylogeny reconstruction methods to analyze deoxyribonucleic acid (DNA) sequence data obtained from all seven extant species and several outgroups. It also indicates that subgenus Pogoyne is paraphyletic, but with poor support while subgenus Hedeomoides is monophyletic with good support.

• The quality of plant genomic DNA sequence analysis was evaluated using seven methods. • Plant genomic DNA sequences were analyzed without DNA and PCR product purification. • Gene-edited plant screening can be easily accomplished using the tested methods. • The sequencing primer should be set within 400 bp 5'-upstream from the DNA sequence. • Genomic DNA and PCR product purification steps could be omitted. Genome-editing technologies are widely used to characterize gene functions and improve the features of agricultural plants. Although sequence analysis of gene editing target DNA is the most reliable method of screening gene-edited plants, the current DNA sequence analysis methods are time consuming and labor intensive because they include genomic DNA and polymerase chain reaction (PCR) product purification. In this study, seven methods were performed for sequence analysis of plant genomic DNA with and/or without genomic DNA and PCR product purification. Consequently, good-quality sequencing chromatograms were obtained using all methods. Results showed that the partial genomic DNA sequence of Nicotiana benthamiana and Arabidopsis thaliana could be sufficiently analyzed without plant genomic DNA and PCR product purification. Furthermore, screening of gene-edited N. benthamiana was successful using the present methods. Therefore, the tested methods could reduce the time, simplify the workflow of plant gene analysis, and help in screening gene-edited plants. [ABSTRACT FROM AUTHOR]

The article demonstrates that Lophiarella (Orchidaceae), as originally circumscribed, is polyphyletic. The morphological characters, nucleotide sequences and coded gaps of two DNA regions, namely plastid rp132-trnL and nrDNA-ITS, were analyzed using parsimony and Bayesian inference. It also details the evolution of several taxonomically, ecologically and bio-geographically relevant characters, such as rostellum shape, vegetative architecture and column structure.

Abstract: In order to assess the phylogenetic relationships and taxonomic position of Tanacetum semenovii Herder (Compositae, Anthemideae), a molecular-phylogenetic analysis based on nrDNA ITS sequences of nine representatives of the subtribe Handeliinae and 26 species of three other subtribes (Anthemidinae, Artemisiinae and Matricariinae) of the tribe Anthemideae was conducted. The central Asian endemic T. semenovii Herder was found being unrelated to members of the genus Tanacetum L. (subtribe Anthemidinae) and sister to a clade formed by three representatives of the genus Richteria Kar. & Kir. of subtribe Handeliinae. Due to the close relationship of the T. semenovii to the Richteria species in a well-supported clade (PP = 1.0, BS = 78), which is supported by morphological features, the inclusion of T. semenovii in the genus Richteria and the new combination Richteria semenovii (Herder) Sonboli & Oberpr. is proposed. [Copyright &y& Elsevier]