4-(2-Chloro­ethoxy)phthalonitrile

In the title compound, C10H7ClN2O, the O and both C atoms of the chloroethoxy group are disordered over two positions, the occupancy factor of the major disorder component refining to 0.54 (2)

Potential Global-Local Inconsistency in Species-Area Relationships Fitting

The Species-Area Relationship (SAR) has been widely employed to assess species diversity and predict species extinction. Thus far, although many functions were proposed to fit SAR based on field observations or simulation results, the shape of SAR curve has been debated extensively over decades. Here we uncover a potential global-local inconsistency in SARs fitting simulation blocked by the limitation of large area sampling before. The results indicated that power and logarithm SAR formulas were good for the fitting if the sampling area range is small which is also the practical sampling interval in the field. However, for the logarithm SAR fitting, a sigmoid curve occurred in the log10 Area - Number of Species plane, and for the power SAR fitting, the curve is convex instead of a straight line as assumed when linear regression was applied. In conclusion, neither the power SAR nor the logarithm SAR fitted to simulated data is linear at large sampling range as commonly assumed in previous studies, no matter the distribution of species abundance is log-normal or negative-binomial, which unmasks the global-local inconsistency in Species-Area Relationships fitting. Thus misestimates of total number of species or other derivation parameters can occur if the fitted relationship is extrapolated beyond the range of the small and intermediate sampling size

A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability

Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to support protoplasts in the form of uniform thin layer, which has three unique properties. • The tissue culture-treated membrane has a good affinity for the plant cell; thus, protoplasts can spread evenly and form a very thin layer. • There are more choices for membrane pore size, depending on the application. • It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol–gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays

Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads.

BackgroundAdapter trimming is a prerequisite step for analyzing next-generation sequencing (NGS) data when the reads are longer than the target DNA/RNA fragments. Although typically used in small RNA sequencing, adapter trimming is also used widely in other applications, such as genome DNA sequencing and transcriptome RNA/cDNA sequencing, where fragments shorter than a read are sometimes obtained because of the limitations of NGS protocols. For the newly emerged Nextera long mate-pair (LMP) protocol, junction adapters are located in the middle of all properly constructed fragments; hence, adapter trimming is essential to gain the correct paired reads. However, our investigations have shown that few adapter trimming tools meet both efficiency and accuracy requirements simultaneously. The performances of these tools can be even worse for paired-end and/or mate-pair sequencing.ResultsTo improve the efficiency of adapter trimming, we devised a novel algorithm, the bit-masked k-difference matching algorithm, which has O(kn) expected time with O(m) space, where k is the maximum number of differences allowed, n is the read length, and m is the adapter length. This algorithm makes it possible to fully enumerate all candidates that meet a specified threshold, e.g. error ratio, within a short period of time. To improve the accuracy of this algorithm, we designed a simple and easy-to-explain statistical scoring scheme to evaluate candidates in the pattern matching step. We also devised scoring schemes to fully exploit the paired-end/mate-pair information when it is applicable. All these features have been implemented in an industry-standard tool named Skewer (https://sourceforge.net/projects/skewer). Experiments on simulated data, real data of small RNA sequencing, paired-end RNA sequencing, and Nextera LMP sequencing showed that Skewer outperforms all other similar tools that have the same utility. Further, Skewer is considerably faster than other tools that have comparative accuracies; namely, one times faster for single-end sequencing, more than 12 times faster for paired-end sequencing, and 49% faster for LMP sequencing.ConclusionsSkewer achieved as yet unmatched accuracies for adapter trimming with low time bound

The effects of the E3 ubiquitin–protein ligase UBR7 of Frankliniella occidentalis on the ability of insects to acquire and transmit TSWV

The interactions between plant viruses and insect vectors are very complex. In recent years, RNA sequencing data have been used to elucidate critical genes of Tomato spotted wilt ortho-tospovirus (TSWV) and Frankliniella occidentalis (F. occidentalis). However, very little is known about the essential genes involved in thrips acquisition and transmission of TSWV. Based on transcriptome data of F. occidentalis infected with TSWV, we verified the complete sequence of the E3 ubiquitin-protein ligase UBR7 gene (UBR7), which is closely related to virus transmission. Additionally, we found that UBR7 belongs to the E3 ubiquitin–protein ligase family that is highly expressed in adulthood in F. occidentalis. UBR7 could interfere with virus replication and thus affect the transmission efficiency of F. occidentalis. With low URB7 expression, TSWV transmission efficiency decreased, while TSWV acquisition efficiency was unaffected. Moreover, the direct interaction between UBR7 and the nucleocapsid (N) protein of TSWV was investigated through surface plasmon resonance and GST pull-down. In conclusion, we found that UBR7 is a crucial protein for TSWV transmission by F. occidentalis, as it directly interacts with TSWV N. This study provides a new direction for developing green pesticides targeting E3 ubiquitin to control TSWV and F. occidentalis

Effects of quantum dots in polymerase chain reaction

The effects of quantum dots (QDs) on the elimination of nonspecific amplification of the polymerase chain reaction (PCR) were investigated. It was found that QDs could increase the specificity of the PCR at different annealing temperatures and with DNA templates of different lengths. The effects of QDs on the efficiency of the PCR were also studied, and the results snowed that there was no enhancement. The mechanisms underlying these effects are discussed. This method could be used to modify the amplification results of the conventional PCR. Furthermore, this technology could make the PCR more widely applicable, especially in the multi-PCR reaction system with different annealing temperatures. This is of great significance for gene diagnosis

Comparative Chloroplast Genomes of Sorghum Species: Sequence Divergence and Phylogenetic Relationships

Sorghum comprises 31 species that exhibit considerable morphological and ecological diversity. The phylogenetic relationships among Sorghum species still remain unresolved due to lower information on the traditional DNA markers, which provides a limited resolution for identifying Sorghum species. In this study, we sequenced the complete chloroplast genomes of Sorghum sudanense and S. propinquum and analyzed the published chloroplast genomes of S. bicolor and S. timorense to retrieve valuable chloroplast molecular resources for Sorghum. The chloroplast genomes ranged in length from 140,629 to 140,755 bp, and their gene contents, gene orders, and GC contents were similar to those for other Poaceae species but were slightly different in the number of SSRs. Comparative analyses among the four chloroplast genomes revealed 651 variable sites, 137 indels, and nine small inversions. Four highly divergent DNA regions (rps16-trnQ, trnG-trnM, rbcL-psaI, and rps15-ndhF), which were suitable for phylogenetic and species identification, were detected in the Sorghum chloroplast genomes. A phylogenetic analysis strongly supported that Sorghum is a monophyletic group in the tribe Andropogoneae. Overall, the genomic resources in this study could provide potential molecular markers for phylogeny and species identification in Sorghum

Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been widely applied in functional genomics research and plant breeding. In contrast to the off-target studies of mammalian cells, there is little evidence for the common occurrence of off-target sites in plants and a great need exists for accurate detection of editing sites. Here, we summarized the precision of CRISPR/Cas9-mediated mutations for 281 targets and found that there is a preference for single nucleotide deletions/insertions and longer deletions starting from 40 nt upstream or ending at 30 nt downstream of the cleavage site, which suggested the candidate sequences for editing sites detection by whole-genome sequencing (WGS). We analyzed the on-/off-target sites of 6 CRISPR/Cas9-mediated Arabidopsis plants by the optimized method. The results showed that the on-target editing frequency ranged from 38.1% to 100%, and one off target at a frequency of 9.8%–97.3% cannot be prevented by increasing the specificity or reducing the expression level of the Cas9 enzyme. These results indicated that designing guide RNA with high specificity may be the preferred factor to avoid the off-target events, and it is necessary to predict or detect off-target sites by WGS-based methods for preventing off targets caused by genome differences in different individuals