Trichomes as dangerous lollipops: Do lizards also use caterpillar body and frass odor to optimize their foraging?

When attacked by herbivores, plants produce toxic secondary metabolites that function as direct defenses, as well as indirect defenses that attract and reward predators of the offending herbivores. These indirect defenses include both nutritive rewards such as extra floral nectar, as well as informational rewards, such as the production and release of volatile compounds that betray the location of feeding herbivores to predators. Herbivory of Nicotiana attenuata by the tobacco hornworm (Manduca larvae) alters the volatile profiles of both the plant and larval headspace. Herbivory-elicited specific changes in the volatile profiles are detected by arthropod predators of Manduca larvae. The known predators that perceive volatile cues induced by Manduca herbivory of N. attenuata are insects that target Manduca at early developmental stages, when the larvae are still small; large, late-instar larvae may have outgrown these predation risks. However, here we offer evidence that branched chain aliphatic acids derived from the digestion of plant O-acyl sugars from trichomes may betray Manduca larvae to lizard predators during late developmental stages as well.     

Application of Genotyping-by-Sequencing on Semiconductor Sequencing Platforms: A Comparison of Genetic and Reference-Based Marker Ordering in Barley

The rapid development of next-generation sequencing platforms has enabled the use of sequencing for routine genotyping across a range of genetics studies and breeding applications. Genotyping-by-sequencing (GBS), a low-cost, reduced representation sequencing method, is becoming a common approach for whole-genome marker profiling in many species. With quickly developing sequencing technologies, adapting current GBS methodologies to new platforms will leverage these advancements for future studies. To test new semiconductor sequencing platforms for GBS, we genotyped a barley recombinant inbred line (RIL) population. Based on a previous GBS approach, we designed bar code and adapter sets for the Ion Torrent platforms. Four sets of 24-plex libraries were constructed consisting of 94 RILs and the two parents and sequenced on two Ion platforms. In parallel, a 96-plex library of the same RILs was sequenced on the Illumina HiSeq 2000. We applied two different computational pipelines to analyze sequencing data; the reference-independent TASSEL pipeline and a reference-based pipeline using SAMtools. Sequence contigs positioned on the integrated physical and genetic map were used for read mapping and variant calling. We found high agreement in genotype calls between the different platforms and high concordance between genetic and reference-based marker order. There was, however, paucity in the number of SNP that were jointly discovered by the different pipelines indicating a strong effect of alignment and filtering parameters on SNP discovery. We show the utility of the current barley genome assembly as a framework for developing very low-cost genetic maps, facilitating high resolution genetic mapping and negating the need for developing de novo genetic maps for future studies in barley. Through demonstration of GBS on semiconductor sequencing platforms, we conclude that the GBS approach is amenable to a range of platforms and can easily be modified as new sequencing technologies, analysis tools and genomic resources develop.     

Identification of the Chromosome Carrying the Factor for Resistance to Meloidogyne incognita in Tobacco

To identify the chromosome carrying the factor for resistance to Meloidogyne incognita in tobacco, crosses were made between resistant tobacco ''NC95'' as pollen parent and each of the 12 tobacco monosomics (A-L) representative of the Tomentosae half of the Nicotiana tabacum chromosome complement. Of the F₁ seedlings, 927 plants were grown for observation. From these, 223 plants were selected as possible monosomics on the basis of morphological characteristics. These plants were self-pollinated, and the resulting F₂ plants were inoculated with both M. incognita acrita and M. incognita incognita. Sixteen F₂ populations, derived from the haplo-G monosome, were completely resistant. All of the F₂ populations derived from the other 11 monosomic crosses segregated into a 3:1 (resistant:susceptible) ratio. These results indicate that the factor for resistance to M. incognita is located on the G chromosome of N. tabacum. This is the first report establishing the N. tabacum chromosome that carries the factor for root-knot resistance. The results are consistant with our earlier evidence that M. incognita resistance in tobacco is derived from N. tomentosa, a species in the section Tomentosae of the subgenus Tabacum, genus Nicotiana. The other 12 chromosomes of N. tabacum have affinities with N. sylvestris, section Alatae, subgenus Petunoides, genus Nicotiana.     

Evaluation of Nicotiana otophora as a Source of Resistance to Meloidogyne incognita Race 4 for Tobacco

No currently available tobacco cultivar possesses resistance to Meloidogyne incognita race 4, nor has any source of resistance been reported within Nicotiana tabacum. The purpose of this study was to evaluate N. otophora acc. La Quinta as a source of resistance to this pathogen. Plants of tobacco cvs. NC 95 and NC 2326, N. otophora La Quinta and N. repanda were inoculated with second-stage juveniles of M. incognita race 4. Gall indices and egg-mass ratings were assessed at 4 and 8 weeks after inoculation. The two N. tabacum cultivars were heavily galled and had numerous egg masses at both rating periods. Nicotiana repanda was only weakly resistant. The galls on this species were very small and present at a low to moderate level; however, egg-mass ratings approaching those of the tobacco cultivars were observed 8 weeks after inoculation. In contrast, low gall indices and egg-mass ratings were found for N. otophora La Quinta at both the 4- and 8-week rating periods. In addition, little variability was observed within this species for either disease rating. Therefore, it appears that the La Quinta accession of N. otophora is a very promising source of M. incognita race 4 resistance for transfer to N. tabacum.     

NGSpeciesID: DNA barcode and amplicon consensus generation from long‐read sequencing data

Third‐generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), have gained popularity over the last years. These platforms can generate millions of long‐read sequences. This is not only advantageous for genome sequencing projects, but also advantageous for amplicon‐based high‐throughput sequencing experiments, such as DNA barcoding. However, the relatively high error rates associated with these technologies still pose challenges for generating high‐quality consensus sequences. Here, we present NGSpeciesID, a program which can generate highly accurate consensus sequences from long‐read amplicon sequencing technologies, including ONT and PacBio. The tool includes clustering of the reads to help filter out contaminants or reads with high error rates and employs polishing strategies specific to the appropriate sequencing platform. We show that NGSpeciesID produces consensus sequences with improved usability by minimizing preprocessing and software installation and scalability by enabling rapid processing of hundreds to thousands of samples, while maintaining similar consensus accuracy as current pipelines.     

GmWRKY53, a water- and salt-inducible soybean gene for rapid dissection of regulatory elements in BY-2 cell culture

Drought is the major cause of crop losses worldwide. Water stress-inducible promoters are important for understanding the mechanisms of water stress responses in crop plants. Here we utilized tobacco (Nicotiana tabacum L.) Bright Yellow 2 (BY-2) cell system in presence of polyethylene glycol, salt and phytohormones. Extension of the system to 85 mM NaCl led to inducibility of up to 10-fold with the water stress and salt responsive soybean GmWRKY53 promoter. Upon ABA and JA treatment fold inducibility was up to 5-fold and 14-fold, respectively. Thus, we hypothesize that GmWRKY53 could be used as potential model candidate for dissecting drought regulatory elements as well as understanding crosstalk utilizing a rapid heterologous system of BY-2 culture.     

Strain engineering for microbial production of value-added chemicals and fuels from glycerol

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.     

Method for quick DNA barcode reference library construction

DNA barcoding has become one of the most important techniques in plant species identification. Successful application of this technology is dependent on the availability of reference database of high species coverage. Unfortunately, there are experimental and data processing challenges to construct such a library within a short time. Here, we present our solutions to these challenges. We sequenced six conventional DNA barcode fragments (ITS1, ITS2, matK1, matK2, rbcL1, and rbcL2) of 380 flowering plants on next‐generation sequencing (NGS) platforms (Illumina Hiseq 2500 and Ion Torrent S5) and the Sanger sequencing platform. After comparing the sequencing depths, read lengths, base qualities, and base accuracies, we conclude that Illumina Hiseq2500 PE250 run is suitable for conventional DNA barcoding. We developed a new “Cotu” method to create consensus sequences from NGS reads for longer output sequences and more reliable bases than the other three methods. Step‐by‐step instructions to our method are provided. By using high‐throughput machines (PCR and NGS), labeling PCR, and the Cotu method, it is possible to significantly reduce the cost and labor investments for DNA barcoding. A regional or even global DNA barcoding reference library with high species coverage is likely to be constructed in a few years.     

An assembly and alignment-free method of phylogeny reconstruction from next-generation sequencing data

Background

Next-generation sequencing technologies are rapidly generating whole-genome datasets for an increasing number of organisms. However, phylogenetic reconstruction of genomic data remains difficult because de novo assembly for non-model genomes and multi-genome alignment are challenging.

Results

To greatly simplify the analysis, we present an Assembly and Alignment-Free (AAF) method (https://sourceforge.net/projects/aaf-phylogeny) that constructs phylogenies directly from unassembled genome sequence data, bypassing both genome assembly and alignment. Using mathematical calculations, models of sequence evolution, and simulated sequencing of published genomes, we address both evolutionary and sampling issues caused by direct reconstruction, including homoplasy, sequencing errors, and incomplete sequencing coverage. From these results, we calculate the statistical properties of the pairwise distances between genomes, allowing us to optimize parameter selection and perform bootstrapping. As a test case with real data, we successfully reconstructed the phylogeny of 12 mammals using raw sequencing reads. We also applied AAF to 21 tropical tree genome datasets with low coverage to demonstrate its effectiveness on non-model organisms.

Conclusion

Our AAF method opens up phylogenomics for species without an appropriate reference genome or high sequence coverage, and rapidly creates a phylogenetic framework for further analysis of genome structure and diversity among non-model organisms.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1647-5) contains supplementary material, which is available to authorized users.     

复杂基因组测序技术研究进展

复杂基因组指的是无法使用常规测序和组装手段直接解析的一类基因组,通常指包含高比例重复序列、高杂合度、极端GC含量、存在难消除异源DNA污染的基因组。为了解决复杂基因组的测序和组装问题,需要分别从基因组测序实验方法、测序技术平台、组装算法与策略3个方面进行深入研究。本文详细介绍了复杂基因组测序组装相关的现有技术与方法,并结合复杂基因组经典实例介绍了复杂基因组测序的技术解决途径和发展历程,可为制订合适的复杂基因组测序策略提供参考。     

Development of Selected Tobacco Cyst Nematode Isolates on Resistant and Susceptible Cultivars of Flue-cured Tobacco

Tobacco cyst nematode (Globodera tabacum solanacearum) isolates were collected from 11 locations in Virginia, 3 in North Carolina, and 1 in Maryland. Isolates from each location were maintained and increased on flue-cured tobacco, Nicotiana tabacum cv K326. Plants of flue-cured tobacco cultivars K326 (susceptible) and NC567 (resistant) were each inoculated with 6,000 G. t. solanacearum eggs/plant. Tests were conducted over one (6 weeks) or two (14 weeks) generations of the nematode. Shoot and root weights and the number of nematodes present within a 1-g subsample of feeder roots were recorded at completion of the tests. Nematode counts were categorized by nematode life stage (vermiform, swollen, pyriform, and adult). Although significant differences in nematode development were detected among isolates, differences were not consistent across experiments. Results indicate similar virulence among G. t. solanacearum isolates on resistant and susceptible flue-cured tobacco cultivars. Therefore, plant breeders may effectively use a single G. t. solanacearum isolate when screening tobacco germplasm for resistance.     

Effects of Continuous Cropping of Resistant and Susceptible Cultivars on Reproduction Potentials of Heterodera glycines and Globodera tabacum solanacearum

The reproductive potentials of Heterodera glycines (mixture of races 3 and 4 and unidentified races) and a tobacco cyst nematode Globodera tabacum solanacearum were studied in the field. The experiments involved four cultivars of soybean Glycine max and four cultivars of Nicotiana tabacum. The reproductive potential of the H. glycines population was high on Essex and Lee 74 soybean but low on Forrest and Bedford over the 3 years (1982-84) of continuous cropping. The reproductive potential of H. glycines was 12% on Forrest and 6% on Bedford in 1982 but increased to 37 and 35% in 1983 and to 71 and 41% in 1984, respectively, on these two cultivars. The reproductive potential of G. tabacum solanacearum was high on McNair 944 and Coker 319 tobacco cultivars and low on VA 81 and PD 4 over the 3 years of cropping. The reproductive potential of G. tabacum solanacearum on VA 81 and PD 4 was 18 and 17% in 1982, 7 and 16% in 1983, and 5 and 5% in 1984, respectively. The changes in reproductive potentials of H. glycines and G. tabacum solanacearum may be related to inherent genetic variability in the systems that control reproduction of the two cyst nematodes and nature of resistance incorporated in the soybean and tobacco cultivars.     

Development of user-friendly markers for disease resistance to black root rot of tobacco through genotyping by sequencing

Black root rot (BRR), a disease caused by the hemibiotrophic fungus Thielaviopsis basicola, seriously compromises yield and leaf quality in tobacco (Nicotiana tabacum). Full resistance to black root rot, conferred by the resistance to BRR 1 (RBRR1) locus from Nicotiana debneyi Domin, was transferred to a burley tobacco cultivar through interspecific hybridization. Some undesirable traits potentially caused by linkage drag restrict wider application of RBRR1 in flue-cured tobacco. Therefore, user-friendly molecular markers are needed to assist selection for resistance to black root rot and to break the unfavorable linkage. Genotyping by sequencing (GBS) is a rapid and robust approach for reduced representation sequencing of multiplexed genomic DNA samples that combines genome-wide molecular marker discovery with genotyping. In the present study, we used GBS to identify single-nucleotide polymorphisms (SNPs) linked to the RBRR1 locus, and PCR-based assays for detection of these SNPs were also developed. Sequence analysis of the SNP markers suggested that RBRR1 is located on chromosome 17, providing a basis for map-based cloning of this valuable gene. Co-dominant CAPS markers that co-segregate with the disease-resistant phenotype offer user-friendly tools for tobacco breeding and variety improvement. Furthermore, tested with diverse N. tabacum germplasm, SS192650 displayed 100% selection accuracy for resistance to BRR, suggesting that this marker can be used in diverse tobacco populations.     

Web Resources for Metagenomics Studies

The development of next-generation sequencing(NGS) platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.     

Metabolomics of forage plants: a review

Background

Forage plant breeding is under increasing pressure to deliver new cultivars with improved yield, quality and persistence to the pastoral industry. New innovations in DNA sequencing technologies mean that quantitative trait loci analysis and marker-assisted selection approaches are becoming faster and cheaper, and are increasingly used in the breeding process with the aim to speed it up and improve its precision. High-throughput phenotyping is currently a major bottle neck and emerging technologies such as metabolomics are being developed to bridge the gap between genotype and phenotype; metabolomics studies on forages are reviewed in this article.

Scope

Major challenges for pasture production arise from the reduced availability of resources, mainly water, nitrogen and phosphorus, and metabolomics studies on metabolic responses to these abiotic stresses in Lolium perenne and Lotus species will be discussed here. Many forage plants can be associated with symbiotic microorganisms such as legumes with nitrogen fixing rhizobia, grasses and legumes with phosphorus-solubilizing arbuscular mycorrhizal fungi, and cool temperate grasses with fungal anti-herbivorous alkaloid-producing Neotyphodium endophytes and metabolomics studies have shown that these associations can significantly affect the metabolic composition of forage plants. The combination of genetics and metabolomics, also known as genetical metabolomics can be a powerful tool to identify genetic regions related to specific metabolites or metabolic profiles, but this approach has not been widely adopted for forages yet, and we argue here that more studies are needed to improve our chances of success in forage breeding.

Conclusions

Metabolomics combined with other ‘-omics’ technologies and genome sequencing can be invaluable tools for large-scale geno- and phenotyping of breeding populations, although the implementation of these approaches in forage breeding programmes still lags behind. The majority of studies using metabolomics approaches have been performed with model species or cereals and findings from these studies are not easily translated to forage species. To be most effective these approaches should be accompanied by whole-plant physiology and proof of concept (modelling) studies. Wider considerations of possible consequences of novel traits on the fitness of new cultivars and symbiotic associations need also to be taken into account.     

En route to a genome-based classification of Archaea and Bacteria?

Given the considerable promise whole-genome sequencing offers for phylogeny and classification, it is surprising that microbial systematics and genomics have not yet been reconciled. This might be due to the intrinsic difficulties in inferring reasonable phylogenies from genomic sequences, particularly in the light of the significant amount of lateral gene transfer in prokaryotic genomes. However, recent studies indicate that the species tree and the hierarchical classification based on it are still meaningful concepts, and that state-of-the-art phylogenetic inference methods are able to provide reliable estimates of the species tree to the benefit of taxonomy. Conversely, we suspect that the current lack of completely sequenced genomes for many of the major lineages of prokaryotes and for most type strains is a major obstacle in progress towards a genome-based classification of microorganisms. We conclude that phylogeny-driven microbial genome sequencing projects such as the Genomic Encyclopaedia of Archaea and Bacteria (GEBA) project are likely to rectify this situation.     

Phenotypic subunit composition of the tobacco (Nicotiana tabacum L.) vacuolar-type H-translocating ATPase

The model plant tobacco (Nicotiana tabacum L.) was chosen for a survey of the subunit composition of the V-ATPase at the protein level. V-ATPase was purified from tobacco leaf cell tonoplasts by solubilization with the nonionic detergent Triton X-100 and immunoprecipitation. In the purified fraction 12 proteins were present. By matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and amino acid sequencing 11 of these polypeptides could be identified as subunits A, B, C, D, F, G, c, d and three different isoforms of subunit E. The polypeptide which could not be identified by MALDI analysis might represent subunit H. The data presented here, for the first time, enable an unequivocal identification of V-ATPase subunits after gel electrophoresis and open the possibility to assign changes in polypeptide composition to variations in respective V-ATPase subunits occurring as a response to environmental conditions or during plant development.