Functional markers in wheat: technical and economic aspects

Speed and cost–effective techniques for evaluation of plant materials to provide information before entering the next cycle of selection are critical for success in plant breeding. Whether or not a ‘new’ technique realizes its potential depends on technical and economic considerations. Biotechnology-based research tools such as doubled haploid technology and molecular markers have already demonstrated their value for application in plant breeding. In the area of genomics, implementation of functional markers (FMs) is currently of particular interest. The pipeline from plant materials to FM data points for any application includes maceration of plant material, DNA isolation and sample preparation. For each step of this pipeline, a number of techniques are available, and no single method is ideally suited for all applications. The challenge is to meet the needs of many different scenarios which are present in a modern breeding programme such as the use of (1) few markers for genotyping hundreds of samples (e.g., marker assisted backcrossing (MAB)), (2) few markers in thousands of samples (e.g., screening for GMO), (3) hundreds to thousands of markers for hundreds to thousands of samples (e.g., genetic characterization of breeding materials (fingerprinting)). This paper compares different techniques for each of the steps from plant material to FM data point, with the main emphasis on SNP detection platforms, assuming that multiple FMs will become available in the near future. We focus on technical and economic aspects and discuss which techniques are most suitable for each of the scenarios using wheat as a model.     

Development of a comprehensive detection method for medicinal and toxic plant species

Pharmacologically active ingredients in plants can cause significant morbidity through their increasingly common use in herbal alternative medicines and dietary supplements. Monitoring consumer products for the presence of toxic plants is encumbered by the lack of rapid and specific assays. To create a sensitive, reliable, fast, and broad-spectrum assay for medicinal or toxic plant species, we tested multiplexed ligation-dependent probe amplification (MLPA), which requires partial genomic DNA sequences from species of plants that are not well represented in currently available genetic databases. Genomic DNA was obtained from 21 species of medicinal and/or toxic plants. The PCR products were amplified from these plants and cloned for sequencing. The MLPA method was successful with DNA samples from many different species. The use of a microarray to facilitate screening of potentially thousands of plants in a single assay also was successful. The combination of the specificity of the MLPA assay with the broad-scale capabilities of microarray technology should make this an especially useful tool in screening in foods and commercial herbal preparations to identify the plant compounds actually present. Other applications could potentially extend to the identification of any plant species in samples for academic botanical studies and for biodefense and forensics applications.     

Structural changes within trophic levels are constrained by within‐family assembly rules at lower trophic levels

Historical contingency broadly refers to the proposition that even random historical events can constrain the ecological and evolutionary pathways of organisms and that of entire communities. Focusing on communities, these pathways can be reflected into specific structural changes within and across trophic levels – how species interact with and affect each other – which has important consequences for species coexistence. Using the registry of the last 2000 years of plant introductions and their novel herbivores encountered in Central Europe, we find that the order of arrival of closely related (but not of distantly related) plant species constrained the structural changes within the trophic level formed by herbivore species across the observation period. Because it is difficult for field and lab experiments to be conducted over hundreds of years to record and replay the assembly history of a community, our study provides an alternative to understand how structural changes have occurred across extensive periods of time.     

Abbott Physicochemical Tiering (APT)--a unified approach to HTS triage

The selection of the highest quality chemical matter from high throughput screening (HTS) is the ultimate aim of any triage process. Typically there are many hundreds or thousands of hits capable of modulating a given biological target in HTS with a wide range of physicochemical properties that should be taken into consideration during triage. Given the multitude of physicochemical properties that define drug-like space, a system needs to be in place that allows for a rapid selection of chemical matter based on a prioritized range of these properties. With this goal in mind, we have developed a tool, coined Abbott Physicochemical Tiering (APT) that enables hit prioritization based on ranges of these important physicochemical properties. This tool is now used routinely at Abbott to help prioritize hits out of HTS during the triage process. Herein we describe how this tool was developed and validated using Abbott internal high throughput ADME data (HT-ADME).     

Advances in functional protein microarray technology

Numerous innovations in high-throughput protein production and microarray surface technologies have enabled the development of addressable formats for proteins ordered at high spatial density. Protein array implementations have largely focused on antibody arrays for high-throughput protein profiling. However, it is also possible to construct arrays of full-length, functional proteins from a library of expression clones. The advent of protein-based microarrays allows the global observation of biochemical activities on an unprecedented scale, where hundreds or thousands of proteins can be simultaneously screened for protein-protein, protein-nucleic acid, and small molecule interactions. This technology holds great potential for basic molecular biology research, disease marker identification, toxicological response profiling and pharmaceutical target screening.     

DNA microarrays: new tools in the analysis of plant defence responses

Large-scale DNA sequencing is providing information on the number and organization of genes and genomes of plant species and their pathogens. The next phase is to identify gene functions and gene networks with key roles in compatible and incompatible plant–pathogen interactions. DNA microarrays can provide information on the expression patterns of thousands of genes in parallel. The application of this technology is already revealing new features of plant–pathogen interactions and will be a key tool for a wide range of experiments in molecular plant pathology.     

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.     

植物园发展战略研究

本文包括 (1)正确认识世界植物园发展的经验和 (2 )我国植物园发展的战略。阐述世界植物园发展的源、流、兴、衰和各国植物园之间因时而异的主、辅、唱、和关系。提出我国植物园新世纪战略目标和综合发展战略 ,认为万种药用植物的迁地保护和栽培化 ,以及城市植物环境的构建可以作为研究的重点     

Identification of characteristic plant co-occurrences in neotropical secondary montane forests

Aims Inferring environmental conditions from characteristic patterns of plant co-occurrences can be crucial for the development of conservation strategies concerning secondary neotropical forests. However, no methodological agreement has been achieved so far regarding the identification and classification of characteristic groups of vascular plant species in the tropics. This study examines botanical and, in particular, statistical aspects to be considered in such analyses. Based on these, we propose a novel data-driven approach for the identification of characteristic plant co-occurrences in neotropical secondary mountain forests.Methods Floristic inventory data were gathered in secondary tropical mountain forests in Ecuador. Vegetation classification was performed by coupling locally adaptive isometric feature mapping, a non-linear ordination method and fuzzy- c -means clustering. This approach was designed for dealing with underlying non-linearities and uncertainties in the inventory data.Important findings The results indicate that the applied non-linear mapping in combination with fuzzy classification of species occurrence allows an effective identification of characteristic groups of co-occurring species as fuzzy-defined clusters. The selected species indicated groups representing characteristic life-form distributions, as they correspond to various stages of forest regeneration. Combining the identified 'characteristic species groups' with meta-information derived from accompanying studies indicated that the clusters can also be related to habitat conditions. In conclusion, we identified species groups either characteristic of different stages of forest succession after clear-cutting or of impact by fire or a landslide. We expect that the proposed data-mining method will be useful for vegetation classification where no a priori knowledge is available.     

Legume diversity as indicator for botanical diversity on Sundaland,South East Asia

The Global Legume Diversity Assessment (GLDA) proposes the legume family (Fabaceae or Leguminosae) – one of the largest and economically important plant families – as a target for a global botanical diversity assessment project. Where in the Neotropics and Africa legumes dominate the rain forest in terms of diversity and abundance, the Dipterocarpaceae claim this role in South East Asia and on Sundaland in particular. This raises the question whether legumes are an indicator for overall botanical diversity on Sundaland? To answer this question we use the largest compiled database of collection records of the region and species distribution modelling techniques. As a proxy for total botanical diversity we selected seven plant families; Dipterocarpaceae, Ericaceae, Fagaceae, Lauraceae, Moraceae, Myristicaceae, and Sapindaceae. Although the legumes were the most diverse family, the predictive power of legume diversity for overall botanical diversity was poor. This related to the fact that the other seven selected families largely represent trees, whereas legume species more equally represent all different growth forms. After assigning individual legume species to different growth habits (tree, liana, herb, miscellaneous) we were able to predict 78% of the variance in botanical diversity on Sundaland. The lianas represent the single growth habit that best predicted (66%) the variance in botanical diversity. The herb- and miscellaneous growth habits had an inverse relationship to botanical diversity. Legumes can be used as a predictor of overall botanical diversity in tropical and seasonal rain forests, but the relationship should be fitted for different biogeographic regions individually.     

A template for the future: digitizing and databasing a taxonomic illustration collection

This is the first iconographic taxonomic database intended as an everyday tool for the practising taxonomist, that allows access to the original drawings used to describe species. We demonstrate the system with water mites (Acari, Parasitengona, Hydrachnidia) but it may be used with any other plant or animal group. Query by genus or species leads to the original drawings and any additional images associated to that particular name. At present, the database includes images from more than 4,000 species (around 38,000 illustrations) and is easily expandable. Copyright does not allow the database to be used in an open WWW context, but it can be an extremely useful tool in private taxonomic applications.     

Spatial patterns of plant association in grazed and ungrazed shrublands in the semi‐arid Karoo,South Africa

Abstract. The investigation of vegetation pattern and plant association by spatial statistics has become increasingly popular among plant ecologists. Recently, Individual‐centered analysis (ICA) has been introduced as a new tool for analysis of multi‐species co‐occurrence patterns. We tested this new technique by applying it to spatial data from grazed and ungrazed shrub communities in the semi‐arid Great Karoo, South Africa. There were substantial but complex and scale‐dependent differences in pattern between grazed and ungrazed vegetation. Unpalatable species that increase in abundance in grazed vegetation possibly play a key role in the change of vegetation pattern. At small scales we found indications of aggregation (< 30 cm) at the ungrazed, but of repulsion (30 – 40 cm) at the grazed site. An additional non‐random pattern at 60 – 170 cm at the grazed site was probably due to the clumped distributions of some species on broader scales. We show that the interpretability of ICA results is improved when the actual observed and expected frequencies of species combinations are added to the program output. The main strength of ICA is that it has the potential to detect association patterns that involve more than two species.     

DNA barcoding Bromeliaceae: achievements and pitfalls

Background

DNA barcoding has been successfully established in animals as a tool for organismal identification and taxonomic clarification. Slower nucleotide substitution rates in plant genomes have made the selection of a DNA barcode for land plants a much more difficult task. The Plant Working Group of the Consortium for the Barcode of Life (CBOL) recommended the two-marker combination rbcL/matK as a pragmatic solution to a complex trade-off between universality, sequence quality, discrimination, and cost.

Methodology/Principal Findings

It is expected that a system based on any one, or a small number of plastid genes will fail within certain taxonomic groups with low amounts of plastid variation, while performing well in others. We tested the effectiveness of the proposed CBOL Plant Working Group barcoding markers for land plants in identifying 46 bromeliad species, a group rich in endemic species from the endangered Brazilian Atlantic Rainforest. Although we obtained high quality sequences with the suggested primers, species discrimination in our data set was only 43.48%. Addition of a third marker, trnH–psbA, did not show significant improvement. This species identification failure in Bromeliaceaecould also be seen in the analysis of the GenBank''s matK data set. Bromeliaceae''s sequence divergence was almost three times lower than the observed for Asteraceae and Orchidaceae. This low variation rate also resulted in poorly resolved tree topologies. Among the three Bromeliaceae subfamilies sampled, Tillandsioideae was the only one recovered as a monophyletic group with high bootstrap value (98.6%). Species paraphyly was a common feature in our sampling.

Conclusions/Significance

Our results show that although DNA barcoding is an important tool for biodiversity assessment, it tends to fail in taxonomy complicated and recently diverged plant groups, such as Bromeliaceae. Additional research might be needed to develop markers capable to discriminate species in these complex botanical groups.     

Image-based chemical screening

Technological advances have made it feasible to conduct high-throughput small-molecule screens based on visual phenotypes of individual cells, using automated imaging and analysis. These screens are rapidly moving from being small, proof-of-principle tests to robust and widespread screens of hundreds of thousands of compounds. Automated imaging screens maximize the information obtained in an initial screen and improve the ability to select high-quality leads. In this Perspective, I highlight the key steps necessary for conducting a high-throughput image-based chemical compound screen.     

BASECO: a floristic and ecological database of Mediterranean French flora

The French Mediterranean zone is one of the richest of the country, with 3200 species and many endemics. Because of its interest as a synthetic tool to store and manage data, an ecological Mediterranean flora database was created. Built around five tables, BASECO allows several queries about the botanical and ecological characteristics of about 1800 plants. The database was implemented in Access, which is a relational database management system. Each species is identified by a code and is characterised by several qualitative traits relating to morphology, reproduction, life forms and biogeographical distribution, including several modalities. Each trait is informed from one or two pre-defined reference botanical handbooks as much as possible. There are many different possible uses of this database, even at a huge scale, allowing to reveal patterns hard to detect with the taxonomic approach alone.     

Rapid plant identification using species- and group-specific primers targeting chloroplast DNA

Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory.     

Plant DNA barcoding: from gene to genome

DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species. Because single‐locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole‐chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies. While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource‐effective and does not yet offer the speed of analysis provided by single‐locus barcodes to unspecialized laboratory facilities. Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super‐barcode. We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single‐locus barcodes and super‐barcodes for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels.     

DNA barcoding in Atlantic Forest plants: What is the best marker for Sapotaceae species identification?

The Atlantic Forest is a phytogeographic domain with a high rate of endemism and large species diversity. The Sapotaceae is a botanical family for which species identification in the Atlantic Forest is difficult. An approach that facilitates species identification in the Sapotaceae is urgently needed because this family includes threatened species and valuable timber species. In this context, DNA barcoding could provide an important tool for identifying species in the Atlantic Forest. In this work, we evaluated four plant barcode markers (matK, rbcL, trnH-psbA and the nuclear ribosomal internal transcribed spacer region - ITS) in 80 samples from 26 species of Sapotaceae that occur in the Atlantic Forest. ITS yielded the highest average interspecific distance (0.122), followed by trnH-psbA (0.019), matK (0.008) and rbcL (0.002). For species discrimination, ITS provided the best results, followed by matK, trnH-psbA and rbcL. Furthermore, the combined analysis of two, three or four markers did not result in higher rates of discrimination than obtained with ITS alone. These results indicate that the ITS region is the best option for molecular identification of Sapotaceae species from the Atlantic Forest.