This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ http://www.plantmethods.com The latest articles from Plant Methods (ISSN 1746-4811) published by BioMed Central Background: High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers. Results: We have developed a high-throughput DNA extraction protocol for conifers using an automated liquid handler and modifying the Qiagen MagAttract Plant Kit protocol. The modifications involve change to the buffer system and improving the protocol so that it almost doubles the number of samples processed per kit, which significantly reduces the overall costs. We describe two versions of the protocol: one for medium-throughput (MTP) and another for high-throughput (HTP) DNA isolation. The HTP version works from start to end in the industry-standard 96-well format, while the MTP version provides higher DNA yields per sample processed. We have successfully used the protocol for DNA extraction and genotyping of thousands of individuals of several spruce and a pine species. Conclusion: A high-throughput system for DNA extraction from conifer needles and seeds has been developed and validated. The quality of the isolated DNA was comparable with that obtained from two commonly used methods: the silica-spin column and the classic CTAB protocol. Our protocol provides a fully automatable and cost effective solution for processing large numbers of conifer samples. http://www.plantmethods.com/content/4/1/20 Stanislav Bashalkhanov and Om P Rajora Plant Methods 2008, 4:20 2008-08-01 doi:10.1186/1746-4811-4-20 Plant Methods 1746-4811 4 20 2008-08-01 Background: Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover. Results: Genomic libraries containing simple sequence repeat (SSR) sequences were constructed using a modified Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) procedure and phpSSRMiner was used to develop the microsatellite markers. SSR motifs were isolated using two biotin-labeled probes, (CA)17 and (ATG)12. The sequences of 6,816 clones were assembled into 1,698 contigs, 32% of which represented novel sequences based on BLASTN searches. Approximately 32%, 28%, and 16% of these SSRs contained hexa-, tri-, and di-nucleotide repeats, respectively. The most frequent motifs were the CA and ATG complementary repeats and the associated compound sequences. Primer pairs were designed for 859 SSR loci based on sequences from these genomic libraries and from GenBank white clover nucleotide sequences. A total of 191 SSR primers developed from the two libraries were tested for polymorphism in individual clones from the parental genotypes GA43 ('Durana'), 'SRVR' and six F1 progeny from a mapping population. Ninety two percent produced amplicons and 66% of these were polymorphic. Conclusion: The combined approach of identifying SSR-enriched fragments by FIASCO coupled with the primer design and in silico amplification using phpSSRMiner represents an efficient and low cost pipeline for the large-scale development of microsatellite markers in plants.The approach described here could be readily adapted and utilized in other non-related species with none or limited genomic resources. http://www.plantmethods.com/content/4/1/19 Yan Zhang, Ji He, Patrick X Zhao, Joseph H Bouton and Maria J Monteros Plant Methods 2008, 4:19 2008-07-16 doi:10.1186/1746-4811-4-19 Plant Methods 1746-4811 4 19 2008-07-16 Background: Medicago truncatula is a model legume species that is currently the focus of an international genome sequencing effort. Although several different oligonucleotide and cDNA arrays have been produced for genome-wide transcript analysis of this species, intrinsic limitations in the sensitivity of hybridization-based technologies mean that transcripts of genes expressed at low-levels cannot be measured accurately with these tools. Amongst such genes are many encoding transcription factors (TFs), which are arguably the most important class of regulatory proteins. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive method currently available for transcript quantification, and one that can be scaled up to analyze transcripts of thousands of genes in parallel. Thus, qRT-PCR is an ideal method to tackle the problem of TF transcript quantification in Medicago and other plants. Results: We established a bioinformatics pipeline to identify putative TF genes in Medicago truncatula and to design gene-specific oligonucleotide primers for qRT-PCR analysis of TF transcripts. We validated the efficacy and gene-specificity of over 1000 TF primer pairs and utilized these to identify sets of organ-enhanced TF genes that may play important roles in organ development or differentiation in this species. This community resource will be developed further as more genome sequence becomes available, with the ultimate goal of producing validated, gene-specific primers for all Medicago TF genes. Conclusion: High-throughput qRT-PCR using a 384-well plate format enables rapid, flexible, and sensitive quantification of all predicted Medicago transcription factor mRNAs. This resource has been utilized recently by several groups in Europe, Australia, and the USA, and we expect that it will become the 'gold-standard' for TF transcript profiling in Medicago truncatula. http://www.plantmethods.com/content/4/1/18 Klementina Kakar, Maren Wandrey, Tomasz Czechowski, Tanja Gaertner, Wolf-Rüdiger Scheible, Mark Stitt, Ivone Torres-Jerez, Yongli Xiao, Julia C Redman, Hank C Wu, Foo Cheung, Christopher D Town and Michael K Udvardi Plant Methods 2008, 4:18 2008-07-08 doi:10.1186/1746-4811-4-18 Plant Methods 1746-4811 4 18 2008-07-08 Background: Transcription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome. This co-ordinate regulation is achieved, in part, through the interaction of transcription factors with conserved cis-regulatory motifs that are in close proximity to the target genes. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs.In Arabidopsis, over-expression of the MYB transcription factor PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) leads to transgenic plants with elevated anthocyanin levels due to the co-ordinated up-regulation of genes in the anthocyanin biosynthetic pathway. In addition to the anthocyanin biosynthetic genes, there are a number of un-associated genes that also change in expression level. This may be a direct or indirect consequence of the over-expression of PAP1. Results: Oligo array analysis of PAP1 over-expression Arabidopsis plants identified genes co-ordinately up-regulated in response to the elevated expression of this transcription factor. Transient assays on the promoter regions of 33 of these up-regulated genes identified eight promoter fragments that were transactivated by PAP1. Bioinformatic analysis on these promoters revealed a common cis-regulatory motif that we showed is required for PAP1 dependent transactivation. Conclusion: Co-ordinated gene regulation by individual transcription factors is a complex collection of both direct and indirect effects. Transient transactivation assays provide a rapid method to identify direct target genes from indirect target genes. Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes. While this type of analysis does not prove a direct interaction between protein and DNA, it does provide a tool to characterise cis-regulatory sequences that are necessary for transcription activation in a complex list of co-ordinately regulated genes. http://www.plantmethods.com/content/4/1/17 Andrew P Dare, Robert J Schaffer, Kui Lin-Wang, Andrew C Allan and Roger P Hellens Plant Methods 2008, 4:17 2008-07-07 doi:10.1186/1746-4811-4-17 Plant Methods 1746-4811 4 17 2008-07-07 We describe an efficient method for the rapid quantitative determination of the abundance of three acidic plant hormones from a single crude extract directly by LC/MS/MS. The method exploits the sensitivity of MS and uses multiple reaction monitoring and isotopically labelled samples to quantify the phytohormones abscisic acid, jasmonic acid and salicylic acid in Arabidopsis leaf tissue. http://www.plantmethods.com/content/4/1/16 Silvia Forcat, Mark H Bennett, John W Mansfield and Murray R Grant Plant Methods 2008, 4:16 2008-06-30 doi:10.1186/1746-4811-4-16 Plant Methods 1746-4811 4 16 2008-06-30 Background: Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results: Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP) fluorescence was possible. Conclusion: The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy. http://www.plantmethods.com/content/4/1/15 Elisabeth Truernit and Jim Haseloff Plant Methods 2008, 4:15 2008-06-23 doi:10.1186/1746-4811-4-15 Plant Methods 1746-4811 4 15 2008-06-23 Background: Quantitative Real Time RT-PCR (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RT)PCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species. Results: Primers were designed and successfully optimized for 57 of the 104 reported genes in the maize plastome plus two nuclear genes. All 59 primer pairs produced single amplicons after end-point reverse transcriptase polymerase chain reactions (RT-PCR) as visualized on agarose gels and subsequently verified by q2(RT)PCR. Primer pairs were divided into several categories based on the optimization requirements or the uniqueness of the target gene. An in silico test suggested the majority of the primer sets should work with other members of the Poaceae family. An in vitro test of the primer set on two unsequenced species (Panicum virgatum and Miscanthus sinensis) supported this assumption by successfully producing single amplicons for each primer pair. Conclusion: Due to the highly conserved chloroplast genome in plant families it is possible to utilize primer pairs designed against one genomic sequence to detect the presence and abundance of plastid genes or transcripts from genomes that have yet to be sequenced. Analysis of steady state transcription of vital system genes is a necessary requirement to comprehensively elucidate gene expression in any organism. The primer pairs reported in this paper were designed for q2(RT)PCR of maize chloroplast genes but should be useful for other members of the Poaceae family. Both in silico and in vitro data are presented to support this assumption. http://www.plantmethods.com/content/4/1/14 Richard M Sharpe, Sade N Dunn and A Bruce Cahoon Plant Methods 2008, 4:14 2008-06-02 doi:10.1186/1746-4811-4-14 Plant Methods 1746-4811 4 14 2008-06-02 Background: We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features. Results: We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to Magnaporthe grisea, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype. Conclusion: We anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals. http://www.plantmethods.com/content/4/1/13 Jeremy D Edwards, Jaroslav Janda, Megan T Sweeney, Ambika B Gaikwad, Bin Liu, Hei Leung and David W Galbraith Plant Methods 2008, 4:13 2008-05-29 doi:10.1186/1746-4811-4-13 Plant Methods 1746-4811 4 13 2008-05-29 Background: Freezing tolerance is an important factor in the geographical distribution of plants and strongly influences crop yield. Many plants increase their freezing tolerance during exposure to low, nonfreezing temperatures in a process termed cold acclimation. There is considerable natural variation in the cold acclimation capacity of Arabidopsis that has been used to study the molecular basis of this trait. Accurate methods for the quantitation of freezing damage in leaves that include spatial information about the distribution of damage and the possibility to screen large populations of plants are necessary, but currently not available. In addition, currently used standard methods such as electrolyte leakage assays are very laborious and therefore not easily applicable for large-scale screening purposes. Results: We have performed freezing experiments with the Arabidopsis accessions C24 and Tenela, which differ strongly in their freezing tolerance, both before and after cold acclimation. Freezing tolerance of detached leaves was investigated using the well established electrolyte leakage assay as a reference. Chlorophyll fluorescence imaging was used as an alternative method that provides spatial resolution of freezing damage over the leaf area. With both methods, LT50 values (i.e. temperature where 50% damage occurred) could be derived as quantitative measures of leaf freezing tolerance. Both methods revealed the expected differences between acclimated and nonacclimated plants and between the two accessions and LT50 values were tightly correlated. However, electrolyte leakage assays consistently yielded higher LT50 values than chlorophyll fluorescence imaging. This was to a large part due to the incubation of leaves for electrolyte leakage measurements in distilled water, which apparently led to secondary damage, while this pre-incubation was not necessary for the chlorophyll fluorescence measurements. Conclusion: Chlorophyll fluorescence imaging is an alternative method to accurately determine the freezing tolerance of leaves. It is quick and inexpensive and the system could potentially be used for large scale screening, allowing new approaches to elucidate the molecular basis of plant freezing tolerance. http://www.plantmethods.com/content/4/1/12 Britta Ehlert and Dirk K Hincha Plant Methods 2008, 4:12 2008-05-27 doi:10.1186/1746-4811-4-12 Plant Methods 1746-4811 4 12 2008-05-27 Background: For omics experiments, detailed characterisation of experimental material with respect to its genetic features, its cultivation history and its treatment history is a requirement for analyses by bioinformatics tools and for publication needs. Furthermore, meta-analysis of several experiments in systems biology based approaches make it necessary to store this information in a standardised manner, preferentially in relational databases. In the Golm Plant Database System, we devised a data management system based on a classical Laboratory Information Management System combined with web-based user interfaces for data entry and retrieval to collect this information in an academic environment. Results: The database system contains modules representing the genetic features of the germplasm, the experimental conditions and the sampling details. In the germplasm module, genetically identical lines of biological material are generated by defined workflows, starting with the import workflow, followed by further workflows like genetic modification (transformation), vegetative or sexual reproduction. The latter workflows link lines and thus create pedigrees. For experiments, plant objects are generated from plant lines and united in so-called cultures, to which the cultivation conditions are linked. Materials and methods for each cultivation step are stored in a separate ACCESS database of the plant cultivation unit. For all cultures and thus every plant object, each cultivation site and the culture's arrival time at a site are logged by a barcode-scanner based system. Thus, for each plant object, all site-related parameters, e.g. automatically logged climate data, are available. These life history data and genetic information for the plant objects are linked to analytical results by the sampling module, which links sample components to plant object identifiers. This workflow uses controlled vocabulary for organs and treatments. Unique names generated by the system and barcode labels facilitate identification and management of the material. Web pages are provided as user interfaces to facilitate maintaining the system in an environment with many desktop computers and a rapidly changing user community. Web based search tools are the basis for joint use of the material by all researchers of the institute. Conclusion: The Golm Plant Database system, which is based on a relational database, collects the genetic and environmental information on plant material during its production or experimental use at the Max-Planck-Institute of Molecular Plant Physiology. It thus provides information according to the MIAME standard for the component 'Sample' in a highly standardised format. The Plant Database system thus facilitates collaborative work and allows efficient queries in data analysis for systems biology research. http://www.plantmethods.com/content/4/1/11 Karin I Köhl, Georg Basler, Alexander Lüdemann, Joachim Selbig and Dirk Walther Plant Methods 2008, 4:11 2008-05-21 doi:10.1186/1746-4811-4-11 Plant Methods 1746-4811 4 11 2008-05-21