A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta

Background  

Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems.     

UCE: A uracil excision (USER™)-based toolbox for transformation of cereals

Background  

Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs. These problems are obviated by "The Uracil Specific Excision Reagent (USER™)" technology (New England Biolabs) which thus offers a new and very time-efficient method for engineering of big and complex plasmids.     

Isolation of a strong <Emphasis Type="Italic">Arabidopsis</Emphasis> guard cell promoter and its potential as a research tool

Background  

A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. An additional drawback of the 35S promoter is that ectopically expressing a gene throughout the organism could cause pleiotropic effects. To improve available methods for targeted gene expression in guard cells, we isolated strong guard cell promoter candidates based on new guard cell-specific microarray analyses of 23,000 genes that are made available together with this report.     

High-throughput <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated barley transformation

Background  

Plant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (<10%) transformation efficiencies.     

Isolation of intact sub-dermal secretory cavities from <Emphasis Type="Italic">Eucalyptus</Emphasis>

Background  

The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils.     

Genome-wide identification of microsatellites in white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) using FIASCO and phpSSRMiner

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.     

An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol

Background  

The Agrobacterium vacuum (Bechtold et al 1993) and floral-dip (Clough and Bent 1998) are very efficient methods for generating transgenic Arabidopsis plants. These methods allow plant transformation without the need for tissue culture. Large volumes of bacterial cultures grown in liquid media are necessary for both of these transformation methods. This limits the number of transformations that can be done at a given time due to the need for expensive large shakers and limited space on them. Additionally, the bacterial colonies derived from solid media necessary for starting these liquid cultures often fail to grow in such large volumes. Therefore the optimum stage of plant material for transformation is often missed and new plant material needs to be grown.     

A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

Background

In vivo detection of protein-bound genomic regions can be achieved by combining chromatin-immunoprecipitation with next-generation sequencing technology (ChIP-seq). The large amount of sequence data produced by this method needs to be analyzed in a statistically proper and computationally efficient manner. The generation of high copy numbers of DNA fragments as an artifact of the PCR step in ChIP-seq is an important source of bias of this methodology.

Results

We present here an R package for the statistical analysis of ChIP-seq experiments. Taking the average size of DNA fragments subjected to sequencing into account, the software calculates single-nucleotide read-enrichment values. After normalization, sample and control are compared using a test based on the ratio test or the Poisson distribution. Test statistic thresholds to control the false discovery rate are obtained through random permutations. Computational efficiency is achieved by implementing the most time-consuming functions in C++ and integrating these in the R package. An analysis of simulated and experimental ChIP-seq data is presented to demonstrate the robustness of our method against PCR-artefacts and its adequate control of the error rate.

Conclusions

The software ChIP-seq Analysis in R (CSAR) enables fast and accurate detection of protein-bound genomic regions through the analysis of ChIP-seq experiments. Compared to existing methods, we found that our package shows greater robustness against PCR-artefacts and better control of the error rate.     

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> and oat

Background  

Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.     

Combining metal oxide affinity chromatography (MOAC) and selective mass spectrometry for robust identification of <Emphasis Type="Italic">in vivo</Emphasis> protein phosphorylation sites

Background  

Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy.     

Rapid metabolic profiling of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> defence responses against <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> using direct infrared laser desorption ionization mass spectrometry and principal component analysis

Background  

Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection.     

High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

Background  

Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression.     

Rapid analysis of seed size in <Emphasis Type="Italic">Arabidopsis</Emphasis> for mutant and QTL discovery

Background  

Arabidopsis thaliana is a useful model organism for deciphering the genetic determinants of seed size; however the small size of its seeds makes measurements difficult. Bulk seed weights are often used as an indicator of average seed size, but details of individual seed is obscured. Analysis of seed images is possible but issues arise from variations in seed pigmentation and shadowing making analysis laborious. We therefore investigated the use of a consumer level scanner to facilitate seed size measurements in conjunction with open source image-processing software.     

A plastome primer set for comprehensive quantitative real time RT-PCR analysis of <Emphasis Type="Italic">Zea mays</Emphasis>: a starter primer set for other <Emphasis Type="Italic">Poaceae</Emphasis> species

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.     

Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0)

Background  

We previously developed a high-throughput system called 'Ice-Cap' for growing Arabidopsis seedlings in a 96-well format and rapidly collecting tissue for subsequent DNA extraction and genotyping. While the originally described Ice-Cap method is an effective tool for high-throughput genotyping, one shortcoming of the first version of Ice-Cap is that optimal seedling growth is highly dependent on specific environmental conditions. Here we describe several technical improvements to the Ice-Cap method that make it much more robust and provide a detailed protocol for implementing the method.     

A high-density Diversity Arrays Technology (DArT) microarray for genome-wide genotyping in <Emphasis Type="Italic">Eucalyptus</Emphasis>

Background  

A number of molecular marker technologies have allowed important advances in the understanding of the genetics and evolution of Eucalyptus, a genus that includes over 700 species, some of which are used worldwide in plantation forestry. Nevertheless, the average marker density achieved with current technologies remains at the level of a few hundred markers per population. Furthermore, the transferability of markers produced with most existing technology across species and pedigrees is usually very limited. High throughput, combined with wide genome coverage and high transferability are necessary to increase the resolution, speed and utility of molecular marker technology in eucalypts. We report the development of a high-density DArT genome profiling resource and demonstrate its potential for genome-wide diversity analysis and linkage mapping in several species of Eucalyptus.