Ripening-related gene expression during fruit ripening in Vitis vinifera L. cv. Cabernet Sauvignon and Clairette blanche

The gene expression patterns in ripening fruit of a high quality wine cultivar (Cabernet Sauvignon) and a poor quality wine cultivar (Clairette blanche) were studied using cDNA-AFLP fingerprinting. Total RNA from "immature" (14-weeks post flowering) and "mature" (18-weeks post flowering) berries were used to study ripening-related gene expression in post-véraison stages of berry development. A total of 1,276 fragments were analysed, of which 175 appeared to be ripening-related. Average pairwise differences of the fragments amplified from "immature" and "mature" Cabernet Sauvignon and Clairette blanche berries, revealed a high level of similarity between the two cultivars. 70 % of the ripening-related fragments were cultivar-specific. The number of cultivar-specific and/or ripening-related fragments amplified, depended on the selective nucleotides of the primers used in the cDNA-AFLP analysis. Reverse slot blot and northern blot analysis confirmed that the expression of the identified genes were ripening-related.

Large-scale Gene Ontology analysis of plant transcriptome-derived sequences retrieved by AFLP technology

<p>Abstract</p> <p>Background</p> <p>After 10-year-use of AFLP (Amplified Fragment Length Polymorphism) technology for DNA fingerprinting and mRNA profiling, large repertories of genome- and transcriptome-derived sequences are available in public databases for model, crop and tree species. AFLP marker systems have been and are being extensively exploited for genome scanning and gene mapping, as well as cDNA-AFLP for transcriptome profiling and differentially expressed gene cloning. The evaluation, annotation and classification of genomic markers and expressed transcripts would be of great utility for both functional genomics and systems biology research in plants. This may be achieved by means of the Gene Ontology (GO), consisting in three structured vocabularies (<it>i</it>.<it>e</it>. ontologies) describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner. In this paper, the functional annotation of about 8,000 AFLP-derived ESTs retrieved in the NCBI databases was carried out by using GO terminology.</p> <p>Results</p> <p>Descriptive statistics on the type, size and nature of gene sequences obtained by means of AFLP technology were calculated. The gene products associated with mRNA transcripts were then classified according to the three main GO vocabularies. A comparison of the functional content of cDNA-AFLP records was also performed by splitting the sequence dataset into monocots and dicots and by comparing them to all annotated ESTs of Arabidopsis and rice, respectively. On the whole, the statistical parameters adopted for the <it>in silico </it>AFLP-derived transcriptome-anchored sequence analysis proved to be critical for obtaining reliable GO results. Such an exhaustive annotation may offer a suitable platform for functional genomics, particularly useful in non-model species.</p> <p>Conclusion</p> <p>Reliable GO annotations of AFLP-derived sequences can be gathered through the optimization of the experimental steps and the statistical parameters adopted. The Blast2GO software was shown to represent a comprehensive bioinformatics solution for an annotation-based functional analysis. According to the whole set of GO annotations, the AFLP technology generates thorough information for angiosperm gene products and shares common features across angiosperm species and families. The utility of this technology for structural and functional genomics in plants can be implemented by serial annotation analyses of genome-anchored fragments and organ/tissue-specific repertories of transcriptome-derived fragments.</p

Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles

BACKGROUND: Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR) and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far. RESULTS: We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i) cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii) intensity of bands is determined by densitometry, (iii) densitometric values are normalized, and (iv) intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment) for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans. CONCLUSION: We developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in reseach on organisms for which microarray analysis is not available or economical

Development of a simple and powerful method, cDNA AFLP-SSPAG, for cloning of differentially expressed genes

A novel plant differentially expressed gene cloning technique called cDNA-AFLP-SSPAG was descrybed. Using this method, Differential mRNA expression was found between high and low heterosis groups of maize. Differential cDNAs were easily obtained from silver stained cDNA-AFLP separated on polyacylamide gels. The cDNA was then reamplified, cloned and fragments were sequenced. Sequenced clones were used as probes in northern dot blot analyses and library screening. Full-length cDNA was cloned from a library suggesting that the cDNA-AFLP technique combined with silverstaining polyacrylamide gels provide a novel and very powerful gene cloning method

Recent developments in primer design for DNA polymorphism and mRNA profiling in higher plants

Primer design is a critical step in the application of PCR-based technologies in gene expression and genetic diversity analysis. As more plant genomes have been sequenced in recent years, the emphasis of primer design strategy has shifted to genome-wide and high-throughput direction. This paper summarizes recent advances in primer design for profiling of DNA polymorphism and mRNA in higher plants, as well as new primer systems developed for animals that can be adapted for plants

An overview of molecular marker methods for plants

The development and use of molecular markers for the detection and exploitation of DNA polymorphism is one of the most significant developments in the field of molecular genetics. The presence of various types of molecular markers, and differences in their principles, methodologies, and applications require careful consideration in choosing one or more of such methods. No molecular markers are available yet that fulfill all requirements needed by researchers. According to the kind of study to be undertaken, one can choose among the variety of molecular techniques, each of which combines at least some desirable properties. This article provides detail review for 11 different molecular marker methods: restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequencerepeats (ISSRs), sequence characterized regions (SCARs), sequence tag sites (STSs), cleaved amplified polymorphic sequences (CAPS), microsatellites or simple sequence repeats (SSRs), expressedsequence tags (ESTs), single nucleotide polymorphisms (SNPs), and diversity arrays technology (DArT)

A microarray-based method for the parallel analysis of genotypes and expression profiles of wood-forming tissues in Eucalyptus grandis

<p>Abstract</p> <p>Background</p> <p>Fast-growing <it>Eucalyptus grandis </it>trees are one of the most efficient producers of wood in South Africa. The most serious problem affecting the quality and yield of solid wood products is the occurrence of end splitting in logs. Selection of <it>E. grandis </it>planting stock that exhibit preferred wood qualities is thus a priority of the South African forestry industry. We used microarray-based DNA-amplified fragment length polymorphism (AFLP) analysis in combination with expression profiling to develop fingerprints and profile gene expression of wood-forming tissue of seven different <it>E. grandis </it>trees.</p> <p>Results</p> <p>A 1578-probe cDNA microarray was constructed by arraying 768 cDNA-AFLP clones and 810 cDNA library clones from seven individual <it>E. grandis </it>trees onto silanised slides. The results revealed that 32% of the spotted fragments showed distinct expression patterns (with a fold change of at least 1.4 or -1.4 and a p value of 0.01) could be grouped into clusters representing co-expressed genes. Evaluation of the binary distribution of cDNA-AFLP fragments on the array showed that the individual genotypes could be discriminated.</p> <p>Conclusion</p> <p>A simple, yet general method was developed for genotyping and expression profiling of wood-forming tissue of <it>E. grandis </it>trees differing in their splitting characteristics and in their lignin contents. Evaluation of gene expression profiles and the binary distribution of cDNA-AFLP fragments on the chip suggest that the prototype chip developed could be useful for transcript profiling and for the identification of Eucalyptus trees with preferred wood quality traits in commercial breeding programmes.</p

Rapid Identification Of Aspergillus Spp. Using A Pcr Based Melting Curve Method And Characterization Of A Novel Chitinase In Insect Resistant Maize Lines

Identification of fungal isolates is critical in studying Aspergillus flavus ecology and for developing methods to reduce aflatoxin contamination. In our efforts to track biocontrol applications of the atoxigenic A. flavus K49 (NRRL 30797), we have developed a rapid and accurate classification system for A. flavus based on PCR product melting temperatures (Tm). Using 18 primers and a total of 59 Aspergilli strains, including all 49 representatives of the Georgian peanut Vegetative Compatibility Groups (VCGs), a decision tree Tm flowchart was generated. The decision tree can classify all 59 strains using only 9 of the SSR primers and an average of 3.4 primers for each definitive classification. To confirm the effectiveness of the decision tree for strain identification, unknown samples isolated from experimental fields inoculated with various A. flavus strains in Stoneville, MS were analyzed. Ninety-six percent of the samples could be placed into a VCG using Tm(s) coupled with the decision tree. This dynamic system is an excellent tool for researchers studying biodiversity of A. flavus