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

Assessment and development of microarray-based DNA fingerprinting in Eucalyptus grandis and related species

DNA micro-array technology is a new and powerful technology that could substantially increase the speed of forest tree breeding programmes. This thesis represents a compilation of investigations that focus on the exploitation of DNA micro-array technology for genetic marker analysis of Eucalyptus trees. The major focus of the studies presented in this thesis was on the assessment and development of micro-array-based DNA fingerprinting in Eucalyptus. A DNA chip for Eucalyptus was not available at start of the study. As a result of this study a 384-prototype chip was developed to evaluate the potential of micro-arrays for fingerprinting closely related Eucalyptus clones, species and hybrids. These studies show that micro-arrays are an efficient DNA marker technology for genome-wide fingerprinting of complex organisms for which no sequence data exist. However, cross-hybridisation and the lack of dedicated software products remain a challenge. The 384-probe array developed in this study was subsequently employed for the detection of putative markers associated with tolerance to Chrysoporthe austroafricana in Eucalyptus grandis. Putative tolerance-associated markers were identified by bulk segregant analysis (BSA) and converted to cleaved amplified polymorphic sequence markers for further characterization in segregating Eucalyptus populations. BSA revealed a total of 109 scorable, polymorphic loci, of which nine appeared to be associated with tolerance or susceptibility. Two DArT markers were converted to cleaved amplified polymorphic sequence (CAPS) markers, which discriminate susceptible and tolerant individuals. These PCR markers can be used for the rapid screening for disease tolerance in Eucalyptus planting and breeding stock. The collection of studies included in this thesis demonstrated that DArT is an efficient DNA marker technology for genome-wide genotyping, particularly for application in less-studies plant genomes. Whole-genome profiling using DArT raises significant opportunities for tree breeding programmes and for future genome analysis of Eucalyptus.Thesis (PhD)--University of Pretoria, 2011.GeneticsUnrestricte