Genetic Variation in the Wood Chemistry of Eucalyptus globulus Fiona S. Poke BSc (Hons) Submitted in fulfilment of the requiremenls for the Degree of Doctor of Philosophy University of Tasmania April, 2006 DECLARATION This thesis contains no material that has been accepted for a degree or diploma by any tertiary institution. To the best of my knowledge and belief, this thesis contains no material previously published or written by another person except where due acknowledgment is made in the text. This thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. Fiona S. Poke ACKNOWLEDGMENTS Many thanks must go to all the people who have helped me during the last three and a half years. To my supervisors Jim Reid, Carolyn Raymond, Rene Vaillancourt and Bruce Greaves, I could not have asked for better guidance. Thanks to the co-authors on my published manuscripts: Brad Potts for his input into Chapter 1 and help with executing the statistical analyses in Chapter 5; Dorothy Steane for her instruction on the phylogenetic analysis in Chapter 7; Darren Martin for his assistance with the recombination detection analysis in Chapter 7; and Judith Wright for the collection of NIR spectra and development of the calibrations in Chapters 2 and 3. I would also like to thank Linda Ballard, Leon Savage and Leigh Johnson for their technical assistance, plus Greg Jordan for his help with the statistical analyses in Chapter 6. A very special thank you goes to my colleagues in the School of Plant Science for making my PhD years so enjoyable. To those I have been sharing an office with, eating lunch with and having a "knock-off' drink with, who have been there for the ride - Corinne, Prue, Beck, Matt and Brett, you guys are the best. Most of all I would like to thank those people who have supported me the most, my friends and family. To my parents, Lance and Kaye, and my brother Rohan thanks for everything. To Simon, thanks for your understanding, support and encouragement, and most of all, thanks for keeping life fun. And lastly, to my wonderful, amazing friends Jacs, Rach, Brad, Justin, Nige, Barn, Deb, Anna, Simon who define my fun times and who will help me celebrate the end of this era! CONTENTS Abstract .............................................................................................................. 1 Preface ................................................................................................................ 3 Chapter 1. Genomic research in Eucalyptus ........................................................ 5 Chapter 2. Predicting extractives and lignin contents in Eucalyptus globulus using near infrared reflectance analysis on ground wood .............................................. 29 Chapter 3. Predicting extractives, lignin and cellulose contents using near infrared spectroscopy on solid wood in Eucalyptus globulus ............................................ 41 Chapter 4. Within-tree variation in lignin, cellulose and extractives in Eucalyptus globulus .............................................................................................................. 55 Chapter 5. Genetic parameters for lignin, extractives and decay in Eucalyptus globulus .............................................................................................................. 71 Chapter 6. The effect of a single amino acid substitution in a lignin biosynthesis enzyme on wood properties in Eucalyptus globulus ............................................ 87 Chapter 7. The impact of intragenic recombination on phylogenetic reconstruction at the sectional level in Eucalyptus when using a single copy nuclear gene (cinnamoyl CoA reductase) ................................................................................................... 105 Conclusions ........................................................................................................ 127 References .......................................................................................................... 131 Appendix 1. Supporting material written during candidature ............................... 164 Abstract ABSTRACT Eucalyptus globulus is grown in temperate regions throughout the world predominantly for the production of kraft pulp. Kraft pulping involves chemically removing most of the lignin and extractives and some of the hemicellulose from the cellulose fibres. The amount of lignin and extractives in the wood is therefore important. The aim of this study was to strengthen knowledge of genetic variation in the chemical wood properties of E. globulus. Methods were developed for fast and simple assessment of these traits. These will benefit quantitative and molecular approaches to breeding, both of which were explored in this thesis. In addition, the utility of a lignin biosynthesis gene for phylogenetic analysis in Eucalyptus was investigated. Measuring wood chemistry using traditional chemical methods is costly and time­ consuming. Near infrared reflectance (NIR) analysis was explored as an alternative to these for the prediction of extractives, lignin and cellulose contents from both ground and solid wood samples. Good calibrations were developed for each of these traits with high correlation coefficients (R2 of 0.62 to 0.93) and standard errors of less than 1.37%. All calibrations were validated using a separate set of samples with strong correlations obtained between predicted and laboratory values (R2 of 0.67 to 0.99), with the exception of acid-soluble lignin content for solid wood (R2 of 0.12). NIR analysis was found to be a reliable predictor of the wood chemistry of E. globulus, with solid wood shown to be a good alternative to ground wood samples. Solid wood NIR calibrations were then used to assess within-tree variation in the wood chemistry of E. globulus. Wood chemistry was found to vary within-tree, with extractives content significantly decreasing with height in the tree, but not varying radially. Bark-t~-pith variation was observed for cellulose and lignin contents, the former decreasing and the latter increasing. However, for cellulose content this was generally not significant, while for lignin content significance was found between 20 and 60% of tree height. These traits showed no significant height variation. Results indicated that current assessment techniques for wood chemistry, using wood cores extracted at breast height, are effective. 1 Abstract Natural variation in the wood chemistry of E. globulus was investigated using NIR predictions. Physical wood traits, growth and decay were also measured. Genetic variation was found for lignin content and decay, with significant locality differences. The only trait to have significant family within locality variation was acid-soluble lignin content, giving a high narrow-sense heritability (0.51 ± 0.26). Family means heritabilities were high for lignin content, extractives content and decay (0.42 - 0.64). This suggested that traditional breeding may be used to improve these traits. Furthermore, the chemical wood traits were highly correlated with each other and with density and microfibril angle. This indicated that selection for the breeding objective traits (basic density, pulp yield and volume) could concurrently produce favourable states in wood chemistry, decay resistance and fibre properties. Having identified natural genetic variation in lignin, molecular variation in the lignin biosynthesis gene, cinnamoyl CoA reductase (CCR), was explored, firstly for its effect on wood properties; and secondly for its use in phylogenetic reconstruction at the sectional level of Eucalyptus. Segregation and quantitative trait loci (QTL) approaches were used to identify the effect of an amino acid substitution at a highly conserved position in CCR, on lignin content, lignin composition (syringyl/guaiacyl ratio) and wood density. The amino acid substitution had no significant influence on these traits nor did CCR collocate with any of the QTL for growth and density found in that cross. The high levels of sequence variation found for CCR in E. globulus suggested it may be useful for testing the monophyly of Eucalyptus sections Exsertaria and Latoangulatae, using section Maidenaria as an outgroup. Latoangulatae and Maidenaria were polyphyletic or paraphyletic, while Exsertaria species formed a clade but included a single Latoangulatae species. Analysis of intragenic recombination, a confounding factor when using nuclear genes for phylogenetic analysis, identified two events involving species from different sections. The occurrence of intragenic recombination may explain the anomalous positions of some species wilhin the phylogenetic tree, and also suggests that levels of linkage disequilibrium will be low, which has implications for association studies. The high level of CCR sequence variation between and within Eucalyptus species suggests that molecular variants may be found in natural populations that will allow selection for improved lignin profiles. 2 Preface PREFACE Eucalyptus globulus is a temperate hardwood species native to south-east Australia. Its wood quality is ideal for pulp production, and because of this it is now grown in temperate areas throughout the world. During kraft pulping, lignin and extractives are chemically removed from the cellulose fibres (Smoak, 1992). If high quality paper is the desired end product, the pulp must then be bleached to remove the residual lignin (Smoak, 1992). These processes have been implicated in contributing