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Lignin is a highly abundant aromatic biopolymer deposited during the final stages of secondary cell wall formation in plants and it constitutes a substantial proportion of the dry weight of woody plant stems. Lignin contributes structural support to xylem cell walls and hydrophobisity to water-conducting vessels and forms a defence mechanism against pathogen invasion. Although being an essential part of normal plant cell development, lignin content and composition are targets for tree improvement, because residual lignin in paper pulp has negative effects on paper quality and lignin therefore has to be removed using treatments that are expensive and often detrimental to the environment. At present, little is known about the amount of allelic diversity in lignin biosynthetic genes and whether such diversity may be associated with variation in lignin content and composition. However, the identification of alleles associated with desirable lignin phenotypes is dependent on a detailed understanding of the molecular evolution and population genetics of these genes. This M. Sc. study was aimed at analysing nucleotide and allelic diversity in two lignin biosynthetic genes of Eucalyptus trees. Additionally, the study aimed to develop single nucleotide polymorphism (SNP) markers that could be used to assay allelic diversity for these genes in populations of two target species, E. grandis and E. smithii. Orthologues of the tobacco LIM-domain1 (NtLIM1) transcription factor gene involved in the regulation of lignin biosynthesis were isolated from E. grandis and E. smithii. Approximately 3 kb of genomic sequence including the promoter and full-length gene regions were isolated for the two orthologues, respectively labeled EgrLIM1 and EsLIM1. The predicted amino acid sequences of EgrLIM1 and EsLIM1 were 99.4% identical to each other and indicated that LIM1 is a small protein of only 188 residues in eucalypt trees and has a predicted molecular weight of 21.0 kDa. Quantitative, real-time RT-PCR analysis confirmed the expression of LIM1 in wood-forming tissues undergoing lignification. Ten putative cis-regulatory elements were observed in the promoter regions of EgrLIM1 and EsLIM1including a GA-dinucleotide microsatellite that appears to be specific to LIM1 promoters of Eucalyptus tree species. The full-length LIM1 gene sequences could subsequently be used in the assessment of nucleotide and allelic diversity, together with the full-length CAD2 sequences that were already available in the public domain. The level of nucleotide and allelic diversity and the distribution and decay of linkage disequilibrium (LD) were surveyed in 5 and 3 derived gene fragments of CAD2 and LIM1 obtained from 20 E. grandis and 20 E. smithii individuals. Each gene displayed a unique genetic diversity profile, but for the most part, nucleotide diversity () was estimated at approximately 0.0010 except for the E. grandis LIM1 gene where lower than 0.0040 was observed. Generally, except for the high amounts of LD observed in the CAD2 gene of E. grandis (> 2.5 kb), LD decayed within 500 bp. A large number (13 to 45) of SNP sites (defined as single nucleotide changes with minor allele frequencies of at least 0.10 in each species) were observed in each gene of each species. The high SNP density (ranging from one per 45 to one per 155 bp) observed in the two genes facilitated the efficient development of SNP markers to be used in future aspects of LD mapping, association genetics and marker-assisted breeding. The allele sequences obtained for the CAD2 and LIM1 genes were used as templates for the development of SNP marker panels (a series of six or seven SNP markers analysed together) for the analysis (tagging) of SNP haplotype diversity in species-wide reference populations (100 E. grandis and 137E. smithii individuals) of the two species. Each tag SNP was assayed using a single base extension assay and capillary gel electrophoresis. High polymorphism information content (average PIC of 0.836) was observed for the SNP marker panels. Four SNPs in the CAD2 and two in the LIM1 genes were found to be polymorphic in E. grandis and E. smithii (i.e. trans-specific SNPs), suggesting a possible ancestral origin for these polymorphisms. Assessment of candidate gene variation in the genomes of forest trees is of importance to ultimately be able to predict the amount and structure of nucleotide diversity available for the future design of SNP assays at the whole-genome level. Such assays will be useful to study differentiation among tree species and populations, to associate nucleotide polymorphisms with desirable phenotypes and to increase the efficiency of tree improvement approaches.
Eucalyptus trees are an important source of wood and fibre. The wood (secondary xylem) of this genus is widely used for pulp and papermaking. However, our understanding of the mechanisms which control the wood formation process (xylogenesis) in Eucalyptus and other woody species is far from complete. One reason is that xylogenesis is a very complex developmental process. The major components of wood are lignin and cellulose. Many genes involved in lignin and cellulose biosynthesis have been characterized. For example, Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) are two important lignin biosynthesis genes. Plant cellulose is synthesized by cellulose synthase enzymes with the aid of some other proteins, such as sucrose synthase (SuSy) and sucrose phosphate synthase (SPS). Another factor which makes it difficult to analyze the function of Eucalyptus wood formation genes in vivo, is the long generation times of Eucalyptus trees and the difficulty to obtain transgenic Eucalyptus plants. Therefore, in this study, we investigated the use of Arabidopsis thaliana as a model system for functional analysis of wood formation genes. We transformed a lignin and a cellulose biosynthesis gene isolated from Eucalyptus to wild-type and mutant genetic backgrounds of Arabidopsis in order to test our ability to modify the cell wall chemistry of Arabidopsis thaliana using tree genes. The Eucalyptus CCR (EUCCR) gene was transformed into wild-type Arabidopsis (Col-0) and irregular xylem 4 (irx4) mutant plants, in which the homolog of EUCCR is mutated. A Eucalyptus cellulose synthase gene (EgCesA1) was also transformed into irregular xylem 1 (irx1) mutant plants, in which the homolog of EgCesA1 is mutated. Transgenics were only obtained from wild-type Col-0 transformed with EUCCR and from irx1 transformed with EgCesA1. We studied the cell wall chemistry of wild-type Arabidopsis plants overexpressing the Eucalyptus CCR gene. Chemical analysis of inflorescence stems revealed the modification of lignin and cellulose content in transgenic plants. Total lignin content was increased in T2 (5%) and T3 (12%) lines as revealed by micro-Klason lignin and thioglycolic acid quantification methods, respectively. High Pressure Liquid Chromatography (HPLC) analysis revealed that cellulose content was significantly decreased (10%) in T2 transgenic plants. This suggested the reallocation of carbon from cellulose to lignin as a result of overexpression of EUCCR in transgenic plants. Interestingly, thioacidolysis analysis revealed that in T2 plants, monomethoxylated guaiacyl (G) monomer was increased (16%) and bimethoxylated syringyl (S) monomer was decreased (21%). Therefore, the S/G lignin monomer ratio was significant decreased (32%). This implied that EUCCR might be specific to G monomer biosynthesis. The results described above confirmed that Arabidopsis thaliana can be used to model the function of wood formation genes isolated from Eucalyptus. Two novel full-length Eucalyptus sucrose synthase (SuSy) genes, EgSuSy1 and EgSuSy3, and one putative pseudogene, EgSuSy2, were also isolated in this study. Degenerate PCR was used to amplify Eucalyptus SuSy fragments from cDNA and genomic DNA. 3 RACE was used to amplify the 3 ends of two Eucalyptus SuSy genes. Genome walking was performed to obtain the 5 ends of EgSuSy1 and EgSuSy2 whereas 5 RACE technology was used to isolate the 5 end of EgSuSy3. However, 3 RACE analysis failed when we tried to identify the 3 end of EgSuSy2. Sequencing results from the genome walking product of EgSuSy2 further revealed that the start codon of this gene was missing, and we hypothesize that this is a psuedogene in the Eucalyptus genome. The EgSuSy1 cDNA was 2498 bp in length with an open reading frame of 2418 bp encoding 805 amino acids with a predicted molecular mass of 92.3 kDa. The 2528 bp full-length EgSuSy3 cDNA contained the same length of open reading frame as EgSuSy1, but encoded a polypeptide with a predicted molecular mass of 92.8 kDa. The results of quantitative real-time RT-PCR, phylogenetic analysis and gene structure of the two genes revealed that both genes might be involved in cellulose biosynthesis in primary and secondary cell walls of Eucalyptus. These two genes, EgSuSy1 and EgSuSy3, could therefore be useful targets for genetic engineering of wood properties in Eucalyptus.