Linkage disequilibrium (LD) mapping using natural populations results in higher resolution of markertrait associations compared to family-based quantitative trait locus (QTL) studies. Depending on the extent of LD, it is possible to identify alleles within candidate genes associated with a trait. Analysis of a natural mutant in Arabidopsis has shown that mutations in cinnamoyl CoA reductase (CCR), a key lignin gene, affect physical properties of the secondary cell wall such as stiffness and strength. Using this gene, we tested whether LD mapping could identify alleles associated with microfibril angle (MFA), a wood quality trait affecting stiffness and strength of wood. We identified 25 common single-nucleotide polymorphism (SNP) markers in the CCR gene in Eucalyptus nitens. Using single-marker and haplotype analyses in 290 trees from a E. nitens natural population, two haplotypes significantly associated with MFA were found. These results were confirmed in two full-sib families of E. nitens and Eucalyptus globulus. In an effort to understand the functional significance of the SNP markers, we sequenced the cDNA clones and identified an alternatively spliced variant from the significant haplotype region. This study demonstrates that LD mapping can be used to identify alleles associated with wood quality traits in natural populations of trees.

The neighbourhood model apportions offspring of individual mother plants to self-fertilization, outcrossing to males within a circumscribed area around the mother plant (the neighbourhood), and outcrossing to males outside the neighbourhood. Formerly the model was applied only to haploid pollen gametes in the offspring of conifers, but is extended so that it can be used with genotypic data from diploid offspring of both angiosperms and gymnosperms. In addition, it is shown that the mating parameters can be estimated without independent estimates of allele frequencies in the pollen pools outside the neighbourhood; thus the model might be applied effectively to natural populations exposed to unknown external pollen sources. Parameters of the neighbourhood mating model were estimated for a 10-year-old seed orchard population of the insect-pollinated tree, Eucalyptus regnans, in southeast Australia, which contained a mixture of two geographical provenances (Victoria and Tasmania). The mating patterns revealed were complex. Crosses between trees of the same provenance occurred three times more often than crosses between trees of different provenances. Levels of self-fertilization and patterns of mating within neighbourhoods were influenced by provenance origin, crop fecundity and orchard position (central vs. edge) of mother trees. Gene dispersal, however, was extensive, with approximately 50% of effective pollen gametes coming from males more than 40 m away from mother trees (average distance between neighbouring trees was 7.4 m). Thus, insect pollinators are efficient promoters of cross-fertilization in this orchard, with the result that the effective number of males mating with each female is large.

Summary In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G‐layer). Gene expression was studied in Eucalyptus nitens branches oriented at 45° using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X‐ray diffraction, chemical and histochemical methods. Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G‐layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin‐like arabinogalactan proteins and a β‐tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G‐layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.

Four microsatellite loci have been characterised in Eucalyptus nitens Maiden and in six other eucalypt species. The dinucleotide repeats were identified by screening a Sau3AI genomic DNA library from E. nitens with (CA)n and (GA)n oligonucleotide probes and sequencing the positive clones. Genetic analysis of 20 unrelated individuals from five populations of E. nitens showed all loci to be highly polymorphic with an average of 9.5 alleles per locus and an average heterozygosity of 0.575. Analysis of four individuals from each of six species from three subgenera showed complete conservation of microsatellite loci between species within the same subgenus, Symphyomyrtus, and conservation of 50% of loci across species between the two main subgenera, Symphyomyrtus and Monocalyptus. None of the primers amplified microsatellite loci in Eucalyptus maculata from the subgenus Corymbia. All microsatellite loci that were detected were polymorphic. Highly polymorphic microsatellite loci that are conserved across species will be useful for mapping quantitative traits, fingerprinting breeding lines, and for within-population studies requiring fine-scale analysis of genetic variation.

A genetic linkage map containing potential candidate loci for wood, fibre and floral traits has been constructed for Eucalyptus globulus (Labill.) based on the segregation of 249 codominant loci in an outbred F(1) population of 148 individuals. The map contains 204 RFLP loci, including 31 cambium-specific expressed sequence tags (ESTs) and 14 known function genes, and 40 microsatellite and five isozyme loci. Independent male and female maps were constructed, and the 98 loci (39%) that segregated in both parents were used to combine the parental maps into an integrated map. The 249 loci mapped to 11 major linkage groups ( n=11 in eucalypts) and a 12th small linkage group containing three loci that segregated in the male parent only. Total map distance is 1375 cM with an average interval of 6 cM. Forty one of the mapped loci identify known proteins (five isozymes) or sequences with known function (14 genes and 22 ESTs). The mapped genes include enzymes involved in lignin and cell-wall polysaccharide biosynthesis, and floral-development genes. This map will be used to locate quantitative trait loci for wood, fibre, and other traits in Eucalyptus.

There are 15 populations of E. caesia Benth. on granite rocks in south-westem Australia which include a total of about 2120 plants. The level of genetic variation at 18 allozyme loci in 13 populations was estimated. Seven loci were monomorphic for all plants assayed. At a majority of the 11 polymorphic loci the level of polymorphism was very low in most populations. Within populations the mean number of alleles per locus was 1.31 and the genetic diversity 6.8%. However, populations differed markedly in allelic frequencies at a number of loci. The genetic diversity within populations was remarkably low for a tree species but the level of population differentiation was the highest reported for any tree species. The data suggest that genetic drift may in part have been responsible for the low overall genetic diversity and the extensive population differentiation. The optimal strategies for conservation of the genetic resources of this valuable ornamental are considered in the light of the results of this study.

SummaryCellulose microfibrils are the major structural component of plant secondary cell walls. Their arrangement in plant primary cell walls, and its consequent influence on cell expansion and cellular morphology, is directed by cortical microtubules; cylindrical protein filaments composed of heterodimers of a-and b-tubulin. In secondary cell walls of woody plant stems the orientation of cellulose microfibrils influences the strength and flexibility of wood, providing the physical support that has been instrumental in vascular plant colonization of the troposphere. Here we show that a Eucalyptus grandis b-tubulin gene (EgrTUB1) is involved in determining the orientation of cellulose microfibrils in plant secondary fibre cell walls. This finding is based on RNA expression studies in mature trees, where we identified and isolated EgrTUB1 as a candidate for association with woodfibre formation, and on the analysis of somatically derived transgenic wood sectors in Eucalyptus. We show that cellulose microfibril angle (MFA) is correlated with EgrTUB1 expression, and that MFA was significantly altered as a consequence of stable transformation with EgrTUB1. Our findings present an important step towards the production of fibres with altered tensile strength, stiffness and elastic properties, and shed light on one of the molecular mechanisms that has enabled trees to dominate terrestrial ecosystems.

Background: There is little information about the DNA sequence variation within and between closely related plant species. The combination of re-sequencing technologies, large-scale DNA pools and availability of reference gene sequences allowed the extensive characterisation of single nucleotide polymorphisms (SNPs) in genes of four biosynthetic pathways leading to the formation of ecologically relevant secondary metabolites in Eucalyptus. With this approach the occurrence and patterns of SNP variation for a set of genes can be compared across different species from the same genus.