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The development of woody tissue, which is comprised largely of the secondary walls of xylem cells, has been fundamental to the evolution of land plants, most notably trees (Spicer & Groover, 2010). Trees are foundation species in ecosystems that cover vast areas of the earth’s surface, and the formation of wood contributes substantially to primary production across climatically diverse environments (Petit & Hampe, 2006). The physiological and ecological importance of wood is reflected in the abundance of natural variation for wood traits among and within tree species and populations, arising from both genetic and environmental factors. Wood is a complex structure and thousands of genes have been identified as being important to its development (Allona et al., 1998; Paux et al., 2005; Pavy et al., 2005; Cato et al., 2006; Yuan et al., 2007; Qiu et al., 2008; Li et al., 2009), yet the specific genetic variants resulting in variation in wood phenotypes are only starting to be understood.
Quantifying genetic variation causal to phenotypic differences has traditionally been achieved by characterizing quantitative trait loci (QTLs) via pedigree based mapping (Grattapaglia & Kirst, 2008). However, the markers identified via QTL studies typically have low resolution with the causative genetic variant, because linkage disequilibrium (LD) in pedigrees is inherently high. Consequently, QTLs have had limited application in breeding and gene functional studies. Genomic selection (GS) is a recently pioneered approach which uses genome scale molecular data to estimate breeding values for trait selection (Hayes et al., 2009). GS has proved to be highly advantageous in breeding programmes in diverse species including trees (Grattapaglia & Resende, 2011; Resende et al., 2012), as it potentially captures all available QTLs, and hence a large proportion of the variance for a given trait. However, this approach similarly relies on populations with extended LD. As such, resolution between the marker and gene is likely to be low, and linkage with the QTL can be lost through recombination in advanced generations. For the same reason, markers identified by GS are often not transferable between populations with different genetic backgrounds (Resende et al., 2012).
LD (or association) mapping applies populations of unrelated individuals to map gene variants (SNPs) that effect phenotype, or are closely linked to the causative variant (Neale & Savolainen, 2004). This is achieved via statistical inference of co-segregation of genotype and phenotype data (Oraguzie et al., 2007). The approach has been applied widely in plants and animals and is well suited to forest tree populations, many of which exhibit abundant genetic diversity and rapidly decaying genome-wide LD (Neale & Savolainen, 2004; Thumma et al., 2005; Külheim et al., 2009). Low LD demands the use of dense SNP marker sets, which in trees have been practicably constructed using a candidate gene approach, resulting in tight linkage of the marker and qualitative trait nucleotide (QTN) (Neale & Savolainen, 2004). This is advantageous for breeding application, as the predictive ability of a marker will be robust through generations and across populations. High-resolution mapping via association studies also provides important insights into gene function. Confirmation of association in one or more additional populations, or ‘validation’, is practised commonly in human association studies (Hinohara et al., 2009; Pasche & Yi, 2010; Konig, 2011), but has to date been reported in only a few studies in plants (Thumma et al., 2005, 2009; Dillon et al., 2010). This approach is a valuable tool in studies where false positive rates as a result of multiple testing, or other biases, are expected to be high, even when statistical corrections are applied (Greene et al., 2009).
To date, SNPs affecting wood properties have been identified in several tree species using this approach. The cinnamoyl CoA reductase gene in Eucalyptus nitens (Thumma et al., 2005) and an MYB transcription factor and pectin methyltransferase in Eucalyptus pilularis (Sexton et al., 2010, 2011) were shown to influence variation in wood quality traits including cellulose microfibril angle, wood collapse and radial shrinkage. Recently, an SNP in a cobra-like gene in E. nitens was found to be part of a cis-acting regulatory element that is directly associated with cellulose content and pulp yield (Thumma et al., 2009). In conifers, SNPs from five cell wall genes have been shown to influence variation in growth, density, microfibril angle (MFA), early wood specific gravity and late wood proportion in Pinus taeda (Yu et al., 2006; Gonzalez-Martinez et al., 2007); two lignin biosynthetic genes, phenylalanine ammonia lyase and phenylcoumaran benzylic ether reductase, were associated with wood density in multiple populations of radiata pine (Pinus radiata) (Dillon et al., 2010); and in a study of over 500 genes in Picea glauca, 13 cell wall genes were associated with nine wood traits (Beaulieu et al., 2011). Recent advances have also been made in Populus, where SNPs from 11 cell wall genes were associated with lignocellulosic traits in Populus trichocarpa (Wegrzyn et al., 2010).
In the present study, we extend the investigation of wood developmental genetics into the genus Corymbia. The spotted gum, Corymbia citriodora subsp. variegata (CCV), is a large tree (growing up to 50 m) that is locally abundant throughout its range. It occurs naturally along the subtropical Australian coast east of the Great Dividing Range between Maryborough (south-east QLD: 25°32′S, 152°42′E) and Taree (northern NSW: 31°46′S, 152°26′E) in a replacement series with several closely related species (Corymbia maculata, Corymbia henryi and Corymbia citriodora subsp. citriodora) (Brooker & Kleinig, 1999; McDonald et al., 2000; Shepherd et al., 2008).
The spotted gums, including CCV, are emerging as important forestry species (Lee et al., 2010). In Queensland, CCV is a source of hardwood, as both natural stands and plantations, and has become the most commonly harvested native in the state (Bacles et al., 2009; Lee et al., 2010). The species has been widely planted as an ornamental in many regions of the world and commercial plantations have been established in South America, southern China, India, Sri Lanka, Congo, Kenya and most countries in southern Africa. Provenance trials indicate that CCV performs better compared with other eucalypts and corymbias for growth, wood properties, survival and tolerance to pests and diseases (Lee et al., 2010). There is considerable interest in developing CCV for plantation forestry in marginal zones because of its tolerance to cold and drought (Larmour et al., 2000; Lee et al., 2010).
Inheritance of wood, growth and disease traits in CCV has been examined to assess its potential for improvement via breeding programmes (Lee et al., 2009; Brawner et al., 2011). High wood density and cellulosic pulp yield indicate that CCV may be well suited for pulpwood production. Inheritance of pulping properties was recently examined in multiple CCV trials, indicating ample phenotypic variation with high heritability, implying a sizeable genetic component (J. T. Brawner et al., unpublished). Growth has been extensively studied in this species, and CCV trees exhibit variation within and between provenances, although heritability for growth is typically lower (0.20) relative to density or pulp yield (0.50 and 0.30) (Lee et al., 2009). Lastly, CCV exhibits heritable variation for resistance to Quambalaria piterika, a fungus causing Quambalaria shoot blight (QSB) that is endemic to coastal forests of eastern Australia (Brawner et al., 2011; Pegg et al., 2011a). QSB infects the leaves, stems and woody tissue of seedlings and young trees. The disease severely affects the growth and form of infected plants and reduces wood quality (Pegg et al., 2011b), and variation in the severity of the disease may be related to variation in wood properties in addition to gene-for-gene mediated resistance.
High heritabilities and ample phenotypic variation suggest that there is potential for improvement of CCV wood phenotypes though breeding. In addition to quantitative selection, there is an opportunity to explore genetic variation underlying traits at the gene level via association mapping, providing insights into the genetic mechanisms underlying wood development and identifying robust markers for phenotypic selection in tree breeding programmes. Using such an approach, we explore SNP variation underlying wood properties, growth and disease resistance for the first time in natural populations of CCV. We examine correlations between SNP variation within candidate genes and phenotypes in first-generation CCV provenance progeny trials established at three locations in southeast Queensland. Candidate genes involved in cambial division, differentiation, photosynthesis, expansion and secondary cell wall biosynthesis were identified from a previous gene expression study in developing xylem in E. nitens (Qiu et al., 2008). The application of three populations allowed verification of detected associations in trees growing under different conditions, and strengthens the argument for gene–phenotype association in several compelling cases.
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Fig. S1 Histogram plot for trait values across the entire Corymbia citriodora subsp. variegata population.
Fig. S2 Provenance means for six traits measured on individuals in the entire Corymbia citriodora subsp. variegata population.
Fig. S3 Scatter and scree plots for first three principal components derived upon six traits (QRES, DEN, DBH, MOE, MFA and KPY) in the discovery population.
Table S1 Summary statistics for calibration (2nd derivative, 15-points, 2nd order polynomial)
Table S2 Trait variances within population, calculated as Σ (x − x)2/(n − 1)
Notes S1 Trait and SNP data from the three study populations.
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