Roger Meder

Identification of a Cis-Acting Regulatory Polymorphism in a Eucalypt COBRA-Like Gene Affecting Cellulose Content

Populations with low linkage disequilibrium (LD) offer unique opportunities to study functional variants influencing quantitative traits. We exploited the low LD in forest trees to identify functional polymorphisms in a Eucalyptus nitens COBRA-like gene (EniCOBL4A), whose Arabidopsis homolog has been implicated in cellulose deposition. Linkage analysis in a full-sib family revealed that EniCOBL4A is the most strongly associated marker in a quantitative trait locus (QTL) region for cellulose content. Analysis of LD by genotyping 11 common single-nucleotide polymorphisms (SNPs) and a simple sequence repeat (SSR) in an association population revealed that LD declines within the length of the gene. Using association studies we fine mapped the effect of the gene to SNP7, a synonymous SNP in exon 5, which occurs between two small haplotype blocks. We observed patterns of allelic expression imbalance (AEI) and differential binding of nuclear proteins to the SNP7 region that indicate that SNP7 is a cis-acting regulatory polymorphism affecting allelic expression. We also observed AEI in SNP7 heterozygotes in a full-sib family that is linked to heritable allele-specific methylation near SNP7. This study demonstrates the potential to reveal functional polymorphisms underlying quantitative traits in low LD populations.

Rapid Determination of the Chemical Composition and Density of Pinus radiata by PLS Modelling of Transmission and Diffuse Reflectance FTIR Spectra

Summary Transmission and diffuse reflectance FTIR spectra of samples of Pinus radiata wood have been used as the basis of PLS-1 (partial least squares) and PLS-2 (projection to latent structures) models for the prediction of extractives, lignin, total carbohydrate and basic density. Very little difference was observed between models based on transmission spectra and those based on diffuse reflectance spectra. Because of the ease of sample preparation and presentation the diffuse reflectance technique was chosen as the method of preference. Similarly very little difference was observed between models prepared using PLS-1 and models prepared using PLS-2 correlations of diffuse reflectance spectra. Multiple correlation coefficients between the four measured properties and the diffuse reflectance spectra using PLS-2 modelling with four principal components are respectively: extractives, 0.87; Klason lignin, 0.84; total carbohydrate, 0.58; and density, 0.87.

Developing and evaluating a multisite and multispecies NIR calibration for the prediction of Kraft pulp yield in eucalypts

Over recent years the application of near infra-red (NIR) spectroscopy to the prediction of wood properties has been demonstrated in many proof-of-concept studies. Previous work has demonstrated that NIR measurements can be used to predict basic density from woodmeal, chainsaw dust and solid wood, as well as microfibril angle and modulus of elasticity in solid samples. For over a decade, the prediction of Kraft pulp yield (KPY) has been a constant research focus, and numerous small studies have demonstrated this potential. However, because of the cost of obtaining calibration samples with known KPY, sample numbers are typically less than 100. While the potential for NIR prediction of KPY is well recognised, the shift to routine commercial use has not occurred. There still remains considerable scepticism in the research and industry communities about the use of NIR. Concern is typically expressed in two areas: (1) the consistency, accuracy and precision of predictions and (2) the need to prepare a separate calibration for each site and/or species group. To elevate NIR from proof-of-concept to a pilot scale, a large multisite, multispecies calibration was developed over iterative cycles to: (1) determine whether KPY in eucalypts can be predicted from a single calibration independent of site and species, and (2) identify the potential limits of accuracy and precision. This paper reports the results of the first seven testing cycles. The NIR calibration was expanded from an initial sample set of 104 mixed eucalypt samples to over 720 samples covering more than 40 species from predominantly temperate sites across Australia. The performance of the final calibration using two independent and contrasting data sets showed that a multisite and multispecies calibration is feasible. The expected potential accuracy and precision that can be expected from NIR predictions is discussed.

Near infrared hyperspectral imaging applied to mapping chemical composition in wood samples

This paper describes a method for the two-dimensional mapping of chemical composition on the transverse face of cross-sectional discs from trees. The method uses an imaging spectrograph coupled to a near infrared (NIR) camera (900–1700 nm) to obtain NIR hyperspectral data sets which are processed using partial least squares regression to visualise the distribution and variation of lignin, galactose and glucose in Pinus radiata discs with R2/standard error of performance values of 0.84/1.48 (lignin), 0.87/0.68 (galactose) and 0.87/0.95 (glucose). The hardware design and software control are described along with a method for calibration based on one dimension spatially resolved predictions of chemical composition from conventional NIR spectroscopy. The NIR imaging system was designed as a rapid and cost-effective means of mapping chemical composition over the entire disc at a spatial resolution of ∼4mm2/pixel. The resulting maps of chemical composition clearly indicate, at high spatial resolution, the extent of heterogeneity that occurs in logs.

Spatially-resolved radial scanning of tree increment cores for near infrared prediction of microfibril angle and chemical composition.

A fibre-optic accessory with a linear drive transport system has been coupled to a near infrared (NIR) instrument to enable solid samples, in this instance increment cores from standing trees, to be scanned at 1 mm increments along the length of the sample. This allows the NIR prediction of wood properties (oven-dry chemical composition and microfibril angle) to be undertaken so that the radial profile of chemistry or microfibril angle can be determined from the pith to the bark. Calibration models provided prediction errors for microfibril angle in Pinus radiata softwood of 4.1° while for Eucalyptus globulus the error is 3.9°. The errors for prediction of chemical composition in Pinus radiata are 0.2% (arabinose) 1.1% (galactose), 2.3% (glucose), 0.7% (mannose), 0.7% (xylose) and 1.6% (lignin).

Association genetics in Corymbia citriodora subsp. variegata identifies single nucleotide polymorphisms affecting wood growth and cellulosic pulp yield

Wood is an important biological resource which contributes to nutrient and hydrology cycles through ecosystems, and provides structural support at the plant level. Thousands of genes are involved in wood development, yet their effects on phenotype are not well understood. We have exploited the low genomic linkage disequilibrium (LD) and abundant phenotypic variation of forest trees to explore allelic diversity underlying wood traits in an association study. Candidate gene allelic diversity was modelled against quantitative variation to identify SNPs influencing wood properties, growth and disease resistance across three populations of Corymbia citriodora subsp. variegata, a forest tree of eastern Australia. Nine single nucleotide polymorphism (SNP) associations from six genes were identified in a discovery population (833 individuals). Associations were subsequently tested in two smaller populations (130-160 individuals), validating our findings in three cases for actin 7 (ACT7) and COP1 interacting protein 7 (CIP7). The results imply a functional role for these genes in mediating wood chemical composition and growth, respectively. A flip in the effect of ACT7 on pulp yield between populations suggests gene by environment interactions are at play. Existing evidence of gene function lends strength to the observed associations, and in the case of CIP7 supports a role in cortical photosynthesis.

Towards the in-forest assessment of Kraft pulp yield: comparing the performance of laboratory and hand-held instruments and their value in screening breeding trials

The non-destructive prediction of Kraft pulp yield in standing trees using near infrared (NIR) spectroscopy has been typically performed by removing 5 mm or 12 mm increment cores from candidate trees in the forest and returning the samples to the laboratory for analysis. It would be desirable to perform in-field prediction using a portable NIR device; for example, to rapidly screen individual trees in a breeding trial prior to selecting trees for further sampling. This paper compares the performance of a laboratory-based full-range Fourier transform NIR system with two portable NIR devices operating at two different spectral ranges (950–1800 nm and 1600–2400 nm). Calibrations, explaining commercially useful amounts of variance in Kraft pulp yield (R2 = 0.85–0.94) and cellulose content (R2 = 0.92–0.96), were developed for the laboratory NIR and both hand-held portable NIR instruments using woodmeal. This demonstrates that the wavelength range and resolution of the portable instruments are suitable for the prediction of Kraft pulp yield and cellulose content. In an initial in-forest trial, calibration of Kraft pulp yield ranged between R2 = 0.4–0.6 across two sites and two sampling periods. While there are still a number of in-forest sampling issues to be resolved around seasonal sampling, the implication is that low-cost, hand-held NIR systems are a feasible alternative to laboratory-based NIR systems for routine assessment of Kraft pulp yield.

Genetic variation of natural durability traits in Eucalyptus cladocalyx (sugar gum)

Abstract• IntroductionWe present a study on genetic variation in natural durability traits of young-aged Eucalyptus cladocalyx, a species adapted to temperate, low rainfall regions. Our motivation was the production of naturally durable posts for applications such as vine trellises, a sector dominated by heavy metal preservative-treated wood in some of the world’s main wine-producing countries.• MethodsStem diameter at breast height over- and under-bark, heartwood proportion, wood density, methanol extractives and fungal decay were assessed in a progeny test on a set of 48 families from eight provenances nested within three regions of provenance (ROP) from the species’ natural range. Near-infrared reflectance (NIR) was examined as an efficient assessment method.• ResultsStrong variation among ROP was indicated for all traits, with low-moderate narrow-sense heritability for growth traits and extractives content and moderate-high heritability of basic density and decay resistance to two of the three fungi. Trait–trait correlations ranged from low to high, with basic density and extractives content being negatively correlated to decay mass loss.• DiscussionNIR was an effective predictor of methanol extractives, moderately effective for basic density, but unsuccessful for fungal decay. Generally, there were no practically adverse correlations between growth and durability traits.• ConclusionSubstantial genetic variation in natural durability traits is indicated, with wide scope for genetic improvement.

Assessing sapwood depth and wood properties in Eucalyptus and Corymbia spp. using visual methods and near infrared spectroscopy (NIR)

Accurate measurement of sapwood depth (DS) is essential for calculating volumetric water use of individual trees and stands. Various methods are available to measure DS but their accuracy is rarely cross-validated. We sampled 15 Eucalyptus and 1 Corymbia species along a gradient of aridity and obtained reference values of DS in fresh wood cores using light microscopy, which represents our reference method. We compared this method to the simpler and widely used macroscopic method: visual assessment of natural or induced colour change from sapwood to heartwood. In a third method, estimation of DS was based on species-specific models that rely on wood properties measured using near infrared spectroscopy (NIR). Microscopy allowed clear identification of DS based on the presence of blocked vessels. Measurement of DS using microscopic methods was possible for 78 of a total of 80 cores and ranged from 3.6xa0mm (E. loxophleba) to 43.8xa0mm (E. viminalis). Macroscopic assessment clearly differentiated sapwood and heartwood in 60 cores. Results from microscopic and macroscopic methods agreed closely (<10% deviation between estimates) in 35 of 78 cores. After elimination of clearly erroneous measurements (>50% deviation between estimates), macroscopic measurement across all species agreed well with microscopic assessment of DS (R2xa0=xa00.92). Models developed for differentiation between sapwood and heartwood using NIR spectroscopy were very robust (high coefficient of determination) for four species, but DS could only be predicted well for one (E. obliqua) of the four species. Even after elimination of apparent false estimates, prediction of DS by NIR across species was not as strong as for macroscopic assessment (R2xa0=xa00.88). DS can accurately be measured using microscopy if vessel occlusion is clearly visible. Although slightly overestimated, DS from macroscopic assessment was generally similar to that measured by microscopy. NIR spectroscopy was unable to predict DS with acceptable accuracy for the majority of species. Further improvements in the prediction of DS using NIR will require more intensive model calibration and validation, and may not be applicable to all species.

A multi-site, multi-species near infrared calibration for the prediction of cellulose content in eucalypt woodmeal.

Calibrations between spectra derived from near infrared (NIR) spectroscopy and chemical analyses can be used to predict cellulose content in samples of ground eucalypt woodmeal. Past calibrations have used small sample sets (<100 samples) often representing a single stand or region and only one species, making their application to wider populations of samples problematic. A robust, nondestructive prediction capability for eucalypt wood cellulose that works across stands, regions and species would find many applications in tree breeding and resource assessment. Here, we describe the development and test the performance of a large (> 1000 samples) multi-site and species NIR calibration for predicting cellulose content of eucalypt woodmeal obtained from increment cores, wood chips and stem cross-sections. Most of the samples came from Eucalyptus globulus and E. nitens. The calibration was tested against four independent sample sets and explained between 65% and 84% of the variance in each set. A higher proportion of variance was explained in those sample sets that had a wider range of cellulose content. Standard errors of prediction were between 0.5% and 1.5% cellulose for the four independent sample sets. The test samples were added to the large calibration and a new calibration with 1260 samples was constructed. Principal components analysis suggests additional wood samples with more diverse chemistries are required to enable the calibration to capture more fully the chemical variation present in the genus.