Luke J. McManus

Pectin Methylesterase Genes Influence Solid Wood Properties of Eucalyptus pilularis

This association study of Eucalyptus pilularis populations provides empirical evidence for the role of Pectin Methylesterase (PME) in influencing solid wood characteristics of Eucalyptus. PME6 was primarily associated with the shrinkage and collapse of drying timber, which are phenotypic traits consistent with the role of pectin as a hydrophilic polysaccharide. PME7 was primarily associated with cellulose and pulp yield traits and had an inverse correlation with lignin content. Selection of specific alleles in these genes may be important for improving trees as sources of high-quality wood products. A heterozygote advantage was postulated for the PME7 loci and, in combination with haplotype blocks, may explain the absence of a homozygous class at all single-nucleotide polymorphisms investigated in this gene.

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.

Genetic association studies in Eucalyptus pilularis Smith (blackbutt)

Summary Breeding for wood quality is limited by the long generation times and the delay before wood quality can be measured reliably. Association studies allow links between phenotype and genotype to be made, and are a prelude to accelerated domestication of trees by molecular breeding approaches. This study uses association genetics to identify DNA polymorphisms that correlate with solid wood properties of Eucalyptus pilularis Smith (blackbutt). We undertook extensive phenotyping of dimensional stability, growth and structural wood properties on a nine-year-old progeny trial established by Forests NSW at Hannam Vale, near Port Macquarie in NSW. A subset of 372 phenotyped individuals representing 284 families collected from 37 provenances was used as the association population for genotypic assessment. Fifty-two out of 127 novel DNA polymorphisms were surveyed within four candidate genes, CCR, CAD, MYB1 and MYB2. Several putative associations between wood quality traits and selected DNA polymorphisms are reported, along with the likely mechanism of action on wood quality. Association studies such as this will facilitate non-destructive DNA tests for heritable wood properties that can be used to enrich breeding populations at any developmental stage with desirable alleles.

The Significance of Single Nucleotide Polymorphisms (SNPS) in 'Eucalyptus globulus' Breeding Programs

Summary Eucalyptus globulus (Labill.) is the most widely planted eucalypt for pulpwood in temperate regions of the world. Breeding to improve pulp properties of this species has been hampered by the long time between planting and pulp trait assessment and the high cost of estimating pulp traits. Identifying and employing allelic variants that associate with superior pulp yield and quality has the potential to assist breeding programs. Before this strategy can deliver benefits, detailed knowledge of population structure, nucleotide diversity, haplotype diversity and linkage disequilibrium (LD) must be collected. To address this, 20 wood quality candidate genes were sequenced in 8 to 28 Eucalyptus globulus individuals. Relative to other tree species where such studies have been conducted, single nucleotide polymorphism (SNP) frequencies were high. Decay of linkage disequilibrium was rapid at all loci tested, with linkage rarely extending beyond 500 base pairs. Regions within many candidate genes exhibited significant positive or negative selection signatures, indicative of purifying or balancing selection, respectively. Our findings have implications for association mapping in Eucalyptus species. The potential for E. globulus pedigree reconstruction and whole-genome association approaches in eucalypts in general are discussed.

Association mapping for wood quality and growth traits in Eucalyptus globulus ssp. globulus Labill identifies nine stable marker-trait associations for seven traits

The moderate to high levels of nucleotide diversity and low linkage disequilibrium found in many forest tree species make them ideal candidates for association mapping. Here, we report candidate gene-based association mapping results for complex wood quality and growth traits in Eucalyptus globulus Labill. ssp. globulus, the most widely grown eucalypt in temperate regions of the world. Ninety-eight single nucleotide polymorphisms (SNPs) from 20 wood quality candidate genes were assayed in a discovery population consisting of 385 trees sourced from a provenance-progeny trial. Twenty-five selected SNPs with significant associations (P < 0.05) in the discovery population were assayed for validation in 296 trees sourced from an independent second-generation breeding trial. To account for background genetic structure, mixed models were used in the association analyses. Two associations identified in the discovery population were independently supported in the validation testing. However, combining the discovery and validation results in a combined analysis, we discovered nine stable marker-trait associations for seven traits. These associations link underlying complex wood and growth phenotypes to earlier putative selection signatures opening new avenues to accelerate the dissection of these traits.

Extraction and characterization of agar from Australian Pterocladia lucida

Polysaccharides were sequentially extracted from Australian Pterocladia lucida at 50 °C, to give the Warm Water (WW) fraction, and at 95 °C. The 95 °C extract was further separated into gelling (GF) and thaw water (TW) fractions by freezing-thawing. Fourier Transform infrared (FTIR) spectroscopy, compositional and linkage analyses, and physico-chemical properties indicated that the GF contained an agar with nearly idealized repeating structure and low levels of sulfate and pyruvate substitution. By contrast, the WW and TW contained heterogeneous, highly sulfated galactans with relatively low levels of 3,6-anhydrogalactose and higher levels of pyruvate and glycosyl branching and impurities, such as starch and protein. The properties of the gels formed from the GF and two commercially available agars (Sigma High Gel Strength agar and Sigma Type A agar) were investigated with a texture analyser. The GF from P. lucida had a gel strength intermediate between that of the commercial agars. The gel setting temperature of a 0.8% (w/v) solution formed from the GF was 2 °C below that of comparable solutions of the two commercial agars.

Candidate gene-based association mapping of growth and wood quality traits in Eucalyptus globulus Labill

Background The identification of polymorphisms that underlie complex phenotypic traits presents new and exciting challenges to molecular genetics. Association mapping, which uses linkage disequilibrium (LD) to map trait variation with nucleotide polymorphisms, has proved suited for this purpose in outcrossed tree species. Association mapping can be undertaken either at a candidate gene level or at the whole-genome level however; until sufficiently dense marker assays are developed the candidate gene approach will remain the most effective way of dissecting complex traits in tree species. To date, this approach has led to the identification of several quantitative trait nucleotides (QTN) that associate with a variety of breeding traits including, early wood specific gravity, percentage latewood [1], microfibril angle [2], cellulose [3]and carbon isotope discrimination [4]. Aim

SNP discovery and association mapping in Eucalyptus pilularis (blackbutt)

Background This research explores the universality of genetic variation in genes controlling wood formation across the genus Eucalyptus. Breeding and deploying Eucalypts for improved wood quality is constrained by the delay before wood traits can be measured reliably. Marker assisted selection (MAS) offers aw ay to make earlier selection of wood properties, by selecting Single Nucleotide Polymorphisms (SNPs) in the DNA which can predict specific phenotypic traits (genotype-phenotype links). SNPs shared between species (trans-specific SNPs) may have broad application to multiple species. When the species are more distantly related, ancient SNPs shared between subgenera (trans-subgeneric SNPs) are likely to be of adaptive importance and persist in separate lineages due to balancing selection [1]. Sample and methods