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In the last decade, outbreaks of bark beetles in coniferous forests of North America have caused unprecedented tree mortality and economic losses (Nowak et al., 2008; van Mantgem et al., 2009; Waring et al., 2009), converting forests that were previously atmospheric carbon sinks into carbon sources (Kurz et al., 2008). Native species of bark beetle rapidly kill healthy trees by aggregating on their hosts, boring into the stem, and vectoring pathogenic fungi that are tolerant of conifer defenses (Paine et al., 1997; Wang et al., 2013). Climate change is thought to have exacerbated tree mortality from bark beetle infestations by increasing the number of beetle generations yr–1, expanding the range of beetles and their associated pathogens, and by weakening host defenses (Raffa et al., 2008; Bentz et al., 2010).
Increasing oleoresin production in conifer stems through breeding and biotechnology may enhance baseline resistance to bark beetles in managed plantations (Phillips & Croteau, 1999). Oleoresin is a viscous mixture of terpenoids stored under positive pressure within resin canals in the stems of conifers (Trapp & Croteau, 2001). Oleoresin that flows from the stem upon wounding obstructs beetle entry and inhibits germination and growth of pathogenic fungi (Franceschi et al., 2005; Kopper et al., 2005). Among pines, survival after a bark beetle infestation is positively correlated with the rate of oleoresin flow (Strom et al., 2002) and the number of resin canals within the stem (Kane & Kolb, 2010). However, increased stem oleoresin production alone may not be sufficient to protect conifer stands from severe bark beetle population eruptions, where healthy trees with the greatest oleoresin flow can become the preferred hosts of beetles (Boone et al., 2011).
The potential to utilize terpenoids in liquid biofuels is an additional incentive to genetically enhance oleoresin production in conifer stems. Whereas the energy content of bioethanol is only 70% of that of gasoline (Peralta-Yahya & Keasling, 2010), advanced biofuels derived from terpenoids have similar energy content to gasoline and diesel, with densities and hygroscopicities that are amenable to blending with fossil fuels (Harvey et al., 2010; Peralta-Yahya et al., 2011). Conifers require fewer inputs of fertilizer and herbicide than annual food crops even under intensive management, and the net energy balance of producing cellulosic ethanol from conifer wood compares favorably with ethanol derived from maize starch (Evans & Cohen, 2009).
The capacity to genetically enhance terpenoid production in conifer stems is largely untapped. Under normal growing conditions, pines accumulate oleoresin to 1–5% of their stem mass, but stem oleoresin contents of 20% have been observed after treatment with chemical elicitors of resinosis (Stubbs et al., 1984; Wolter & Zinkel, 1984). Although it remains to be demonstrated whether these high terpenoid concentrations can be achieved through genetics, previous studies indicate that variation in oleoresin flow is heritable and positively correlated with growth (Squillace & Bengtson, 1961; Roberds et al., 2003; Romanelli & Sebbenn, 2004).
Developing a detailed knowledge of how allelic variation in conifer breeding populations relates to phenotypic variation in oleoresin flow can accelerate selective breeding for enhanced oleoresin production. Conifer breeding has traditionally relied on phenotypic characterization of the breeding population near the harvest age to infer genetic merit for selection, requiring ≥ 20 yr to complete one breeding cycle (White & Carson, 2004). Genomic selection, or the prediction of breeding values from the summed effects of a panel of genetic markers in linkage disequilibrium with alleles controlling a trait (Meuwissen et al., 2001), circumvents the need to phenotype the breeding population for each generation. When combined with top-grafting to induce early seed development, genomic selection could reduce the breeding cycle of Pinus taeda from 12–20 yr to 4–7 yr (Resende et al., 2012a).
Genetic engineering is an alternative strategy to rapidly increase oleoresin production in conifer stems. This approach may include overexpressing and increasing the catalytic efficiency of terpenoid biosynthetic enzymes (Aharoni et al., 2006; Leonard et al., 2010), down-regulating competing pathways (e.g. lignin biosynthesis), and reprogramming stem development to favor resin canal formation (Zulak & Bohlmann, 2010). While many genes in the terpenoid biosynthetic pathway have been cloned in conifers (Kim et al., 2008, 2009; Schmidt & Gershenzon, 2008; Hamberger et al., 2011; Keeling et al., 2011), the genes involved in the regulation of terpenoid synthesis (e.g. transcriptional and post-translational regulation), the development of resin canals, and the transport of oleoresin into resin canals have yet to be characterized. Coordinated up-regulation of the terpenoid biosynthetic pathway and resin canal development may be achieved through transgenic manipulation of regulatory genes, as has generally been suggested for the metabolic engineering of plant defense pathways (Jirschitzka et al., 2012).
The genomic selection and biotechnological approaches to increase oleoresin production in conifers are potentially complicated by genotype × environment (G × E) interactions. Substantial G × E in oleoresin flow was observed among families of Pinus sylvestris and Pinus elliotti planted at different sites (Bridgen, 1980; Romanelli & Sebbenn, 2004). For traits in which G × E is prevalent, the effects of alleles may depend on the environment (Gillespie & Turelli, 1989), which reduces the prediction accuracy of genomic selection models (Resende et al., 2012a), and leads to uncertainty about the performance of transgenic varieties in diverse field environments (Zeller et al., 2010).
We measured oleoresin flow in a pedigreed population of loblolly pine that was clonally replicated at three sites in the southeastern United States and estimated heritability, site × genotype interactions, and genetic correlations with growth. By comparing genetic correlations in oleoresin flow across years vs between sites, we assessed the relative effect of weather vs climate and soils, respectively, on the genetic control of oleoresin flow. We then used an association genetic approach to discover genes underlying additive genetic variation in oleoresin flow and compared their allelic effects among sites. We specifically tested for associations with single nucleotide polymorphisms (SNPs) in genes with potential roles in terpenoid biosynthesis. Finally, we used cross-validation to assess how accurately the significantly associated loci could predict additive genetic variation within and across sites.