Discovering candidate genes that regulate resin canal number in Pinus taeda stems by integrating genetic analysis across environments, ages, and populations.

Genetically improving constitutive resin canal development in Pinus stems may enhance the capacity to synthesize terpenes for bark beetle resistance, chemical feedstocks, and biofuels. To discover genes that potentially regulate axial resin canal number (RCN), single nucleotide polymorphisms (SNPs) in 4027 genes were tested for association with RCN in two growth rings and three environments in a complex pedigree of 520 Pinus taeda individuals (CCLONES). The map locations of associated genes were compared with RCN quantitative trait loci (QTLs) in a (P. taeda × Pinus elliottii) × P. elliottii pseudo-backcross of 345 full-sibs (BC1). Resin canal number was heritable (h(2) ˜ 0.12-0.21) and positively genetically correlated with xylem growth (rg ˜ 0.32-0.72) and oleoresin flow (rg ˜ 0.15-0.51). Sixteen well-supported candidate regulators of RCN were discovered in CCLONES, including genes associated across sites and ages, unidirectionally associated with oleoresin flow and xylem growth, and mapped to RCN QTLs in BC1. Breeding is predicted to increase RCN 11% in one generation and could be accelerated with genomic selection at accuracies of 0.45-0.52 across environments. There is significant genetic variation for RCN in loblolly pine, which can be exploited in breeding for elevated terpene content.

Association genetics of oleoresin flow in loblolly pine: discovering genes and predicting phenotype for improved resistance to bark beetles and bioenergy potential.

Rapidly enhancing oleoresin production in conifer stems through genomic selection and genetic engineering may increase resistance to bark beetles and terpenoid yield for liquid biofuels. We integrated association genetic and genomic prediction analyses of oleoresin flow (g 24 h(-1)) using 4854 single nucleotide polymorphisms (SNPs) in expressed genes within a pedigreed population of loblolly pine (Pinus taeda) that was clonally replicated at three sites in the southeastern United States. Additive genetic variation in oleoresin flow (h(2) ≈ 0.12-0.30) was strongly correlated between years in which precipitation varied (r(a) ≈ 0.95), while the genetic correlation between sites declined from 0.8 to 0.37 with increasing differences in soil and climate among sites. A total of 231 SNPs were significantly associated with oleoresin flow, of which 81% were specific to individual sites. SNPs in sequences similar to ethylene signaling proteins, ABC transporters, and diterpenoid hydroxylases were associated with oleoresin flow across sites. Despite this complex genetic architecture, we developed a genomic prediction model to accelerate breeding for enhanced oleoresin flow that is robust to environmental variation. Results imply that breeding could increase oleoresin flow 1.5- to 2.4-fold in one generation.

Association genetics of the loblolly pine (Pinus taeda, Pinaceae) metabolome.

The metabolome of a plant comprises all small molecule metabolites, which are produced during cellular processes. The genetic basis for metabolites in nonmodel plants is unknown, despite frequently observed correlations between metabolite concentrations and stress responses. A quantitative genetic analysis of metabolites in a nonmodel plant species is thus warranted. Here, we use standard association genetic methods to correlate 3563 single nucleotide polymorphisms (SNPs) to concentrations of 292 metabolites measured in a single loblolly pine (Pinus taeda) association population. A total of 28 single locus associations were detected, representing 24 and 20 unique SNPs and metabolites, respectively. Multilocus Bayesian mixed linear models identified 2998 additional associations for a total of 1617 unique SNPs associated to 255 metabolites. These SNPs explained sizeable fractions of metabolite heritabilities when considered jointly (56.6% on average) and had lower minor allele frequencies and magnitudes of population structure as compared with random SNPs. Modest sets of SNPs (n = 1-23) explained sizeable portions of genetic effects for many metabolites, thus highlighting the importance of multi-SNP models to association mapping, and exhibited patterns of polymorphism consistent with being linked to targets of natural selection. The implications for association mapping in forest trees are discussed.

Clonal variation in crown structure, absorbed photosynthetically active radiation and growth of loblolly pine and slash pine.

Crown structure, absorbed photosynthetically active radiation (APAR) and growth were analyzed in 300 replicated loblolly (Pinus taeda L.) and slash pine (Pinus elliottii Engelm. var. elliotti) clones to: (1) quantify genetic variation in crown structural traits, growth and APAR at the species, family and clonal levels; and (2) estimate within-family genetic and environmental influences on measured variables. Species and family-within-species differences were found in some growth traits, crown size, leaf area, APAR and branch angle. Loblolly pine developed larger crowns, exposed more leaf area with an acute angle, and intercepted more radiation than slash pine. Significant differences among clones within-family were found for stem volume and crown architecture. Loblolly pine and slash pine within-family, individual-tree broad-sense heritabilities ranged from 0.00 to 0.41 for growth and crown structural traits and most were between 0.10 and 0.25 when estimated from a combined analysis across families. Genetic correlations of crown size, leaf area and APAR with volume increment generally ranged from 0.60 to 0.75. This knowledge of the genetic interactions among growth and crown structural traits improves our understanding of how crown morphology affects light interception and stand development, and ultimately how these attributes can be incorporated in the selection of families or clones for the development of new crop tree ideotypes.

Association mapping of quantitative disease resistance in a natural population of loblolly pine (Pinus taeda L.).

Genetic resistance to disease incited by necrotrophic pathogens is not well understood in plants. Whereas resistance is often quantitative, there is limited information on the genes that underpin quantitative variation in disease resistance. We used a population genomic approach to identify genes in loblolly pine (Pinus taeda) that are associated with resistance to pitch canker, a disease incited by the necrotrophic pathogen Fusarium circinatum. A set of 498 largely unrelated, clonally propagated genotypes were inoculated with F. circinatum microconidia and lesion length, a measure of disease resistance, data were collected 4, 8, and 12 weeks after inoculation. Best linear unbiased prediction was used to adjust for imbalance in number of observations and to identify highly susceptible and highly resistant genotypes ("tails"). The tails were reinoculated to validate the results of the full population screen. Significant associations were detected in 10 single nucleotide polymorphisms (SNPs) (out of 3938 tested). As hypothesized for genes involved in quantitative resistance, the 10 SNPs had small effects and proposed roles in basal resistance, direct defense, and signal transduction. We also discovered associated genes with unknown function, which would have remained undetected in a candidate gene approach constrained by annotation for disease resistance or stress response.

Validating a QTL region characterized by multiple haplotypes.

Validation of a quantitative trait locus (QTL) for outcrossing perennial plants is rarely reported due to complexity of plausible genetic models and reliance on field designs already available. Here, a particular marker-QTL haplotype exerted a large, positive effect on height for Pinus taeda and its origin could be traced to a founder, GP(3), in a three-generation QTL pedigree. To validate this QTL effect, we used an extended GP(3)-based pedigree. In the validation cross, each of the 46 offspring was clonally propagated from developing seeds using somatic embryogenesis technology. Subsequent analyses were conducted separately for seedlings and for other somatic emblings. For seedlings, the original QTL effect could not be fully validated. For somatic emblings, a strong negative QTL effect was detected in the validation cross; some evidence from another cross-supported the original positive QTL effect. From this part of the analysis, three distinct marker-QTL haplotypes at a single locus could be inferred. Validating QTL haplotypes in readily available field tests was feasible despite the genetic model complexity inherent to outcrossing long-lived perennials.

Genetic dissection of fusiform rust and pitch canker disease traits in loblolly pine.

Loblolly pine (Pinus taeda L.) exhibits genetic resistance to fusiform rust disease (incited by the biotrophic fungus, Cronartium quercuum f. sp. fusiforme) and pitch canker disease (incited by the necrotrophic fungus, Fusarium circinatum). In this study, a total of 14,015 loblolly pine cuttings from 1,065 clones were screened in controlled greenhouse conditions to identify phenotypes of clones, families, and parents that guide a genetic dissection of disease traits associated with pitch canker and fusiform rust. A total of 23,373 phenotypic data points were collected for lesion length (pitch canker) and gall score, gall length, and gall width (fusiform rust). We verified heritable fusiform rust and pitch canker traits and calculated parental, clonal, and full-sib family rankings for both diseases. Genetic correlations revealed that traits associated with fusiform rust are genetically distinct from one another, and that the genetic mechanisms underlying pitch canker and fusiform rust resistance are independent. The disease phenotyping described here is a critical step towards identifying specific loci and alleles associated with fusiform rust and pitch canker resistance.