Enhanced Resistance Pines for Improved Renewable Biofuel and Chemical Production
Authors:
Mallory Morgan*1 Mallory.Morgan@ufl.edu, Matthew Lane2, Salvador Gezan3, Chris Dervinis1; Daniel Ence1, David Kainer4, Mirko Pavicic4, Manesh Shah4, Daniel Jacobson4, Gary F. Peter1
Institutions:
1University of Florida; 2University of Tennessee; 3VSN International, Hemel Hempstead, UK; 4Oak Ridge National Laboratory
Goals
The goal is to genetically increase constitutive terpene defenses of loblolly and slash pine to enhance protection against pests and pathogens and simultaneously expand terpene supplies for renewable biofuels and chemicals.
Abstract
The constitutive and inducible oleoresin defense network in loblolly (Pinus taeda) and slash (Pinus elliottii var elliottii) pine provides physical and chemical resistance to insects and pathogens and the chemical composition of oleoresin can be used as a renewable source of biofuels harvested directly from live tree stems. Increasing pine terpenes is well aligned with the needs of the developing bioeconomy, as the southeastern United States hosts the world’s largest biomass supply chain, annually delivering 17% of global wood products, and has the potential to expand the U.S. pine chemicals industry by increasing biofuels from pine terpenes, which is limited by relatively low average wood terpene content. The focus is to increase constitutive terpene production to enhance loblolly and slash pine resistance to pests and pathogens and to simultaneously increase biofuel feedstocks in these commercial pine species.
Pine terpenes evolved as a primary chemical and physical defense system and are a main component of a durable, quantitative defense mechanism against pests and pathogens. In previous research it was demonstrated that terpene defense traits are under genetic control and behave as quantitative traits and have used genetic engineering to validate 12 genes that can significantly increase wood terpene content. In objective one, researchers are integrating existing and new genome wide association studies (GWAS) genetic results with RNA expression, quantitative trait locus (QTL) mapping, and allele frequency information in known high oleoresin flow selections and the project’s breeding populations to discover and validate loblolly and slash pine alleles/genes that are important for resistance.
GWAS analyses of constitutive oleoresin flow, wood diterpenoid content, and resin canal number with ~83,000 biallelic single nucleotide polymorphisms (SNPs) were completed for the project’s CCLONES population and constitutive oleoresin flow, mono- and diterpene content are complete and complete and resin canal number is in progress for the project’s ADEPT2 population. In the ADEPT2 population, researchers simultaneously measured constitutive and induced oleoresin flow after treating clones with methyl-jasmonate (MeJA). While the goal is to increase constitutive terpene defenses, researchers use MeJA to induce defense responses to identify the genes and genetic architecture of resinosis. In the ADEPT2 population, the team found the clonal repeatability of constitutive oleoresin flow and inducible oleoresin flow to be 0.31, suggesting these traits are under moderate genetic control.
The team’s estimate of clonal repeatability for constitutive oleoresin flow in the ADEPT2 population is consistent with what was previously published in the CCLONES population, and the estimate of clonal repeatability for inducible oleoresin flow in the ADEPT2 population is the first estimate of genetic control for this trait. Importantly, researchers observed a strong genetic correlation (0.82) between induced and constitutive oleoresin flow, suggesting the genetic architecture between these traits is shared.
Researchers conducted association analyses—with constitutive and inducible oleoresin flow, wood monoterpene content and composition, and diterpenoid content obtained in the ADEPT2 population—using linear mixed models and multi-locus linear mixed models in ASRgwas and GAPIT packages using two sets of SNP markers totaling ~2.28 million biallelic SNPs.
Significant SNPs are being mapped to genes and compared with those for constitutive oleoresin flow found in the CCLONES population. In the pseudo-backcross population between one F1 slash x loblolly hybrid genotype backcrossed to slash and loblolly genotypes, researchers collected constitutive and induced oleoresin flow and needle tissue for future QTL mapping.
To identify early, mid, and late genes expressed in differentiating resin ducts, team members induced axial resin canal formation in the cambial meristem by applying methyl-jasmonate (MeJA), which is a known inducer of traumatic resin canal formation in the Pinaceae family. The team conducted a time course experiment where 78 RNAseq libraries were created from cambial zone tissue collected from days 0, 1 to 14, 17, and 21 after MeJA treatment. Researchers also constructed 43 RNAseq libraries from 10 transgenic pine lines from four different constructs with significantly elevated wood terpene content. They pooled all libraries from the time course and from the transgenic lines and sequenced to a 30x read depth with the NovaSeq Illumina NGS platform.
Team members mapped the reads to an improved de novo loblolly pine transcriptome that includes 64,671 genes composed of existing EST contigs, PacBio reads, and predicted transcripts from loblolly pine reference genome v2.01. Researchers used DESeq2 to identify thousands of significantly differentially expressed genes across the time course and in transgenic pines compared with wild type. With these differentially expressed genes researchers created a Predictive Expression Network (PEN) using iterative Random Forest Leave-One- Out Prediction to illustrate higher-order interactions between genes and to determine the gene-to-gene relationships that are the most highly predictive of each other. To identify and prioritize genes across the PEN that are involved in axial resin canal formation, researchers applied random walk with restart (RWR) algorithms based on a set of literature-curated seed genes that included known orthologous regulators of xylem formation and development, which are suppressed while resin canal formation is increased. The RWR approaches allowed us to identify mechanistically associated genes that did not appear in GWAS due to a lack of statistical power or genetic variation but are still important components of resinosis. Researchers are continuing to annotate the network to identify genes whose expression supports involvement in resin canal formation and terpene synthesis.
In objective two, team members are using information from objective one to accelerate breeding for increased resistance in loblolly and slash pine through marker-assisted introgression and will develop and test genomic selection models to accelerate breeding of resistant slash pine.