Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Enhanced Resistance Pines for Improved Renewable Biofuel and Chemical Production

Authors:

Gary F. Peter1*, Mallory Morgan1 (Mallory.Morgan@ufl.edu), Matthew Lane2, Salvador Gezan3, Chris Dervinis1; Daniel Ence1, David Kainer4, Mirko Pavicic4, Manesh Shah4, Daniel Jacobson4

Institutions:

1University of Florida; 2Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee; 3VSN International; 4Oak Ridge National Laboratory

Goals

The team’s 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 U.S. currently 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 currently limited by relatively low average wood terpene content. The team’s focus is to increase constitutive terpene production to enhance loblolly and slash pine’s 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 researchers 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 1, researchers are integrating existing and new genome wide association (GWAS) genetic results with RNA expression, quantitative trait loci (QTL) mapping, and allele frequency information in known high oleoresin flow selections and researchers’ 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 Comparing Clonal Lines ON Experimental Sites (CCLONES) population. Constitutive and inducible oleoresin flow along with mono- and diterpene content were completed and resin canal number is in progress for the team’s Allele Discovery of Economic Pine Traits 2 (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, the group used MeJA to induce defense responses to identify the genes and genetic architecture of resinosis. In the ADEPT2 population, researchers found the clonal repeatability of constitutive oleoresin flow and inducible oleoresin flow to be 0.31, suggesting these traits are under moderate genetic control. In the ADEPT2 population researchers observed a strong genetic correlation (0.82) between induced and constitutive oleoresin flow, suggesting the genetic architecture of these traits is strongly 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 multilocus linear mixed models in ASRgwas and Genome Association and Prediction Integrated Tool packages using two sets of SNP markers totaling ~2.28 million biallelic SNPs. After controlling for multiple testing, researchers identified 146 significant SNPs (p<0.05) for 10 oleoresin traits, including constitutive oleoresin flow, monoterpene composition and content. Two of the significant SNPs for wood limonene content are in an α-pinene synthase gene. To validate significant SNPs the team quantified oleoresin flow in a pseudo-backcross population between one F1 slash x loblolly hybrid genotype backcrossed to slash and loblolly genotypes and are now completing genotyping 982 individuals with the Pita50k chip for future QTL mapping.

To identify genes regulating resin duct differentiation and function, researchers induced new axial resin canal formation in the cambial meristem by applying MeJA, a known inducer of traumatic resin canal formation in the Pinaceae family. Researchers conducted a time course experiment where the team created 78 RNA sequencing (RNAseq) libraries from vascular cambial zone tissue collected from days 0, 1 to 14, 17, and 21 after MeJA treatment. Pooled libraries were sequenced to a 30x read depth with the NovaSeq Illumina next-generation sequencing platform and reads were mapped to an improved de novo loblolly pine transcriptome that includes 64,671 genes that researchers constructed with existing expressed sequence tags contigs, Pacific Biosciences reads, and predicted transcripts from loblolly pine reference genome v2.01. DESeq2 analysis identified significantly 1,890 up and 4,634 down differentially expressed genes across the time course compared with wild-type controls. With these 6,524 differentially expressed genes the team 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 the team 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. This identified 119 transcripts in the top 200 based on lines of evidence. Researchers are continuing to annotate the network to identify genes whose expression supports involvement in resin canal formation and terpene synthesis.

In Objective 2, the group is using information from Objective 1 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.

Funding Information

This research was supported by the DOE Office of Science, BER program–U.S. Department of Agriculture Biomass Feedstocks DE-SC0 019099.