Assessments of genes associated with plant host defense responses can be challenging as the defensive mechanisms that enable the host-mediated defense can be the very compounds that make gene expression assays particularly challenging. RNA extraction from woody plant tissues presents significant challenges due to endogenous phenolics, secondary metabolites, and stem polysaccharides. We have established an improved extraction protocol for Fraxinus species, yielding superior results to commercial kits. Our optimized approach, validated across diverse tissue types from over 10 Fraxinus species, consistently produces high-purity RNA with exceptional concentrations (>3000 ng/μL) and integrity (RIN scores 8.0-10.0). The RNA quality we have achieved allows us to detect and analyze rare transcripts that may play crucial roles in emerald ash borer resistance mechanisms. Our approach enables us to quantify copy numbers of defense genes triggered during insect attacks, shedding new light on the molecular basis of resistance pathways in ash trees. By employing digital droplet PCR and RNA-seq calibrated, we can determine the key defense genes' exact transcript copy numbers, including those encoding protease inhibitors, phenolic compounds, and terpenoid synthases central to anti-herbivory responses. We have validated a stable reference gene suite with reliable quality control and consistent expression benchmarks. These references serve as crucial yardsticks when measuring expression patterns across ash trees with varying levels of EAB vulnerability. By determining actual transcript numbers, we can make more meaningful comparisons between ash species and genotypes, helping us identify the critical expression thresholds needed for effective resistance. This work strengthens our partnership with the Chicago Region Tree Initiative, supporting efforts to build more resilient urban forests and protect endangered ash species. Through our detailed analysis of gene expression profiles across diverse ash populations, we are working to pinpoint the genetic signatures that confer EAB resistance. These findings will enable the development of efficient molecular screening tools (e.g., SNP marker panels, transcriptomic signature profiles, or RNA expression ratio tests) for large-scale population assessment and accelerate efforts for this important genus. (Co-authored by Dr. Nathan Maren, Woody Plant Breeder and Genomics Specialist at The Morton Arboretum).
