Chevron report (microarray analysis), CJ Tsai Leaf Chemistry: Leaf phenolics analysis was not proposed in the original work plan, but was initiated as supplemental study to determine whether the genotypes selected for xylem gene expression analysis also differ in their foliar phenolics. The analysis focused on two major nonstructural phenolics pools in Populus, namely condensed tannins (CT) and phenolic glycosides (PG). CT and PG play important roles in defense and stress management of Populus. Their levels vary among Populus species and genotypes, and can constitute up to 35% leaf dry weight in some cases. Indeed, even with the relatively small collection of 12 genotypes, considerable variation in foliar phenolics was observed (Fig. 1). CT levels ranged from ~5% to ~12%, whereas PG levels varied between 0 and ~4%. The data suggest that leaf chemistry offers promising trait for future association genetics study to identify marker genes Fig. 1. Foliar phenolics vary between genotypes. important for tree growth and fitness. Microarray analysis: Developing xylem tissues (as well as leaves described above) from 36 poplar trees derived from 12 unrelated genotypes were harvested at the GreenWood Resources on August 15, 2007. The sampling took place within a 3-hour window (11 am to 2 pm) to minimize environmental variation. RNA was extracted from xylem tissues by the CTAB protocol and Cy3-labeled cRNA targets were prepared using Agilent’s Low RNA Input Fluorescent Linear Amplification Kit. A total of 36 whole-genome microarray hybridizations were performed using the Agilent poplar array that contains ~45,000 features and represents 43,665 nuclear gene models, 130 organelle genes, 6 common reporter genes and ~1400 Agilent controls for various quality control purposes. The overall hybridization quality was good. Hybridization signals from ~20,000 probes were detected in all 36 samples, with ~10,700 probes passing a within-clone coefficient-of-variance (CV) cutoff of 50%. Approximately 8,000 probes showed statistically significant differences in their expression (false discovery rate p = 0.05), when compared between genotypes. Interestingly, the two genotypes showing the most between-tree variation in foliar chemistry (clones 9586 and 9919, Fig. 1) also exhibited more variation in xylem gene expression levels between replicates (Fig. 2). The data are in support of a systems-level association between gene expression and phenotypic attributes. Fig. 2. Principal component analysis of the 36 samples (12 clones x 3 replicates), based on expression values of 8,089 significant genes. Associations between xylem gene expression and MBMS-derived wood chemistry traits (e.g., lignin content and lignin S-to-G ratio) were explored. One limitation for such analysis at the present time is the relatively small collection of genotypes for which microarray data were obtained, and the relatively narrow range of variation in their wood chemistry traits relative to the large population (>1,000) sampled for MBMS analysis. For instance, lignin content varied from 15.8% to 27.5% within the large population, but ranged between 20.8% to 23.3% among the 12 genotypes used for microarray analysis. Likewise, the lignin S-to-G ratio differed by two-fold (1.2 to 2.4) within the large population, but ranged between 1.7 and 2.2 among the 12 clones. Nevertheless, candidate genes exhibiting a significant Pearson correlation (p < 0.05) with lignin content or S-to-G ratio were identified. Examples include a fasciclin-like arabinogalactan protein Involved in cell wall remodeling, one-carbon metabolism pathway genes (e.g., Sadenosylmethionine decarboxylase and serine hydroxymethyltransferase) involved in lignin biosynthesis, and various transcription factors. The microarray dataset thus represents a rich resource for genetical genomics-based identification of marker genes.