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.