Large-scale qRT-PCR of soybean TF genes
As a test of the validity of the Illumina Solexa data to accurately measure soybean gene
expression, one might normally turn to the large number of soybean Affymetrix microarray
hybridization experiments available in the public databases. However, as demonstrated by
Libault et al. (2010), the Affymetrix arrays used in these studies are considerably compromised
by the fact that 75% of soybean genes are duplicated (Schmutz et al., 2010). Therefore, to
provide independent validation for the expression levels deduced from the Illumina Solexa data,
we utilized the previously published qRT-PCR primer set library designed against more than
1000 soybean regulatory genes including 652 TF genes (Libault et al., 2009b). In these
experiments, we extended our analysis to eleven different tissues [i.e. untreated root hair cells,
root tip, stripped root (root lacking root hairs), root, nodulated root, stem, leaves, SAM, flower,
green pods and green seeds; Supplemental Table 15]. These experiments resulted in a similar
distribution of the tissues based on the similarities of their expression patterns to that obtained by
analysis of the Illumina Solexa sequences (Supplemental Figure 5; i.e. root tip, stripped root, root
hairs and, roots shared a similar regulatory gene expression pattern; SAM, pod, seeds, stem, leaf
and flower were part of a second group; the nodulated root transcriptome did not share strong
correlation with any other transcriptome, except the root transcriptome). In addition, the
transcriptomes from pairs of tissues, such as root and stem, stripped root and stem and leaf and
flower showed significant correlation. A deeper analysis of the qRT-PCR data led to the
identification of 93 soybean TF genes expressed preferentially in one tissue versus the ten others
(≥3 fold-change; Supplemental Table 16). Among the 624 TF genes expressed preferentially in
different soybean tissues based on Solexa sequencing (≥3 fold-change), 65 TF genes were
represented in the qRT-PCR library. Among these, 22 TF genes were preferentially regulated in
the exact same soybean tissues based on qRT-PCR while 13 TF genes were found preferentially
expressed in different tissues than those initially highlighted by Solexa sequencing. A closer
examination of these discrepancies suggested that the fact that a larger number of conditions
were used to quantify TF gene expression by qRT-PCR led to a refinement in the tissue
expression specificity of some TF genes. For example, Glyma10g28820 and Glyma09g37930,
identified as preferentially expressed in the SAM based on Solexa sequencing, were defined
based on qRT-PCR as preferentially expressed in the stem. Likewise, Glyma03g36710,
Glyma08g42300, Glyma07g04210 and Glyma13g16890, initially identified as expressed in the
pods based on sequencing, were defined as seed-specific genes according to qRT-PCR analyses
(Supplemental Table 16). The use of qRT-PCR technology also led to the identification of 58
new TF genes expressed preferentially in a single soybean tissue. Surprisingly, these genes were
not identified as preferentially expressed based on Illumina Solexa sequencing. However, we
noticed that a significant number of these 60 genes defined as tissue-specific by qRT-PCR also
showed a trend toward tissue-specificity based on sequencing but not sufficiently so to be
considered as tissue-specific genes (e.g. 10 genes were clearly identified as root hair-specific
based on qRT-PCR but not based on Solexa sequencing despite their preferential expression in
root hairs). These results likely point to the greater accuracy of the qRT-PCR analysis. For
example, the use of the qRT-PCR method with the large number of different tissues clearly did
identify some new tissue specific TF genes, including Glyma15g15110 and Glyma10g28290
preferentially expressed in stripped roots and Glyma08g24340, Glyma18g29400,
Glyma08g38190, Glyma03g01000, Glyma19g40070, Glyma06g47000, Glyma11g01850 and
Glyma03g31630 preferentially expressed in immature seeds (Supplemental Table 15).