First, we selected genes containing the GCN4, ACGT, and ATGA motifs in their 1-kb promoter regions from the RAP2 rice database.
Therefore, we selected genes containing 7-mer or 8-mer seed matched 3′UTR sites as miR-203 target candidates and obtained a collection of 100 potential miR-203 targets.
Here, as a proof of concept for a new strategic component of sequencing a large complex and highly repetitive crop plant genome in a clone-by-clone approach, we report the pool sequencing of 91 barcoded, randomly selected, gene containing barley BACs by the 454 technology.
We have clustered these 159 cases using several gene lists that were obtained by selecting genes contained in specific Gene Ontology categories [ 70] that were associated with the parameter "relapse" with a P value below 0.001.
We selected the genes containing the largest number of intronic miRNAs, CLCN5 and HTR2C.
To validate our results, we randomly selected ten genes containing 106 SNP sites for PCR-Sanger sequencing using the AB 3730XL.
To limit the sample size, we selected only genes containing a Gli2a peak or located immediately downstream of it, but also included prdm8b, based on its recent identification as a target of Gli1 (Peterson et al., 2012).
We selected genes encoding MCPs containing periplasmic domains (e.g TAR, PRK09793, PAS and HAMP domains).
All selected gene models contain start and stop codons, unless stated otherwise, as well as putative upstream targeting sequences as determined by SignalP v3.0 and ChloroP v1.1 [34].
To validate the microarray results, a real competitive PCR (rcPCR) approach was applied to five selected genes: Flavin-containing monooxygenase 1 (FMO1), transcription factor GATA-4 (GATA4), 17β-hydroxysteroid dehydrogenase 13 (HSD17B13), 17β-hydroxysteroid dehydrogenase 2 (HSD17B2) and N-acetyltransferase 12 (NAT12).
Since the selected gene fragment contains several repetitions of short sequences, a careful optimization of the PCR conditions had to be performed to circumvent the amplification of non-specific fragments from wheat flour.
