PCR assays used in this study. (A) Flanking primer (FP) assay employs primers immediately flanking the splice site, so PCR products from both the unspliced and spliced Zmbzip60 are amplified. (B) Specific primer unspliced (SPU) assay uses a primer that crosses the exon-intron boundary and is specific for detecting the unspliced form of bzip60. Specific primers spliced (SPS) assay uses a primer that crosses the exon-exon boundary and is specific for detecting the spliced form of ZmbZIP60. The specificity of the two SP assays was demonstrated by amplifying the plasmid-cloned cDNA derived from the spliced and unspliced bzip60.