Quantitative analysis of transcript accumulation from genes duplicated by polyploidy using cDNA-SSCP

Repeated rounds of polyploidy have been commonplace in the lineages leading to modern eukaryotic genomes, giving rise to widespread gene duplication. Genes duplicated by polyploidy, or "homoeologs," may continue to be expressed at equal levels following polyploidization or their expression may be dramatically altered. In this report, we describe how SSCP analysis of RT-PCR products can be used to evaluate the expression status (presence and relative quantity) of highly similar homoeologous gene pairs from an allotetraploid genome. This cDNA-SSCP approach was used to evaluate transcript abundance in "synthetic tetraploid" mRNA pools (i.e., mixtures of diploid products) and three natural homoeologous gene pairs expressed in tetraploid cotton (Gossypium hirsutum) ovules. Results from replicated tests show that cDNA-SSCP reliably separates duplicated transcripts with 99% sequence identity. Most significantly, the method yields quantitative estimates Of transcript ratios in template pools that range from equimolar to approximately 100:1.