Development of a qRT&Allele Specific-PCR Assay to Study Allele Specific Expression: A Case Study of Genes Involved in Drought Stress Response in Sunflower Hybrids

Despite the recent genomic advances, the molecular basis of heterosis remains poorly understood. Specially, we required high-throughput and cost-effective tools to study the specific contribution of both parental genomes in hybrids.

In this study, we developed a quantitative Real-Time and Allele-Specific PCR (qRT&AS-PCR) assay, using SYBR Green, to measure allele-specific expression. To validate our data, we used the PyroMark Q24 (QIAGEN) assay as the golden standard. We found a high correlation between the both assays and validated that the qRT&AS-PCR method is accurate, inexpensive (compared with current methods) and also scalable to interesting throughput using microfluidics.

Our first results on a selection of genes changing their global expression during drought stress in sunflower showed that in most cases there is an imbalance of allelic expressions in the hybrid. This imbalance is consistent with the expression levels of the gene in each of the two parental inbred lines. For one gene, the allelic ratio changed significantly according to the environment again this was consistent with the parental expression responses to a drought stress. Since in the hybrid, both alleles benefit of the same transcription factor environment, we suggest that the observed allele expression imbalances are mainly due to variations in the promoter sequences of the studied genes. 

The new qRT&AS-PCR assay appears to be a promising tool to study the allelic ratio of transcript expression in hybrids and therefore a step forward the understanding of the gene regulatory network properties at the basis of heterosis.