Quantification of allele-specific expression of a gene encoding strawberry polygalacturonase-inhibiting protein (PGIP) using Pyrosequencing™

Recent studies indicate that allele-specific differences in gene expression are a common phenomenon. The extent to which differential allelic expression exists might be underestimated, due to the limited accuracy of the methods used so far. To demonstrate allele-specific expression, we investigated the transcript abundance of six individual, highly homologous alleles of a polygalacturonase-inhibiting protein gene (FaPGIP) from octoploid strawberry (Fragaria x ananassa). We applied the highly quantitative Pyrosequencing method which, for the gene under study, detected allele frequency differences as small as 4.0 +/- 2.8%. Pyrosequencing of RT-PCR products showed that one FaPGIP allele was preferentially expressed in leaf tissue, while two other alleles were expressed in a fruit-specific way. For fruits that were inoculated with Botrytis cinerea a strong increase in overall FaPGIP gene expression was observed. This upregulation was accompanied by a significant change in FaPGIP allele frequencies when compared with non-treated fruits. Remarkably, in the five cultivars tested, the allele frequency in cDNA from the inoculated fruits was similar to that in genomic DNA, suggesting uniform upregulation of all FaPGIP alleles present as a result of pathogenesis-related stress. The results demonstrate that when Pyrosequencing of RT-PCR products is performed, novel allele-specific gene regulation can be detected and quantified.