Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of...

Striga is a root parasitic weed that attacks many of the staple crops in Africa, India and Southeast Asia, inflicting tremendous losses in yield and for which there are few effective control measures. Studies of parasitic plant virulence and host resistance will be greatly facilitated by the recent emergence of genomic resources that include extensive transcriptome sequence datasets spanning all life stages of S. hermonthica. Functional characterization of Striga genes will require detailed analyses of gene expression patterns. Quantitative real-time PCR is a powerful tool for quantifying gene expression, but correct normalization of expression levels requires identification of control genes that have stable expression across tissues and life stages. Since no S. hermonthica housekeeping genes have been established for this purpose, we evaluated the suitability of six candidate housekeeping genes across key life stages of S. hermonthica from seed conditioning to flower initiation using qRT-PCR and high-throughput cDNA sequencing. Based on gene expression analysis by qRT-PCR and RNA-Seq across heterogeneous Striga life stages, we determined that using the combination of three genes, UBQ1, PP2A and TUB1 provides the best normalization for gene expression throughout the parasitic life cycle. The housekeeping genes characterized here provide robust standards that will facilitate powerful descriptions of parasite gene expression patterns.