Reference genes for reliable potyvirus quantitation in cassava and analysis of Cassava brown streak virus load in host varieties.

A reliable method for detection and quantitation of viruses associated with cassava brown streak disease (CBSD) is essential to determine their presence in material used for field propagation as well as for precise evaluation of CBSD resistance in the cassava germplasm. Quantitative RT-PCR (RT-qPCR) is a well-established method for precise quantitation of viral RNA amount in infected tissues. The method requires host reference genes with stable expression patterns under experimental conditions as internal controls for correct data normalization. Using the Genevestigator Refgene tool with Arabidopsis microarray data from Potyvirus-infected Arabidopsis as input data, candidate reference genes with stable expression pattern were selected as potential internal controls for the cassava -Cassava brown streak virus (CBSV; genus Ipomovirus; family Potyviridae) pathosystem. Primer pairs were designed for the cassava orthologs and their expression was analyzed in different tissues of three different CBSV-infected cassava varieties. The expression patterns of PP2A, UBQ10 and GTPb appeared to be the most stable in different CBSV-infected tissues and cassava varieties. The reference genes can therefore be used as internal controls for normalization of gene expression data in all types of cassava samples as well as in different cassava varieties infected by CBSV. The selected reference genes were used as internal controls to quantify CBSV in various symptomatic and asymptomatic plant organs to establish a correlation between virus load and symptom severity.