Comparative Expression Analysis of Reference Genes in Field-Grown Cassava

Due to its accuracy, sensitivity and reproducibility, real-time PCR has emerged as a key technique to measure changes in expression of target genes. But to obtain reliable results using real-time PCR, gene transcripts that do not alter its expression under different conditions need to be identified. These transcripts can then be used as references to normalize the expression of target genes. Only a few studies have been conducted so far in the identification of such reference genes in plants. In an effort to identify appropriate reference genes, cassava, an important food source in the tropics and an excellent crop against famine and drought, was assessed for its capacity to express eight housekeeping genes in leaves, stems and roots at four different stages of development under field conditions. The eight candidate genes tested were rRNA18S(18S), β-tubulin(TUB), actin 11(ACT), elongation factor 1α(EF1), translation initiation factor 5A(F5A), ubiquitin protein ligase E3-2a(UBI), ubiquitin conjugating enzyme E2-10(U10) and ubiquitin conjugating enzyme E2-35(U35). Data analysis was performed using four approaches commonlyemployed in identifying the reference genes and consensus rankings were generated using the output of each independent approach. EF1 and TUB were the most stable genes when all tissues and developmental stages were analyzed together. At different stages of development, UBI/18S, EF1/U35, TUB/U35werethe most stable genes in roots, leaves and stem tissues, respectively. Our results suggest the use of more than one reference gene in gene expression studies involving different tissues at different developmental stages.