Selection of Reference Genes in Ganoderma pseudoferreum for Studying a Rubber Tree Disease

  Objective   Stability of the reference genes of Ganoderma pseudoferreum under stresses was determined for studying the gene functions and mechanism of the red root rot disease on rubber trees.

  Methods  RNA of G. pseudoferreum mycelia under a stress of temperature, salt, oxidation, pH, drought, or a biocontrol bacteria invasion were isolated and reverse-transcribed into cDNA. RT-qPCR was applied to amplify 6 candidate reference genes, i.e., UBC, ACT, RPL6, β-TUB, APT, and 28s. Software geNorm, NormFinder, BestKeeper, and RefFinder were used to determine the stability of the genes under stresses.

  Results   The RNA of all samples displayed two clear bands and melting curves with a single peak. Analyses by geNorm, NormFinder, Bestkeeper, and RefFinder collectively ranked the expression stability of the genes under a temperature stress as UBC ＞ACT ＞RPL6 ＞β-TUB ＞28s ＞APT; that under a salt stress, ACT ＞RPL6 ＞UBC ＞APT ＞β-TUB ＞28s; that under an oxidative stress, UBC ＞ ACT ＞28s ＞APT ＞β-TUB ＞RPL6; that under a pH stress, RPL6 ＞UBC ＞APT＞ACT ＞ β-TUB ＞28s; that under a drought stress, ACT ＞ UBC ＞β-TUB ＞RPL6 ＞APT ＞28s; and that under the biotic stress, UBC ＞ACT ＞RPL6 ＞ 28s ＞APT ＞β-TUB.

  Conclusion  It appeared that ACT and UBC to be the choice reference genes for normalizing the gene expression under drought, oxidation, temperature, and biotic stresses, while ACT and RPL6 under a salt stress, and UBC and RPL6 under a pH stress.