Determination of the reference genes for qRT-PCR normalization and expression levels of MAT genes under various conditions in Ulocladium.

Abstract

The genus Ulocladium is thought to be strictly asexual. One of the possible reasons for the lack of sexuality in Ulocladium species is the absence of the stimulus of environmental factors. Sexual reproduction in ascomycetes is controlled by a specific region in the genome referred to as mating-type locus (MAT) that consists of two dissimilar DNA sequences in the mating partners, termed MAT1-1 and MAT1-2 idiomorphs. To identify the response of MAT loci to environmental conditions, the mRNA transcription level of MAT1-1-1 and MAT1-2-1 genes was tested using qRT-PCR under different temperatures (−20 °C, −10 °C, 0 °C, 10 °C, 20 °C, 30 °C and 40 °C), culture medias (CM, OA, HAY, PCA, PDA and V8), photoperiods (24 h light, 24 h dark, 12 h light/12 h dark, 10 h light/14 h dark and 8 h light/16 h dark), and CO2 concentrations (0.03%, 0.5%, 1%, 5%, 10%, 15% and 20%). For obtaining reliable results from qRT-PCR, the most stable internal control gene and optimal number of reference genes for normalization were determined under different treatments. The results showed that there is no universal internal control gene that is expressed at a constant level under different experimental treatments. In comparison to various incubation conditions, the relative expression levels of both MAT genes were significantly increased when fungal mycelia were grown on HAY culture media at 0–10 °C with a light/dark cycle, indicating that temperature, culture media, and light might be the key environmental factors for regulating the sexuality in Ulocladium. Moreover, MAT1-1-1 and MAT1-2-1 genes showed similar expression patterns under different treatments, suggesting that the two MAT genes might play an equally important role in the sexual evolutionary process.