Evaluation of reference genes for accurate normalization of qPCR data under biotic stresses in mulberry (Morus indica L.)

Biotic stresses adversely affect the leaf quality as well as the yield of mulberry (Morus spp.) which is used to feed mulberry silkworm (Bombyx mori L.). The different biotic stresses which are of economic importance in mulberry are tukra caused by mealybug insect, root knot disease by nematode, powdery mildew, and root rot by fungi. Reverse transcription-quantitative PCR (RT-qPCR) is a preferred technique to assess the transcript abundance of target genes associated with the diseases using a suitable reference gene as a normalizer. However, no study has been conducted to identify suitable reference genes during different biotic stresses in mulberry. In the present work, six candidate reference genes were quantified through qPCR during different biotic stresses in mulberry and their stability was compared using geNorm, NormFinder and RefFinder algorithms. The analysis of data revealed that actin was the preferred gene during root rot and root knot nematode infection for normalizing gene expression. Ubiquitin gene was the best-scoring gene for mealybug infestation resulting in tukra disease. PP2A (Protein phosphatase 2A) and RPL3 (60S ribosomal protein L3) were highly stable expressing genes during powdery mildew fungi infection. In the combined analysis of all tested biotic stresses, actin gene was the most preferred one followed by ubiquitin and PP2A as reference genes. This study identified the best scoring reference genes in various biotic stresses which will contribute for the precise quantification of target genes by RT-qPCR in mulberry as a tool to unravel the disease resistance mechanism.