Validation of Internal Reference Genes for Accurate Gene Expression Analysis in Soybean Roots Interacted with Heterodera glycines and Bacillus megaterium

Soybean cyst nematode (SCN), Heterodera glycines and Bacillus induced gene expression alterations in plant host play a vital role in root invasion. The true differences in the level of genes of interest were accurately measured by real-time PCR (RTqPCR). However, internal reference genes were indispensable for the namalization of transcription. Reference genes for the use of RT-qPCR in soybean under H. glycines and Bacillus interactions have been lacking in soybean. In the study, ten candidate reference genes (ELF1A, ELF1B, CYP2, UBC4, UBQ10, TUB4, G6PD, ACT2/7, ACT11, and CONS4) were evaluated and their expression stability was analyzed in Bacillus megaterium Sneb207 strain coated soybean roots and susceptible and resistant varieties of soybean roots infected by SCN. All the data were statistically analyzed using geNorm, NormFinder, BestKeeper and RefFinder. The results showed that ELF1B and CONS4 were the most stable genes among soybean-Sneb207-nematode interactions. Furthermore, ELF1A and TUB4 were the most stable among soybean-nematode interactions. Moreover, ACT11, G6PD, ELF1B, and CONS4 could also be used as stable reference genes in different soybean varieties interactions with H. glycines and additional Bacillus. In addition, the relative expression of the PR-2 gene was examined in susceptible soybean roots infected with SCN. This confirmed the results of the chosen reference genes. The results provide a basis for RT-qPCR gene expression analysis of soybean-Bacillus-nematode.