Assessment of Appropriate Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Normalisation in Magnolia sieboldii K. Koch across Various Experimental Conditions
Xiujun Lu 2,3
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College of Horticulture, Shenyang Agricultural University, Shenyang 110866, Liaoning, China
College of Forestry, Shenyang Agricultural University, Shenyang 110866, Liaoning, China
Key Laboratory of Forest Tree Genetics, Breeding and Cultivation of Liaoning Province, China
Xiujun Lu   

College of Forestry, Shenyang Agricultural University, Shenyang 110866, Liaoning, China, China
Submission date: 2021-08-25
Final revision date: 2021-10-17
Acceptance date: 2021-10-31
Online publication date: 2022-02-14
Publication date: 2022-04-06
Pol. J. Environ. Stud. 2022;31(3):2103–2116
Magnolia sieboldii K. Koch is a famous ornamental plant. The deep dormancy of the seeds makes breeding difficult and prevents cultivation for economic purposes. Gene expression profiles of germination-related genes can provide clues for understanding the molecular mechanism of dormancy breaking. The accuracy of results acquired through quantitative real-time polymerase chain reaction (RT-qPCR), which was developed for investigating the gene expression profiles, is determined by the suitability of the selected reference gene. Nevertheless, appropriate RT-qPCR reference genes have not been determined in M. sieboldii. In the present study, seven potential reference genes in diverse M. sieboldii sample subgroups of different organ tissues, seed developmental stages, and seed subjected to imbibition and stratification treatments were checked through RT-qPCR. The gene expression stability was assessed and analysed through three statistical algorithms, namely, geNorm, NormFinder, and BestKeeper. Cyclophilin (CYC) and ubiquitin-conjugating enzyme (UCE) were identified as the best reference genes for diverse organs. CYC and UCE were evaluated as suitable reference genes in seeds in diverse periods of development. Copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) served as optimal reference genes during seed imbibition treatment, whereas Cu/Zn-SOD and 30S ribosomal protein S13 (RPS13) were optimal during seed stratification treatment. In M. sieboldii, Cu/Zn-SOD was recommended for RT-qPCR normalisation, whereas DBP was inappropriate for gene expression analysis. The expression pattern of DELAY OF GERMINATION1 (DOG1) was analysed for validating reference gene creditability better. The present study provides a useful guideline for appropriate reference gene selection for RT-qPCR at diverse experiment conditions for M. sieboldii.