Validation of reference genes for real-time quantitative PCR normalisation in non-heading Chinese cabbage
Dong Xiao A B , Ning-Wen Zhang B , Jian-Jun Zhao B C , Guusje Bonnema B D and Xi-Lin Hou A D
+ Author Affiliations
- Author Affiliations
A State Key Laboratory of Crop Genetics and Germplasm Enhancement; Horticultural College, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
B Laboratory of Plant Breeding, Wageningen University, The Netherlands.
C Horticultural College, Hebei Agricultural University, Baoding, Hebei, China.
D Corresponding author. Emails: guusje.bonnema@wur.nl; hxl@njau.edu.cn
Functional Plant Biology 39(4) 342-350 https://doi.org/10.1071/FP11246
Submitted: 29 October 2011 Accepted: 7 March 2012 Published: 24 April 2012
Abstract
Non-heading Chinese cabbage is an important vegetable crop that includes pak choi, caixin and several Japanese vegetables like mizuna, mibuna and komatsuna. Gene expression studies are frequently used to unravel the genetics of complex traits and in such studies the proper selection of reference genes for normalisation is crucial. We assessed the expression of 13 candidate reference genes including ACTIN, ACTIN-1, ACTIN-2, GAPDH, Tub_α, CyP, EF1-α, 18S rRNA, UBQ, UBC30, PPR, PP2A and MDH. Their expression stabilities were analysed using two programs, geNorm and NormFinder, in 20 different samples that represent four strategic groups. Results showed that no single gene was uniformly expressed in all tested samples. ACTIN and CyP are proposed as good reference genes when studying developmental stages. CyP, Tub_α and UBC30 are good reference genes when studying different tissues (from flowering to seed set). CyP and Tub_α are the most stable reference genes under biotic stress treatments using the fungi Peronospora parasitica and Alternaria brassicicola. UBC30, EF1-α and ACTIN are recommended for normalisation in abiotic stress studies, including hormone, salt, drought, cold and heath treatments. Moreover, at least five reference genes (ACTIN, CyP, UBC30, EF1-α and UBQ) are required for accurate qRT–PCR data normalisation when studying gene expression across all tested samples.
Additional keywords: Brassica rapa ssp. chinensis, gene expression, qRT-PCR, reference genes.
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