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Non-heading Chinese cabbage ( Brassica rapa  ssp.  chinensis  Makino) is an important vegetable member of  Brassica rapa  crops. It exhibits a typical sporophytic self-incompatibility (SI) system and is an ideal model plant to explore the mechanism of SI. Gene expression research are frequently used to unravel the complex genetic mechanism and in such studies appropriate reference selection is vital. Validation of reference genes have neither been conducted in  Brassica rapa  flowers nor in SI trait. In this study, 13 candidate reference genes were selected and examined systematically in 96 non-heading Chinese cabbage flower samples that represent four strategic groups in compatible and self-incompatible lines of non-heading Chinese cabbage. Two RT-qPCR analysis software, geNorm and NormFinder, were used to evaluate the expression stability of these genes systematically. Results revealed that best-ranked references genes should be selected according to specific sample subsets.  DNAJ, UKN1 , and  PP2A  were identified as the most stable reference genes among all samples. Moreover, our research further revealed that the widely used reference genes,  CYP  and  ACP , were the least suitable reference genes in most non-heading Chinese cabbage flower sample sets. To further validate the suitability of the reference genes identified in this study, the expression level of  SRK  and  Exo70A1  genes which play important roles in regulating interaction between pollen and stigma were studied. Our study presented the first systematic study of reference gene(s) selection for SI study and provided guidelines to obtain more accurate RT-qPCR results in non-heading Chinese cabbage.

Identification and Validation of Reference Genes for RT-qPCR Analysis in Non-Heading Chinese Cabbage Flowers