Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree

Real-time reverse transcriptase PCR is a powerful tool to investigate relevant changes in gene expression during plant somatic embryogenesis (S.E.); however, this method lacks ideal reference genes. To select the most stable reference genes for S.E. studies, the expression profiles of seven frequently used reference genes (18S RNA, eIF-4a, UBQ, ACTB, EF-1a, Histone H3, and 2-TUB) and functional genes (Fe-SOD, Cu/Zn-SOD, and Mn-SOD) were tested in synchronized longan tree embryogenic cultures at different developmental stages at temperatures of 20 °C, 25 °C, and 30 °C. The expression of the 10 candidate genes showed little variation during longan S.E. at 25 °C. The most stable combination for gene expression analysis was UBQ and EF-1a or Fe-SOD and UBQ. However, the expression of these genes varied considerably at different developmental stages at different temperatures. Comprehensive analysis of the results using the software packages geNorm, BestKeeper, and NormFinder revealed that UBQ and Fe-SOD together could be used as internal controls for gene expression analysis in a wide variety of stages of longan S.E. cultured under different temperatures.