Abstract：The selection of a suitable reference gene is an important prerequisite for successful gene expression analysis by quantitative Real-time PCR (qRT-PCR). In this study, expression stability of 22 endogenous candidate genes including 16 new candidate reference genes form transcriptome data of tree peony, including AMPDS, ARFA1C, ERVTP, FCF2PRP, GATA24, GRF9, MBF1A, MHCRP, PAD1FP, PIN1AT, PKSP, PP2A, PP2CFP, PTRP, PUF1639, RPS9 and 6 traditional housekeeping genes, including ARP2, IWS1, GAPDH, TBCC, TUA5 and UBC28 were assessed by qRT-PCR in a diverse set of 33 tissue samples representing different floral development stages of ‘Feng Dan’(Paeonia ostii ‘Feng Dan’), ‘Xi Shi’(Paeonia ostii ‘Xi Shi’) and ‘Que Hao’(Paeonia ostii ‘Que Hao’), different color of ‘Feng Dan’, different samples (stems, leaves, sepals and flowers), different development stages of seeds. The results showed that the most suitable candidate reference genes of tree peony had many differences in the various experimental condition groups. Though there were some minor differences, the results of two programs were similar in the same experimental condition group. ERVTP, PP2CFP, FCF2PRP and UBC28 had the highest expression stability in flowers of different floral development stages in Feng Dan, Xi Shi and Que Hao. RPS9 and ARFA1C were the best genes in flowers of different color of Feng Dan. AMPDS, PUF1639 and MBF1A were the most stable reference genes which were suitable for different samples (stems, leaves, sepals, petals). In seeds of different development stages, RPS9 and PUF1639 had the stable expression. Further, four newly selected reference genes (PUF1639, MBF1A, PP2CFP and RPS9) of tree peony were superior to traditional ones in terms of their expression stability. Identifying stably expressed genes for candidate reference genes from transcriptome database was found to be reliable and efficient. The reference genes selected in this study will be helpful to improve the quality of gene expression studies in tree peony.
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