Screening and Validation of Reference Genes for qPCR Analysis of Flower Color Synthesis Genes in Prunus mume
YE Yong1, Henry Lusekelo INGWE1, ZHENG Zi-Fei1, CHEN Ying-Zhi1, ZHAO Hong-Bo1,2, DONG Bin1,2,*
1 School of Landscape Architecture, Zhejiang A&F University, Hangzhou 311300, China; 2 Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants, Zhejiang A&F University, Hangzhou 311300, China
Abstract：Prunus mume, one of the top 10 traditional famous flowers in China, is widely used in gardens. With the development of molecular biology research of P. mume, it is very important to screen reference genes suitable for qPCR analysis of flower color synthesis genes in P. mume. In this study, a total of 6 P. mume cultivars of 3 different color lines were selected for flower color phenotypic analysis and anthocyanin content determination, and it was found that anthocyanin content was positively correlated with the color of P. mume petals. Meanwhile, the expression of 14 candidate reference genes was detected among different color varieties of P. mume using qPCR, and their expression stability was analyzed using 3 software (including geNorm, NormFinder, BestKeeper) and ΔCt method. Finally, the best reference gene was synthesized using the RefFinder program and validated using genes related to anthocyanin synthesis. The experimental results showed that the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin 1 (ACT1) were the most stable and ACT3 was the least stable. The validation results showed that when GAPDH and ACT1 were used as reference genes, the expression patterns of anthocyanin synthesis-related genes were similar, and more genes showed significant differential expression; when ACT3 was used as reference gene, the expression patterns of anthocyanin synthesis-related genes were different from the results of GAPDH and ACT1 as reference genes, and only a few genes showed significant differential expression. This study provides basic data for in-depth analysis of the expression patterns of genes related to flower color synthesis.
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