IDENTIFICATION AND VERIFICATION OF THE OPTIMAL REFERENCE GENES FOR THE FLORAL DEVELOPMENT OF LUCULIA GRATISSIMA (RUBIACEAE) BY qRT-PCR

Luculia gratissima is a perennial short-day woody plant with important ornamental value, of which flowering is affected by photoperiod. Currently, the studies on the perennial woody plants remains scarce, especially on the short-day woody plants. However, the expression analysis of genes related to flowering is the basis of understanding the molecular mechanism of flowering regulated by photoperiod. Selecting the appropriate reference genes is an essential prerequisite for the qRT-PCR which is extensively applied into gene expression analysis. Therefore, based on the transcriptome data of the floral development of L. gratissima, seven housekeeping genes with low RPKWI variation coefficient were chosen as candidate genes, and four softwares were applied to evaluate the optimal reference genes. The result of the study indicated that Actin, EF-1 alpha and TUB had good stability in geNorm and NormFinder analysis. SKIP, UBQ and TUB had good stability in BestKeeper analysis. In RefFinder analysis, it was found that Actin, EF-1 alpha and TUB had good stability, totally different with that of BestKeeper. In order to further verify the stability of the selected reference genes, the expressions of LgPRR7 and LgFKF1 in different floral development stages were standardized. The result indicated that when three individual genes (Actin, EF-1 alpha and TUB) or the combination of five genes (Actin, EF-1 alpha, TUB, UBQ and SKIP) were used as reference genes, the expression trends of LgPRR7 and LgFKF1 were basically consistent. According to the analysis of economic cost and correction accuracy of qRT-PCR, 2 or 3 gene combinations in Actin, EF-1 alpha and TUB, as reference genes, can accurately correct the expression of genes involved in the floral development of L. grandissima. The results can provide a correction basis for the gene expression analysis on the floral development of L. grandissima in the future.