Reference Gene Selection for Real-time Quantitative Reverse-transcription Polymerase Chain Reaction in Flower Buds of Marigold

Marigold (Tagetes erecta) is an important commercial plant because of its ornamental, industrial, and medicinal values. Male-sterile two-type lines are important for heterosis utilization and breeding of marigold. Mining of fertility-related genes may help to elucidate the mechanisms underlying male sterility. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a popular and useful tool for analyzing the expression level of a specific gene. Notably, identifying a suitable reference gene is important for data normalization because it affects the accuracy of quantitative analysis. However, at present, no reference genes are available for marigold. During the current study, 10 candidate reference genes were selected and their expression levels in different samples were analyzed by qRT-PCR. The expression level of each gene was analyzed across different developmental stages of male-sterile and male-fertile flower buds by four software programs (geNorm, NormFinder, BestKeeper, and RefFinder). The results showed that different reference genes are required for male-sterile and male-fertile samples, even if they belong to the same line. For male-sterile samples, the ribosomal protein S5/18S ribosomal RNA (RPS5/18S) gene pair was the best reference for qRT-PCR normalization, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be used as an alternative. For male-fertile samples, elongation factor 1-alpha (EF1 alpha) and RPS5 were the most suitable reference genes, and Ubiquitin-conjugating enzyme (UBC) could be used as an alternative. Beta-actin (ACTB), tubulin beta (TUB), and phenylalanine ammonia-lyase (PAL) should not be used as reference genes because they were the most unstable genes in flower buds of marigold. The results of the current study may facilitate the selection of reference genes for analyzing the expression patterns of genes involved in flower development related to male sterility in marigold.