The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that EF1-α (elongation factor 1-alpha), 18S (18s ribosomal RNA), and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin (TUB) was the least stable. ACT5 (actin), RPL3, 18S, and EF1-α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle. Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1-α, 18S, ACT5, RPL3, and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle.

Rhododendron molle G. Don occupies an important phylogenetic node in the genus rhododendron with unique yellow flower and medicinal functions. However, only limited genetic resources and their genome information are available for the generation of rhododendron flowers. The next generation sequencing technologies enables generation of genomic resources in a short time and at a minimal cost, and therefore provide a turning point for rhododendron research. Our goal is to use the genetic information to facilitate the relevant research on flowering and flower color formation in R. molle. In total, 66,026 unigenes were identified, among which 31,298 were annotated in the NCBI non-redundant protein database and 22,410 were annotated in the Swiss-Prot database. Of these annotated unigenes, 9490 and 18,680 unigenes were assigned to clusters of orthologous groups and gene ontology categories, respectively. A total of 7177 genes were mapped to 118 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. In addition, 8266 simple sequence repeats (SSRs) were detected, and these SSRs will undoubtedly benefit rhododendron breeding work. Metabolic pathway analysis revealed that 32 unigenes were predicted to be involved in carotenoid biosynthesis. Our transcriptome revealed 32 engines that encode key enzymes in the carotenoid biosynthesis pathway, including PSY, PDS, LCYB, LCYE, etc. The content of β-carotene was much higher than the other carotenoids throughout the flower development. It was consistent with the key genes expression level in the carotenoid biosynthesis pathway by the Illumina expression profile analysis and the qRT-PCR analysis. Our study identified genes associated with carotenoid biosynthesis in R. molle and provides a valuable resource for understanding the flowering and flower color formation mechanisms in R. molle.

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