Selection and validation of reference genes for quantitative real-time PCR in different tissues of Clematis lanuginosa

Abstract

BackgroundThe lack of reference genes makes it difficult to conduct molecular biology research on the plant genus, Clematis L. Clematis lanuginosa belongs to Sect. Viticella DC of Clematis L. It is also an important ornamental cultivated variety parent of the early and late large-flowered groups. Studying the reference genes of C. lanuginosa in different tissues will provide a theoretical basis for the reference selection of early and late large-flowered groups of Clematis, which could promote research progress on molecular biology of ornamental Clematis. ResultsThe roots, stems, leaves, sepals, stamens, and carpels of C. lanuginosa were used as research materials, and seven candidate reference genes were used for quantitative real-time PCR analysis. Comprehensive stability analysis using geNorm, NormFinder, BestKeeper, and RefFinder software showed that suitable reference genes in C. lanuginosa root, stem, and leaf were PP2A-2 and UBC34; and in floral tissue were UBC34, PP2A-2, and ARP7. These reference genes can be used as internal reference either alone or in combination. The pairwise variation value evaluated with geNorm software showed that two internal reference genes were needed for gene expression correction in the tissues. In the floral organs, three reference genes were required for gene expression correction. ConclusionsOur results provide a foundation for future gene expression analysis of C. lanuginosa and guidance for the screening of reference genes in Clematis.