Identification of the Optimal Quantitative RT-PCR Reference Gene for Paper Mulberry (Broussonetia papyrifera)

Paper Mulberry (Broussonetia papyrifera) possesses medicinal, economic, and ecological significance and is extensively used for feed production, papermaking, and ecological restoration due to its ease of propagation, rapid growth rate, and strong stress resistance. The recent completion of the sequencing of the Paper Mulberry genome has prompted further research into the genetic breeding and molecular biology of this important species. A highly stable reference gene is essential to enhance the quantitative analysis of functional genes in Paper Mulberry; however, none has been identified. Accordingly, in this study, the leaves, stems, roots, petioles, young fruits, and mature fruits of Paper Mulberry plants were selected as experimental materials, and nine candidate reference genes, namely, α-TUB1, α-TUB2, β-TUB, H2A, ACT, DnaJ, UBQ, CDC2, and TIP41, were identified by RT-qPCR. Their stability was assessed using the geNorm, Normfinder, Delta Ct, BestKeeper, and RefFinder algorithms, identifying ACT and UBQ as showing the greatest stability. The expression of BpMYB090, which regulates the production of trichomes, was examined in the leaves of plants of the wild type (which have more trichomes) and mutant (which have fewer trichomes) at various developmental stages to validate the results of this study. As a result, their identification addresses a critical gap in the field of Paper Mulberry research, providing a solid foundation for future research that will concentrate on the characterization of pertinent functional genes in this economically valuable species.