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

Abstract

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.

Keywords: Broussonetia papyrifera; real-time quantitative PCR; reference gene.

Figure 1

Different Paper Mulberry tissues. (A) leaves, (B) young fruit, (C) mature fruit, (D) stems, (E) roots, and (F) petioles. Scale bar: 1 cm.

Figure 2

Ct values for the nine candidate reference genes in different sample types. (A) Average Ct values. (B) Distributions of Ct values in the test samples. Medians are represented by the horizontal lines, upper quartiles by the top of the boxes, and lower quartiles by the bottom of the boxes, while the upper lines represent the maximum Ct values and the outermost lines the minimum values.

Figure 3

The M values for the nine candidate reference genes were analyzed using the geNorm algorithm on leaves, stems, roots, young fruits, mature fruits, petioles, and overall samples.

Figure 4

Optimal reference gene numbers, assessed by paired variation analyses (V_n/n+1). The V_n/n+1 values were determined using geNorm using a critical V_n/n+1 value of 0.15, where n represents the optimal number of references.

Figure 5

Venn diagrams showing the five reference genes according to the BestKeeper, geNorm, NormFinder, and Delta Ct analyses. Blue, purple, green, and pink ovals, respectively, indicate the five genes with the greatest stability in samples from (A) leaves, (B) stems, (C) young fruits, (D) petioles, (E) roots, (F) mature fruits, and (G) all samples. Genes in overlapping regions of these diagrams were identified as being among the five most stable according to more than one of the utilized algorithms.

Figure 6

Patterns of BpMYB090 expression. (A–F) The front and back of the topmost unfolded and mature leaves of wild-type Paper Mulberry; (G) Microscopic observation of trichomes in the topmost leaves of wild-type Paper Mulberry (Magnification: ×100.0); (H–M) The front and back of the topmost, unfolded, and mature leaves of the mutant Paper Mulberry; (N) Microscopic observation of trichomes in the topmost leaves of the mutant Paper Mulberry (Magnification: ×100.0); (O) RT-qPCR was used to assess BpMYB090 expression in different Paper Mulberry tissues, normalizing gene expression using ACT, UBQ, or a combination of the two. Genes with the lowest stability, H2A, and CDC2, were also included as a comparison. Gene expression was analyzed via the. 2^−ΔΔCt approach.