Abstract
Perennial tree Dalbergia odorifera T. Chen could form the precious heartwood used to produce chinese traditional medicine, rosewood furniture and fragrances. However the formation of heartwood is time-consuming and low efficient, leading to the severe destruction of its wild resources. Thus, it is urgent to study the molecular mechanism of heartwood formation in D. odorifera. But till now, there is no report about the reference gene selection in this species. In this study, the expression stability of nine candidate reference genes were evaluated across different tissues and stems treated by wound and chemical stimulators. Four algorithms were applied to obtain the robust genes. The results support HIS2, GAPDH, and CYP to be the most stable reference genes in samples under different wound treatments while DNAj was the least stable. In different tissues, HIS2, UBQ, and RPL were the most stable reference genes while DNAj was the least stable. The selected reference genes were validated through the normalization of the qRT-PCR data of six heartwood related genes in terpene biosynthesis pathway and ethylene signal pathway. The results showed that their expression levels were accurate when they were normalized by the most stable reference gene HIS2, or by the combination of the two or three most stable reference genes. These results demonstrated that these selected reference genes are reliable.
Highlights
The formation of heartwood is regulated temporally and spatially, which is a long and complex physiological process[13,14]
Even though several large-scale gene expression tools have been developed, the quantitative real-time PCR assay with its advantages on the high sensitivity, accuracy, specificity, and good reproducibility is normally used as the small-scale tool in gene expression analysis[19,20]
Candidate reference genes were selected according to the previous reports in other plants or the expression pattern in D. odorifera transcriptome data[31,32,33,34]
Summary
The formation of heartwood is regulated temporally and spatially, which is a long and complex physiological process[13,14]. Www.nature.com/scientificreports the trunk begins to form heartwood after growing for 8–10 years. It takes at least 30 years before it can be used as timber. The geNorm software conducts pair-wise comparison and calculates the M value of all candidate genes[27]. The deltaCt method compares relative expression of pairs of reference genes within each sample[30]. No report is available concerning reference gene selection in D. odorifera, especially for its heartwood formation by different wound treatments. The expression stability of nine candidate reference genes were assessed in D. odorifera samples exposed to different wound treatments or in different tissues using geNorm, Normfinder, Bestkeeper, and deltaCt algorithms. The relative expression of six genes including genes in the terpene biosynthesis pathway and ethylene signal pathway were used to validate the reliability of the selected reference genes and to compare the effect of different wound treatments
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