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Dendrobium sinense is a tropical plant endemic to Hainan Island with great ornamental and medicinal values. However, unascertained reference genes restrict the quantitative analysis of gene expression in the species. In this study, using our previous transcriptome data of D. sinense, the 164 candidate genes were identified with low variance (CV≤0.2) and moderate expressions (TPM between 10-300). By GO clustering, 24 candidate genes were selected from different functional groups to avoid the effect of co-expression on further stability analysis. In order to avoid the false positive of genomic DNA that may remain in the template during RT-qPCR amplification, primers across introns were designed in this study, and amplification primers that met the requirements of 15 candidate reference genes were screened by electrophoresis. To ensure the specificity of RT-qPCR amplification, LightCycler 96 was used for melting curve analysis. According to the RT-qPCR data of 15 candidate genes, four tools were used to evaluate the expression stability. The results of multiple algorithm analysis showed that CLP1 & SEC23, SEC23 & CPY71, CLP1 & SEC23 and RNG1L & PECT were the best reference genes under drought stress. After comprehensive sorting, SEC23 and CLP1 were selected as the most stable reference genes under drought stress. In different tissues of D.sinense, ADF11 & IBR5, PRP19 & CLP1, ADF11 & CLP1, ACBP2 & IBR5 were identified as the best reference genes, respectively. After comprehensive analysis, ADF11 and ACBP2 were identified as the most stable reference genes in different tissues. Combined with the above drought stress and different tissue analysis data, IBR5 and CPY71 were finally determined as stable reference genes under comprehensive conditions. The validation of internal reference genes showed that compared with the traditional Actin internal reference, the internal reference genes screened in this study could obtain more accurate RT-qPCR calibration results, indicating that the generated internal reference genes had extremely high stability. In summary, this study provides ideas for identifying stable reference genes using transcriptome data. For the first time, stable reference genes were identified in the whole genome of D.sinense, which laid a foundation for further study of gene expression and function.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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