The Genome-Wide Identification of Stable Internal Reference Genes Related to Delayed Spoilage for Accurate qRT-PCR Normalization in Ethephon-Treated <i>Pueraria thomsonii</i> Benth.
2023
Yujia Liu | Ya Li | Binrong He | Baiyin Yu | Boting Liu | Chongjian Ma | Jie Chen | Tianhua Zhang | Hongrui Zhang | Yuanlong Liu | Xiang Li
<i>Pueraria thomsonii</i> Benth. is a perennial leguminous vine with medicinal and nutritional value. However, rapid postharvest physiological deterioration (PPD) reduces its quality and market value. To detect gene expression levels, the quantitative reverse transcription polymerase chain reaction (qRT-PCR) technique requires stable internal reference genes (IRGs). Our findings indicated that an ethephon (C<sub>2</sub>H<sub>6</sub>ClO<sub>3</sub>P) treatment delayed PPD in <i>P. thomsonii</i> tuberous roots and an RNA-seq analysis revealed a significant number of differentially expressed genes (DEGs). To find stable IRGs for the further identification of the genes associated with delayed PPD in <i>P. thomsonii</i>, eight candidate IRGs of the tuberous roots were screened and assessed using qRT-PCR. The expression stability of these genes was determined and ranked using five different algorithms, including NormFinder, BestKeeper, ΔCt, GeNorm, and ReFinder. Consequently, we identified two genes, <i>PtUBC10</i> and <i>PtACT7</i>, as the best candidate IRGs for qRT-PCR normalization in <i>P. thomsonii</i>, both exposed to ethephon treatment and in different tissues. Moreover, <i>PtUBC10</i> was found to be the most stably expressed IRG of <i>P. thomsonii</i> during the ethephon treatment. The findings of this investigation furnish significant insights for future gene expression analyses concerning the delay of PPD via ethephon administration, which could also be used in other tuberous plants.
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