Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the preferred method for gene expression research, but normalization based on suitable reference genes (RGs) is the key to obtaining reliable gene expression results. In this study, we selected six “commonly used” RGs, two “traditional” housekeeping genes (HKGs), and four novel genes as candidate RGs based on 54 publicly available peel transcriptome libraries that include data from development, bagging, and post-harvest cryopreservation studies of four pear cultivars. The results of this multifaceted assessment from transcriptome and qRT-PCR analyses consistently revealed that ACT6/7/8/9 had the best expression stability among all candidate RGs, and expression of the novel RGs showed greater stability compared with the other “commonly used” RGs and “traditional” HKGs. Among the candidate RGs, ACT6/7/8/9 and Nucleosome Assembly Protein 1 (NAP1) showed superior expression stability and abundance, and they were recommended as the optimal RG combination for gene expression normalization in pear peel. These genome-wide findings provide more reasonable RG usage specifications, and additional useful and reliable RGs as resources for accurate study of gene expression in pear peel studies of different cultivars.

中文翻译：

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梨皮基因表达标准化最佳参考基因的全基因组鉴定和验证

定量逆转录聚合酶链反应 (qRT-PCR) 是基因表达研究的首选方法，但基于合适的参考基因 (RG) 的标准化是获得可靠基因表达结果的关键。在本研究中，我们基于 54 个公开的果皮转录组文库（包括发育、装袋和收获后的数据）选择了六个“常用”RG、两个“传统”管家基因 (HKG) 和四个新基因作为候选 RG。四个梨品种的冷冻保存研究。转录组和 qRT-PCR 分析的多方面评估结果一致表明 ACT6/7/8/9 在所有候选 RGs 中具有最好的表达稳定性，并且与其他“常用”RGs 相比，新型 RGs 的表达表现出更高的稳定性RG 和“传统”HKG。在候选 RGs 中，ACT6/7/8/9 和核小体组装蛋白 1 (NAP1) 表现出优异的表达稳定性和丰度，它们被推荐为梨果皮基因表达标准化的最佳 RG 组合。这些全基因组的发现提供了更合理的 RG 使用规范，以及额外有用和可靠的 RG，作为在不同品种的梨皮研究中准确研究基因表达的资源。