Functional genomics in whitefly, Bemisia tabaci is gaining impetus due to its polyphagous nature, worldwide distribution and recently sequenced whole genome. These studies require an in-depth evaluation and validation of reference genes in different development stages and variable experimental setups. Normalization with reference genes is an essential step in the gene expression studies. Rather than selecting a reference gene empirically, the suitability of these genes must be validated for an individual organism, its specific stage or even for particular experimental conditions. The Quantitative real-time polymerase chain reaction (RT-qPCR) has evolved as an efficient and widely used technique for precise monitoring of gene expression. The prime focus of this study was to identify candidate reference genes in different developmental stages (adults, nymphs, eggs), sex (male and female), hosts (Gossypium hirsutum, G. arboreum), and under insecticidal and starvation stress. Expression stability of these genes in different experimental samples was evaluated by employing five different computational algorithms such as NormFinder, BestKeeper, Comparative delta-CT, geNorm and RefFinder. Our results identified a different set of reference genes under each experimental setup such as electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), ubiquitin (UBIQ) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as best reference genes in adult whitefly. In addition, glutathione S-transferase (GST) in eggs, cyclophilin (CYCLOPH) in red eyed nymph, GAPDH in third instar, tubulin (Tub) in female, ubiquitin ribosomal protein S27 (UBIRPS2) in male, succinate dehydrogenase complex subunit B (SDHB) under insecticidal stress, ETF-QO under starvation stress, UBIRPS2 under host influence were the top most stable genes. Our studies report the importance of selection of specific reference genes for accurate gene expression studies under various experimental setups in B. tabaci.

粉虱，烟粉虱的功能基因组学由于其多食性，遍布全球的分布以及最近对整个基因组进行测序，该技术正获得动力。这些研究需要在不同的开发阶段和可变的实验设置中对参考基因进行深入评估和验证。参考基因的标准化是基因表达研究中必不可少的步骤。并非凭经验选择参考基因，这些基因的适用性必须针对单个生物，其特定阶段甚至特定实验条件进行验证。实时定量聚合酶链反应（RT-qPCR）已发展成为一种高效且广泛使用的技术，用于精确监控基因表达。这项研究的主要重点是鉴定处于不同发育阶段（成人，若虫，卵），性别（男性和女性），宿主（陆地棉（Gossypium hirsutum，G。arboreum），并在杀虫和饥饿胁迫下。通过采用五种不同的计算算法（例如NormFinder，BestKeeper，Comprehensive delta-CT，geNorm和RefFinder）评估这些基因在不同实验样品中的表达稳定性。我们的结果在每个实验设置下确定了一组不同的参考基因，例如电子转移黄素蛋白-泛醌氧化还原酶（ETF-QO），泛素（UBIQ）和甘油醛3-磷酸脱氢酶（GAPDH）是成年粉虱的最佳参考基因。此外，鸡蛋中的谷胱甘肽S-转移酶（GST），红眼若虫GAPDH中的亲环蛋白（CYCLOPH）在三龄幼虫中，雌性中的微管蛋白（Tub），雄性中的泛素核糖体蛋白S27（UBIRPS2），杀虫胁迫下的琥珀酸脱氢酶复合亚基B（SDHB），饥饿胁迫下的ETF-QO，宿主影响下的UBIRPS2是最稳定的基因。我们的研究报告了选择特定参考基因对于烟粉虱各种实验设置下准确基因表达研究的重要性。