Sequencing and Validation of Reference Genes to Analyze Endogenous Gene Expression and Quantify Yellow Dwarf Viruses Using RT-qPCR in Viruliferous Rhopalosiphum padi

Abstract

The bird cherry-oat aphid (Rhopalosiphum padi), an important pest of cereal crops, not only directly sucks sap from plants, but also transmits a number of plant viruses, collectively the yellow dwarf viruses (YDVs). For quantifying changes in gene expression in vector aphids, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a touchstone method, but the selection and validation of housekeeping genes (HKGs) as reference genes to normalize the expression level of endogenous genes of the vector and for exogenous genes of the virus in the aphids is critical to obtaining valid results. Such an assessment has not been done, however, for R. padi and YDVs. Here, we tested three algorithms (GeNorm, NormFinder and BestKeeper) to assess the suitability of candidate reference genes (EF-1α, ACT1, GAPDH, 18S rRNA) in 6 combinations of YDV and vector aphid morph. EF-1α and ACT1 together or in combination with GAPDH or with GAPDH and 18S rRNA could confidently be used to normalize virus titre and expression levels of endogenous genes in winged or wingless R. padi infected with Barley yellow dwarf virus isolates (BYDV)-PAV and BYDV-GAV. The use of only one reference gene, whether the most stably expressed (EF-1α) or the least stably expressed (18S rRNA), was not adequate for obtaining valid relative expression data from the RT-qPCR. Because of discrepancies among values for changes in relative expression obtained using 3 regions of the same gene, different regions of an endogenous aphid gene, including each terminus and the middle, should be analyzed at the same time with RT-qPCR. Our results highlight the necessity of choosing the best reference genes to obtain valid experimental data and provide several HKGs for relative quantification of virus titre in YDV-viruliferous aphids.

Reference genes for gene expression studies by RT-qPCR in Brevipalpus yothersi (Acari: Tenuipalpidae), the mite vector of citrus leprosis virus

“…At the same viruliferous and non-viruliferous comparison, we identified Act and RPL32 as the least stable genes. Both genes were revealed as the most reliable ones in Rhopalosphum padi non-viruliferous and viruliferous for Barley yellow dwarf virus 45 , supporting the notion that gene stability must be checked for each specie and condition. The pairwise variation analysis revealed that two reference genes perform trustfully (V2/3 < 0.15) across mite samples with and without CiLV-C, and hence adding more genes would not increase the confidence of the analysis (Fig.…”

Selection and Validation of Reference Genes For qRT-PCR Analysis of Rhopalosiphum padi (Hemiptera: Aphididae)

“…Accordingly, the stability of reference gene expression levels in R. padi under diverse experimental conditions should be evaluated. In a previous study, the expression stability of only four housekeeping genes was assessed for viruliferous winged and wingless R. padi adults (Wu et al, 2014). In the current study, we examined the stability of 11 candidate reference gene expression levels in R. padi in response to various conditions.…”

“…Considering the importance of reference genes for qRT-PCR, previous studies validated reference gene sets in various insect species, including Lymantria dispar (Yin et al, 2020), Drosophila melanogaster (Kim et al, 2020), Pagiophloeus tsushimanus (Chen C. et al, 2020), Phenacoccus solenopsis (Zheng et al, 2019), Chilo partellus (Adeyinka et al, 2019), Harmonia axyridis (Yang et al, 2018), Henosepilachna vigintioctomaculata (Lu et al, 2018), Liriomyza trifolii (Chang et al, 2017), Myzus persicae (Kang et al, 2017), Bradysia odoriphaga (Shi et al, 2016), Lipaphis erysimi (Koramutla et al, 2016), Helicoverpa armigera (Shakeel et al, 2015), Sesamia inferens (Lu et al, 2015), and Spodoptera exigua (Zhu et al, 2014). In a previous study, the gene expression stability of four potential housekeeping genes was evaluated for viruliferous winged and wingless R. padi adults (Wu et al, 2014), which is an insufficient number of genes.…”

Integrative proteomics to understand the transmission mechanism of Barley yellow dwarf virus-GPV by its insect vector Rhopalosiphum padi

“…A laboratory population of nonviruliferous aphids of R. padi was reared on winter wheat ( Triticum aestivum L., cv. Beijing 837), under controlled conditions at 18 °C in a growth chamber 60 .…”