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.

Figure 1. Box and whisker plots of Cq values for the 4 candidate reference genes in 6 experimental groups (one of three viruses in either winged or wingless adults of Rhopalosiphum padi).

Each box shows the lower 25^th and upper 75^th percentiles with median Cq values; the whiskers mark the lower 5^th and upper 95^th percentiles of the Cq values in each data set. Experimental groups from left to right: WYDV-GPV in winged adult, BYDV-PAV in winged adult, BYDV-GAV in winged adult, WYDV-GPV and wingless adult, BYDV-PAV and wingless adult, and BYDV-GAV and wingless adult.

Figure 2. Determination of optimal reference genes for normalization by GeNorm.

The optimal number of reference genes was determined by pairwise variation V (A) between two sequential normalization factors containing an increasing number of reference genes (n and n +1 reference genes). The pairwise variation begins with the first two and three (V2/3) most stable candidate reference genes (B), terminating with the addition of the least stable one, here V3/4. The cut-off V value is set at 0.15 by the program GeNorm, below which an additional reference gene does not need to be included for calculating the normalization factor.

Figure 3. Mean relative expression values (± SE, n = 3) for endogenous aphid genes ago-1a-1 (A), ago-1a-2 (B), ago-1a-3 (C) and dcr1 (D) in BYDV-GAV-viruliferous winged adults of Rhopalosiphum padi after different virus-feeding durations.

A–D, Expression values for each gene or each region of one gene at each duration were normalized with the reference gene(s) selected by GeNorm and then compared with a one-way ANOVA (p) among these durations with each normalization condition. 2re = 2 best reference genes; 3re = 3 best reference genes; 4re = all 4 reference genes; bad2re = 2 least stable reference genes; EF-1α = EF-1α as the reference gene; 18S = 18S rRNA as the reference gene.

Figure 4. Mean relative expression values (± SE, n = 3) for endogenous aphid genes ago-1a-1 (A), ago-1a-2 (B), ago-1a-3 (C) and dcr1 (D) in BYDV-GAV-viruliferous wingless adults of Rhopalosiphum padi after different virus-feeding durations.

A–D, Expression values for each gene or each region of one gene at each duration were normalized with the reference gene(s) selected by GeNorm and then compared with a one-way ANOVA (p) among these durations with each normalization condition. 2re = 2 best reference genes; 3re = 3 best reference genes; 4re = all 4 reference genes; bad2re = 2 least stable reference genes; EF-1α = EF-1α as the reference gene; 18S = 18S rRNA as the reference gene.

Figure 5. Mean relative expression values (± SE, n = 3) for endogenous aphid genes ago-1a-1 (A), ago-1a-2 (B), ago-1a-3 (C) and dcr1 (D) in BYDV-PAV-viruliferous winged adults of Rhopalosiphum padi after different virus-feeding durations.

A–D, Expression values for each gene or each region of one gene at each duration were normalized with the reference gene(s) selected by GeNorm and then compared with a one-way ANOVA (p) among these durations with each normalization condition. 2re = 2 best reference genes; 3re = 3 best reference genes; 4re = all 4 reference genes; bad2re = 2 least stable reference genes; EF-1α = EF-1α as the reference gene; 18S = 18S rRNA as the reference gene.

Figure 6. Mean relative expression values (± SE, n = 3) for endogenous aphid genes ago-1a-1 (A), ago-1a-2 (B), ago-1a-3 (C) and dcr1 (D) in BYDV-PAV-viruliferous wingless adults of Rhopalosiphum padi after different virus-feeding durations.

A–D, Expression values for each gene or each region of one gene at each duration were normalized with the reference gene(s) selected by GeNorm and then compared with a one-way ANOVA (p) among these durations with each normalization condition. 2re = 2 best reference genes; 3re = 3 best reference genes; 4re = all 4 reference genes; bad2re = 2 least stable reference genes; EF-1α = EF-1α as the reference gene; 18S = 18S rRNA as the reference gene.

Figure 7. Mean relative expression values (± SE, n = 3) for endogenous aphid genes ago-1a-1 (A), ago-1a-2 (B), ago-1a-3 (C) and dcr1 (D) in BYDV-GPV-viruliferous winged adults of Rhopalosiphum padi after different virus-feeding durations.

A–D, Expression values for each gene or each region of one gene at each duration were normalized with the reference gene(s) selected by GeNorm and then compared with a one-way ANOVA (p) among these durations with each normalization condition. 2re = 2 best reference genes; 3re = 3 best reference genes; 4re = all 4 reference genes; bad2re = 2 least stable reference genes; EF-1α = EF-1α as the reference gene; 18S = 18S rRNA as the reference gene.

Figure 8. Mean relative expression values (± SE, n = 3) for endogenous aphid genes ago-1a-1 (A), ago-1a-2 (B), ago-1a-3 (C) and dcr1 (D) in BYDV-GPV-viruliferous wingless adults of Rhopalosiphum padi after different virus-feeding durations.

A–D, Expression values for each gene or each region of one gene at each duration were normalized with the reference gene(s) selected by GeNorm and then compared with a one-way ANOVA (p) among these durations with each normalization condition. 2re = 2 best reference genes; 3re = 3 best reference genes; 4re = all 4 reference genes; bad2re = 2 least stable reference genes; EF-1α = EF-1α as the reference gene; 18S = 18S rRNA as the reference gene.

Figure 9. Mean relative titre of YDVs (± SE, n = 3) in viruliferous winged adults of Rhopalosiphum padi after different virus-feeding durations.

Virus titre is illustrated by relative expression values of the CP (A, C and E) and RTD (B, D and F) gene of YDVs. Expression values for the CP or RTD gene at each duration were normalized with the reference gene(s) selected by GeNorm and then compared with a one-way ANOVA (p) among these durations with each normalization condition. A and B: WYDV-GPV; C and D: BYDV-PAV; E and F: BYDV-GAV. 2re = 2 best reference genes; 3re = 3 best reference genes; 4re = all 4 reference genes; bad2re = 2 least stable reference genes; EF-1α = EF-1α as the reference gene; 18S = 18S rRNA as the reference gene.

Figure 10. Mean relative titre of YDVs (± SE, n = 3) in viruliferous wingless adults of Rhopalosiphum padi after different virus-feeding durations.

Virus titre is illustrated by relative expression values of the CP (A, C and E) and RTD (B, D and F) gene of YDVs. Expression values for the CP or RTD gene at each duration were normalized with the reference gene(s) selected by GeNorm and then compared with a one-way ANOVA (p) among these durations with each normalization condition. A and B: WYDV-GPV; C and D: BYDV-PAV; E and F: BYDV-GAV. 2re = 2 best reference genes; 3re = 3 best reference genes; 4re = all 4 reference genes; bad2re = 2 least stable reference genes; EF-1α = EF-1α as the reference gene; 18S = 18S rRNA as the reference gene.