The optimal number of reference genes for normalization by geNorm analysis.

<p>Optimal number of reference genes required for accurate normalization of gene expression under biotic (A) and abiotic (B) conditions. Average pairwise variations (V) were calculated between the normalization factors NF_n and NF_n+1 to indicate whether inclusion of an extra reference gene adds to the stability of the normalization factor.</p

Ranking of candidate reference genes in order of their expression stability as calculated by RefFinder.

a<p>GM refers to the Geometric mean.</p><p>Ranking of candidate reference genes in order of their expression stability as calculated by RefFinder.</p

Identification and Validation of Reference Genes for Quantitative Real-Time PCR in <i>Drosophila suzukii</i> (Diptera: Drosophilidae)

<div><p>To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. <i>Drosophila suzukii</i>, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (<i>RPL18</i>, <i>RPS3</i>, <i>AK</i>, <i>EF-1β</i>, <i>TBP</i>, <i>NADH</i>, <i>HSP22</i>, <i>GAPDH</i>, <i>Actin</i>, <i>α-Tubulin</i>), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysis of the ten candidate reference genes identified <i>α-Tubulin</i>, <i>TBP</i> and <i>AK</i> as the most stable candidates, while <i>HSP22</i> and <i>Actin</i> showed the lowest expression stability. We used three most stable genes (<i>α-Tubulin</i>, <i>TBP</i> and <i>AK</i>) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes <i>α-Tubulin</i>, <i>TBP</i> and AK, which show good stability, while the result of <i>HSP22</i> has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in <i>D.suzukii</i>.</p></div

Primers used for qRT-PCR analysis.

a<p>the Spotted Wing Fly Base (<a href="http://spottedwingflybase.oregonstate.edu" target="_blank">http://spottedwingflybase.oregonstate.edu</a>);</p>b<p>F and R refer to forward and reverse primers, respectively;</p>c<p>R² refers to the coefficient of determination.</p><p>Primers used for qRT-PCR analysis.</p

Venn diagrams showing the most stable genes identified by geNorm.

<p>The most stable genes were identified using data from various biotic and abiotic conditions. Each circle with a distinct color represents a different condition.</p

Expression levels of candidate reference genes in different samples of <i>D.suzukii</i>.

<p>Expression levels are displayed as cycle threshold (Ct) values of the candidate reference genes used in this study. Data represent mean values ± S.E.M. (n = 150).</p

The expression levels of P-glycoprotein by qRT-PCR.

<p>The transcript levels of P-glycoprotein gene in abamectin-resistant and sensitive strains by qRT-PCR using four different reference genes. Data represent mean values ± S.E.M, and those in the columns followed by the different letters mean significant difference (<i>p</i> = 0.05, Duncan's multiple range test).</p

Ranking of candidate reference genes in order of their expression stability as calculated by BestKeeper.

a<p>SD refers to the standard deviation.</p><p>Ranking of candidate reference genes in order of their expression stability as calculated by BestKeeper.</p

CRN effectors disrupt regions of collinear gene order.

<p>Filled red arrows represent two orthologous CRN genes. Collinear orthologous gene pairs are connected by black (direct) or red (inverted) lines.</p

Recombination patterns contribute to sequence divergence.

<p>(A) CRN genes with different C-terminal regions were shown to have nearly identical N-terminal sequences. (B) CRN genes with different N-terminal regions were shown to have nearly identical C-terminal sequences. (C) CRN genes were generated by combining the N- and C-terminal sequences of the other two CRN genes. (D) Bootscanning plot of PsCRN19 gene sequence against PsCRN170 and PsCRN178 is depicted, with region from nucleotides 44–341 highlighted. Plot is modeled on a pairwise alignment with a step size of 20 and 1000 bootstrap replicates.</p