Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug

<div><p>The brown marmorated stink bug (<i>Halyomorpha halys</i>) has emerged as one of the most important invasive insect pests in the United States. Functional genomics in <i>H</i>. <i>halys</i> remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (<i>RPS26</i>, <i>EF1A</i>, <i>FAU</i>, <i>UBE4A</i>, <i>ARL2</i>, <i>ARP8</i>, <i>GUS</i>, <i>TBP</i>, <i>TIF6</i> and <i>RPL9</i>) for their stability across various treatments in <i>H</i>. <i>halys</i>. Our treatments included two biotic factors (tissues and developmental stages) and two stress treatments (RNAi injection and starvation). Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). The qRT-PCR results indicated <i>ARP8</i> and <i>UBE4A</i> exhibit the most stable expression across tissues and developmental stages, <i>ARL2</i> and <i>FAU</i> for dsRNA treatment and <i>TBP</i> and <i>UBE4A</i> for starvation treatment. Following the dsRNA treatment, all genes except <i>GUS</i> showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in <i>H</i>. <i>halys</i>, we performed RNAi by administering dsRNA of target gene (catalase) through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed.</p></div

Description of candidate reference genes for qRT-PCR studies in brown marmorated stink bug.

<p>Description of candidate reference genes for qRT-PCR studies in brown marmorated stink bug.</p

RefFinder ranking of eight candidate reference genes for for qRT-PCR studies in brown marmorated stink bug.

<p>RefFinder ranking of eight candidate reference genes for for qRT-PCR studies in brown marmorated stink bug.</p

Average expression stability (M) and ranking of candidate reference genes in brown marmorated stink bug as calculated by geNorm.

<p>M values and rankings are presented for brown marmorated stink bug under different experimental conditions as indicated in each figure. Details on candidate reference genes and primer sequences are provided in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152730#pone.0152730.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152730#pone.0152730.t002" target="_blank">2</a>, respectively.</p

Ct values±S.D. (standard deviation) obtained for candidate reference genes in brown marmorated stink bug under different experimental conditions.

<p>Each data point represents the Mean±S.D. of Ct values for three biological replications in each treatment. Details on candidate reference genes are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152730#pone.0152730.t001" target="_blank">Table 1</a>.</p

Recommended Reference Genes for Quantitative PCR Analysis in Soybean Have Variable Stabilities during Diverse Biotic Stresses

<div><p>For real-time reverse transcription-PCR (qRT-PCR) in soybean, reference genes in different tissues, developmental stages, various cultivars, and under stress conditions have been suggested but their usefulness for research on soybean under various biotic stresses occurring in North-Central U.S. is not known. Here, we investigated the expression stabilities of ten previously recommended reference genes (<i>ABCT</i>, <i>CYP</i>, <i>EF1A</i>, <i>FBOX</i>, <i>GPDH</i>, <i>RPL30</i>, <i>TUA4</i>, <i>TUB4</i>, <i>TUA5</i>, and <i>UNK2</i>) in soybean under biotic stress from <i>Bean pod mottle virus</i> (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two‐spotted spider mite (TSSM). BPMV, PMD, SBA, and TSSM are amongst the most common pest problems on soybean in North-Central U.S. and other regions. Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Reference genes showed variability in their expression as well as stability across various stressors and the best reference genes were stress-dependent. <i>ABCT </i>and <i>FBOX </i>were found to be the most stable in soybean under both BPMV and SBA stress but these genes had only minimal to moderate stability during PMD and TSSM stress. Expression of <i>TUA4</i> and <i>CYP</i> was found to be most stable during PMD stress; <i>TUB4</i> and <i>TUA4</i> were stable under TSSM stress. Under various biotic stresses on soybean analyzed, <i>GPDH</i> expression was found to be consistently unstable. For all biotic stressors on soybean, we obtained pairwise variation (V_2/3) values less than 0.15 which suggested that combined use of the two most stable reference genes would be sufficient for normalization. Further, we demonstrated the utility of normalizing the qRT-PCR data for target genes using the most stable reference genes validated in current study. Following of the recommendations from our current study will enable an accurate and reliable normalization of qRT-PCR data in soybean under biotic stress.</p></div

Stability values of candidate reference genes in brown marmorated stink bug as calculated by NormFinder.

<p>Stability values are indicated for brown marmorated stink bug under different experimental conditions as indicated in each Fig</p

Pairwise variation (V) analysis of the candidate reference genes in brown marmorated stink bug using geNorm.

<p>The pairwise variation (V_n/n+1) between the normalization factors NF_n and NF_n+1 (shown along x-axis) is calculated to determine the optimal number of reference genes for normalization in brown marmorated stink bug under different experimental conditions as indicated. Each bar indicates change in normalization when adding reference genes stepwise according to rankings in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152730#pone.0152730.g002" target="_blank">fig 2</a>.</p

BestKeeper ranking of eight candidate reference genes for for qRT-PCR studies in brown marmorated stink bug.

<p>BestKeeper ranking of eight candidate reference genes for for qRT-PCR studies in brown marmorated stink bug.</p

Usefulness of identified reference genes in detecting target gene expression.

<p>Relative expression levels (Mean± Standard Deviation) of two target genes (Glyma.05g184500 and Glyma.06g147100) in soybean under biotic stress by soybean aphid (SBA) are shown. Expression values of both target genes were normalized with respect to the most stable gene pair (<i>ABCT</i> and <i>FBOX</i>) and the least stable gene pair (<i>TUA4</i> and <i>GPDH</i>) during SBA stress in soybean (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134890#pone.0134890.t004" target="_blank">Table 4</a>). The asterisk (*) represents a significant difference (<i>P</i> <u><</u> 0.05) in SBA-infested soybean compared to uninfested control. N.S: No significant difference (<i>P</i> > 0.05).</p