Selection and Validation of Reference Genes for Normalization of RT-qPCR Analysis in Developing or Abiotic-Stressed Tissues of Loquat (<i>Eriobotrya japonica</i>)

Loquat (Eriobotrya japonica Lindl.) is a subtropical evergreen fruit tree that produces fruits with abundant nutrients and medicinal components. Confirming suitable reference genes for a set of loquat samples before qRT-PCR experiments is essential for the accurate quantification of gene expression. In this study, eight candidate reference genes were selected from our previously published RNA-seq data, and primers for each candidate reference gene were designed and evaluated. The Cq values of the candidate reference genes were calculated by RT-qPCR in 31 different loquat samples, including 12 subgroups of developing or abiotic-stressed tissues. Different combinations of stable reference genes were screened according to a comprehensive rank, which was synthesized from the results of four algorithms, including the geNorm, NormFinder, BestKeeper and ΔCt methods. The screened reference genes were verified by normalizing EjLGA1 in each subgroup. The obtained suitable combinations of reference genes for accurate normalization were GAPDH, EF1α and ACT for floral development; GAPDH, UBCE and ACT for fruit setting; EF1α, GAPDH and eIF2B for fruit ripening; ACT, EF1α and UBCE for leaves under heat stress; eIF2B, UBCE and EF1α for leaves under freezing stress; EF1α, TUA and UBCE for leaves under salt stress; ACT, EF1α and eIF2B for immature pulp under freezing stress; ACT, UBCE and eIF2B for immature seeds under freezing stress; EF1α, eIF2B and UBCE for both immature pulp and seeds under freezing stress; UBCE, TUB and TUA for red-fleshed fruits under cold-storage stress; eIF2B, RPS3 and TUB for white-fleshed fruits under cold-storage stress; and eIF2B, UBCE and RPS3 for both red- and white-fleshed fruits under cold-storage stress. This study obtained different combinations of stable reference genes for accurate normalization in twelve subgroups of developing or abiotic-stressed tissues in loquat. To our knowledge, this is the first report to obtain stable reference genes for normalizing gene expression of abiotic-stressed tissues in E. japonica. The use of the three most stable reference genes could increase the reliability of future quantification experiments.     

Validation of Reference Genes for Accurate Normalization of Gene Expression in Lilium davidii var. unicolor for Real Time Quantitative PCR

Lilium is an important commercial market flower bulb. qRT-PCR is an extremely important technique to track gene expression levels. The requirement of suitable reference genes for normalization has become increasingly significant and exigent. The expression of internal control genes in living organisms varies considerably under different experimental conditions. For economically important Lilium, only a limited number of reference genes applied in qRT-PCR have been reported to date. In this study, the expression stability of 12 candidate genes including α-TUB, β-TUB, ACT, eIF, GAPDH, UBQ, UBC, 18S, 60S, AP4, FP, and RH2, in a diverse set of 29 samples representing different developmental processes, three stress treatments (cold, heat, and salt) and different organs, has been evaluated. For different organs, the combination of ACT, GAPDH, and UBQ is appropriate whereas ACT together with AP4, or ACT along with GAPDH is suitable for normalization of leaves and scales at different developmental stages, respectively. In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression. This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes.     

Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis)

The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a β-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis.     

Selection of reference genes for gene expression studies in astrocytomas

This study was aimed to test a panel of six housekeeping genes (GAPDH, HPRT1, POLR2A, RPLP0, ACTB, and H3F) so as to identify and validate the most suitable reference genes for expression studies in astrocytomas. GAPDH was the most stable and HPRT1 was the least stable reference gene. The effect of reference gene selection on quantitative real-time polymerase chain reaction data interpretation was demonstrated, normalizing the expression data of a selected gene of interest. Thus, GAPDH may be recommended for data normalization in gene expression studies in astrocytomas. Nevertheless, a preliminary validation of reference gene stability is required prior to every study.     

Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.     

Reference gene selection for normalizing gene expression using quantitative real-time PCR in Haemaphysalis longicornis

The Asian longhorned tick, Haemaphysalis longicornis, the dominant species of Ixodidae in Korea, has a wide distribution in East Asia, far-East Russia, and Western Pacific countries, and has recently been discovered in the Eastern states of the United States of America. H. longicornis transmits various pathogens, including Babesia ovate, Rickettsia japonica, and severe fever with thrombocytopenia syndrome virus (SFTSV). Considering its medical importance, in order to understand the physiology of H. longicornis, it is crucial to determine the expression of the genes of interest. Although quantitative real-time PCR (qRT-PCR) has been widely used to analyze gene expression, stably-expressed internal reference genes across samples of different conditions should be selected for the accurate normalization of target gene expression levels. Therefore, in this study, we investigated the expression levels of five candidate reference genes, namely ACT, RPP0, RPL23, TUB, and GAPDH, in H. longicornis under different conditions, including different collection months, developmental stages, and SFTSV infection status. Using four software programs, namely, NormFinder, BestKeeper, geNorm, and RefFinder, their expression stabilities were evaluated. Subsequently, a single gene between RPL23 and RPP0 was validated, which was found to be most stable reference gene after comparing the expression levels of HSP70 determined using different normalization methods.     

Reference gene selection for transcriptional profiling by RT-qPCR in the 28-spotted larger potato ladybird

Henosepilachna vigintioctomaculata is one of the most serious defoliates attacking potatoes. However, studies on functional genes have greatly been limited due to the insufficiency of effective and stable endogenous references to normalize RT-qPCR data. In this report, nine housekeeping genes (RPL4, RPL6, RPL13, RPL32, RPS18, ACT, EF1α, GAPDH and α-TUB) involved in different biological processes were selected. Their expression levels under diverse experimental conditions including developmental stages, tissues, temperatures and host plants were determined using RT-qPCR technology. The tested candidate genes were comprehensively ranked based on five alternative stability analysis methods (Ct value, geNorm, NormFinder, BestKeeper and ReFinder). The results revealed that the optimal internal reference genes varied under different experimental conditions. Any gene pair among the five candidates (RPL4, RPL13, RPL32, RPS18 and EF1α) was a suitable reference gene set under different temperatures and on different host plants. A combination of RPL6 and RPL13 was recommended as the best reference gene set across different developmental stages. A pair of RPS18 and EF1α was ranked as the optimal reference gene combination within different tissues. The most suitable reference genes were RPS18 and RPL13 under four different experimental conditions. Our findings not only establish an accurate and reliable normalization of RT-qPCR data, but also lay a solid foundation for further functional gene researches in H. vigintioctomaculata.     

Is success in detection of Agrilus planipennis related to forest edges?

The emerald ash borer (Agrilus planipennis) is a destructive invasive beetle that has caused mortality of millions of Fraxinus spp. trees in North America. The extended interval between insect establishment, detection and management has allowed this pest to spread over large parts of North America. Artificial purple canopy traps are currently used in national detection surveys for this beetle. The effectiveness of purple canopy traps at low‐to‐moderate A. planipennis population densities in relation to road proximity was evaluated in 2013 and 2014. Transects of traps were established at set distances from roads in northern Michigan near an isolated A. planipennis infestation. It was hypothesized that trap effectiveness is influenced by road proximity, and that traps placed closer to roads were more likely to detect A. planipennis. A significant relationship was established between the mean number of A. planipennis captured on traps and road proximity (nearness), while no significant relationship was observed between detection success and road proximity. These findings suggest establishing traps further from roads (which can be less economically efficient) provides no greater likelihood of detection than establishing traps on or near the road edge. Basal area of non‐ash and ash species, beetle population density, vigour rating, ash tree species and sampling duration were shown to significantly influence the number of A. planipennis captured on traps.     

Reference gene selection for real-time quantitative polymerase chain reaction normalization in “Swingle” citrumelo under drought stress

We describe the first systematic evaluation of reference genes for use in real-time quantitative polymerase chain reaction (qPCR) for water deficit stress studies in the citrus rootstock “Swingle” citrumelo. The expression levels of seven reference genes—cyclophilin (CYP), cathepsin (CtP), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), β-tubulin (TUB), and ADP ribosylation factor (ADP)—during drought stress were tested using geNorm and NormFinder programs. Results from four experimental conditions indicated that EF1α and ADP were the most stable reference genes. Relative expression levels of Δ1-pyrroline-5-carboxylate synthetase (P5CS) was used for reference gene validation.     

黄花大苞姜花药发育qRT-PCR内参基因筛选

qRT-PCR技术具有定量准确、灵敏度高、重复性好等特点,被广泛用于基因表达分析。内参基因的稳定性对于准确分析实验结果非常重要。该研究以黄花大苞姜(Caulokaempferia coenobialis)花粉母细胞时期(PMC)、四分体时期(TET)、成熟花粉时期(MP)的花药组织为材料,基于3个阶段花药转录组表达谱数据以及常用传统内参基因,筛选出Glyceraldehyde 3-phosphate dehydrogenase(GAPDH)、Malate dehydrogenase(MDH)、α-tubulin3(TUA3)、β-tubulin7(TUB7)和Actin6(ACT6)作为候选内参基因,进行qRT-PCR分析;并运用BestKeeper、geNorm和Normfinder软件综合分析5个候选内参基因在黄花大苞姜花药发育过程中的表达稳定性。结果表明:MDH和TUB7的表达最稳定,ACT6的稳定性最差;分别以MDH和TUB7作为内参,分析GBE1在黄花大苞姜花药发育中的表达模式,并与该基因在花药转录组中的表达模式做相关系数分析,3种表达模式结果一致,进一步验证了MDH和TUB7的表达稳定性。这说明MDH和TUB7适合作为qRTPCR分析黄花大苞姜花药发育过程中相关基因表达模式的内参基因。该研究结果为黄花大苞姜花药发育分子机制相关研究奠定了基础,也为姜科花药发育相关内参基因的选择提供了参考。