Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida)

<p>Abstract</p> <p>Background</p> <p>Gene expression studies are a prerequisite for understanding the biological function of genes. Because of its high sensitivity and easy use, quantitative PCR (qPCR) has become the gold standard for gene expression quantification. To normalise qPCR measurements between samples, the most prominent technique is the use of stably expressed endogenous control genes, the so called reference genes. However, recent studies show there is no universal reference gene for all biological questions. Roses are important ornamental plants for which there has been no evaluation of useful reference genes for gene expression studies.</p> <p>Results</p> <p>We used three different algorithms (BestKeeper, geNorm and NormFinder) to validate the expression stability of nine candidate reference genes in different rose tissues from three different genotypes of <it>Rosa hybrida </it>and in leaves treated with various stress factors. The candidate genes comprised the classical "housekeeping genes" (<it>Actin, EF-1α, GAPDH</it>, <it>Tubulin </it>and <it>Ubiquitin</it>), and genes showing stable expression in studies in <it>Arabidopsis </it>(<it>PP2A, SAND, TIP </it>and <it>UBC</it>). The programs identified no single gene that showed stable expression under all of the conditions tested, and the individual rankings of the genes differed between the algorithms. Nevertheless the new candidate genes, specifically, <it>PP2A </it>and <it>UBC</it>, were ranked higher as compared to the other traditional reference genes. In general, <it>Tubulin </it>showed the most variable expression and should be avoided as a reference gene.</p> <p>Conclusions</p> <p>Reference genes evaluated as suitable in experiments with <it>Arabidopsis thaliana </it>were stably expressed in roses under various experimental conditions. In most cases, these genes outperformed conventional reference genes, such as <it>EF1-α </it>and <it>Tubulin</it>. We identified <it>PP2A</it>, <it>SAND </it>and <it>UBC </it>as suitable reference genes, which in different combinations may be used for normalisation in expression analyses via qPCR for different rose tissues and stress treatments. However, the vast genetic variation found within the genus <it>Rosa</it>, including differences in ploidy levels, might also influence expression stability of reference genes, so that future research should also consider different genotypes and ploidy levels.</p

Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest

THE THERMAL AGING AND TWO WAY MEMORY EFFECT (TWME) IN Cu-Al-Be SHAPE MEMORY ALLOY

In this study T.T.P. (Temperature, Time, Precipitation) curves of β-Cu-Al-Be have been determined by Differential Scanning Calorimetry technique. This alloy have been found to shows a remarkable thermal stability. The training experiments and the obtained two way memory effect (TWME) have shown that this alloy exhibits a good ability to memory properties comparable to the ones obtained on Cu-Zn-Al and Cu-Al-Ni. The aging kinetics of TWME have been drawn for several temperatures. This work has confirmed the hypothesis already advanced concerning Cu-Zn-Al and Cu-Al-Ni alloys, namely that the TWME aging is mainly due to the dislocation recovery, and in this case is independent of the precipitation phenomenon

Temperature dependence of the second order elastic constants of Cu-Zn-Al shape memory alloy in its martensitic and beta phases

The temperature dependence of the sound velocities for 13 propagation modes has been measured in a single crystal of Cu-Zn-Al monoclinic 1 8 R martensite, using the pulse-echo method. By numerical procedure the complete set of nine second-order adiabatic elastic constants ( C ij ) of the closest orthorhombic reference phase, their relative thermal variation ( Γ ij ) , and the Debye temperature ( θ D ) have been obtained. The values found in the martensitic phase have been compared to data avalaible for the high-temperature bcc β -phase in the same alloy system. The velocity surfaces in the corresponding crystallographic directions of both phases have also been computed at different temperatures. It has been shown that the mechanical stability of the lattice for some particular distortions decreases as the transformation temperature is approached in both the martensitic as well as in the β phase

IN SITU STUDY OF THE MARTENSITIC TRANSFORMATION OF Cu-Zn-Al SHAPE MEMORY ALLOY SINGLE CRYSTALS

We have studied by synchrotron white beam X-Ray Topography in situ and in real time, the thermoelastic transformation of the Cu-Zn-Al shape memory alloy. The evolution of the first martensite lath has been followed during the temperature variation and its interaction with the crystalline defects of the parent phase has been shown

Dynamic Study of the Thermoelastic Transformation of Cu-Zn-Al single crystals by X-Ray diffraction

The martensitic transformation of Cu-Zn-Al- single crystals has been followed in situ and in real time by synchrotron X-ray topography. We have shown that at the Ms temperature, the transformation proceeds by nucleation and growth of transformation variants associated by self-accomodating pairs. The nucleation of these variants is triggered by the crystal substructure. When the transformation is multivariant we have displayed the development of elastic stresses, accompanying the growth of variants, inducing a reversible curvature of the lattice planes