213185880
Identifying and validating housekeeping hybrid Prunus spp. genes for root gene-expression studies
Prunus rootstock belonging to subgenera Amygdalus (peach), Prunus (plum) and Cerasus (cherry) are either from the same species as the scion or another one. The number of inter-species (including inter-subgenera) hybrids has increased as a result of broadening the genetic basis for stress (biotic and abiotic) resistance/tolerance. Identifying genes associated with important traits and responses requires expression analysis. Relative quantification is the simplest and most popular alternative, which requires reference genes (housekeeping) to normalize RT-qPCR data. However, there is a scarcity of validated housekeeping genes for hybrid Prunus rootstock species. This research aims to increase the number of housekeeping genes suitable for Prunus rootstock expression analysis. Twenty-one candidate housekeeping genes were pre-selected from previous RNAseq data that compared the response of root transcriptomes of two rootstocks subgenera to hypoxia treatment, ‘Mariana 2624’ (P. cerasifera Ehrh.× P. munsoniana W. Wight & Hedrick), and ‘Mazzard F12/1’ (P. avium L.). Representing groups of low, intermediate or high levels of expression, the genes were assayed by RT-qPCR at 72 hours of hypoxia treatment and analyzed with NormFinder software. A sub-set of seven housekeeping genes that presented the highest level of stability were selected, two with low levels of expression (Unknown 3, Unknown 7) and five with medium levels (GTB 1, TUA 3, ATPase P, PRT 6, RP II). The stability of these genes was evaluated under different stress conditions, cold and heat with the hybrid ‘Mariana 2624’ and N nutrition with the hybrids ‘Colt’ (P. avium × P. pseudocerasus Lindl.) and ‘Garnem’ [P. dulcis Mill.× (P. persica L.× P. davidiana Carr.)]. The algorithms of geNorm and BestKeeper software also were used to analyze the performance of these genes as housekeepers. Stability rankings varied according to treatments, genotypes and the software for evaluation, but the gene GBT 1 often had the highest ranking. However, most of the genes are suitable depending on the stressor and/or genotype to be evaluated. No optimal number of reference genes could be determined with geNorm software when all conditions and genotypes were considered. These results strongly suggest that relative RT-qPCR should be analyzed separately with their respective best housekeeper according to the treatment and/or genotypes in Prunus spp. rootstocks.
86596729
Precocious, Dwarfing, and Productive—How Will New Cherry Rootstocks Impact the Sweet Cherry Industry?
ADDITIONAL INDEX WORDS. Prunus avium, Prunus hybrids, high density, virus sensitivity, orchard management SUMMARY. Sweet cherries (Prunus avium L.) can be one of the most profitable tree fruits cultivated in temperate climates. While cherry trees grow naturally to relatively tall heights (≈35 ft (≥10 m)), new size-controlling cherry rootstocks similar to those used in high-density apple (Malus domestica Borkh.) orchards are now a reality. The Gisela (GI.) and Weiroot (W.) series from Germany, the Gran Manier (GM.) series from Belgium, the P-HL series from Czech Republic, 'Tabel Edabriz' from France, and others of international origin are at various stages of scientific and field testing in North America, with some now being used for commer- cial fruit production. These stocks confer several advantageous traits besides vigor control, including precocious fruiting and high productivity. While these beneficial traits are exciting, serious problems also have been documented on occasion, such as small fruit size and tree decline. As many of these rootstocks are interspecific Prunus L. hybrids, might there be significant limitations for fruit quality and orchard longevity? What is known about their tolerance to various soil types and/or climatological stresses? What is known about their susceptibilities to pathogens and pests? Further, with the U.S. and worldwide orchard area planted to fresh-market sweet cherries already expanding to record levels throughout the 1990s and a time-honored agricultural tendency toward overproduction until grower profits are minimized (e.g., recent international apple markets), what might be the future impact of such precocious, productive rootstocks on sweet cherry profitability and sustainable produc- tion? This overview addresses these topics, providing some answers and some areas for future scientific investigation and industry discussion.
89744511
Environmental and orchard bases of peach fruit quality
Abstract This article provides an overview of preharvest factors that affect peach fruit quality attributes. Improvement of peach fruit quality is impossible postharvest. Hence, optimum peach quality at harvest and during postharvest and subsequently, consumer satisfaction, is achievable through understanding the influence of preharvest environmental and orchard factors. ‘Quality’ definition for producers, packers, shippers and consumers is discussed, with a description of the most important peach quality attributes and the physiological mechanisms that affect them. The effect of cultivar, rootstock, harvest time, crop load management, light manipulation, fruit position in the canopy, irrigation, fertilization and the impact of the growing environment on peach and nectarine harvest quality are reviewed. The development of new technologies that help clarify the biological and horticultural bases of preharvest factors on peach fruit quality can help increase peach consumption.
41806549
Vegetable Grafting: History, Use, and Current Technology Status in North America
Grafting of vegetable seedlings is a unique horticultural technology practiced for many years in East Asia to overcome issues associated with intensive cultivation using limited arable land. This technology was introduced to Europe and other countries in the late 20th century along with improved grafting methods suitable for commercial production of grafted vegetable seedlings. Later, grafting was introduced to North America from Europe and it is now attracting growing interest, both from greenhouse growers and organic producers. Grafting onto specific rootstocks generally provides resistance to soilborne diseases and nematodes and increases yield. Grafting is an effective technology for use in combination with more sustainable crop production practices, including reduced rates and overall use of soil fumigants in many other countries. Currently, over 40 million grafted tomato seedlings are estimated to be used annually in North American greenhouses, and several commercial trials have been conducted for promoting use of grafted melon seedlings in openfields.Nevertheless, thereareissuesidentifiedthatcurrently limitadoptionofgraftedseedlings inNorthAmerica. One issue unique to North America is the large number of seedlings needed in a single shipment for large-scale, open-field production systems. Semi- or fully-automated grafting robots were invented by several agricultural machine industries in the 1990s, yet the available models are limited. The lack of flexibility of the existing robots also limits their wider use. Strategies to resolve these issues are discussed, including the use of a highly controlled environment to promote the standardized seedlings suitable for automation and better storage techniques. To use this technology widely in North American fresh vegetable production, more information and locally collected scientific and technical data are needed. Grafting of herbaceous seedlings is a unique horticultural technology practiced for many years in East Asia to overcome issues associated with intensive cultivation using limited arable land for vegetable pro- duction. According to Lee and Oda (2003), a self-grafting technique to produce a large gourd fruit by increasing root-to-shoot ratio through multiple graftings was described in an ancient book written in China in the 5th century and in Korea in the 17th century. The first record of interspecific, herbaceous graft- ing as a yield increase and pest/disease con- trol strategy was for watermelon (Citrullus lanatus (Thunb.) Matsum.& Nakai), using a squash rootstock (Cucurbita moschata Duch.), reportedly developed by a water- melon farmer in Japan (Tateishi, 1927). This watermelon grafting technique was quickly disseminated to farmers through extension research programs of regional agricultural experimental stations in Japan, and then later into Korea, during the late 1920s and early 1930s. Use of grafted seedlings in commer- cial vegetable production occurred as early as the 1930s in Japan for watermelon grafted on Lagenaria siceraria (Mol.) Standl. (Oda, 2002). Research on grafting cucumber (Cucumis sativus L.) also started in the late 1920s,butwidercommercialapplicationsdid not happen until 1960 (Sakata et al., 2008). For members of the Solanaceae, the first record was of eggplant (Solanum melongena L.) grafted on scarlet eggplant (Solanum integrifolium Poir.) in the 1950s (Oda, 1999). Grafting tomato (Lycopersicon escu- lentum Mill.) was introduced commercially in the1960s(LeeandOda,2003).Alongwiththe rapid development of intensive protected cul- tivation technologies using high tunnels and greenhouses, which presumably prevented farmers from continuing traditional crop rota- tion, vegetable grafting became a crucial tool to overcome soilborne diseases and other pests. In the 1990s, nearly 60% of open fields and greenhouses in Japan producing musk- melon (Cucumis melo L.), watermelon, cucumber, tomato, and eggplant were report- edly planted with grafted seedlings (National Research Institute of Vegetables, Ornamental
32330407
Comparative mapping and marker-assisted selection in Rosaceae fruit crops.
The development of saturated linkage maps using transferable markers, restriction fragment length polymorphisms, and micro-satellites has provided a foundation for fruit tree genetics and breeding. A Prunus reference map with 562 such markers is available, and a further set of 13 maps constructed with a subset of these markers has allowed genome comparison among seven Prunus diploid (x = 8) species (almond, peach, apricot, cherry, Prunus ferganensis, Prunus davidiana, and Prunus cerasifera); marker colinearity was the rule with all of them. Preliminary results of the comparison between apple and Prunus maps suggest a high level of synteny between these two genera. Conserved genomic regions have also been detected between Prunus and Arabidopsis. By using the data from different linkage maps anchored with the reference Prunus map, it has been possible to establish, in a general map, the position of 28 major genes affecting agronomic characters found in different species. Markers tightly linked to the major genes responsible for the expression of important traits (disease/pest resistances, fruit/nut quality, self-incompatibility, etc.) have been developed in apple and Prunus and are currently in use for marker-assisted selection in breeding programs. Quantitative character dissection using linkage maps and candidate gene approaches has already started. Genomic tools such as the Prunus physical map, large EST collections in both Prunus and Malus, and the establishment of the map position of high numbers of ESTs are required for a better understanding of the Rosaceae genome and to foster additional research and applications on fruit tree genetics.
2309287
Monitoring gene expression: quantitative real-time rt-PCR.
Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time.RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification or absolute quantification compared to standards that are run in parallel can be performed.This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addition, tips for choosing primers and performing analyses are provided to help the beginner in understanding the technique.
17782461
Real-time RT-PCR normalisation; strategies and considerations
Real-time RT-PCR has become a common technique, no longer limited to specialist core facilities. It is in many cases the only method for measuring mRNA levels of vivo low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, large dynamic range, the potential for high throughout as well as accurate quantification. To achieve this, however, appropriate normalisation strategies are required to control for experimental error introduced during the multistage process required to extract and process the RNA. There are many strategies that can be chosen; these include normalisation to sample size, total RNA and the popular practice of measuring an internal reference or housekeeping gene. However, these methods are frequently applied without appropriate validation. In this review we discuss the relative merits of different normalisation strategies and suggest a method of validation that will enable the measurement of biologically meaningful results.
5943765
Reference genes in real-time PCR
This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.
18307320
Sets Normalization , Applied to Bladder and Colon Cancer Data Estimation Approach to Identify Genes Suited for Transcription-PCR Data : A Model-Based Variance Normalization of Real-Time Quantitative Reverse
Accurate normalization is an absolute prerequisite for correct measurement of gene expression. For quantitative real-time reverse transcription-PCR (RT-PCR), the most commonly used normalization strategy involves standardization to a single constitutively expressed control gene. However, in recent years, it has become clear that no single gene is constitutively expressed in all cell types and under all experimental conditions, implying that the expression stability of the intended control gene has to be verified before each experiment. We outline a novel, innovative, and robust strategy to identify stably expressed genes among a set of candidate normalization genes. The strategy is rooted in a mathematical model of gene expression that enables estimation not only of the overall variation of the candidate normalization genes but also of the variation between sample subgroups of the sample set. Notably, the strategy provides a direct measure for the estimated expression variation, enabling the user to evaluate the systematic error introduced when using the gene. In a side-by-side comparison with a previously published strategy, our modelbased approach performed in a more robust manner and showed less sensitivity toward coregulation of the candidate normalization genes. We used the model-based strategy to identify genes suited to normalize quantitative RT-PCR data from colon cancer and bladder cancer. These genes are UBC, GAPD, and TPT1 for the colon and HSPCB, TEGT, and ATP5B for the bladder. The presented strategy can be applied to evaluate the suitability of any normalization gene candidate in any kind of experimental design and should allow more reliable normalization of RT-PCR data.
206978197
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes
BackgroundGene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem.ResultsWe outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data.ConclusionsThe normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
977404
Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations
The stability of standard gene expression is an elementary prerequisite for internal standardisation of target gene expression data and many so called housekeeping genes with assumed stable expression can exhibit either up- or down-regulation under some experimental conditions. The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index. The index can be compared with further ten target genes to decide, whether they are differentially expressed under an applied treatment. All data processing is based on crossing points. The BestKeeper software tool was validated on four housekeeping genes and 10 members of the somatotropic axis differentially expressed in bovine corpora lutea total RNA. The BestKeeper application and necessary information about data processing and handling can be downloaded on http://www.wzw.tum.de/gene-quantification/bestkeeper.html
9016148
Transcriptome sequencing of Prunus sp. rootstocks roots to identify candidate genes involved in the response to root hypoxia
Root hypoxia in fruit trees affects growth, vegetative development, and reproductive development, which is reflected in low productivity, poor fruit quality, and premature decay of trees. Using Illumina Hiseq2000, we performed transcriptome analysis of roots from two different rootstocks, ‘Mariana 2624’ and ‘Mazzard F12/1,’ which are tolerant and sensitive to hypoxia, respectively. Transcriptomes from control and hypoxia-stressed plants (6, 24, and 72 h) were compared, using Prunus persica (L.) as reference genome. Hypoxic conditions altered the transcription in both genotypes. There were a high number of common differentially expressed genes (DEG) between the two genotypes for each sampling time, but also exclusive DEG for each genotype, with a few DEG that presented opposite modes of regulations during the hypoxia treatment. An important group of DEGs exclusively upregulated in the tolerant genotype are associated to enzymes of posttranslational protein modifications, such as leucine-rich repeat (LRR), kinases and ubiquitin-protein ligases, regulation of transcription, and process of oxide reduction. Singular enrichment analysis of gene ontology (GO), detected at least 115 GOs involved in the response to root hypoxia in the sensitive and/or tolerant genotypes. At least 25 GOs were identified as part of the baseline differences between the genotypes, most GO were disturbed in the sensitive genotype. The contribution from the baseline gene expression to the differential response between the Prunus genotypes is evidence that the resistant genotype is already “prepared” for a hypoxia event. An example are GO BP:0042221 of response to chemical stimulus; BP:0006979 of response to oxidative stress; MF:0016209 of antioxidant activity; MF:0016684 of oxidoreductase activity, acting on peroxide as acceptor; and MF:0004601 of peroxidase activity, which were disturbed only in the sensitive genotype, but not in the tolerant.
1901000
Selection of reliable reference genes for gene expression studies in peach using real-time PCR
BackgroundRT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach.ResultsIn this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT), cyclophilin (CYP2), RNA polymerase II (RP II), phospholipase A2 (PLA2), ribosomal protein L13 (RPL13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), tubblin beta (TUB), tubblin alpha (TUA), translation elongation factor 2 (TEF2) and ubiquitin 10 (UBQ10). All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results.ConclusionOur study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls. GAPDH and ACT also performed poorly and were less stable in our analysis. To achieve accurate comparison of levels of gene expression, two or more reference genes must be used for data normalization. The combinations of TEF2/UBQ10/RP II and TEF2/RP II were suggested for use in all samples and subsets, respectively.
14516227
Class 1 non-symbiotic and class 3 truncated hemoglobin-like genes are differentially expressed in stone fruit rootstocks (Prunus L.) with different degrees of tolerance to root hypoxia
Root hypoxia produced by flooding or over-irrigation limits stone fruit tree development, particularly in orchards established on soils with restricted drainage. To overcome this problem, stone fruit trees are usually grafted on rootstocks (species or hybrid of the Prunus L. genus) with different degrees of tolerance to root hypoxia. However, the molecular base of such variability is largely unknown. In Arabidopsis thaliana (Heynh.), as well as in a number of crops and tree species, hemoglobin (Hb)-like genes stand out among hypoxia-related genes, but no such studies have been done with the Prunus species used as rootstocks. In this study, we analyzed the expression pattern of class 1 non-symbiotic Hb-like (nsHb) and class 3 truncated Hb-like (trHb) genes in Prunus rootstock roots with different responses to this stress. We observed that the putative Prunus nsHb and trHb genes were induced by root hypoxia in all analyzed Prunus genotypes, independently of their tolerance to hypoxia. However, Prunus nsHb and trHb genes had higher expression levels in roots of tolerant rootstocks. Prunus nsHb and trHb genes were also regulated by other abiotic stresses, such as salt stress and low temperatures. Our results suggest that changes in nsHb and trHb expressions could be part of the adaptive mechanisms that have evolved in the Prunus species to survive under hypoxia or other types of environmental stress that commonly challenge stone fruit tree orchards.
13010819
Jasmonic acid transient accumulation is needed for abscisic acid increase in citrus roots under drought stress conditions.
Phytohormones are central players in sensing and signaling numerous environmental conditions like drought stress. In this work, an experimental system based on severe drought was established and hormone profiling together with gene expression of key enzymes involved in abscisic acid (ABA) and jasmonic acid (JA) biosynthesis was studied in roots of citrumelo CPB 4475 (a commercial citrus rootstock) plants. JA concentration transiently increased after a few hours of stress, returning to control levels 30 h after the onset of the condition. A more progressive ABA accumulation was observed, with the onset of this increase at the same time or right after the JA transient accumulation. Molecular data suggested that, at least, part of the hormonal regulation takes place at the biosynthetic level. These observations also pointed to a possible involvement of JA on ABA biosynthesis under stress. To test this hypothesis, JA and ABA biosynthesis were chemically inhibited and subsequently phenotypes rescued by the addition of exogenous hormones. Results showed that the early JA accumulation was necessary for the subsequent ABA increase in roots under stress whereas the opposite could not be stated. The model includes a burst of JA in roots of citrus under severe drought stress conditions that leads to a more progressive ABA accumulation that will induce later plant responses. The present work adds a new level of interaction between JA and ABA at the biosynthetic level that together with the previously described interaction between signal transduction cascades of the two hormones would allow plants to fine-tune specific responses to different stimuli.
12057843
Arabidopsis Nitrate Transporter NRT1.9 Is Important in Phloem Nitrate Transport[W][OA]
The nitrate transporter NRT1.9 is expressed in root companion cells and has an important role in the transport of nitrate in plant phloem tissue. Mutants of NRT1.9 show reduced downward nitrate transport in phloem and increased root-to-shoot nitrate transport in xylem. This suggests that phloem transport serves as fine-tuning for nitrate redistribution. This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport.
9253369
Differential analysis of gene regulation at transcript resolution with RNA-seq
Differential analysis of gene and transcript expression using high-throughput RNA sequencing (RNA-seq) is complicated by several sources of measurement variability and poses numerous statistical challenges. We present Cuffdiff 2, an algorithm that estimates expression at transcript-level resolution and controls for variability evident across replicate libraries. Cuffdiff 2 robustly identifies differentially expressed transcripts and genes and reveals differential splicing and promoter-preference changes. We demonstrate the accuracy of our approach through differential analysis of lung fibroblasts in response to loss of the developmental transcription factor HOXA1, which we show is required for lung fibroblast and HeLa cell cycle progression. Loss of HOXA1 results in significant expression level changes in thousands of individual transcripts, along with isoform switching events in key regulators of the cell cycle. Cuffdiff 2 performs robust differential analysis in RNA-seq experiments at transcript resolution, revealing a layer of regulation not readily observable with other high-throughput technologies.
27061085
Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.
High-throughput mRNA sequencing (RNA-Seq) promises simultaneous transcript discovery and abundance estimation. However, this would require algorithms that are not restricted by prior gene annotations and that account for alternative transcription and splicing. Here we introduce such algorithms in an open-source software program called Cufflinks. To test Cufflinks, we sequenced and analyzed >430 million paired 75-bp RNA-Seq reads from a mouse myoblast cell line over a differentiation time series. We detected 13,692 known transcripts and 3,724 previously unannotated ones, 62% of which are supported by independent expression data or by homologous genes in other species. Over the time series, 330 genes showed complete switches in the dominant transcription start site (TSS) or splice isoform, and we observed more subtle shifts in 1,304 other genes. These results suggest that Cufflinks can illuminate the substantial regulatory flexibility and complexity in even this well-studied model of muscle development and that it can improve transcriptome-based genome annotation.
32110254
The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization.
hasbecome a routine technique for gene expression analysis,allowing accurate high-throughput RNA quantiWcationover a wide dynamic range at a relatively low cost.Although the technique has become well establishedover the past few years, data normalization remainsproblematic and is subject to frequent criticism. Normal-ization is essential to control for experimental errorbetween samples that can be introduced at a number ofstages throughout the procedure. Several di Verent strate-gies can be applied to the normalization of the expres-sion of an RNA of interest (for a review, see [1]). Themost common strategy is to use a reference gene as aninternal standard that is assumed to remain constantbetween experimental groups. It is now clear, however,that in most experimental situations the use of conven-tional reference genes, such as glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) and -actin, isinappropriate due to their variability [1,2]. If the refer-ence gene chosen has a large expression error, the noiseof the assay is increased and detection of small changesbecomes unfeasible. Far worse, if expression of the refer-ence gene is altered by the experimental condition or thedisease under study, the results obtained may be entirelyincorrect. Therefore, it is essential to validate potentialreference genes to establish whether they are appropriatefor a speciWc experimental purpose. We have shown pre-viously that human acidic ribosomal protein (HuPO)was the least variable reference gene investigated in ourmodel of pulmonary tuberculosis (PTB) [3].Despite the fact that many validation exercises havebeen undertaken conWrming the variability of convention-ally used reference genes [1], it has been argued that over-all study conclusions remain unchanged because thisvariability is often similar in the control and interventiongroups [4]. Furthermore, reports that address the net e Vectof reference gene variability on experimental conclusionsare sparse and do not provide the Wnal proof that refer-ence gene variability may have a variable inXuence onstudy conclusions. To address the impact of a validationexercise on intergroup comparisons, we normalized diVer-ent sets of data with diVering reference genes, includingconventionally chosen genes. This report demonstratesthat experimental results are highly dependent on the ref-erence gene chosen. Moreover, whether statistically sig-niWcant diVerences between study groups were present orabsent depended on which reference gene was used.In the study, 21 patients with proven PTB and 21matched healthy volunteers were recruited. E Vector T-cellIFN- ELISPOT responses to TB-speciWc antigens wereused to conWrm that control subjects were not latentlyinfected with
9952932
Defining reference genes in Oryza sativa using organ, development, biotic and abiotic transcriptome datasets
BackgroundReference genes are widely used to normalise transcript abundance data determined by quantitative RT-PCR and microarrays. However, the approaches taken to define reference genes can be variable. Although Oryza sativa (rice) is a widely used model plant and important crop specie, there has been no comprehensive analysis carried out to define superior reference genes.ResultsAnalysis of 136 Affymetrix transcriptome datasets comprising of 373 genome microarrays from studies in rice that encompass tissue, developmental, abiotic, biotic and hormonal transcriptome datasets identified 151 genes whose expression was considered relatively stable under all conditions. A sub-set of 12 of these genes were validated by quantitative RT-PCR and were seen to be stable under a number of conditions. All except one gene that has been previously proposed as a stably expressed gene for rice, were observed to change significantly under some treatment.ConclusionA new set of reference genes that are stable across tissue, development, stress and hormonal treatments have been identified in rice. This provides a superior set of reference genes for future studies in rice. It confirms the approach of mining large scale datasets as a robust method to define reference genes, but cautions against using gene orthology or counterparts of reference genes in other plant species as a means of defining reference genes.
17263393
Genome-Wide Identification of New Reference Genes for qRT-PCR Normalization under High Temperature Stress in Rice Endosperm
qRT-PCR is one of the most popular approaches to analyze specific gene expression level, and stably expressed reference genes are essential to obtain reliable results. However, many reference genes are only stable under certain circumstances and different reference genes might be required in different experiments. High temperature is a common stress that affects rice endosperm development and it has become a hot topic recently. Although study about reference genes at different developmental stages in rice has been reported, these genes may not be suitable to study high temperature mediated responses especially in endosperm. In our quest for proper reference genes to quantify gene expression in rice endosperm under high temperature, we studied 6 candidate genes selected from the transcriptome data and 11 housekeeping genes. All genes were analyzed with qRT-PCR and the expression stability was assessed with software geNorm and NormFinder. Fb15 and eIF-4a were identified as the two most stable genes in endosperm at different developmental stages, while high temperature treatment has a least effect on expression of Fb15 and UBQ5 in rice endosperm. Our results provide some good candidate reference genes for qRT-PCR normalization in rice endosperm under different temperatures.
16831406
Selection and Validation of Reference Genes for Real-Time Quantitative PCR in Hyperaccumulating Ecotype of Sedum alfredii under Different Heavy Metals Stresses
Real-time Quantitative PCR (RT-qPCR) has become an effective method for accurate analysis of gene expression in several biological systems as well as under different experimental conditions. Although with high sensitivity, specificity and broad dynamic range, this method requires suitable reference genes for transcript normalization in order to guarantee reproducible and meaningful results. In the present study, we evaluated five traditional housekeeping genes and five novel reference genes in Hyperaccumulating ecotype of Sedum alfredii, a well known hyperaccumulator for heavy metals phytoremediation, under Cd, Pb, Zn and Cu stresses of seven different durations. The expression stability of these ten candidates were determined with three programs - geNorm, NormFinder and BestKeeper. The results showed that all the selected reference genes except for SAND could be used for RT-qPCR normalization. Among them UBC9 and TUB were ranked as the most stable candidates across all samples by three programs together. For the least stable reference genes, however, BestKeeper produced different results compared with geNorm and NormFinder. Meanwhile, the expression profiles of PCS under Cd, Pb, Zn and Cu stresses were assessed using UBC9 and TUB respectively, and similar trends were obtained from the results of the two groups. The distinct expression patterns of PCS indicated that various strategies could be taken by plants in adaption to different heavy metals stresses. This study will provide appropriate reference genes for further gene expression quantification using RT-qPCR in Hyperaccumulator S. alfredii.
20298407
Using RNA-seq data to select reference genes for normalizing gene expression in apple roots
Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.
91861918
Evaluation of stability and validation of reference genes for real time PCR expression studies in leaves and roots of Prunus spp. rootstocks under flooding
Abstract Molecular oxygen (O2) is involved in several biochemical reactions of plants. The low availability of oxygen to cells can lead to an energy crisis, since O2 is the main substrate of respiratory energy metabolism in aerobic organisms, acting as the final acceptor of electrons in the mitochondrial transport chain. When oxygen becomes insufficient for respiration, the plants begin a hypoxia condition. This situation can occur naturally due to excessive rainfall and the existence of poorly drained soils, causing soil flooding. This condition occurs in the South region of Rio Grande do Sul State, in Brazil, which is the largest Brazilian producer of stone fruits, mainly peaches, therefore, its production is adversely affected by flooding. Since the production of stone fruits plants (Prunus spp.) in the region occurs using rootstocks, the aim of this study was to identify potential reference genes for normalization of RT-qPCR data in leaves and roots of four rootstock of Prunus spp. (Capdeboscq, NR0170401, Marianna 2624 and Myrabolan 29-C) in the soil flooding condition. Eight candidate genes were evaluated: PLA2 (phospholipase A2), CYP2 (cyclophilin 2), Ef1α (elongantion factor 1-alpha), RPL13 (ribosomal protein L13), TUA (tubulin alpha), TUB (tubulin beta), RPII (RNA polymerase II), and 18S rRNA (18S ribosomal RNA). Some algorithms for the stability analysis were used, such as the ΔCt comparative method, BestKeeper, NormFinder, geNorm, and the RefFinder tool. As result, the CYP2 and RPII genes presented greater stability in the peach roostocks, whereas RPII and RPL13 were more stable in the plum rootstocks. The Ef1α gene showed no expression stability in the rootstocks ‘NR0170401’ (both tissues) and ‘Myrabolan 29-C’ (roots). The 18S rRNA gene was shown to be unstable for ‘Capdeboscq’ and ‘Marianna 2624’ leaves, while the TUB and PLA2 genes were not stable for roots of these cultivars, respectively. Analysis of the expression of LDH1 (Lactate Dehydrogenase) and ADH1 (alcohol Dehydrogenase) proved the importance of validation of the reference genes to obtain accurate results in RT-qPCR. Taken together, this study selected the reference genes suitable for the reliable normalization of gene expression data in Prunus spp. under flooding.
83165963
Structure-function relationship in P-type ATPases--a biophysical approach.
P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane. P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions. So far, a crystal structure with atomic resolution is available only for one species, the SR Ca-ATPase. However, biochemical and biophysical studies provide an abundance of details on the function of this class of ion pumps. The aim of this review is to summarize the results of preferentially biophysical investigations of the three best-studied ion pumps, the Na,K-ATPase, the gastric H,K-ATPase, and the SR Ca-ATPase, and to compare functional properties to recent structural insights with the aim of contributing to the understanding of their structure-function relationship.
15631449
PRT6/At5g02310 encodes an Arabidopsis ubiquitin ligase of the N‐end rule pathway with arginine specificity and is not the CER3 locus
The eukaryotic N‐end rule pathway mediates ubiquitin‐ and proteasome‐dependent turnover of proteins with a bulky amino‐terminal residue. Arabidopsis locus At5g02310 shows significant similarity to the yeast N‐end rule ligase Ubr1. We demonstrate that At5g02310 is a ubiquitin ligase and mediates degradation of proteins with amino‐terminal Arg residue. Unlike Ubr1, the Arabidopsis protein does not participate in degradation of proteins with amino‐terminal Phe or Leu. This modified target specificity coincides with characteristic differences in domain structure. In contrast to previous publications, our data indicate that At5g02310 is not identical to CER3, a gene involved in establishment of a protective surface wax layer. At5g02310 has therefore been re‐designated PROTEOLYSIS 6 (PRT6), in accordance with its ubiquitin ligase function.
12556540
SPT6L encoding a putative WG/GW-repeat protein regulates apical-basal polarity of embryo in Arabidopsis.
In eukaryotes, a protein motif consisting of WG/GW repeats, also called the Argonaute (AGO) hook, is thought to be essential for binding AGO proteins to fulfill their functions in RNA-mediated gene silencing. Although a number of WG/GW-containing proteins have been computationally identified in Arabidopsis, their roles in plant growth and development are unknown. Here, we show that the Arabidopsis Suppressor of Ty insertion 6-like (SPT6L) gene, which encodes a protein with C-terminal WG/GW repeats, plays critical roles in embryonic development. SPT6L is evolutionarily conserved only in vascular plants, with varying numbers of C-terminal WG/GW repeats, which are plant-species specific. spt6l mutants formed embryos with an aberrant apical-basal axis, showing insufficient development of the basal domain and embryonic lethality. Expression domains of the class-III homeodomain-leucine zipper (HD-ZIP III) genes PHABULOSA (PHB) and PHAVOLUTA (PHV) were expanded in the spt6l embryo. In contrast, the PLETHORA1 (PLT1) gene, which acts antagonistically to the HD-ZIP III genes in specification of basal fate, was severely down-regulated in the spt6l mutant. Furthermore, the phb phv double mutations partially rescued aberrant basal development in the spt6l background and restored PLT1 expression. Collectively, our results indicate that SPT6L is essential for specification of the apical-basal axis, partly by controlling the HD-ZIP III genes in embryos.
25928903
Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs.
Plant basic helix-loop-helix (bHLH) transcription factors play essential roles in abiotic stress tolerance. However, most bHLHs have not been functionally characterized. Here, we characterized the functional role of a bHLH transcription factor from Arabidopsis, AtbHLH112, in response to abiotic stress. AtbHLH112 is a nuclear-localized protein, and its nuclear localization is induced by salt, drought and abscisic acid (ABA). In addition, AtbHLH112 serves as a transcriptional activator, with the activation domain located at its N-terminus. In addition to binding to the E-box motifs of stress-responsive genes, AtbHLH112 binds to a novel motif with the sequence 'GG[GT]CC[GT][GA][TA]C' (GCG-box). Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with salt and drought tolerance. AtbHLH112 mediates stress tolerance by increasing the expression of P5CS genes and reducing the expression of P5CDH and ProDH genes to increase proline levels. AtbHLH112 also increases the expression of POD and SOD genes to improve reactive oxygen species (ROS) scavenging ability. We present a model suggesting that AtbHLH112 is a transcriptional activator that regulates the expression of genes via binding to their GCG- or E-boxes to mediate physiological responses, including proline biosynthesis and ROS scavenging pathways, to enhance stress tolerance.
2941880
Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots.
Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.