scholarly journals Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover (Trifolium repens L.) Involved in Five Abiotic Stresses

Plants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 996
Author(s):  
Qi Pu ◽  
Zhou Li ◽  
Gang Nie ◽  
Jiqiong Zhou ◽  
Lin Liu ◽  
...  
Keyword(s):  
Real Time ◽  
White Clover ◽  
Trifolium Repens ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Grass Species ◽  
Stable Gene ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Trifolium Repens L

White clover (Trifolium repens L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC, and ABC) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that 60SrRNA and ACT101 were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, SAMDC, UBQ, 60SrRNA, and ACT101 were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of Cyt-Cu/Zn SOD and CAT confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.

Selection of optimal reference genes for quantitative RT-PCR transcript abundance analysis in white clover (Trifolium repens L.)

2018 ◽  
Vol 45 (7) ◽  
pp. 737 ◽  
Author(s):  
Rafael Narancio ◽  
Ulrik John ◽  
John Mason ◽  
German Spangenberg
Keyword(s):  
White Clover ◽  
Trifolium Repens ◽  
Reference Genes ◽  
Mrna Level ◽  
Housekeeping Genes ◽  
Transcript Abundance ◽  
Mrna Levels ◽  
Qrt Pcr ◽  
Trifolium Repens L ◽  
Suitable Reference

Quantitative reverse transcription PCR (qRT-PCR) is a widely used method for transcript abundance analyses in plants. Relative quantification by qRT-PCR requires the use of a stably expressed reference gene. There are many ‘housekeeping’ genes reported in different plant species that are used as reference genes. However, it is important that the steady-state mRNA levels of these housekeeping genes are confirmed across different conditions and tissues in each species studied. Prior to this study, no comprehensive work had been performed in identifying optimal reference genes in white clover (Trifolium repens L.). To identify suitable reference genes in white clover, we analysed the transcript abundance stability of seven candidate genes in two organs (leaves and stolons) across two treatments (water-limited and well-watered). ΔCt, NormFinder and ANOVA tests were carried out to evaluate the mRNA level stability of candidate reference genes. According to the ΔCt results, the genes with the most stable mRNA levels were EF1α and ACT11. When stability among groups was evaluated by NormFinder, UBQ was the most stable across all organs and treatments. By multiple criteria, EF1α, followed by ACT11 and UBQ, was the most stably-expressed gene across organs and treatments, and each of these are recommended as reference genes for transcript abundance studies in white clover.

scholarly journals Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Madhab Kumar Sen ◽  
Kateřina Hamouzová ◽  
Pavlina Košnarová ◽  
Amit Roy ◽  
Josef Soukup
Keyword(s):  
Reference Gene ◽  
Herbicide Resistance ◽  
Reference Genes ◽  
Gene Selection ◽  
High Yield ◽  
Suitable Reference Gene ◽  
Grass Species ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

scholarly journals Egg Production by Hens Fed with Homemade or Commercial Concentrate in a Cage-Free System

2021 ◽  
Vol 13 (12) ◽  
Author(s):  
Fernando González-Cerón ◽  
María Alejandra Padilla-Jiménez ◽  
Arturo Pro-Martínez ◽  
José Isidro Alejos de la Fuente ◽  
Leodan Tadeo Rodríguez-Ortega ◽  
...  
Keyword(s):  
Rhode Island ◽  
White Clover ◽  
Trifolium Repens ◽  
Egg Production ◽  
Egg Mass ◽  
Productive Performance ◽  
Experimental Conditions ◽  
Free System ◽  
Trifolium Repens L ◽  
System Methodology

Objective: To evaluate two types of concentrate (homemade and commercial), in laying percentage (LP, %), weight (EW, g), and egg mass (EM, g bird-1 d-1), among hens in a cage-free system. Methodology: Sixty hens aged 37 weeks (Rhode Island Red and Barred Plymouth Rock) were assigned two treatments: COM, 150 g of commercial concentrate bird-1 d-1and CAS, 150 g of homemade concentrate bird-1 d-1. The birds were managed in a cage-free system with access to a meadow of white clover (Trifolium repens L.). LP, EW and EM were evaluated for 11 weeks. Results: LP was different between treatments (P<0.05) in the last four weeks of observation. In this time, the COM birds laid 17 to 24% more than CAS birds. The EW produced by birds from the COM group (59.1 to 60.7 g) was greater (P<0.05) than that of the CAS birds (55.0 to 57.0 g). In the second half of the study period, a lower EM (P<0.05) was observed in the CAS treatment (24.7 to 31.8 g bird-1 d-1) compared to the COM treatment (39.7 to. 41.8 bird-1 d-1). Study Implications: The results obtained are only valid for the types of concentrate evaluated and under the specified experimental conditions. Conclusions: The homemade concentrate reduces the productive performance of hens in a cage-free system in terms of LP, EW, and EM, when compared to the commercial concentrate.

scholarly journals Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Forests ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 193
Author(s):  
Dandan Li ◽  
Sen Yu ◽  
Minzhen Zeng ◽  
Xiao Liu ◽  
Jia Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Real Time ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Stable Gene ◽  
Larix Olgensis ◽  
Qrt Pcr ◽  
Study Gene Expression ◽  
Selection Of

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

Assessment of genetic diversity in cultivars of white clover (Trifolium repens L.) detected by SSR polymorphisms

Genome ◽  
2006 ◽  
Vol 49 (8) ◽  
pp. 919-930 ◽  
Author(s):  
Julie George ◽  
Mark P Dobrowolski ◽  
Eline van Zijll de Jong ◽  
Noel O.I Cogan ◽  
Kevin F Smith ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Multidimensional Scaling ◽  
White Clover ◽  
Trifolium Repens ◽  
Simple Sequence Repeat ◽  
Nonmetric Multidimensional Scaling ◽  
Grass Species ◽  
Sequence Repeat ◽  
Trifolium Repens L ◽  
Simple Sequence

White clover (Trifolium repens L.) is an important temperate pasture legume that plays a key role as a companion to grass species, such as perennial ryegrass (Lolium perenne L.). Due to the outbreeding nature of white clover, cultivars are highly heterogeneous. Genetic diversity was assessed using 16 elite cultivars from Europe, North and South America, Australia, and New Zealand. Fifteen simple sequence repeat markers that detect single, codominant polymorphic genetic loci were selected for the study. The genetic relationships among individuals were compared using phenetic clustering, and those among cultivars were compared using nonmetric multidimensional scaling. Intrapopula tion variability exceeded interpopulation variability, with substantial overlap among populations and weak interpopula tion differentiation. No obvious or significant differentiation was observed on the basis of morphology or geographic origin of the cultivars. The number of parental genotypes used to derive each cultivar was not a major determinant of genome-wide genetic diversity. The outcomes of this assessment of genetic variation in elite white clover germplasm pools have important implications for the feasibility of molecular marker-based cultivar discrimination, and will be used to assist the design of linkage disequilibrium mapping strategies for marker-trait association.Key words: white clover, allotetraploid, genetic diversity, polymorphism, simple sequence repeat, cluster analysis, nonmetric multidimensional scaling.

scholarly journals Selection and validation of reference genes for real-time qRT-PCR normalization in different tissues of Eucalyptus tereticornis

2012 ◽  
Vol 61 (1-6) ◽  
pp. 280-286
Author(s):  
B. Karpaga Raja Sundari ◽  
M. Ghosh Dasgupta
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Genomic Research ◽  
Initiation Factor ◽  
Primary Cell Wall ◽  
Stable Gene ◽  
Eucalyptus Tereticornis ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Different Tissues

AbstractReference genes are generally used as endogenous normalization factor for relative quantification of target genes in quantitative real-time PCR (qRT-PCR). The present work aimed at identifying suitable reference genes for normalization of qRT-PCR data in tissues of Eucalyptus tereticornis. The expression levels of housekeeping genes like Actin (EtAct2), Isocitrate dehy - drogenase (EtIDH), ribosomal RNA (Et18s rRNA), SAND family protein (EtSAND), Histone protein (EtH2B), α-Tubulin (EtTUB), and eukaryotic initiation factor (EteIF4B) were studied to characterize their normalization stability in different tissues including young leaves, internodes, developing and mature xylem. The expression level of these genes was analyzed using different algorithms like geNorm, NormFinder and Best- Keeper. Among the seven reference genes analyzed, EtAct2 was expressed with less variance and was found to be the most stable reference gene across different tissues using all the three programs, while the least stable gene identified was EtH2B. Further, the normalization efficiency of the reference genes were assessed to predict the expression levels of three primary cell wall specific cellulose synthase transcripts (EtCesAs) in E. tereticornis tissues. The relative expression of EtCesA4, EtCesA5 and EtCesA6 was determined to be 3-19 fold higher in leaf and internode tissues when compared to developing and mature xylem tissues. This study will allow accurate normalization of qRT-PCR experiments across different tissues in E. tereticornis for future genomic research in this tropical eucalypt species.

Selection of reference genes for quantitative real-time PCR normalization in the coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae)

2021 ◽  
pp. 1-11
Author(s):  
Qianqian Meng ◽  
Benshui Shu ◽  
Shiwei Sun ◽  
Ying Wang ◽  
Mei Yang ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Stem Borer ◽  
Internal Reference ◽  
Experimental Conditions ◽  
Tissue Samples ◽  
Abiotic Conditions ◽  
Qrt Pcr

Abstract The coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is a major destructive pest of Coffea arabica L. (Gentianales: Rubiaceae), widely planted in many Asian countries, including China. Quantitative real-time polymerase chain reaction (qRT-PCR) is a common method for quantitative analysis of gene transcription levels. To obtain accurate and reliable qRT-PCR results, it is necessary to select suitable reference genes to different experimental conditions for normalizing the target gene expression. However, the stability of the expression of reference genes in X. quadripes has rarely been studied. In this study, the expression stability of nine candidate reference genes were investigated under biotic and abiotic conditions for use in qRT-PCR's normalization. By integrating the results of four algorithms of NormFinder, BestKeeper, geNorm, and RefFinder, the optimal reference gene combinations in different experimental conditions were performed as follows: RPL10a and EIF3D were the optimal reference genes for developmental stage samples, EIF4E, RPL10a, and RPS27a for tissue samples, V-ATP and EF1α for the sex samples, EIF3D and V-ATP for temperature treatment, RPS27a and RPL10a for insecticide stress, and RPL10a, RPS27a, and EF1α for all the samples. This study will help to obtain the stable internal reference genes under biotic and abiotic conditions and lay the foundation for in-depth functional research of target genes or genomics on olfactory molecular mechanisms, temperature adaptability, and insecticide resistance in X. quadripes.

Selection and validation of reference genes for quantitative real-time PCR analysis of Nitraria tangutorum

2020 ◽  
Author(s):  
Bo Wang ◽  
Lirong WANG ◽  
Huirong Duan ◽  
Peifang Chong ◽  
Shiping Su ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Expression Stability ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Nitraria Tangutorum ◽  
Desert Areas ◽  
Polymerase Chain ◽  
Suitable Reference

Abstract Background: Suitable reference genes can be used to calibrate the error in quantitative real‑time polymerase chain reaction (qRT-PCR) experiments and make the results more credible. However, reference genes suitable for different species and different experimental conditions do not exist. Nitraria tangutorum Bobr. is a typical plant in desert areas and desert plains, which is drought-resistant, saline-alkali resistant, barren-resistant, and has extremely strong adaptability. Due to insufficient understanding of the importance of this germplasm in the past, it is still unclear which genes can be used as reference genes to calibrate qRT-PCR data of N. tangutorum .Results: In this study, we analyzed the expression levels of 10 candidate reference genes (ACT, GAPDH, TUA, TUB, CYP, UBC, His, PP2A, HSP, and EF1-α) in three tissues (root, stem and leaf) and under five abiotic stresses (salt, drought, heat, cold, and ABA) of N. tangutorum seedlings by qRT-PCR. Three analysis software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate expression stability of ten genes. Comprehensive analysis showed that EF1-α and His had the best expression stability, whereas HSP was the least suitable as a reference gene. The expression profile of NtCER7, a gene related to the regulation of the waxy synthesis of N. tangutorum, verified the accuracy of the experimental results.Conclusion: Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This study provides the first data on stable reference genes in N. tangutorum, which will be beneficial to study of the gene expression of N. tangutorum and other Nitraria species in the future.

geNorm assessment of the efficacy of novel reference genes ALG9, TFCP2 and ZNF410 compared to GAPDH for real time normalization in GI carcinoids

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20052-20052
Author(s):  
G. Eick ◽  
M. Kidd ◽  
S. Mane ◽  
B. Nadler ◽  
M. Champaneria ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Large Scale ◽  
Target Genes ◽  
Marker Genes ◽  
Stable Gene ◽  
Rt Pcr ◽  
Experimental Conditions ◽  
Expression Levels ◽  
Accurate Normalization

20052 Background: Robust quantitation of potential clinical marker genes using quantitative real-time PCR (Q RT-PCR) is critically dependent on accurate normalization. Although GAPDH has historically been used for normalization, its expression has been shown to vary widely between different tissues and experimental conditions. Additionally, conventional normalization strategies based on a single housekeeping gene can lead to large normalization errors. The determination of a panel of genes that have robust expression in the experimental system being studied is therefore essential to ensure accurate normalization and interpretation of results. Methods: Based upon the availability of large-scale gene databases, we developed methodology to identify highly expressed genes (mean log-transformed expression levels: 4–8 in all samples) with low variability (S.D. < 0.22) in a Affymetrix U133A dataset of 36 gastrointestinal tumors and normal tissues. Eight novel candidate reference genes were identified and their expression levels established by Q RT-PCR in an independent set of GI tissue samples (n = 24). The geNorm tool was used to identify the most stably expressed set of genes amongst the 8 candidate genes. The expression levels of 3 potential GI tumor marker genes, namely the adhesin MAGE-D2, the metastasis-associated MTA1, and the mitosis regulator, NAP1L1, were normalized to GAPDH or geNorm and compared. Results: geNorm identified 3 genes, ALG9, TFCP2 and ZNF410, as the most robustly expressed control genes. GAPDH, in contrast, exhibited the highest variability and was considered the least stable gene of the nine evaluated. Two previously-identified target genes, MAGE-D2 and MTA1, were significantly elevated (p < 0.05) in malignant tumor samples (vs normal GI samples) when normalized by geNormATZ but not when normalized using GAPDH. NAP1L1 was only over-expressed in small intestinal carcinoids (normalized to geNormATZ). Expression levels of this gene were high in normal gastric mucosa. Conclusions: We provide a robust basis for the establishment of a reference gene set using GeneChip data and provide evidence for the clinical utility of identifying reference genes using the geNorm approach in GI neuroendocrine tumors. No significant financial relationships to disclose.

scholarly journals Identification of the Most Suitable Reference Gene for Gene Expression Studies With Development and Abiotic Stress Response in Bromus Sterilis

2021 ◽  
Author(s):  
Madhab Kumar Sen ◽  
Katerina Hamouzova ◽  
Pavlina Kosnarova ◽  
Amit Roy ◽  
Josef Soukup
Keyword(s):  
Reference Gene ◽  
Herbicide Resistance ◽  
Reference Genes ◽  
Gene Selection ◽  
High Yield ◽  
Suitable Reference Gene ◽  
Grass Species ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Suitable Reference

Abstract Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide-resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can contribute to elucidation of the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes were evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used softwares for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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