National Center for Citrus Improvement

Changsha, China

National Center for Citrus Improvement

Changsha, China
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Yan J.,Hunan Agricultural University | Yan J.,National Center for Citrus Improvement | Yan J.,Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization | Yuan F.,Hunan Agricultural University | And 8 more authors.
Molecular Biology Reports | Year: 2012

Quantitative real-time reverse transcription polymerase chain reaction (qPCR) has become the preferred method for studying low-abundant mRNA expression. Appropriate application of qPCR in such studies requires the use of reference gene(s) as an internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. Expression of the reference gene should be independent from development stage, cell/tissue types, treatments and environmental conditions. Recognizing the importance of reference gene(s) in normalization of qPCR data, various reference genes have been evaluated for stable expression under specific conditions in various organisms. In plants, only a few of them have been investigated, and very few reports about such reference genes in citrus. In the present study, seven candidate reference genes (18SrRNA, ACTB, rpII, UBQI, UBQ10, GAPDH and TUB) were tested, and three of them (18SrRNA, ACTB and rpII) proved to be the most stable ones among six leaf samples of different citrus genotypes. The three candidate reference genes were further analyzed for their stability of expression in five different tissues, and the results indicated that they were not completely stable. It is commonly accepted that gene expression studies should be normalized using more than one reference gene. Based on our results, we propose the use of the mean result rendered by18SrRNA, ACTB and rpII as reference genes to normalize mRNA levels in qPCR analysis of diverse cultivars and tissues of citrus. These results may provide a guideline for future works on gene expression in citrus by using qPCR. © 2011 Springer Science+Business Media B.V.

Sun H.-P.,Hunan Agricultural University | Li F.,Hunan Agricultural University | Li F.,National Center for Citrus Improvement | Ruan Q.-M.,Hunan Agricultural University | Zhong X.-H.,Hunan Agricultural University
Plant Physiology and Biochemistry | Year: 2016

Reference gene evaluation and selection are necessary steps in gene expression analysis, especially in new plant varieties, through reverse transcription quantitative real-time PCR (RT-qPCR). Hedera helix L. is an important traditional medicinal plant recorded in European Pharmacopoeia. Research on gene expression in H. helix has not been widely explored, and no RT-qPCR studies have been reported. Thus, it is important and necessary to identify and validate suitable reference genes to for normalizing RT-qPCR results. In our study, 14 candidate protein-coding reference genes were selected. Their expression stability in five tissues (root, stem, leaf, petiole and shoot tip) and under seven abiotic stress conditions (cold, heat, drought, salinity, UV-C irradiation, abscisic acid and methyl jasmonate) were evaluated using geNorm and NormFinder. This study is the first to evaluate the stability of reference genes in H. helix. The results show that different reference genes should be chosen for normalization on the basis of various experimental conditions. F-box was more stable than the other selected genes under all analysis conditions except ABA treatment; 40S was the most stable reference gene under ABA treatment; in contrast, EXP and UBQ were the most unstable reference genes. The expressions of HhSE and Hhβ-AS, which are two genes related to the biosynthetic pathway of triterpenoid saponins, were also examined for reference genes in different tissues and under various cold stress conditions. The validation results confirmed the applicability and accuracy of reference genes. Additionally, this study provides a basis for the accurate and widespread use of RT-qPCR in selecting genes from the genome of H. helix. © 2016 Elsevier Masson SAS

Sun H.-P.,Hunan Agricultural University | Li F.,Hunan Agricultural University | Li F.,National Center for Citrus Improvement | Cao X.-J.,Hunan Agricultural University | And 3 more authors.
Biochemical Systematics and Ecology | Year: 2015

Hedera plants are ornamental vines which are regarded as traditional medicinal plants. These plants are widely cultivated worldwide; moreover, new species and varieties have been discovered. For the first time, molecular markers based on sequence-related amplified polymorphism (SRAP) were used in this study to classify species and to analyse the genetic relationship of 21 Hedera plant varieties which are commonly cultivated in China. We also employed high-performance liquid chromatography (HPLC) to evaluate the medicinal value of these plants by measuring their hederacoside C contents. Results of SRAP-PCR and cluster analyses show that the SRAP molecular markers can successfully be applied in Hedera plants and are available. In addition, HPLC detection results indicate that the Chinese wild species in group I contain less than 30 mg/g hederacoside C and do not comply with the European Pharmacopoeia standards. By contrast, groups II and III exhibit high medicinal value and accord with these standards. © 2015 Elsevier Ltd.

Ge H.,Hunan Agricultural University | Ge H.,National Center for Citrus Improvement | Li Y.,Hunan Agricultural University | Li Y.,National Center for Citrus Improvement | And 10 more authors.
Plant Cell, Tissue and Organ Culture | Year: 2015

Xanthomonascitri subsp. citri (Xcc) is the causal agent of citrus canker, a quarantine disease worldwide. This disease is hard to be completely controlled by chemical sprays, and the selection of citrus genotypes resistant to the pathogen becomes one of the most important ways to control the disease. The limited genotypes that have been found up to now tolerant to Xcc are not commercially cultivated. Sweet orange [Citrus sinensis (L.) Osbeck] is one of the most important citrus species widely cultivated in world and all the orange cultivars are susceptible to canker disease. Therefore, this study aims at the establishment of an efficient protocol for in vitro ethyl methane sulphonate (EMS) mutagenesis and the selection of ‘Bingtang’ sweet orange somaclones tolerant to citrus canker disease. Mutation was introduced by treating the cell suspension of embryogenic callus with 1.5 % of EMS for 1 h (the lethal concentration). A crude extract produced from the pathogen solution was used as the selection agent. Sweet orange leaves inoculated by Xcc-crude extract showed the symptoms similar to those inoculated by Xcc bacterial inoculum. The young shoots of susceptive genotypes cultured in 10 % of Xcc-crude extract solution become brown and died in 1 week, while the resistant citron C-05 grew normally under the same conditions. After two steps (cell suspension and plants) of selection by Xcc-crude extract solution, the survival plants were tested by in vitro and in vivo inoculation with Xcc. One somaclone, named DG-2, was identified to be resistant to the canker disease. The results suggested that the established protocol for somaclones variation by treating sweet orange callus with EMS and in vitro selection of the somaclones tolerant to canker disease by the pathogen crude extract was effective. The gained sweet orange somaclone tolerant to X. citri subsp. citri will go into further cultivar selection procedures. © 2015, Springer Science+Business Media Dordrecht.

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