Dataset Information

Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR.

ABSTRACT: Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes.

SUBMITTER: Hu M

PROVIDER: S-EPMC4871855 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR.

Hu Meizhen M Hu Wenbin W Xia Zhiqiang Z Zhou Xincheng X Wang Wenquan W

Frontiers in plant science 20160519

Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, ...[more]

PMID: 27242878

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Project description:External application of acetic acid has been recently reported to enhance the survival to drought in plants such as Arabidopsis, rapeseed, maize, rice and wheat, but the effects of acetic acid application on increased drought tolerance in woody plants such as a tropical crop “cassava” remain elusive. A molecular understanding of acetic acid-induced drought avoidance in cassava will contribute to the development of technology that can be used to enhance drought tolerance without resorting to transgenic technology or advancements in cassava cultivation. In the present study, morphological, physiological and molecular responses to drought were analyzed in cassava after the treatment with acetic acid. Results indicated that the acetic acid-treated cassava plants had a higher level of drought avoidance than water-treated, control plants. Specifically, higher leaf relative water content, and chlorophyll and carotenoid levels were observed as soils dried out during the drought treatment. Leaf temperatures in acetic acid-treated cassava plants were higher relative to leaves on plants pretreated with water and the increase of ABA content was observed in leaves of acetic acid-treated plants, suggesting that stomatal conductance and the transpiration rate in leaves of acetic acid-treated plants decreased to maintain relative water contents and avoid drought. Transcriptome analysis revealed that the acetic acid treatment increased the expression of ABA signaling-related genes, such as OPEN STOMATA 1　(OST1) and protein phosphatase 2C; as well as drought response and tolerance-related genes, such as outer membrane tryptophan-rich sensory protein (TSPO), and heat shock proteins. Collectively, the external application of acetic acid enhances drought avoidance in cassava through the upregulation of ABA signaling pathway genes and several stress response- and tolerance-related genes. These data support the idea that adjustments of the acetic acid application to plants is useful to enhance drought tolerance in order to minimize the growth inhibition in the agricultural field.

Project description:The external application of acetic acid has recently been reported to enhance survival of drought in plants such as Arabidopsis, rapeseed, maize, rice, and wheat, but the effects of acetic acid application on increased drought tolerance in woody plants such as a tropical crop "cassava" remain elusive. A molecular understanding of acetic acid-induced drought avoidance in cassava will contribute to the development of technology that can be used to enhance drought tolerance, without resorting to transgenic technology or advancements in cassava cultivation. In the present study, morphological, physiological, and molecular responses to drought were analyzed in cassava after treatment with acetic acid. Results indicated that the acetic acid-treated cassava plants had a higher level of drought avoidance than water-treated, control plants. Specifically, higher leaf relative water content, and chlorophyll and carotenoid levels were observed as soils dried out during the drought treatment. Leaf temperatures in acetic acid-treated cassava plants were higher relative to leaves on plants pretreated with water and an increase of ABA content was observed in leaves of acetic acid-treated plants, suggesting that stomatal conductance and the transpiration rate in leaves of acetic acid-treated plants decreased to maintain relative water contents and to avoid drought. Transcriptome analysis revealed that acetic acid treatment increased the expression of ABA signaling-related genes, such as OPEN STOMATA 1 (OST1) and protein phosphatase 2C; as well as the drought response and tolerance-related genes, such as the outer membrane tryptophan-rich sensory protein (TSPO), and the heat shock proteins. Collectively, the external application of acetic acid enhances drought avoidance in cassava through the upregulation of ABA signaling pathway genes and several stress responses- and tolerance-related genes. These data support the idea that adjustments of the acetic acid application to plants is useful to enhance drought tolerance, to minimize the growth inhibition in the agricultural field.

Project description:BackgroundProteomics is increasingly becoming an important tool for the study of many different aspects of plant functions, such as investigating the molecular processes underlying in plant physiology, development, differentiation and their interaction with the environments. To investigate the cassava (Manihot esculenta Crantz) proteome, we extracted proteins from somatic embryos, plantlets and tuberous roots of cultivar SC8 and separated them by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).ResultsAnalysis by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) yielded a total of 383 proteins including isoforms, classified into 14 functional groups. The majority of these were carbohydrate and energy metabolism associated proteins (27.2%), followed by those involved in protein biosynthesis (14.4%). Subsequent analysis has revealed that 54, 59, 74 and 102 identified proteins are unique to the somatic embryos, shoots, adventitious roots and tuberous roots, respectively. Some of these proteins may serve as signatures for the physiological and developmental stages of somatic embryos, shoots, adventitious roots and tuberous root. Western blotting results have shown high expression levels of Rubisco in shoots and its absence in the somatic embryos. In addition, high-level expression of alpha-tubulin was found in tuberous roots, and a low-level one in somatic embryos. This extensive study effectively provides a huge data set of dynamic protein-related information to better understand the molecular basis underlying cassava growth, development, and physiological functions.ConclusionThis work paves the way towards a comprehensive, system-wide analysis of the cassava. Integration with transcriptomics, metabolomics and other large scale "-omics" data with systems biology approaches can open new avenues towards engineering cassava to enhance yields, improve nutritional value and overcome the problem of post-harvest physiological deterioration.

Project description:BACKGROUND: Cassava (Manihot esculenta Crantz), a starchy root crop grown in tropical and subtropical climates, is the sixth most important crop in the world after wheat, rice, maize, potato and barley. The repertoire of simple sequence repeat (SSR) markers for cassava is limited and warrants a need for a larger number of polymorphic SSRs for germplasm characterization and breeding applications. RESULTS: A total of 846 putative microsatellites were identified in silico from an 8,577 cassava unigene set with an average density of one SSR every 7 kb. One hundred and ninety-two candidate SSRs were screened for polymorphism among a panel of cassava cultivars from Africa, Latin America and Asia, four wild Manihot species as well as two other important taxa in the Euphorbiaceae, leafy spurge (Euphorbia esula) and castor bean (Ricinus communis). Of 168 markers with clean amplification products, 124 (73.8%) displayed polymorphism based on high resolution agarose gels. Of 85 EST-SSR markers screened, 80 (94.1%) amplified alleles from one or more wild species (M epruinosa, M glaziovii, M brachyandra, M tripartita) whereas 13 (15.3%) amplified alleles from castor bean and 9 (10.6%) amplified alleles from leafy spurge; hence nearly all markers were transferable to wild relatives of M esculenta while only a fraction was transferable to the more distantly related taxa. In a subset of 20 EST-SSRs assessed by fluorescence-based genotyping the number of alleles per locus ranged from 2 to 10 with an average of 4.55 per locus. These markers had a polymorphism information content (PIC) from 0.19 to 0.75 with an average value of 0.55 and showed genetic relationships consistent with existing information on these genotypes. CONCLUSION: A set of 124 new, unique polymorphic EST-SSRs was developed and characterized which extends the repertoire of SSR markers for cultivated cassava and its wild relatives. The markers show high PIC values and therefore will be useful for cultivar identification, taxonomic studies, and genetic mapping. The study further shows that mining ESTs is a highly efficient strategy for polymorphism detection within the cultivated cassava gene pool.

Project description:Drought stress is one of the potent abiotic stress limiting cassava (Manihot esculenta) yield globally, but studies addressing both physiological and proteomic responses that how cassava crops can adjust their growth and metabolism under drought conditions are lacking. Combining leaf physiological and proteomic characteristics strongly allied with drought tolerance should results in enhanced drought tolerance in cassava crop. Therefore, the aims of this study were to explore the plant physiological and proteomic mechanisms involved in drought adaptation in cassava. Xinxuan 048 (XX048) was exposed to well-watered control (CK, relative soil water content (RSWC) as 80 ± 5%), mild drought stress (LD, RSWC as 65 ± 5%), moderate drought stress (MD, RSWC as 50 ± 5%) and severe drought stress (SD, RSWC as 35 ± 5%) from 30 days after planting. Under drought stress conditions, cassava plant showed a substantial decline in plant height, stem diameter, leaf number, leaf water content, the ratio of free water content to bound water content of leaf (FW/BW), net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs) and transpiration rate (Tr) compared with well watered plants. However, compared with control, leaf water content, SPAD value, cell membrane permeability, malondialdehyde (MDA), soluble sugar, protein proline content SOD and CAT activity were at peak under drought stress. The proteomic analysis revealed that among 3 339 identified proteins, drought stress increased and decreased abundance of 262 and 296 proteins, respectively, compared with control condition. These proteins were involved in carbohydrate energy metabolism, protein homeostasis, transcription, cell structure, cell membrane transport, signal transduction, stress and defense responses. These data not only provides a comprehensive dataset on overall proteomic changes in cassava leaves under drought stress, but also highlights the mechanisms by which euphorbiaceae plants can adapt to drought conditions.

Project description:The mealybug, Phenacoccus manihoti, is a leading pest of cassava (Manihot esculenta Crantz), damaging this crop globally. Although the biological control of this mealybug using natural predators has been established, resistance breeding remains an important means of control. Understanding plant responses to insect herbivory, by determining and identifying differentially expressed genes (DEGs), is a vital step towards the understanding of molecular mechanisms of defence responses in plants and the development of resistant cultivars by gene editing. Morphological and molecular analysis confirmed the mealybug identity as Phenacoccus manihoti (Matile-Ferrero). The transcriptome response of the green mite resistant cassava genotype AR23.1 was compared to P40/1 with no known resistance at 24 and 72 hours of mealybug infestation compared to non-infested mock. A total of 301 and 206 genes were differentially expressed at 24 and 72 of mealybug infestation for AR23.1 and P40/1 genotypes respectively, using a log2 fold change and P-value ? 0.05. Gene ontology functional classification revealed an enrichment of genes in the secondary metabolic process category in AR23.1 in comparison with P40/1, while genes in the regulation of molecular function, cellular component biogenesis and electron carrier categories were more significantly enriched in P40/1 than in AR23.1. Biological pathway analysis, based on KEGG, revealed a significant enrichment of plant-pathogen interaction and plant hormonal signal transduction pathways for a cohort of up-regulated and down-regulated DEGs in both genotypes. Defence-related genes such as 2-oxogluterate, gibberellin oxidase and terpene synthase proteins were only induced in genotype AR23.1 and not in P40/1, and subsequently validated by RT-qPCR. The study revealed a difference in response to mealybug infestation in the two genotypes studied, with AR23.1 showing a higher number of differentially expressed transcripts post mealybug infestation at 24 and 72 hours. Candidate defence-related genes that were overexpressed in the AR23.1 genotype post mealybug infestation will be useful in future functional studies towards the control of mealybugs.

Dataset Information

Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR.

Publications

Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR.

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