Selection and validation of reference genes for qPCR analysis of miRNAs and their targets during somatic embryogenesis in tamarillo (Solanum betaceum Cav.)

Cordeiro, Daniela; Rito, Miguel; Borges, Filipe; Canhoto, Jorge; Correia, Sandra

doi:10.1007/s11240-020-01901-7

Cited by 10 publications

(7 citation statements)

References 44 publications

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“…MiR164, MiR168 and MiR396 can regulate the expression levels of plant genes and ultimately affect plant growth and development and respond to (a)biotic stresses [36][37][38][39][40]. A large number of studies have shown significant differences in the expression of target genes after normalisation with stable and unstable RGs [9,41,42].…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, cas-miR166d showed the most unstable expression in C. fortunei under most experimental conditions. This RG also has unstable expression patterns in cold-treated tomato ( Solanum lycopersicum ) seedlings [ 34 ] and winter turnip rape ( Brassica rapa ) leaves and roots [ 35 ], while miR166a is the most appropriate for normalisation in tamarillo ( Solanum betaceum ) callus samples [ 36 ]. These results can be clarified by the fact that RGs exhibit specific expression patterns, with large differences in applicability.…”

Section: Discussionmentioning

confidence: 99%

“…It has been reported that miR396, miR164 and miR168 play an important role in plant growth and development and in response to (a)biotic stresses [ 36 , 37 , 38 , 39 , 40 , 45 ]. Thus, three miRNAs (aly-miR164c-5p, aly-miR168a-5p and smo-miR396) were selected as target genes; the primers used are shown in Table 1 .…”

Section: Methodsmentioning

confidence: 99%

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Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (Cryptomeria fortunei) under Multiple Stresses

Zhang

Xue

Zhu

et al. 2021

IJMS

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MicroRNA (miRNA) expression analysis is very important for investigating its functions. To date, no research on reference genes (RGs) for miRNAs in gymnosperms, including Cryptomeria fortunei, has been reported. Here, ten miRNAs (i.e., pab-miR159a, cln-miR162, cas-miR166d, pab-miR395b, ppt-miR894, cln-miR6725, novel1, novel6, novel14 and novel16) and three common RGs (U6, 5S and 18S) were selected as candidate RGs. qRT-PCR was used to analyse their expressions in C. fortunei under various experimental conditions, including multiple stresses (cold, heat, drought, salt, abscisic acid and gibberellin) and in various tissues (roots, stems, tender needles, cones and seeds). Four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) were employed to assess the stability of candidate RG expression; the geometric mean and RefFinder program were used to comprehensively evaluate RG stability. According to the results, novel16, cln-miR6725, novel1 and U6 were the most stable RGs for studying C. fortunei miRNA expression. In addition, the expression of three target miRNAs (aly-miR164c-5p, aly-miR168a-5p and smo-miR396) was examined to verify that the selected RGs are suitable for miRNA expression normalisation. This study may aid further investigations of miRNA expression/function in the response of C. fortunei to abiotic stress and provides an important basis for the standardisation of miRNA expression in other gymnosperm species.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (Cryptomeria fortunei) under Multiple Stresses

Zhang

Xue

Zhu

et al. 2021

IJMS

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“…Samples with the mix were pooled in a 96-well qPCR plate and measured in C1000 TouchTM Thermal Cycler (Bio-Rad Laboratories, Lda., Amadora, Portugal). For reliable quantitative PCRs, two reference genes were also chosen in order to normalize the data of Ef1α and IRON SUPEROXIDE DISMUTASE, FeSOD [ 61 ]. All the primers ( Table 3 ), with the exception of TIR1 gene primers (designed for Solanum lycopersicum GQ370812.1), were designed for Solanum betaceum transcript sequences obtained from embryogenic cell RNAseq libraries (data not published), using the NCBI primer design tool.…”

Section: Methodsmentioning

confidence: 99%

Induction of Somatic Embryogenesis in Tamarillo (Solanum betaceum Cav.) Involves Increases in the Endogenous Auxin Indole-3-Acetic Acid

Caeiro

Correia

et al. 2022

Plants

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Somatic embryogenesis (SE) is a complex biological process regulated by several factors, such as the action of plant growth regulators, namely auxins, of which the most physiologically relevant is indole-3-acetic acid (IAA). In tamarillo, an optimized system for induction of SE creates, after an induction process, embryogenic (EC) and non-embryogenic callus (NEC). In this work the endogenous levels of auxin along the induction phase and in the calli samples were investigated using chemical quantifications by colorimetric reactions and HPLC as well as immunohistochemistry approaches. Differential gene expression (IAA 11, IAA 14, IAA 17, TIR 1, and AFB3) analysis during the induction phase was also carried out. The results showed that the endogenous IAA content is considerably higher in embryogenic than in non-embryogenic calli, with a tendency to increase as the dedifferentiation of the original explant (leaf segments) evolves. Furthermore, the degradation rates of IAA seem to be related to these levels, as non-embryogenic tissue presents a higher degradation rate. The immunohistochemical results support the quantifications made, with higher observable labeling on embryogenic tissue that tends to increase along the induction phase. Differential gene expression also suggests a distinct molecular response between EC and NEC.

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“…Furthermore, it has also been shown that although both cell lines can be maintained indefinitely, embryogenic competence is somewhat time-dependent, as long-term callus lines begin to show genetic abnormalities and lower rates of somatic embryo conversion after being maintained in a culture for periods of more than 2 years [15,16]. The different phenotypes that arise from explants of the same genotype constitute an important feature of this system that has been optimized in several areas of application, namely genetics and epigenetics, and explored in several studies where the SE process was analyzed with complementary omics, namely proteomics [17,18].…”

Section: Introductionmentioning

confidence: 99%

Primary Metabolite Screening Shows Significant Differences between Embryogenic and Non-Embryogenic Callus of Tamarillo (Solanum betaceum Cav.)

Caeiro

Jarak

Correia

et al. 2023

Plants

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Tamarillo is a solanaceous tree that has been extensively studied in terms of in vitro clonal propagation, namely somatic embryogenesis. In this work, a protocol of indirect somatic embryogenesis was applied to obtain embryogenic and non-embryogenic callus from leaf segments. Nuclear magnetic resonance spectroscopy was used to analyze the primary metabolome of these distinct calli to elucidate possible differentiation mechanisms from the common genetic background callus. Standard multivariate analysis methods were then applied, and were complemented by univariate statistical methods to identify differentially expressed primary metabolites and related metabolic pathways. The results showed carbohydrate and lipid metabolism to be the most relevant in all the calli assayed, with most discriminant metabolites being fructose, glucose and to a lesser extent choline. The glycolytic rate was higher in embryogenic calli, which shows, overall, a higher rate of sugar catabolism and a different profile of phospholipids with a choline/ethanolamine analysis. In general, our results show that a distinct primary metabolome between embryogenic and non-embryogenic calli occurs and that intracellular levels of fructose and sucrose and the glucose to sucrose ratio seem to be good candidates as biochemical biomarkers of embryogenic competence.

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Selection and validation of reference genes for qPCR analysis of miRNAs and their targets during somatic embryogenesis in tamarillo (Solanum betaceum Cav.)

Cited by 10 publications

References 44 publications

Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (Cryptomeria fortunei) under Multiple Stresses

Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (Cryptomeria fortunei) under Multiple Stresses

Induction of Somatic Embryogenesis in Tamarillo (Solanum betaceum Cav.) Involves Increases in the Endogenous Auxin Indole-3-Acetic Acid

Primary Metabolite Screening Shows Significant Differences between Embryogenic and Non-Embryogenic Callus of Tamarillo (Solanum betaceum Cav.)

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