Validation of Reference Genes for Quantitative Real-Time PCR during Bicolor Tepal Development in Asiatic Hybrid Lilies (Lilium spp.)

Xu, Liang; Xu, Hua; Cao, Yang; Yang, Panpan; Feng, Yue; Tang, Yuchao; Yuan, Suxia; Ming, Jun

doi:10.3389/fpls.2017.00669

Cited by 40 publications

(27 citation statements)

References 45 publications

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“…This difference might be due to the different statistical models used in the various algorithms. Differences in the ranking orders obtained with these algorithms have also been observed in other studies ( Tong et al, 2009 ; Reddy et al, 2016 ; Xu et al, 2017 ).…”

Section: Discussionsupporting

confidence: 77%

Tobacco rattle virus-inducedPHYTOENE DESATURASE(PDS)andMg-chelatase H subunit(ChlH) gene silencing inSolanum pseudocapsicumL.

Yang

et al. 2018

PeerJ

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Virus-induced gene silencing (VIGS) is an attractive tool for determining gene function in plants. The present study constitutes the first application of VIGS in S. pseudocapsicum, which has great ornamental and pharmaceutical value, using tobacco rattle virus (TRV) vectors. Two marker genes, PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH), were used to test the VIGS system in S. pseudocapsicum. The photobleaching and yellow-leaf phenotypes of the silenced plants were shown to significantly correlate with the down-regulation of endogenous SpPDS and SpChlH, respectively (P ≤ 0.05). Moreover, the parameters potentially affecting the efficiency of VIGS in S. pseudocapsicum, including the Agrobacterium strain and the inoculation method (leaf syringe-infiltration, sprout vacuum-infiltration and seed vacuum-infiltration), were compared. The optimized VIGS parameters were the leaf syringe-infiltration method, the Agrobacterium strain GV3101 and the growth of agro-inoculated plants at 25°. With these parameters, the silencing efficiency of SpPDS and SpChlH could reach approximately 50% in S. pseudocapsicum. Additionally, the suitability of various reference genes was screened by RT-qPCR using three candidate genes, and the results demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) can serve as a suitable reference for assessing the gene expression levels of VIGS systems in S. pseudocapsicum. The proven application of VIGS in S. pseudocapsicum and the characterization of a suitable reference gene in the present work will expedite the functional characterization of novel genes in S. pseudocapsicum.

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Section: Discussionsupporting

confidence: 77%

Tobacco rattle virus-inducedPHYTOENE DESATURASE(PDS)andMg-chelatase H subunit(ChlH) gene silencing inSolanum pseudocapsicumL.

Yang

et al. 2018

PeerJ

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“…Gene-specific primers for qRT-PCR were designed with Primer 6.0 ( Supplemental Table S2 ). The LilyActin primer was used as an internal control [ 66 ], and SYBR ® Green Master Mix (No Rox) (Yeasen, Shanghai, China) was used in the reaction mixture according to the manufacturer’s instructions. qRT-PCR was conducted using the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA), with an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s and extension at 72 °C for 1 min.…”

Section: Methodsmentioning

confidence: 99%

Cytokinin Type-B Response Regulators Promote Bulbil Initiation in Lilium lancifolium

Yang

Cao

et al. 2021

IJMS

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The bulbil is an important vegetative reproductive organ in triploid Lilium lancifolium whose development is promoted by cytokinins. Type-B response regulators (RRs) are critical regulators that mediate primary cytokinin responses and promote cytokinin-induced gene expression. However, the function of cytokinin type-B Arabidopsis RRs (ARRs) in regulating bulbil formation is unclear. In this study, we identified five type-B LlRRs, LlRR1, LlRR2, LlRR10, LlRR11 and LlRR12, in L. lancifolium for the first time. The five LlRRs encode proteins of 715, 675, 573, 582 and 647 amino acids. All of the regulators belong to the B-I subfamily, whose members typically contain a conserved CheY-homologous receiver (REC) domain and an Myb DNA-binding (MYB) domain at the N-terminus. As transcription factors, all five type-B LlRRs localize at the nucleus and are widely expressed in plant tissues, especially during axillary meristem (AM) formation. Functional analysis showed that type-B LlRRs are involved in bulbil formation in a functionally redundant manner and can activate LlRR9 expression. In summary, our study elucidates the process by which cytokinins regulate bulbil initiation in L. lancifolium through type-B LlRRs and lays a foundation for research on the molecular mechanism of bulbil formation in the lily.

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“…[ 16 ], Achyranthes bidentata Blume [ 17 ], Kentucky bluegrass [ 18 ], Salix matsudana [ 19 ], Rhododendron molle G. Don [ 20 ], Sapium sebiferum [ 21 ], Petroselinum crispum [ 22 ], Lilium spp. [ 23 ], Hibiscus cannabinus L. [ 24 ] and Dendrobium officinale [ 25 ].…”

Section: Introductionmentioning

confidence: 99%

Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data

et al. 2018

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BackgroundQuantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several reliable reference genes have been reported in plants of Traditional Chinese Medicine, but none have been identified for Euscaphis konishii Hayata.ResultsIn this study, 12 candidate reference genes, including 3 common housekeeping genes and 9 novel genes based on E. konishii Hayata transcriptome data were selected and analyzed in different tissues (root, branch, leaf, capsule and seed), capsule and seed development stages. Expression stability was calculated using geNorm and NormFinder, the minimal number of reference genes required for accurate normalization was calculated by Vn/Vn + 1 using geNorm. EkEEF-5A-1 and EkADF2 were the two most stable reference genes for all samples, while EkGSTU1 and EkGAPDH were the most stable reference genes for tissue samples. For seed development stages, EkGAPDH and EkEEF-5A-1 were the most stable genes, whereas EkGSTU1 and EkGAPDH were identified as the two most stable genes in the capsule development stages. Two reference genes were sufficient to normalize gene expression across all sample sets.ConclusionResults of this study revealed that suitable reference genes should be selected for different experimental samples, and not all the common reference genes are suitable for different tissue samples and/or experimental conditions. In this study, we present the first data of reference genes selection for E. konishii Hayata based on transcriptome data, our data will facilitate further studies in molecular biology and gene function on E. konishii Hayata and other closely related species.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0311-x) contains supplementary material, which is available to authorized users.

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Validation of Reference Genes for Quantitative Real-Time PCR during Bicolor Tepal Development in Asiatic Hybrid Lilies (Lilium spp.)

Cited by 40 publications

References 45 publications

Tobacco rattle virus-inducedPHYTOENE DESATURASE(PDS)andMg-chelatase H subunit(ChlH) gene silencing inSolanum pseudocapsicumL.

Tobacco rattle virus-inducedPHYTOENE DESATURASE(PDS)andMg-chelatase H subunit(ChlH) gene silencing inSolanum pseudocapsicumL.

Cytokinin Type-B Response Regulators Promote Bulbil Initiation in Lilium lancifolium

Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data

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