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Reference gene selection for RT-qPCR analysis of Chrysanthemum lavandulifolium during its flowering stages

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Abstract

Quantitative real-time RT-PCR (RT-qPCR) is a technology that can be used to analyze the abundance of gene expression. Reference genes, which are assumed to remain at constant levels in different tissues at various developmental stages and photoperiodic treatments, were selected to analyze the expression levels of flowering time genes and floral development genes. Using digital gene expression technology, nine reference genes with moderate expression in the leaves of Chrysanthemum lavandulifolium at the juvenile phase (CK1) and the squaring stage (W1) were selected as the candidate reference genes for further study. A total of 115 biological samples of C. lavandulifolium were analyzed, including different tissues under various developmental stages and leaves with varied photoperiodic treatments. The stability of the nine reference genes was slightly variable across the samples, but MTP, SKIP16 and PGK were the most stable genes overall. In addition, the relative expression level of ClFT in different tissues of plants with the competence to flower was analyzed to verify the reference genes selected in this study. These studies provide a guide for selecting reference genes for analyzing the expression pattern of flowering time genes and floral development genes in C. lavandulifolium.

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Abbreviations

ACT :

Actin

CTAB:

Cethyltrimethylammonium bromide

Cq:

Quantification cycle

DD:

Constant darkness

EF1b :

Elongation factor 1-beta

FT :

Flowering Locus T

LD:

Long-day

LL:

Constant light

MTP :

Metalloprotease

NF:

Normalization factor

PGK :

Phosphoglycerate kinase

psaA :

Photosynthesis-related plastid gene representing photosystem I

RPKM:

Reads Per Kb per Million reads

RT-qPCR:

Quantitative real-time RT-PCR

SAND :

SAND family protein

SD:

Short-day

SKIP16 :

SKP1/ASK-interacting protein 16

TUA :

α-Tubulin

TIP41 :

TIP41-like family protein

T m :

Melting temperature

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (30871726 and 31071823), the Forestry Administration of China National Public Project (200904050) and the Chinese 863 Project (2007AA021403).

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Authors and Affiliations

  1. College of Landscape Architecture, Beijing Forestry University, Beijing, 100083, People’s Republic of China

    Jianxin Fu, Yi Wang, He Huang, Chao Zhang & Silan Dai

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  1. Jianxin Fu
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  2. Yi Wang
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  3. He Huang
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  5. Silan Dai
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Correspondence to Silan Dai.

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Fu, J., Wang, Y., Huang, H. et al. Reference gene selection for RT-qPCR analysis of Chrysanthemum lavandulifolium during its flowering stages. Mol Breeding 31, 205–215 (2013). https://doi.org/10.1007/s11032-012-9784-x

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