Reference Gene Selection for RT-qPCR Analysis of Flower Development in Chrysanthemum morifolium and Chrysanthemum lavandulifolium

Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley (GAPDH, ACTIN, eIF-4α, SAND, UBC, TIP41, EF-1α, and TUB) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1α and TUB were the most stable genes for abiotic stresses, whereas EF-1α, GAPDH, and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley.

Section: Discussionmentioning

confidence: 99%

Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli

Song

Wang

et al. 2016

“…et al, 2016). In general, different algorithms are usually combined together for qRT-PCR evaluation and normalization (Demidenko et al, 2011; Qi et al, 2016; Reddy et al, 2016). …”

Section: Introductionmentioning

confidence: 99%

Selection and Validation of Appropriate Reference Genes for qRT-PCR Analysis in Isatis indigotica Fort.

Li¹,

Jing²,

Lu³

et al. 2017

Due to its sensitivity and specificity, real-time quantitative PCR (qRT-PCR) is a popular technique for investigating gene expression levels in plants. Based on the Minimum Information for Publication of Real-Time Quantitative PCR Experiments (MIQE) guidelines, it is necessary to select and validate putative appropriate reference genes for qRT-PCR normalization. In the current study, three algorithms, geNorm, NormFinder, and BestKeeper, were applied to assess the expression stability of 10 candidate reference genes across five different tissues and three different abiotic stresses in Isatis indigotica Fort. Additionally, the IiYUC6 gene associated with IAA biosynthesis was applied to validate the candidate reference genes. The analysis results of the geNorm, NormFinder, and BestKeeper algorithms indicated certain differences for the different sample sets and different experiment conditions. Considering all of the algorithms, PP2A-4 and TUB4 were recommended as the most stable reference genes for total and different tissue samples, respectively. Moreover, RPL15 and PP2A-4 were considered to be the most suitable reference genes for abiotic stress treatments. The obtained experimental results might contribute to improved accuracy and credibility for the expression levels of target genes by qRT-PCR normalization in I. indigotica.

“…Chrysanthemum morifolium , one of the 10 most popular Chinese traditional flowers, is a typical example of radiate species with inflorescence composed of both zygomorphic and actinomorphic symmetric flowers (Teixeira da Silva, 2003; Teixeira da Silva et al, 2013; Qi et al, 2016). The contemporary chrysanthemum cultivars are characterized by the substantial variation in petal types and the complicated ploidy levels (from 2n = 4x = 36, to 6 n = 54, 72, up to 90) (Liu et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Identification and Characterization of CYC-Like Genes in Regulation of Ray Floret Development in Chrysanthemum morifolium

Huang

Sun

et al. 2016

Chrysanthemum morifolium, one of the most economically important ornamental crops worldwide, is well-known for the elaborate and complex inflorescence which is composed of both bilaterally symmetrical ray florets and radially symmetrical disc florets. Despite continuing efforts, the molecular mechanisms underlying regulation of the two flower types are still unclear so far. CYC-like proteins have been shown to control flower symmetry or regulate flower-type identity in several angiosperm plant lineages. In this study, we conducted comparative analysis of the CmCYC2 genes in two chrysanthemum cultivars and their F1 progenies with various whorls of ray florets. Six CmCYC genes were identified and sequenced, all of which were grouped into the CYC2 subclade. All the six CmCYC2 genes were predominantly expressed in reproductive organs, and in particular in the petal of ray florets. Of these genes, the transcription level of CmCYC2c was highly up-regulated in ray florets of the double-ray flowered heads. In addition, the result that CmCYC2c was highly expressed at key developing stages indicates its role in regulating petal development. Furthermore, overexpression of CmCYC2c in C. lavandulifolium, one of the original species of C. morifolium, led to significant increase in flower numbers and petal ligule length of ray florets. Besides CmCYC2c, the expression of CmCYC2f was also significantly up-regulated in transgenic lines, implying a possible role in regulating development of ray florets. Both results of expression patterns and transgenic phenotypes suggest that CmCYC2c is involved in regulating ray floret identity in the chrysanthemum. This study will be useful for genetic manipulation of flower shape in chrysanthemum and hence promote the process of molecular breeding.