Research Article: microRNAs as reference genes for quantitative PCR in cotton

Date Published: April 17, 2017

Publisher: Public Library of Science

Author(s): Anna Karoline Silva Fausto, Tatiane da Franca Silva, Elisson Romanel, Maite F. S. Vaslin, Sara Amancio.


Cotton (Gossypium hirsutum) is the most important non-food plant in the world. Studies concerning the fiber quality and plant fitness of cotton at molecular level depend on high sensitive and reproducible gene-expression assays. However, only a few reports have described genes suitable for normalizing gene expression data. In this study, we report for the first time that microRNAs (miRNAs) are reliable reference genes (RGs) for cotton gene expression normalization in quantitative real-time reverse transcription (RT)-PCR. The stability of cotton miRNAs was assayed in root, stem, leaf and flower samples from three different cultivars [FiberMax (FM966), Delta Opal (DO) and Cedro] and under conditions of biotic stress caused by infection with Cotton leafroll dwarf virus (CLRDV). The stability of mRNAs already described as reference genes in cotton was also assessed. The geNorm, NormFinder, BestKeeper and ΔCt algorithms were used to select the best reference genes. In 8 of the 12 sets tested, miRNAs (miR172, 168 and 390) were found to be the best RGs. To validate the best selected RGs, miR159, miR164, miR2118, miR2910, miR3476, GhDCL2 and GhDCL4 expression levels were evaluated under biotic stress conditions, and miR164 and a putative myo-inositol oxigenase gene (GhMIOX) were assessed in leaves and flowers. The RGs selected in this work proved to be excellent reference genes in the two cases studied. Our results support the use of miRNAs as reference genes for miRNA and protein-coding genes.

Partial Text

Cotton is one of the most important crop in the world. It is commercially grown in the temperate and tropical regions of more than 60 countries and represents the main source of renewable textile fibers. Moreover, cotton seed is an important source of oil, and its use as a fuel is currently growing. The allotetraploid upland cotton (AD)1 (Gossypium hirsutum L.) represents more than 90% of cultivated cotton worldwide. Based on the importance of cotton and the high demand for agricultural sources as pesticides and others to achieve high production levels, there is a steady need for the biotechnological improvement of this crop.

To select reliable miRNA RGs for cotton, six miRNA candidates (miRNA159ab, miR166e, miR167g, miR168b, miR172f and miR390a) were selected from the G. hirsutum NGS studies [37] and compared to five previously described cotton reference protein-coding genes: ACT4, EF1α-5, PP2A1, UBQ14 [35] and 18S (Table 1). Cotton Illumina small RNA libraries from uninfected and Cotton leafroll dwarf virus (CLRDV)-infected leaves [37] were used for the cotton miRNA selection. The six cotton miRNAs selected belong to different families in different functional classes based on Arabidopsis miRNA studies [48]. miRNA159 functions in the regulation of signaling pathways and development. miR166 is involved in leaf and vein development in Arabidopsis, while miR167 in involved in signaling pathways and flower development. miR168 plays a role in the biotic stress response and signal transduction during development; miR172 is involved in signaling, development and the stress response, and miR390 is involved in plant development.

qPCR is widely used to investigate gene expression. However, for accurate analysis and interpretation of the results, the choice of suitable genes to normalize the data is a crucial factor. Detailed analyses of various commonly used reference genes, including ACT, GAPDH and 18S rRNA, have revealed that they can be significantly influenced by the experimental conditions [35, 50–53]. Therefore, it is necessary to validate reference genes for each plant species being studied and for each specific experimental condition [54]. Reference candidate genes must be evaluated and validated each time a new experimental condition will be analyzed.




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