Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses.

Weifang Wu,Zhongyuan Hu,Pibiao Shi,Jinghua Yang,Qin Deng,Mingfang Zhang,Xiu-Qing Li

doi:10.1371/journal.pone.0164725

Abstract

Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants.

Highlights

Nitrogen (N) and phosphorus (P) are the two most important macronutrients for the growth and development of plants
The amplification efficiencies (E%) for all primer pairs were determined by Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) standard curve assays, since optimal amplification conditions are indispensable for robust and precise quantification of samples
The efficiency of a qRT-PCR should be 100%, as the cDNA template doubles at every cycle during the exponential phase [17]

Summary

Introduction

Nitrogen (N) and phosphorus (P) are the two most important macronutrients for the growth and development of plants. N is an important constituent of genetic components such as nucleic acids, amino acids, various metabolic compounds including ATP, as well as electrontransfer molecules such as NAD(P) or FAD. It is involved in the composition of critical elements for photosynthesis: proteins, chlorophyll and pigment. The other crucial component of plant cell structure is phosphorus, participating in energy metabolism and signal transduction cascades and regulating the enzymes activities. The availability of the nutrients in the soil solution does not always meet the requirement of plants, leading to a detrimental effect on plant growth and development [2,3,4,5]

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