Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri.

Shiheng Lyu,Shirong Xu,Weiwei Cai,Dongming Pan,Wenqin She,Jianjun Chen,Guixin Chen,Ying Yu

doi:10.3390/genes11010017

Abstract

MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.

Highlights

Citrus is one of the most important fruit crops due to its high nutritional and economic value.Citrus crops are cultivated in a wide range of regions with a total fruit production over 130 million tons in 2015, ranking first in quantity in world fruit production (FAO, 2017)
For reference genes expression in sweet orange, the analysis revealed that the V3/4 value was 0.168, the normalization factor should preferably contain at least the four best reference genes
The present study identified appropriate reference genes for quantifying miRNA expression in two citrus species infected by X. citri subsp. citri (Xcc)

Summary

Introduction

Citrus is one of the most important fruit crops due to its high nutritional and economic value.Citrus crops are cultivated in a wide range of regions with a total fruit production over 130 million tons in 2015, ranking first in quantity in world fruit production (FAO, 2017). Citrus is one of the most important fruit crops due to its high nutritional and economic value. Citrus canker is caused by X. citri subsp. Citri (Xcc) with symptoms of water-soaked eruptions, circular lesions, and pustule-like lesions on all plant tissues, resulting in heavy economic losses [2,3]. Research into citrus canker has been focused on the screening of genetic resources for disease resistance [4,5], transcriptomic analysis of plant responses to pathogen infection [6,7,8], induced resistance for disease control [3], and genome editing of specific genes [9]. Plant defense response is comprised of complex molecular networks regulating gene expression at both transcriptional and posttranscriptional levels [10]. MicroRNAs (miRNAs) are a class of non-coding small RNA (sRNA)

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Conclusion