Sugar Tech

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Suitable Reference Genes/miRNAs for qRT-PCR Normalization of Expression Analysis in Sugarcane Under Sorghum mosaic virus Infection

  • Hui Ling
  • Ning Huang
  • Liping Xu
  • Qiong Peng
  • Feng Liu
  • Yuting Yang
  • Youxiong QueEmail author
Research Article


Sugarcane mosaic disease poses a serious threat to the sugarcane industry. Many studies have aimed to unravel the molecular mechanism related to sugarcane and mosaic virus interaction. Quantitative reverse transcription (qRT)-PCR in combination with suitable internal reference genes has been widely used for gene expression analysis. In this study, the expression of 33 candidate reference genes and 12 candidate reference miRNAs was analyzed for first time in the leaf samples of three sugarcane genotype infected with Sorghum mosaic virus using the geNorm, NormFinder and deltaCt (deltaCq) algorithms. A comparison of the expression of eIF-4E and three virus-derived siRNA (vsiR9230S, vsiR9058A and miR16) with the normalized unstable and stable reference genes or miRNA indicated that PP2A and miR159 constituted the best single reference gene/miRNA under SrMV infection. We also suggested that both CUL + CAC and miR171+ miR1520 could serve as the most suitable reference gene/miRNA combination. The use of reliable reference genes and miRNAs should improve the accuracy of gene expression analysis in sugarcane leaves under SrMV stress.


Sugarcane Reference gene miRNA Mosaic virus qRT-PCR 



Sugarcane mosaic disease


Sugarcane mosaic virus


Sorghum mosaic virus


Sugarcane streak mosaic virus


Quantitative reverse transcription polymerase chain reaction


Glyceraldehyde-3-phosphate dehydrogenase


Eukaryotic elongation factor 1-alpha


Eukaryotic elongation factor 4-alpha


Abscisic acid


Salicylic acid


Methyl jasmonate acid


Clathrin adaptor complex




GTP-binding protein gene


Phosphatase 2A gene


SAND family protein gene


Ubiquitin-conjugating enzyme 18 gene


Uridylate kinase


Candidate reference genes


Candidate reference miRNA


Eukaryotic translation initiation factor 4E


Stability value


Comprehensive stability value




Histone protein H1




Selenium-binding protein 1-like


Ubiquitin-conjugating enzyme E2


Cytochrome P450-like protein




E3 ubiquitin-protein ligase ARI8-like


60S ribosomal protein L29


RMD5 homolog A-like


Flowering time control protein FCA-like


Transport protein particle component Bet3


Glutamate dehydrogenase 2-like


Replication protein A 32 kDa subunit B-like


Tu, mitochondrial-like


Protein IN2-1 homolog B-like


Protein IN2-1 homolog B-like




O-methyltransferase-like protein


Minimum information for publication of quantitative real-time PCR experiments


Quantification cycle



The National Natural Science Foundation of China (30871581 and 31801424) and Sugar Crop Research System (CARS-17) supported this work.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (PDF 1102 kb)
12355_2019_712_MOESM2_ESM.doc (85 kb)
Supplementary material 2 (DOC 85 kb)
12355_2019_712_MOESM3_ESM.doc (16 kb)
Supplementary material 3 (DOC 15 kb)
12355_2019_712_MOESM4_ESM.doc (18 kb)
Supplementary material 4 (DOC 17 kb)


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Copyright information

© Society for Sugar Research & Promotion 2019

Authors and Affiliations

  1. 1.Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of AgricultureFujian Agriculture and Forestry UniversityFuzhouPeople’s Republic of China
  2. 2.Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of EducationFujian Agriculture and Forestry UniversityFuzhouChina
  3. 3.College of Life SciencesFujian Agriculture and Forestry UniversityFuzhouChina

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