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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
  • 14 Downloads

Abstract

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.

Keywords

Sugarcane Reference gene miRNA Mosaic virus qRT-PCR 

Abbreviations

SMD

Sugarcane mosaic disease

SCMV

Sugarcane mosaic virus

SrMV

Sorghum mosaic virus

SCSMV

Sugarcane streak mosaic virus

qRT-PCR

Quantitative reverse transcription polymerase chain reaction

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

eEF-1a

Eukaryotic elongation factor 1-alpha

eIF-4α

Eukaryotic elongation factor 4-alpha

ABA

Abscisic acid

SA

Salicylic acid

MeJA

Methyl jasmonate acid

CAC

Clathrin adaptor complex

CUL

Cullin

GTBP

GTP-binding protein gene

PP2A

Phosphatase 2A gene

SAND

SAND family protein gene

UBC18

Ubiquitin-conjugating enzyme 18 gene

UK

Uridylate kinase

CRGs

Candidate reference genes

CRmiRNA

Candidate reference miRNA

eIF-4E

Eukaryotic translation initiation factor 4E

SV

Stability value

CSV

Comprehensive stability value

CV

Covariance

H1

Histone protein H1

αTUB

α-Tubulin

SBP

Selenium-binding protein 1-like

UBCE2

Ubiquitin-conjugating enzyme E2

CYP

Cytochrome P450-like protein

Nup

Nucleophosmin

ARI

E3 ubiquitin-protein ligase ARI8-like

L29

60S ribosomal protein L29

RMD5

RMD5 homolog A-like

FCA

Flowering time control protein FCA-like

Bet3

Transport protein particle component Bet3

GLUD

Glutamate dehydrogenase 2-like

RPAb

Replication protein A 32 kDa subunit B-like

EFTuM

Tu, mitochondrial-like

IN2-1

Protein IN2-1 homolog B-like

IN2-1

Protein IN2-1 homolog B-like

Pep

Peptidase

OMTA

O-methyltransferase-like protein

MIQE

Minimum information for publication of quantitative real-time PCR experiments

Cq

Quantification cycle

Notes

Funding

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)
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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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