title: Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR.
abstract: Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling 712100, China.
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Yin
authors.forename: Xiwang
authors.initials: X
authors.lastname: Ke
authors.forename: Dingxuan
authors.initials: D
authors.lastname: Huang
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Gao
authors.forename: Ralf T
authors.initials: RT
authors.lastname: Voegele
authors.forename: Zhensheng
authors.initials: Z
authors.lastname: Kang
authors.forename: Lili
authors.initials: L
authors.lastname: Huang
mesh_terms: D001203:Ascomycota / Q000235:genetics*; D002681:China; D003001:Cloning, Molecular; D021983:Cyclophilins / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D020043:Genes, Essential; D005800:Genes, Fungal; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics*; D027845:Malus / Q000382:microbiology; D011096:Polygalacturonase / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics
chemical_list: D012269:Ribosomal Proteins; D037521:Virulence Factors; D005954:Glucosephosphate Dehydrogenase; D011096:Polygalacturonase; D021983:Cyclophilins