Identification and Validation of New Reference Genes for Normalization of Gene Expression in Flower and Berry Developmental Stages of Interspecific Grape Hybrid V. vinifera (L.) × V. labrusca (L.)

Abstract

In quantitative reverse transcription polymerase chain reaction (qRT-PCR), normalizing target gene expression using a reference gene is an indispensable step to control the variability of RNA extraction yield, RNA integrity, reverse transcription efficiency, and PCR amplification. In the present study, we identified candidate reference genes with stable expression during grapes’ flowering and berry development stages. Ten genes, including ACT, CYP5, RLI, TUB, UBC, UBC17, UBC60, UFD1, VAG, and ZNF with relatively stable expression, were selected based on RNAseq data generated earlier in grape hybrid ‘ARI 516’. The expression of these candidate genes was tested at different stages of flowering and grape berry development. Five different algorithms such as RefFinder, geNorm, NormFinder, BestKeeper, and the comparative ΔCq method were used to test the expression stability of candidate genes. A comprehensive ranking obtained by RefFinder showed that UBC17, RLI, and ZNF were the most stable reference genes during flower and berry development stages. UBC17, RLI, and ZNF were calibrators to normalize the expression of VvAGL11 as a target gene to validate the worthiness of identified reference genes. The result demonstrated that newly identified reference genes could be successfully used to normalize the expression of the target gene accurately. These reference genes will provide more choices for selecting appropriate reference genes to normalize gene expression in grapes.

Keywords

• algorithms
• expression stability
• relative expression
• quantitative rt-pcr
• ubc17
• rli
• znf