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Reference genes are used for the correction of qRT-PCR data, and it is necessary to investigate the optimum reference gene under certain conditions. The expression levels of seven traditional reference genes <i>ACT1</i>, <i>ACT2</i>, <i>GAPDH</i>, <i>18S rRNA</i>, <i>UBQ</i>, <i>TUB</i> and <i>CYP</i> were analyzed using qRT-PCR in different varieties, tissues, developmental stages and hormone (or pollen polysaccharide) treatments in kiwifruit. Gene expression stability was assessed with the help of three common software (geNorm, NormFinder, BestKeeper), and the minimum number of reference genes necessary for normalization was also determined. <i>GAPDH</i>, <i>ACT1</i> and <i>ACT2</i> were selected as reference genes for different genotypes of kiwifruit. <i>GAPDH</i> and <i>UBQ</i> were the best combinations of reference genes for root, stem, leaf, flower and fruit. <i>GAPDH</i> and <i>ACT1</i> could be the preferred reference genes for normalization of qRT-PCR data during fruit development. The pairing of <i>ACT1</i> and <i>UBQ</i> constituted the optimal combination of reference genes in kiwifruit treated with different hormones (or pollen polysaccharide). This study provides a new and reliable option for the use of reference genes in the analysis of gene expression patterns of interest in kiwifruit.