Cloning and Stability Analysis of Reference Genes for Expression Studies by Quantitative Real-Time PCR in Litchi

The full-length sequences of β-Actin,GAPDH and 18S rRNA which are frequently used as reference genes in qPCR analysis were obtained from the pericarp of litchi (Litchi chinensis Sonn.cv.Feizixiao) by gene cloning and sequencing. Their sequence lengths were 1 273,1 368 and 1 712 bp,respectively. Sequence analysis showed that the cloned sequences had very high similarity with other species. Furthermore,the stability of six genes,including β-Actin,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18S rRNA,ubiquitin(UBQ),elongation factor (eEF) and 25S rRNA,was determined in litchi pericarp under different developmental stages and regulator treatments in this study. Three different analytic calculation methods including geNorm,NormFinder and BestKeeper were compared. Among the six genes tested,β-Actin appeared to be the most stable single gene under three different calculation methods.