甜瓜属异源多倍体不同倍性材料内参基因的筛选及评估OA北大核心CSTPCD

[Objectives]Polyploidy is widespread in plants kingdom.However,the lack of reference genes has severely limited the research progress of polyploidy-related gene expression.The present study aimed to improve the accuracy and reproducibility of gene expression analyses in polyploids by establishing a set of stable reference genes.[Methods]The abundance of 10 candidate reference genes(UBI-ep,ACT,ACT3,TUA,EF-1α,CACS,TIP41,F-box,CYP and UBQ)were compared by real-time fluorescence quantitative analysis(qPCR)at different ploidy levels of Cucumis including artificial allotetraploid,their diploid parents and triploid offsprings.The stability of these candidate reference genes expression was analyzed by geNorm and NormFinder software.Furthermore,the correlation of relative transcript abundance estimated by qPCR analysis and transcriptome sequencing(RNA-seq)data was calculated to evaluate the stability of candidate reference genes.[Results]The qPCR results showed that the reference gene CYP in cucumber had the highest expression with a CT value of 15.8,and the reference gene F-box in tetraploid material had the lowest expression with a CT value of 30.5.Four stable reference genes were selected by combining the results of geNorm and NormFinder,which were F-box,TIP41,ACT3 and ACT.Among them,F-box and TIP41 had a lower stability value M than ACT3 and ACT genes at the polyploid level,but their expression abundance were low.When ACT3 was used as the internal reference,the relative transcript abundance correlation coefficient between qPCR and RNA-seq was extremely significant(R2=0.844,P＜0.01).But the correlation was minimum when F-box was an internal parameter.[Conclusions]Attention should be paid to the selection of reference genes when comparing transcript abundance across different ploidy levels.TIP41 could be selected as the internal reference gene for the low abundance expression genes of multiple ploidy materials in melon.For highly expressed genes,ACT3 could be used as an internal reference gene.