Reference gene selection for qRT-PCR normalization in alfalfa under UV-B irradiation

[Objectives]In order to acquire accurate results of the expression of genes during different metabolism processes in plants,it is critical to perform the selection of suitable reference genes under different conditions.[Methods]We analyze the stabilities of 10 reference genes(Actin2,GAPDH,MSC27,18S RNA,β-tublin,b ZIP,UBQ,PPPrep,Ms03_50f03 and Ms03_69f07) within different tissues(roots,stems and leaves) of alfalfa(Medicago sativa L.) through ge Norm and Norm Finder softwares,which were collected at different times(samples were collected after UV-B irradiation for 0,1,2 and 5d).[Results]The results illustrated that MSC27 and UBQ were suitable for alfalfa roots analysis upon UV-B irradiation,as revealed by their lowest stability values.Furthermore,when another reference gene was added,corresponding variation(V) value(V3 /4) was not significantly altered when compared with that of V2 /3,which only contained two reference genes.These results further suggested that two reference genes were enough in alfalfa roots analysis under UVB stress.MSC27 and Actin2 was appropriated for the alfalfa stems analysis since the lowest M values were obtained.Meanwhile,the analysis of alfalfa leaves was advised to use Actin2 and GAPDH as internal genes under UV-B stress.The variation(V) value(V3 /4) was higher than that of V2 /3 when another reference gene was added,The 18 S RNA and β-tublin had the hightest M values in the analysis of all three alfalfa tissues,which suggested that they were not suitable for reference genes.The results analyzed by Normfinder were almost the same as that of ge Norm.The differences between these two softwares might be their different algorithms.[Conclusions]In alfalfa seedlings,MSC27 and UBQ were suitable in root analysis; MSC27 and Actin2 were appropriate in stem analysis; while we used Actin2 and GAPDH as internal genes to get more precise gene expression in leaf analysis.The results of the study have important practical value in the analysis of key genes under UV-B irradiation.