Abstract：In order to study the optimal reference genes stablely expressed in different tissues and
under different abiotic stresses in different development stages of soybean, 15 soybean samples were
used as experimental materials, including roots and leaves in JN28 V1 stage, pods in R3 and R4
stages, grains in R5 and R8 stages, and roots and leaves under drought and low temperature salt ABA
stress. Eight candidate reference genes (Actin, β -actin, CYP2, EF1- α, Fbox, GAPDH, TUB4,
18SrRNA) were selected for real-time quantitative PCR detection, and the expression stability of the eight candidate reference genes was analyzed. After analysis with the geNorm NormFinder Best‐
Keeper software, the appropriate internal reference genes were selected by RefFinder calculation.The
results showed that the analysis results of the 4 software were not the same. The comprehensive analy‐
sis results of RefFinder showed that the root of stage V1, leaf of stage V1 and root of stage R3 under
ABA stress, the most suitable internal reference gene was Actin.EF1-α was the most suitable refer‐
ence gene for stem of stage V1 and pod of stage R4 and grain of stage R8.In R5 stage, Fbox was the
most suitable reference gene for leaves under drought stress.The optimal reference gene for roots un‐
der drought stress was CYP2. The optimal reference gene for leaves under ABA stress was 18SrRNA.
β-actin was the most suitable reference gene for leaves under low temperature stress.For roots under
low temperature stress, the optimum reference gene were EF1-α and 18SrRNA.In leaves under salt
stress, the most suitable reference genes were β-actin and 18SrRNA.The comprehensive analysis re‐
sults of the 4 software were consistent in all tissues and stresses. The most suitable reference gene
was Actin in all tissues, and the most suitable reference gene was EF1-α in all stresses.
