Identification and Validation of Reference Genes in the Adzuki Bean (Vigna angularis) Under Iron Deficiency Using Quantitative Real-Time PCR

Abstract

Adzuki bean (Vigna angularis var. angularis), an important food legume, is grown in more than 30 countries, especially in East Asia. Almost 25% of the world population is affected by iron deficiency. Iron is very important on adzuki bean yield and quality. The identification of suitable reference genes in adzuki bean is important for exploring gene expression and the molecular mechanisms of gene, yet few reference genes have been reported for adzuki bean. This research systematically evaluated the stability of 11 candidate reference genes (ACT, ACT1, CYP, CYP2, SKIP16, ELF1B, HDC, Lectin, TUA, PEPKR1, and 18SrRNA) using the geNorm, NormFinder, BestKeeper, and RefFinder protocols under iron-deficient stress. The best combined number of reference genes for all samples was two. ELF1B/Lectin was best for qRT-PCR analysis of roots and combined analysis of roots, stems, and leaves under iron deficiency, ELF1B/CYP was optimum for leaves, and SKIP16/PEPKR1 was optimum for stems. The identification of these reference genes will enable accurate quantification of gene expression under iron deficiency and enhance our comprehension of reference genes in adzuki bean.