Selection and validation of garlic reference genes for quantitative real-time PCR normalization

A stable reference gene is a prerequisite to obtaining reliable quantitative real-time PCR (qPCR) analysis results. Despite the global distribution and nutritional, medicinal and economic value of garlic, reference genes for qPCR have not been reported. Eight garlic housekeeping genes, 18S ribosomal RNA (18S), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor (EF-1 alpha), the alpha-tubulin (TUA) and beta-tubulin (TUB), polyubiquitin (UBQ), and cyclophilin (CYP), were chosen as candidate reference genes. Their stability was evaluated by GeNorm, NormFinder, BestKeeper, the comparative Delta Ct method, and RefFinder. The results demonstrated that TUA, GAPDH, UBQ, CYP, and ACT are the most suitable of the eight reference genes for different garlic genotypes, organs, developmental stages, abiotic stresses and media treatments, respectively. To further validate the stability of these reference genes, the expression levels of the superoxide dismutase gene (SOD) were analyzed. The relative quantification of SOD varied according to the stability of the reference gene, thus highlighting the importance of using suitable reference genes in qPCR. Our results also provide a molecular biological basis for studying the mechanisms of development and stress tolerance in garlic.