The stability and applicability of reference genes in alfalfa vary significantly depending on the cultivar， growth and development stages， and environmental conditions. In this study， alfalfa （Medicago sativa L.） cultivar ‘Gongnong No.1’ was used as material， and eight candidate reference genes， glyceraldehyde-3-phosphate dehydrogenase GAPDH， ubiquitin protein ligase 2a UBL-2a， Retention in Endoplasmic Reticulum Rer1， eukaryotic elongation factor-1 alpha eEF-1α， actin-depolymerizing factor ADF， ubiquitin carrier protein UBC， tubulin beta-1 chain TUB， and Actin were selected to evaluate their expression stability of different tissues in seedling and adult plant， as well as under waterlogging stress. The analysis employed quantitative real-time PCR （qRT-PCR） and four softwares， including geNorm， NormFinder， BestKeeper， and RefFinder. The results showed that， at the seedling stage， Rer1， Actin， and UBC were the most stable reference genes. At the tissue level， the most stable reference genes for leaves， stems， and roots were Actin， Rer1， and GAPDH， respectively. At the adult stage， ADF， Actin， and TUB exhibited the highest stability， while the most stable reference genes for leaves， stems， roots， and flowers were eEF-1α， Rer1， Actin， and UBL-2a， respectively. Under waterlogging stress， the three most stable reference genes in seedling leaves were TUB， ADF， and eEF-1α， whereas in seedling roots they were ADF， UBL-2a， and Rer1. In the adult stage， the most stable genes were ADF， Actin， and TUB in leaves， and UBL-2a， UBC， and ADF in roots. Overall， Rer1 and UBL-2a are recommended as reference genes for studies of tissue-specific expression， as their stability was less affected by developmental stage or tissue type. ADF and TUB are suitable reference genes for studies involving waterlogging stress.

Alfalfa  /  Reference genes  /  Abiotic stress  /  Expression stability