EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions

<i>Salix suchowensis</i> is an ideal model organism for investigating nitrogen (N) transport mechanisms due to its low N-input requirements. Accurate quantification of gene expression is essential for elucidating these processes, with quantitative real-time PCR (RT-qPCR) being the preferred method. However, the identification of stable reference genes for normalization in <i>Salix suchowensis</i> under varying N conditions remains unresolved. In this study, thirteen commonly employed candidate reference genes were evaluated across root, stem, and leaf tissues, under four N treatments (NH₄NO₃, NH₄⁺, NO₃^-, and N deficiency). Five genes (<i>UBQ1</i>, <i>UBQ3</i>, <i>18S</i>, <i>H2A2</i>, and <i>H2B2</i>) were excluded due to poor amplification efficiency or irregular melting curves. The remaining eight genes were further assessed for expression stability using the geNorm, NormFinder, and BestKeeper algorithms. Integrated ranking via RefFinder identified <i>EF1α</i>, <i>EFβ</i>, and <i>αTUB</i> as the most stable reference genes. GeNorm analysis suggested that two reference genes were sufficient for reliable normalization. Validation using the N-responsive gene <i>SsAMT1</i> and <i>SsNRT2</i> confirmed the stability of <i>EF1α</i>, <i>EFβ</i>, and <i>αTUB</i> as suitable reference genes. Based on comprehensive stability assessments and experimental validation, we recommended <i>EF1α</i> + <i>αTUB</i> as optimal reference gene pairs for RT-qPCR normalization under varying N conditions. Furthermore, the consistent expression of <i>EF1α</i> and <i>αTUB</i> across nine willow genotypes highlighted their broader applicability within <i>Salix</i> species. This study provides valuable methodological guidance for advancing molecular research on N transport in woody perennial plants.