Screening and Validation of qRT-PCR Reference Genes in Different Tissues and Autumn Leaf Coloration Period of Euonymus maackii

Euonymus maackii is an important ornamental tree species valued for its autumn foliage in northern China. To precisely elucidate the molecular mechanisms underlying the distinct leaf coloration types in E. maackii during autumn, this study aimed to identify the optimal reference genes for qRT-PCR normalization across different tissues, developmental stages, and autumn leaf coloration types. Using 10 different tissues of E. maackii as experimental materials, 10 candidate reference genes were comprehensively evaluated for expression stability using three software tools, including geNorm, and the RefFinder online platform, with structural genes of the pigment biosynthesis pathway employed for validation. The comprehensive evaluation revealed that TIP41 was the most stable reference gene across different tissues, developmental stages, and the full sample set; EF-1α exhibited the highest stability among samples representing different autumn leaf coloration types; and GAPDH was the least stable in all sample groups. Quantitative validation of target genes demonstrated that when EF-1α and TIP41 were used in combination for normalization, the expression patterns of pigment biosynthesis structural genes were highly consistent with the phenotypic changes observed in autumn leaf coloration. In summary, this study recommends TIP41 as a universal reference gene for expression analysis across different tissues and developmental stages of E. maackii; for studies involving different autumn leaf coloration types, a dual-reference normalization approach combining EF-1α and TIP41 is recommended.