Identification and Validation of Reliable Reference Genes for Gene Expression Studies in Postharvest Atemoya Pulp (Annona cherimola Mill × A. squamosa L.) Subjected to Different Preservation Treatments

Quantitative real-time PCR (qRT-PCR) is a widely used technique for quantifying gene expression. However, accurate data normalization requires stable reference genes that exhibit constant expression levels across different experimental conditions. To date, no suitable reference genes have been validated for atemoya fruits (Annona cherimola Mill × A. squamosa L.). This study aimed to evaluate the expression stability of candidate reference genes in atemoya across distinct tissues, postharvest storage stages, and various treatment conditions. To this end, we designed specific primers for six reference genes: ubiquitin carrier-like protein (UBC), actin 7 (ACT7), actin 11 (ACT11), elongation factor 1α (EF1α), 18S ribosomal RNA (18S), and β-tubulin (TUB). Total RNA was extracted from atemoya tissues (e.g., petals, young fruits, and roots), for pulp across three post-harvest time points (0, 2, and 4 days) and under different conditions: 28 °C, 15 °C, ethylene, or 1-MCP treatment. The geNorm tool was subsequently used to identify the most stable reference transcripts under each experimental condition. ACT7 was the most stable reference gene across atemoya tissues, while UBC ranked first under all postharvest conditions tested (28 °C storage, 15 °C cold storage, ethylene, and 1-MCP). Furthermore, the suitability of these reference genes under ethylene and 1-MCP treatments was validated by examining the expression patterns of AaPG and AaERF. Collectively, the combined use of ACT7, UBC, and 18S proved to be the most reliable normalization strategy across all experimental conditions. This validated multi-gene approach provides a robust foundation for future investigations into gene expression and the postharvest ripening mechanisms of atemoya.