Objective Walnut (Juglans regia L.). is a monoecious, dichogamous species, and a major global nut crop. However, mismatched ratios of male and female flower buds limit fruit set and reduce orchard profitability. This study investigated the molecular mechanism of walnut flower bud sex differentiation using transcriptome sequencing to identify candidate genes regulating bud differentiation and flowering, thereby laying a foundation for targeted manipulations of sex differentiation and improved yield.

Method Male and female flower buds at multiple developmental stages were collected for RNA-seq. Differentially expressed genes (DEGs) were identified and functionally annotated via GO enrichment and KEGG pathway analyses. Selected DEGs were validated by quantitative real-time PCR (RT-qPCR) to refine the candidate set.

Result Transcriptomic analysis revealed genes that were consistently up- or down-regulated across four developmental stages of male and female flower buds. And we selected the 100 genes showing the most significant sex-related expression during the physiological differentiation phase and predicted their functions. Combined with GO and KEGG enrichment analysis, a total of 18 genes that may regulate flower bud differentiation were screened by referring to the literature related to flower development genes. Subsequent RT-qPCR validation, integrated with transcriptome data, narrowed this set down to 10 candidate genes.

Conclusion Through the analysis of transcriptome data results, a total of 10 candidate genes were screened based on RT-qPCR results.