Background/Objectives: Macadamia integrifolia is a valuable subtropical fruit tree, yet genomic studies on its cultivars are limited. This study aims to elucidate the chloroplast genome features, variations, and phylogenetic relationships of three main cultivars (‘Guilin No. 1’, ‘Nanya No. 1’, ‘Qian’ao No. 1’) to support germplasm identification and breeding. Methods: chloroplast genomes of three M. integrifolia cultivars from Guangxi, Guangdong, and Guizhou were sequenced using Illumina technology, followed by assembly, annotation, and comparative analyses of structure, repeats, and codon usage. Phylogenetic relationships were reconstructed using complete genome sequences. Results: The three chloroplast genomes displayed typical quadripartite structures, with lengths of 159,714 bp, 159,195 bp, and 159,508 bp, and GC contents of 38.12%, 38.16%, and 38.14%, respectively. Each encoded 135 genes. Codon usage was biased towards A/U-ending codons. We identified 81, 87, and 80 SSRs and 26, 21, and 20 long repeats, respectively. IR boundary regions were highly conserved. Phylogenetically, the cultivars showed close relationships with M. integrifolia, Macadamia tetraphylla, and Macadamiaternifolia, forming a sister clade to Platanus occidentalis. Conclusions: This study provides essential chloroplast genomic resources for three M. integrifolia cultivars, revealing conserved structures and specific variations. The findings offer crucial insights for the genus's genetic diversity, supporting future germplasm evaluation and phylogenetic research.

Evodia rutaecarpa is a valuable medicinal plant, yet its non-medicinal tissues remain largely underexplored. Here, we integrated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based widely targeted metabolomics and RNA sequencing (RNA-seq) transcriptomics to systematically profile the metabolic and transcriptional landscapes of roots, stems, leaves, and flowers of Evodia rutaecarpa (Juss.) Benth. Our aim was to characterize tissue-specific metabolic differentiation and its underlying transcriptional regulatory mechanisms. Metabolomic analysis, employing principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) with robust model parameters (R²Y > 0.9, Q² > 0.5), identified 3090 differential metabolite features (variable importance in projection, VIP > 1.0; p < 0.05) across the four tissues, which exhibited distinct tissue-specific clustering patterns. Integrated Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and weighted gene co-expression network analysis (WGCNA) revealed that roots specifically accumulated quinolone alkaloids and flavonoid glycosides, accompanied by the coordinated upregulation of genes involved in flavonoid and phenylpropanoid biosynthetic pathways. In contrast, stems, leaves, and flowers were enriched in indole alkaloids (evodiamine and rutaecarpine) and volatile oil precursors, with concurrent upregulation of genes involved in tryptophan metabolism and indole alkaloid biosynthesis (e.g., tryptophan decarboxylase, TDC; s N-methyltransferase, NMT). Notably, leaves and flowers displayed particularly high accumulation levels of these bioactive alkaloids, suggesting their potential as alternative sources for industrial and pharmaceutical applications. WGCNA further identified multiple transcription factors and structural gene modules tightly correlated with evodiamine accumulation, offering promising candidate regulators for future biosynthetic pathway engineering. Collectively, this multi-omics integration study systematically elucidates the tissue-partitioned secondary metabolism of Evodia rutaecarpa (Juss.) Benth. and provides a solid scientific foundation for full-plant resource utilization, targeted development of non-medicinal tissues, and future metabolic engineering of indole alkaloid production.