Despite Macadamia’s global economic importance as a nutrient-dense nut crop, the structural and evolutionary characteristics of its chloroplast genomes (plastomes) remain underexplored. Here, we present the first comprehensive plastomic analysis of 185 cultivated Macadamia accessions from a Chinese germplasm collection. These plastomes exhibited conserved quadripartite structures (159,195–159,726 bp), comprising a large single-copy (LSC, 87,651–88,107 bp), a small single-copy (SSC, 18,743–18,819 bp), and inverted repeats (IRs, 26,378–26,422 bp). All plastomes maintained 115 functional genes (81 protein-coding, 30 tRNA, 4 rRNA) and were classified into 23 unique haplotypes. We identified 573 SNPs, 577 long repeats, and five hypervariable regions (trnS–trnG-exon1, ndhD–psaC, trnH–psbA, petA–psbL, and psbC–psbZ), with nucleotide diversity (Pi) values > 0.01. Phylogenetic reconstruction based on whole plastomes resolved maternal lineages and revealed distinct clustering patterns between ancestral and modern cultivars, with first-generation hybrids demonstrating significant plastomic uniformity. Notably, three accessions (‘Bailahe1’, ‘JingG6’, ‘Jing40’) showed ambiguous phylogenetic placement, warranting further investigation. Our findings provide critical plastomic resources for cultivar authentication, evolutionary studies, and the strategic expansion of genetic diversity in Macadamia breeding programs, particularly valuable given the crop’s recent domestication history and narrow genetic base.

To characterize the transcriptomic dynamics underlying shell hardening in Macadamia integrifolia, RNA sequencing was performed on pericarp and shell tissues collected at three key developmental stages. A total of 18 libraries were generated, yielding 125.19 Gb of clean data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that husk-associated DEGs were mainly enriched in photosynthesis-related functions, whereas shell-associated DEGs were enriched in phenylpropanoid biosynthesis, secondary metabolism, and cell wall organization. Weighted gene co-expression network analysis (WGCNA) identified module M8 as being strongly associated with the middle and late stages of shell development. Genes in this module were mainly enriched in phenylpropanoid metabolism, redox-related processes, and extracellular region functions. The hub genes within M8 included those encoding a glutaredoxin family protein, a WNK-type protein kinase, a tRNA methyltransferase-related protein, an EFR3 membrane-associated protein, a pectin modification-related protein, and a WAT1-related protein. Collectively, these results reveal clear tissue-specific transcriptomic patterns during pericarp and shell development and identify candidate co-expression networks that may contribute to shell hardening.

Palmitoleic acid (C16:1) is rare among woody oil crops yet accumulates to high levels in macadamia kernels, where its inter-accession variation and relationship with fruit traits remain poorly characterized. We profiled 22 fatty acids by GC-MS, quantified crude fat, crude protein and total soluble sugar by NIRS, and measured nine fruit traits in 139 accessions. The major fatty acids ranked as C18:1 > C16:1 > C16:0. C16:1 varied widely among accessions (mean 10.66%, CV = 27.7%, range 2.78–19.05%), about 12-fold the analytical RSD, whereas C18:1 was stable (CV = 5.3%). C16:1 was strongly negatively correlated with C18:1 (r = −0.921, p < 0.001) and positively correlated with C16:0 (r = 0.534). Correlation, partial correlation and PCA consistently indicated that fatty acid composition was independent of fruit traits; after controlling for C18:1, C16:1 retained only a weak positive association with single dry kernel weight (r = 0.170, p < 0.05). Entropy-weighted TOPSIS with five indicators selected five accessions combining high C16:1 with desirable fruit traits. These accessions provide breeding material for C16:1-enriched oil, and the independence of C16:1 from fruit traits means that both can be improved simultaneously.