Abstract
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.