Gyrinops versteegii (Gilg.) Domke, an endangered agarwood-producing tree, faces threats from overexploitation and genetic erosion in Indonesia. This study sought to evaluate the intraspecific genetic diversity and molecular markers for conservation via sequencing and assembling of the chloroplast genomes of six G. versteegii genotypes from four regions using Oxford Nanopore Technology (ONT). The assemblies ranged from 32,442 to 138,782 bp, and the genotype CLD2 produced the largest and most complete chloroplast draft assembly among the six genotypes (138,782 bp; 126 genes). Comparative analysis of nine chloroplast genes revealed notable variations, particularly in the atpA (Pi = 0.09178). The highest genetic distance emerged between genotypes NTB2 (from Nusa Tenggara) and CLD1 (from West Java). The phylogenetic analysis suggested clustering of G. versteegii genotypes into three subclusters, separated from other species of the family Thymelaeaceae. Additionally, 235 chloroplast simple sequence repeats (cpSSRs) entailed identification, predominantly A/T motifs, including several novel loci not reported in the reference genomes. These cpSSRs provided valuable resources for species authentication, population monitoring, and conservation breeding. The results revealed intraspecific variation among G. versteegii genotypes and demonstrated the utility of ONT sequencing for developing molecular markers in tropical tree species. The genomic insights will support effective conservation strategies and sustainable management of agarwood-producing tree resources.

Keywords: G. versteegii, chloroplast genomes, genetic distance, genetic diversity, conservation, intraspecific variation, microsatellites, nucleotide diversity

Key findings: This study explored the crucial intraspecific variations in G. versteegii genotypes as the endangered agarwood-producing tree, offering insights essential for developing effective conservation strategies for this endangered species.