Forensic botany clues: Development of a novel Osmanthus fragrans STR multiplex system

Comprehensive research on human DNA genetic markers shows that short tandem repeats (STRs) have found extensive applications in individual identification and kinship testing. Botanical STRs are less commonly employed in forensic science due to the distinctive structure of plant cells and the incomplete state of genome sequencing. This study developed a novel 12-plex system for the individual identification of Osmanthus fragrans (O. fragrans), a species widely distributed across the world's temperate zones, to furnish essential botanical evidence for forensic investigations at crime scenes. Initially, a method for extracting plant DNA from trace samples was improved, which utilized glass beads to break cell walls and yielded significantly higher DNA quantities compared to the traditional method. Subsequently, five novel highly polymorphic STR loci (GHSTR1, GHSTR2, GHSTR4, GHSTR5, and GHSTR6) with trinucleotide repeats were identified from the O. fragrans reference genome (GCA_019395295.1) and characterized (expected heterozygosity > 0.8, polymorphic information content > 0.8, and power of discrimination > 0.9). These loci were integrated with seven previously reported loci (OF5, OF7, OF8, OF9, OF13, OF19, and OSM063) to establish a multiplex system. Developmental validation was conducted in accordance with ISFG and SWGDAM guidelines. The system demonstrated substantial species specificity, high sensitivity (optimal input: 50-500 pg), robust tolerance to common PCR inhibitors (e.g., ≤500 µM hematin and ≤400 ng/µL humic acid), and reliable performance in mixture studies (detecting minor contributors at 1:19-19:1 ratios). PCR conditions were further optimized to 58°C annealing, 20 min elongation, and 28 cycles. The system showed high precision (standard deviation <0.1 bp), accuracy (size deviation within ±0.5 bp), and inter-laboratory concordance. Population analysis of 273 samples revealed the high power of the system effectiveness (combined power of discrimination = 1 - 1.0045 ×10^-19 and combined probability of exclusion = 0.993111589879955) despite observed Hardy-Weinberg equilibrium deviations. Ultimately, the system established definitive links between botanical evidence from suspects/victims and primary crime scenes in two real-world cases. In conclusion, the findings of the present study demonstrate the applicability of a novel STR multiplex system for individual identification of O. fragrans trace samples in forensic science. Without these forensic botany clues, these cases may not have been resolved.