Rootstock authentication and phylogenetic studies in <i>Myristica</i> sp. using novel chloroplast DNA-based sequence length polymorphism (SLP) markers

Abstract Late-phase graft incompatibility and subsequent yield reduction pose significant threats to sustainable nutmeg ( Myristica fragrans ) production in India, primarily due to the widespread use of wild Myristica species as rootstocks. Accurate rootstock authentication at the seedling stage is critical to prevent these long-term losses, but current methodologies are insufficient. This study develops novel chloroplast DNA (cpDNA)-based markers for species authentication and phylogenetic analysis in Myristica . Utilizing a comparative chloroplast genomics approach, we identified hypervariable regions to create robust sequence length polymorphism markers. Marker validation across 33 diverse Myristica accessions, including all major Indian cultivated germplasm, revealed a unique 25 bp deletion in the ycf4-cemA region. This deletion serves as a highly reliable diagnostic marker to differentiate cultivated nutmeg from wild rootstock species. The validated markers were successfully used to authenticate rootstocks in both commercial nurseries and established plantations, offering a critical tool to prevent long-term graft incompatibility. Intraspecific variation within M. fragrans cultivars was observed in the rpoB_trnC-GCA, trnS-UGA _ psbZ and trnR-ACG_rrn5S intergenic spacer regions. UPGMA clustering distinctly separated the Myristica accessions into three discrete phylogenetic groups. Beyond rootstock management, the identified M. fragrans -specific marker can reliably authenticate nutmeg in commercial products, distinguishing it from potential adulterants like M. magnifica and M. andamanica . Furthermore, we report a species-specific marker for the endemic and IUCN red-listed vulnerable species, M. andamanica , which will be invaluable for its precise identification and conservation efforts. Our research provides an invaluable, easy-to-use toolkit for Myristica germplasm management, breeding programs and quality control of nutmeg products.