Molecular identification of Korean Broussonetia × kazinoki using cleaved amplified polymorphic sequence (CAPS) marker

Broussonetia × kazinoki, a hybrid of B. monoica and B. papyrifera, is a main resource from which traditional paper is made and medicine is obtained. Due to extensive morphological variation, identification from parental species, using morphological characteristics alone is difficult, especially during non-flowering seasons. This study developed a 26 S rDNA-based cleaved amplified polymorphic sequence (CAPS) marker to enable precise identification even when morphological characteristics are limited. CAPS marker was developed using the restriction enzyme BstN1. The marker clearly identified 6 B. monoica, 4 B. papyrifera, and 79 B. × kazinoki individuals when applied to 89 Broussonetia accessions collected across South Korea. Molecular identification revealed inconsistencies with initial field identifications based on morphology, leading to the reclassification of several misidentified specimens. Genome size measurement further revealed that the B. × kazinoki accessions consisted of 49 diploids and 30 triploids. The developed CAPS marker successfully identified the hybrid species from its parents regardless of ploidy levels, even when morphological features were unavailable. This molecular identification system provides an accurate, rapid, and cost-effective method for the identification of Korean Broussonetia. The system is expected to improve the reliability of resource management and lay a foundational framework for the conservation and industrial utilization of Broussonetia genetic resources.