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Broussonetia × hanjiana (Moraceae) microsatellite markers for species and ploidy determination developed using next-generation sequencing



Broussonetia × hanjiana has been considered a hybrid owing to its morphology, which is intermediate between that of B. papyrifera (L.) L’Her. ex Vent. and B. kazinoki Siebold. A recent study demonstrated the hybrid origin of B. × hanjiana in Korea using molecular markers. In this study, we developed microsatellite markers for B. × hanjiana using next-generation sequencing and cross-species transferability analysis.

Methods and results

A total of 432 primers were designed from 205,819 contigs. Among them, 24 microsatellite markers showing polymorphisms were used to evaluate the population genetic characteristics. The observed heterozygosity (HO) and expected heterozygosity (HE) were 0.835 and 0.628, respectively. The cross-species transferability of these markers was evaluated in two closely related species of Broussonetia; all 24 markers showed cross-species amplification. Using flow cytometry, diploid and triploid individuals were identified in B. × hanjiana. In particular, the BR137 marker showed evidence of two parent species (B. papyripera and B. kazinoki), with a hybrid pattern observed in B. × hanjiana, demonstrating its utility for species identification and ploidy assessment.


The new B. × hanjiana microsatellite markers can be useful in genetic studies of closely related B. papyripera, B. kazinoki, and B. × hanjiana.

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Data availability

All data supporting the conclusions of this study are available from the corresponding author upon request.


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This study was supported by the National Institute of Forest Science, Republic of Korea.


This study was funded by National Institute of Forest Science of the Republic of Korea grant FP0802-2019-01.

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Authors and Affiliations



J-Y A and J-WL, conceptualization and experimental design; H-IL, J-YA, H-NS, and S-BC, formal analysis, investigation, and sampling; H-IL, project administration; H-IL, J-YA, and H-NS, original draft; H-IL and J-YA, review and editing.

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Correspondence to Ji-Young Ahn.

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Lim, HI., Ahn, JY., Seo, HN. et al. Broussonetia × hanjiana (Moraceae) microsatellite markers for species and ploidy determination developed using next-generation sequencing. Mol Biol Rep 49, 9089–9094 (2022).

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  • Broussonetia × hanjiana
  • Hybrid
  • Microsatellite marker
  • NGS
  • Ploidy
  • Polymorphism