Genetic Diversity of Broussonetia monoica and Congeneric Species by Novel EST-SSR Markers

Broussonetia monoica (Moraceae) is an ecologically, medicinally and economically valuable species but lacks adequate molecular resources for genetic studies. Here, we developed the first set of expressed sequence tag-derived simple sequence repeat (EST-SSR) markers for B. monoica and applied them to characterize genetic diversity and Broussonetia species relationships. Transcriptome sequencing of B. monoica generated 185,167 unigenes, from which 8921 candidate polymorphic EST-SSRs were mined. Twenty-one primer pairs (21/27, 77.78%) were successfully validated across 100 B. monoica plants. Cross-species transferability was high (14/18, 77.78%), enabling comparative analysis revealing significant genetic differentiation among the Broussonetia species (FST = 0.284). Seventeen polymorphic loci revealed moderate to high genetic diversity in six B. monoica populations, with significant population structure (FST = 0.119) and sub-structuring into three clusters. Bayesian clustering, principal coordinate analysis, and neighbor-joining trees consistently resolved three Broussonetia species boundaries. These novel EST-SSRs provide robust tools for population genetics, germplasm conservation, and molecular breeding in Broussonetia species, addressing a critical resource gap for this genus.