Hyosig Won1p, Joo-Hwan Kim2, and Jun-Hyoung Jang2

1Department of Biological Science, Daegu University, Gyungsan, Gyungbuk, South Korea; 2Maru Biological Resource Research Institute, Daegu, South Korea

Chestnut (Castanea, Fagaceae) comprises ecologically and economically important nut tree species with a cultivation history in Korea exceeding 1,500 years as a vital food source. Despite widespread cultivation, detailed nucleotide sequence variation patterns across natural and cultivated populations of Korean chestnuts remain poorly understood, impeding effective germplasm conservation and breeding efforts. In Korea and Japan, Castanea crenata Siebold & Zucc. (Korean/Japanese chestnut, locally “bamnamu”) is distributed, whereas C. mollissima Blume (Chinese chestnut, locally “yakdanbam”) is native to China. In northern Korea, natural hybrids between these species, known as “Yakbamnamu” occur due to the predominantly insect-pollinated, outcrossing nature of chestnuts and overlapping distributions. In 1958, severe damage from the chestnut gall wasp (Dryocosmus kuriphilus) caused widespread mortality of native and cultivated trees in Korea. To recover production, blight- and gall wasp-resistant cultivars were imported from Japan and integrated with Korean local germplasm in extensive breeding programs. This study characterized the genetic features of Korean native C. crenata using chloroplast and nuclear markers from over 100-year-old individuals. We sequenced three chloroplast regions (rbcL, matK, and trnL intron/trnL-trnF intergenic spacer) and the nuclear ribosomal ITS (nrITS) region, with cloning to resolve intragenomic polymorphisms in nrITS. Results identified two distinct chloroplast haplotypes in Korean C. crenata, clearly differentiated from those of C. mollissima. nrITS analysis detected at least three sequence types per species, with some Korean C. crenata individuals containing additional C. mollissima-type sequences, indicating introgression from C. mollissima, likely through historical natural hybridization events (e.g., Yakbamnamu ancestry). These findings facilitate reliable identification of hybrid-origin individuals (Yakbamnamu) and inference of maternal (cpDNA-uniparental) and paternal/biparental (nrITS) lineages. This integrated molecular approach offers valuable insights into the origins of Korean chestnut populations, supports conservation of native genetic resources, and informs future breeding for resilient, high-quality cultivars.