Cytogenomic profiling of Panax ginseng cultivars and in vitro root cultures through multi-color PLOP-FISH and flow cytometry Reveals Somaclonal Variations

Panax ginseng Meyer (Korean ginseng) is valued as the “King of medicinal plants” for its broad therapeutic properties. Cytogenomic research that defines chromosome-level genome organization provides important information for breeding and cultivar authentication. However, despite the development of many ginseng cultivars, comparative chromosome-level cytogenomic data for identifying and distinguishing them remain limited. Recently, fluorescence in situ hybridization using pre-labeled oligonucleotide probes (PLOP-FISH) and flow cytometry (FC) have enabled efficient chromosome-level genome characterization. Multi-color PLOP-FISH using three universal tandem repeat probes (5S rDNA, 45S rDNA and Arabidopsis -type telomeric DNA) and four ginseng genome-specific repeats ( Pg167TRa, Pg167TRb, PgDel1 and PgDel2) was performed to elucidate the detailed chromosomal composition of nine ginseng cultivars and four culture lines. Genome size variations were assessed by flow cytometry. All nine cultivars and three in vitro root culture lines retained the standard chromosome number (2 n = 48), whereas the transgenic hairy root line TYP1 showed aneuploidy (2 n = 44), indicating loss of four chromosomes. Pg167TR signals revealed cultivar- and line-specific insertions, deletions, and transpositions, supporting Pg167TRa/b as informative cytogenetic markers. In TYP1, repeat-distribution patterns verified tetra-monosomy of chromosomes 8, 13, 16, and 17. Chromosome loss occurred more frequently in the PgDel2 -rich subgenome than in the PgDel2 -poor subgenome. Flow cytometry corroborated a significant reduction in nuclear DNA content in TYP1. This study provides comparative cytogenomic insights into P. ginseng cultivars and in vitro lines, highlighting Pg167TRa/b as effective markers and demonstrating that PLOP-FISH with FC can track chromosomal variation relevant to breeding, metabolite production, and genomic studies.