ISSR markers as a tool to differentiate genotypes of Cinchona hybrids propagated in vitro and ex vitro

The differentiation of Cinchona spp. hybrids genotypes are challenging due to their shared origins and morphological similarities. This study utilized ISSR (Inter Simple Sequence Repeat) markers to distinguish between genotypes of Cinchona spp.: LF40, LC29, LF74, LF74GB, and LF211 maintained in vitro and ex vitro in Colombia. Genomic DNA was extracted from the plant materials, and eight ISSR primers were used for PCR amplification. In total 61 loci were amplified, of which 37 (60.92%) were polymorphic. The total number of loci per primer ranged from 5 to 12, with an average of 7.62, and polymorphic loci varied from 3 to 6, averaging 4.62 per primer. Cluster analysis based on the unweighted pair group method with arithmetic mean (UPGMA) grouped the genotypes into distinct clusters, showing genetic differences. Principal coordinate analysis (PCA) confirmed the clustering patterns, further distinguishing the genotypes despite their shared origins. The primer ISSR4 was the most effective, with the highest polymorphism rate (75%) and PIC value (0.473), followed by ISSR6 which had a polymorphism rate of 71% and a PIC value of 0.426, both primers allowed the identification of a group of plants under field conditions from in vitro cultures, with unknown genotype origin. As a result, it was possible to confirm a cluster of plants belonging to the LF40 genotype using only two primers. The results demonstrate the genetic distinctiveness of the selected Cinchona genotypes and underscore the utility of ISSR markers as a reliable tool for identifying genetic differences of in vitro and ex vitro Cinchona plant selections.