Genome-wide in silico and in vitro mining to develop a novel cyclotide-based marker system in plants

Cyclotides are cysteine-rich cyclic peptides that have various activities and applications. Mining at the whole-genome scale facilitates the profiling and development of cyclotide-based markers. However, the characterization of cyclotide subfamilies at a genome-scale in plants is an untapped area that needs more studies. Here we developed four degenerate cyclotide subfamily-specific (CSS) markers based on their subfamily’s semi-conserved sequence for identifying each subfamily separately. Initially, an in silico cyclotide mining analysis was performed on 15 plant genomes/transcriptomes. The in silico PCR produced 476 amplicons in the Bracelet, Moebius, Hybrid and Trypsin Inhibitor (TI) subfamilies. In silico profiling results identified 903 cyclotide mature domains (CMDs) in the analyzed genomes/transcriptomes for the four subfamilies. In addition, the prevalence of each subfamily correlated positively with the genome size, particularly in Hybrid, followed by TI and Bracelet subfamilies. The CSS markers were validated in vitro on 19 genotypes/cultivars. The CSS markers generated a total of 175 bands, with a polymorphic percentage of 98%–100%. Cluster analysis showed distinct clusters for cultivars/genotypes of each plant species. The marker efficiency parameters demonstrated the capability of CSS markers to determine the genetic diversity among the studied plant species. This study represents the first report to perform genome/transcriptome-wide mining to characterize the Cyclotide subfamilies in plants. This study proposed potential applications of the novel CSS markers as an effective system for screening cyclotide subfamilies across multiple plant species with no prior sequencing knowledge requirement. Additionally, it proposes an innovative approach for identifying the appropriate cyclotide subfamily used in drug design.