Compact Candidate 23-SNP Panel Differentiates Capsicum annuum Germplasm in Computational Study

A computational investigation has converted a public genotyping-by-sequencing resource into a compact candidate Kompetitive Allele-Specific PCR panel designed for germplasm fingerprinting in Capsicum annuum L. While dense genome-wide variant datasets are increasingly available for crop germplasm collections, routine identity control generally requires only a limited number of robust loci. To address this operational requirement, the research team developed an assay-oriented selection workflow that successfully identified a 23-marker single nucleotide polymorphism panel representing all 12 chromosomes. This compact candidate panel achieved complete discrimination across all 61,075 accession pairs present in the 350-accession discovery matrix.

The marker selection pipeline began with a public discovery matrix containing 350 accessions and 10,195 variant records. To extract reliable diagnostic loci from this extensive resource, the researchers implemented a multi-stage bioinformatic filtering procedure. The filtering criteria specifically evaluated candidate records for explicit biallelic single nucleotide polymorphisms, genotype completeness, minor allele frequency thresholds, and chromosome anchoring. Following the strict application of these filtering stages, the initial pool was narrowed down to 2,692 single nucleotide polymorphisms anchored across chromosomes 1 through 12, all of which were retained for subsequent assay-oriented screening.

The workflow performed a primer-feasibility analysis on all 2,692 chromosome-anchored loci. During the primer design process, known neighboring variants were masked. This evaluation retained 2,543 primer-feasible loci from the chromosome-anchored pool. Among these retained candidates, 2,363 loci met strict primer design criteria, while 180 loci required relaxed design criteria.

To establish the minimal marker set required for complete identification, the researchers performed mixed-integer optimization on the pool of 2,543 primer-feasible loci. The optimization algorithm identified a primary 23-locus panel distributed across all 12 chromosomes that successfully distinguished all 61,075 accession pairs in the discovery set. Notably, every accession pair differed at a minimum of two selected loci, establishing built-in discrimination redundancy. Computational leave-one-marker-out analysis confirmed that complete discrimination was retained after the removal of any single marker, while two additional non-overlapping 17-locus sets independently reproduced 100 percent pairwise discrimination across all 350 accessions.

The resulting workflow integrates technical assay feasibility prior to final marker selection and provides direct evidence of failure tolerance through leave-one-marker-out and independent-set evaluations. However, the study emphasized that the selected loci currently remain computational candidate Kompetitive Allele-Specific PCR assays. Consequently, the authors concluded that independent wet-laboratory validation is required before this 23-marker panel or the alternative 17-marker sets can be deployed for routine diagnostic use in Capsicum annuum germplasm collections.

Subject of Research: Computational development of a candidate KASP-SNP panel for Capsicum annuum fingerprinting

Article Title: Genome-informed development of a compact candidate KASP-SNP panel for Capsicum annuum germplasm fingerprinting

Article References: Coşkun, Ö. F. (2026). Genome-informed development of a compact candidate KASP-SNP panel for Capsicum annuum germplasm fingerprinting. Plant Biosystems, 160(6), Article 316. https://doi.org/10.1007/s44473-026-00352-w

Image Credits: AI Generated

DOI: 10.1007/s44473-026-00352-w

Keywords: Capsicum annuum, KASP assays, single nucleotide polymorphisms, germplasm fingerprinting, genotyping-by-sequencing, mixed-integer optimization, leave-one-marker-out analysis, accession discrimination, crop identity control, primer feasibility, biallelic SNPs, chromosome anchoring, minor allele frequency, marker failure tolerance, variant filtering, computational genomics