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Abstract Background The widely cultivated pepper (Capsicum spp.) is one of the most diverse vegetables; however, little research has focused on characterizing the genetic diversity and relatedness of commercial varieties grown in China. In this study, a panel of 92 perfect single-nucleotide polymorphisms (SNPs) was identified using re-sequencing data from 35 different C. annuum lines. Based on this panel, a Target SNP-seq genotyping method was designed, which combined multiplex amplification of perfect SNPs with Illumina sequencing, to detect polymorphisms across 271 commercial pepper varieties. Results The perfect SNPs panel had a high discriminating capacity due to the average value of polymorphism information content, observed heterozygosity, expected heterozygosity, and minor allele frequency, which were 0.31, 0.28, 0.4, and 0.31, respectively. Notably, the studied pepper varieties were morphologically categorized based on fruit shape as blocky-, long horn-, short horn-, and linear-fruited. The long horn-fruited population exhibited the most genetic diversity followed by the short horn-, linear-, and blocky-fruited populations. A set of 35 core SNPs were then used as kompetitive allele-specific PCR (KASPar) markers, another robust genotyping technique for variety identification. Analysis of genetic relatedness using principal component analysis and phylogenetic tree construction indicated that the four fruit shape populations clustered separately with limited overlaps. Based on STRUCTURE clustering, it was possible to divide the varieties into five subpopulations, which correlated with fruit shape. Further, the subpopulations were statistically different according to a randomization test and F st statistics. Nine loci, located on chromosomes 1, 2, 3, 4, 6, and 12, were identified to be significantly associated with the fruit shape index (p |
