STARP Marker Development for Cadmium Accumulation Mutant Loci of the CaHMA1 Gene and Construction of a DNA Fingerprinting Map in Pepper (Capsicum annuum L.)

Pepper (Capsicum annuum L. ) is a significant vegetable crop, and its fruits tend to accumulate cadmium (Cd). The background value of soil Cd in the main pepper-producing area (southwest China) is relatively high, which results in a high risk of Cd contamination in pepper and its products in this area. Therefore, the cultivation of pepper varieties with low Cd accumulation is vital for ensuring food safety and the development of the pepper industry. A prior genome-wide association study (GWAS) identified the heavy-metal-transporting ATPase gene (CaHMA1) as a crucial gene that facilitates Cd accumulation in pepper fruits. Herein, three semi-thermal asymmetric reverse PCR (STARP) molecular markers (STARP1, STARP2, and STARP3) were designed according to three single-nucleotide polymorphism (SNP) loci (Chr02₁54361710, Chr02₁54362005, and Chr02₁54367255) identified in the intronic region of CaHMA1. Subsequently, these STARP molecular markers were validated using 70 pepper core germplasms with known genotypes. The results indicated that the STARP markers exhibited an identity of over 95% with the corresponding SNP markers. By utilizing the aforementioned STARP markers, the pepper population was divided into two haplotypes (Hap) (Hap1 and Hap2). Under Cd stress, the average Cd content in the fruits of Hap2 pepper was 27. 01% lower than that of Hap1. Collectively, these three STARP markers can rapidly and accurately identify the Cd accumulation capacity of pepper varieties with different haplotypes. Furthermore, 24 SNPs were additionally screened from 150 core SNPs according to the criteria of minor allele frequency (MAF) > 0. 40, polymorphism information content (PIC) > 0. 35, observed heterozygosity (OH) < 0. 6, and uniform distribution across 12 chromosomes. These 24 SNPs were combined with the 3 SNPs from the STARP marker developed in the intron region of CaHMA1, and a pepper DNA fingerprinting map was successfully constructed. This DNA fingerprinting map achieved a 100% identification efficiency for 216 pepper germplasm accessions and was able to distinguish the Cd accumulation capacities among different pepper germplasm accessions. In conclusion, this study provides reliable STARP markers for the marker-assisted selection (MAS) breeding of pepper varieties with low Cd accumulation. Moreover, the constructed DNA fingerprinting map possesses dual functions, identifying varieties and evaluating Cd accumulation traits that have high practical value in pepper breeding.