ASSBT Biennial Meeting – Feb. 24 – Feb 27, 2025 in Long Beach, CA
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Development of DNA STARP marker platform for flexible SNP genotyping in sugarbeet.

Abstract

Whole-genome sequencing enables high-throughput detection of SNP (single nucleotide polymorphism) covering the whole genome among plant individuals simply by comparing DNA sequences, which greatly accelerates the identification of associations between specific SNP markers with the target traits through genome-wide association studies (GWAS). Tightly associated SNPs thus can be efficiently used for marker-assisted selection (MAS) during trait integration or locating candidate genes for gene isolation. This requires developing a SNP genotyping system to flexibly detect specific SNP alleles at a few associated loci. Several PCR-based platforms for individual SNP genotyping were developed but most of them used only the fluorescence signal to differentiate SNP alleles. The allele discrimination through fluorescence signal could be less accurate if the strong background signal noise was presented. A newly developed STARP (semi-thermal asymmetric reverse PCR) marker system increased the sensitivity of discriminating SNP alleles by introducing more polymorphic nucleotides in its forward primers, which thus increases the specificity of PCR products from corresponding alleles and the polymorphisms can be detected using either fluorescence signal or the traditional gel electrophoresis system that relies on DNA fragment size difference. The STARP marker system will be more preferrable for flexible SNP genotyping in sugarbeet since the widely spread repetitive sequences and a high level of heterozygosity in the sugarbeet genome produced strong background signal noise. In this study, genotype-by-sequencing (GBS) was used to detect SNPs among sugarbeet lines, and the selected SNPs were further verified through sequencing PCR products amplified from the corresponding regions. STARP primers were designed according to sequences harboring SNPs, and the resulting PCR products clearly discriminated SNP alleles between sugarbeet lines and their F1 hybrids using both methods of fluorescence and gel electrophoresis. This study proved the efficacy of the STARP markers for sugarbeet SNP allele discrimination and developed a flexible SNP genotyping platform in sugarbeet.