Development of STARP Marker Platform for Flexible SNP Genotyping in Sugarbeet

Abstract

Single nucleotide polymorphisms (SNPs) have been widely used for gene identification. Allelic discrimination for an individual SNP with high reliability and flexibility is critical for the accurate detection of beneficial genes linked to specific SNP sites. Several SNP genotyping platforms have been developed but most exclusively rely on fluorescence signals for allelic differentiation. Genotyping via a fluorescence signal can have a lower accuracy if strong background signal noise is present, a common challenge associated with crop genetics. The semi-thermal asymmetric reverse PCR (STARP) marker system introduces extra SNPs in its forward primers to ensure specificity of the PCR reaction and adds a 4-nucleotide insertion into one universal primer to create fragment length polymorphism among STARP markers, which makes SNP allelic discrimination possible through either fluorescence signals or traditional gel electrophoresis. The STARP marker system is preferable for SNP genotyping in crops such as sugarbeet (Beta vulgaris ssp. Vulgaris L.) that exhibit strong background signal noise during PCR reactions due to an abundant repetitive sequence and high levels of heterozygosity in the genome. In this study, SNPs among sugarbeet lines were detected through genotype-by-sequencing (GBS) and confirmed by sequencing PCR products containing SNP sites. STARP primers were designed, and they generated STARP markers clearly discriminated by SNP alleles among sugarbeet plants either through a fluorescence signal or fragment length polymorphism. In addition, by prolonging 5-nucleotide in an allele-specific forward primer F2 that increased fragment length polymorphism of STARP markers from 4-bp to 9-bp, genotyping individual SNPs can be performed using user-friendly agarose gels. This research resulted in the development of a STARP marker platform for the flexible genotyping of individual SNPs of sugarbeet as well as an improved STARP technique for easy SNP allelic discrimination that also has utilities in other plant species.