Development of STARP marker platform for flexible SNP genotyping in sugarbeet

Authors: TEHSSEN, MUHAMMAD MASSUB - North Dakota State University; ZHENG, YAOJIE - North Dakota State University; Wyatt, Nathan; Bolton, Melvin; Yang, Shengming; Xu, Steven; LI, XUEHUI - North Dakota State University; Chu, Chenggen

Submitted to: Agronomy Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/11/2023
Publication Date: 5/12/2023
Citation: Tehssen, M., Zheng, Y., Wyatt, N.A., Bolton, M.D., Yang, S., Xu, S.S., Li, X., Chu, C.N. 2023. Development of STARP marker platform for flexible SNP genotyping in sugarbeet. Agronomy Journal. 13(5):1359. https://doi.org/10.3390/agronomy13051359.
DOI: https://doi.org/10.3390/agronomy13051359

Interpretive Summary: Single nucleotide polymorphisms (SNPs) are a type of mutation that occur in DNA. Since they are abundant in plant genomes, SNPs can be exploited for plant breeding because they are often closely associated with genes of interest. Consequently, the plant breeding community has made considerable efforts to develop ways to identify SNPs in the last decades. Here, we developed a new strategy to identify SNPs in the sugarbeet genome. First, we sequenced the genomes of several sugarbeet varieties and used the resulting sequences to identify SNP mutations among the varieties. We optimized a technique to identify SNPs among the sugarbeet varieties using high-tech instruments in the laboratory or simplified techniques that utilize readily available and affordable lab equipment. Besides sugarbeet, this technique can be adopted for other cropping systems to provide plant breeders a useful tool for crop improvement.

Technical 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 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 fluorescence signal can have 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 that exhibit strong background signal noise during PCR reactions due to 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 SNP alleles among sugarbeet plants either through 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 done using user-friendly agarose gels. This research resulted in the development of a STARP marker platform for flexible genotyping individual SNPs of sugarbeet as well as an improved STARP technique for easy SNP allelic discrimination that also has utility in other plant species.