Direct assessment of genetic purity in Zea mays using innovative next-generation sequencing on samples of multiple seeds rather than traditional single seed assays

Seed genetic identity and purity testing are crucial for identifying varietal off-types caused by segregation, outcrossing or seed mixing, and to uphold high quality standards in maize seed production. In this study, we present a novel Next Generation Sequencing (NGS)-based testing method that directly sequences samples of multiple seeds to assess genetic purity, as opposed to traditional single seed assays. By sequencing DNA mixtures of maize inbred and hybrid lines, we determined that targeted genotyping by sequencing (tGBS) is sufficiently quantitative to effectively differentiate purity levels from 95 to 100% for inbred lines, which is a suitable range for foundation seed production. Further work was conducted using artificial seed mixtures. Impurity types, including variants and seed mixes, were evaluated to compare our sequencing method against the TaqMan assay, which served as the reference method, revealing a strong correlation between the two approaches. While sequencing offers high flexibility in single nucleotide polymorphism (SNP) multiplexing, its quantitative nature also allows the estimation of impurities, enabling a single test on a sample of multiple seeds, instead of genotyping each seed individually. This approach improves testing efficiency and enables a significantly increased sample size for greater representativeness in seed genetic purity testing.