Background

Over the last decades, marker-assisted selection (MAS) has become integral to most plant breeding systems because quantitative trait loci (QTL) and molecular markers linked to desired traits have enhanced the selection efficiency of desirable plants and shortened breeding cycles [1]. Unlike commercial breeding companies with highly automated and streamlined MAS and trait-discovery programs, public-sector breeding programs and small-scale genotyping laboratories rely on laborious and time-consuming tissue sampling and DNA extraction procedures [2]. Compared to the proprietary seed-based MAS programs utilized by industry scientists, public-sector genotyping systems that rely heavily on leaf-based DNA genotyping may be particularly resource- and time-demanding [1] as the marker genotyping process must be coupled to the sowing and germinating of seeds, caring for seedlings, tracking of live plants to static samples, and the immediate interpretation of data in order to identify, retain, and rear the desired, growing plants for their intended purpose. Thus, to improve the efficiency of MAS and related approaches, there is a need to adopt seed-based genotyping as a separate, pre-planting process, which reduces overall labor costs and saves space and time through selection of desirable genotypes during the off-season [1,2]. During trait discovery, seed-based DNA genotyping ensures that seeds with desirable traits are selected and planted, thus saving time and costs associated with greenhouse use and winter fields. In addition to tissue sampling, optimizing rapid and cost-effective DNA extraction methods to screen large populations will be essential for a successful MAS and genotyping pipeline.

Seed-based DNA genotyping methods have been implemented in small-scale maize breeding programs and genotyping laboratories but are still ineffective based on safety and scalability [2,3]. One seed-based DNA genotyping method is based on soaking maize kernels in water before endosperm chipping [2]. The caveats of this procedure include susceptibility to fungal contamination and low seed germination if kernels are not dried and stored well. Another common method uses a razor blade for seed chipping [3], which presents injury hazards and is time-consuming, thus making it impractical to quickly chip a large population. In this study, we used clipping pliers, a safe alternative to previous chipping methods that is scalable to high-throughput applications. This seed-chipping method did not significantly impact germination rates for maize B73 and Mo17 seeds, although seedlings from chipped seeds had reduced vigor that was evident for 14 days after planting, after which they appeared comparable to their non-chipped sibling seedlings. In addition, we tested four DNA extraction methods, based on each of cetyltrimethylammonium bromide (CTAB) [4], sodium dodecyl sulfate (SDS) [5], urea [6], and guanidine hydrochloride [7], and optimized a cost- and time-efficient DNA extraction protocol for maize endosperm samples.