SeedDNA: Optimized Seed-Based Genomic DNA Extraction Protocol for High-throughput Genotyping and Sequencing in Legume Crops

Background: Efficient and cost-effective genomic DNA extraction from legume seeds, such as groundnut, chickpea, and pigeonpea, is important for molecular biology studies, large-scale genotyping, and marker-assisted breeding programs. These seeds are rich in inhibitory compounds such as polysaccharides, polyphenols, and secondary metabolites, which make DNA extraction challenging. The DNA isolation from leaves is very common; however, achieving high-quality DNA from seeds remains a challenge in many plant species, although it is very useful in saving resources and time. Results: In this context, we optimized the protocol for high-quality DNA extraction from seeds of these three legume species. This optimized protocol yielded DNA with high purity (A260/A280 ratio of ~1.8–2.0) and yielded up to 60 ng/µL for all three legumes. As compared to the protocol used for leaf samples, key modifications in this protocol include the use of proteinase K, repeating the phase separation and washing process twice, and overnight incubation with RNAse at 37°C to remove the RNA contamination. This protocol improved DNA yield and purity while minimizing contaminants, with a minimal isolation cost of $ 0.35 per sample. PCR amplification with crop-specific primers performed well on the genomic DNA extracted from both seeds and leaves. The results were identical, showing the reliability of seed-derived DNA for genotyping and other downstream applications. Furthermore, the PCR amplification confirmed that the DNA isolated from seeds using this protocol can be used for genetic purity assessment in seed lots and for genotyping applications in marker-assisted selection. Conclusion : A high-throughput DNA extraction protocol was developed and validated for legume seeds, such as groundnut, chickpea, and pigeonpea. The optimized protocol is reliable, cost-effective, and can be applied for large-scale genotyping and marker-assisted breeding programs. Using this protocol, one person can process four 96-well plates per day, with an estimated cost of $ 0.35 per sample, including consumables.