An optimized SDS-based protocol for high-quality genomic DNA extraction from brown midrib (BMR) pearl millet

Abstract

Extraction of high-quality DNA from brown-midrib (BMR) pearl millet (Cenchrus americanus (L.) Morrone) is challenging due to high levels of phenolics, lignin derivatives, and polysaccharides that interfere with cell lysis and downstream molecular analyses. In this study, four SDS-based lysis buffers (SLB-1 to SLB-4) and a standard CTAB method were evaluated for DNA isolation from BMR leaf tissues. Among these, SLB-3 (2% SDS, 3% PVP-40, 900 mM NaCl, 100 mM Tris-HCl, 25 mM EDTA, and 0.5% β-mercaptoethanol) produced significantly higher DNA yield (649.1 ± 123.2 ng/μL), improved purity (A260/280 = 1.87 ± 0.03; A260/230 = 2.13 ± 0.10), and intact high-molecular-weight DNA. Successful amplification of the extracted DNA with the SSR marker confirmed the integrity and amplifiability of the DNA, indicating its suitability for downstream molecular analyses. The SLB-3 protocol demonstrated advantages over CTAB and other SDS buffers in terms of simplicity, cost efficiency, and reproducibility. This optimized method may be suitable for routine molecular breeding and genetic studies in phenolic-rich and recalcitrant pearl millet tissues.