Optimization of CTAB Based Plant Genomic DNA Extraction Protocol for Genotyping by Sequencing

Abstract

High-quality DNA is essential for next-generation sequencing, yet CTAB-based extractions from polysaccharide- and metabolite-rich plant tissues often yield fragmented, contaminated DNA with high levels of residual chaotropic agents. To address these limitations, this study presents a minimal-reagent, optimised CTAB protocol incorporating three key refinements: extended gentle incubation with 3X CTAB buffer at 60–65 °C to improve cell lysis, enhanced salt precipitation using 0.5×6 M NaCl, 0.1×3 M sodium acetate and ethanol to increase DNA yield and remove polysaccharides and secondary metabolites, and dual 70% ethanol washes with low-volume pipetting to eliminate residual salts. The optimised CTAB method yielded genomic DNA in Indian bean (Lablab purpureus) at 1481.65 ± 673.98 ng/µL, with purity ratios of A260/280 = 2.1 ± 0.07 and A260/230 = 2.09 ± 0.27. The optimised protocol consistently yielded high-quality genomic DNA with A260/280 and A260/230 ratios >1.8 across most samples, even after RNase treatment and storage without further clean-up. Qubit quantification showed an average DNA concentration of 34.36 ± 16.76 ng/µL, demonstrating suitability for downstream sequencing applications. Genotyping by sequencing (GBS) using MspI/PstI digestion of gDNA from 145 parental and recombinant inbred lines generated 87.5 M and 86.0 M reads per Ion 540 chip, with approximately 1.09 ± 0.57 M raw reads per sample (Phred ≥15) and approximately 0.68 ± 0.36 M high-quality reads retained after preprocessing (Phred ≥25). The protocol requires minor adjustments for recalcitrant tissues but remains a reliable, cost-effective and scalable option for SNP discovery, particularly in resource-limited genomics laboratories. Overall, the optimised CTAB protocol provides a cost-effective and scalable method for high-quality DNA isolation from diverse plant tissues and is suitable for next-generation sequencing applications.