Optimization of CTAB Based Plant Genomic DNA Extraction Protocol for Genotyping by Sequencing
Dipendra Kumar Ayer
*
Department of Genetics and Plant Breeding, NM College of Agriculture, Navsari Agricultural University, Navsari-396450, Gujarat, India and Institute of Agriculture and Animal Science, Tribhuvan University, Nepal.
L. M. Megha
Home Facility Laboratory, Department of Genetics and Plant Breeding, NM College of Agriculture, Navsari Agricultural University, Navsari-396450, Gujarat, India.
Kaushal Modha
Department of Genetics and Plant Breeding, NM College of Agriculture, Navsari Agricultural University, Navsari-396450, Gujarat, India.
Vipulkumar Patel
Department of Basic Science and Humanity, College of Forestry, Navsari Agricultural University, Navsari-396450, Gujarat, India.
Isha Mendapara
Home Facility Laboratory, Department of Genetics and Plant Breeding, NM College of Agriculture, Navsari Agricultural University, Navsari-396450, Gujarat, India.
Ritesh Patel
Department of Genetics and Plant Breeding, NM College of Agriculture, Navsari Agricultural University, Navsari-396450, Gujarat, India.
Digvijay Chauhan
Pulses and Castor Research Station, Navsari Agricultural University, Navsari-396450, Gujarat, India.
Alok Shrivastava
Department of Agricultural Statistics, NM College of Agriculture, Navsari Agricultural University, Navsari-396450, Gujarat, India.
*Author to whom correspondence should be addressed.
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.
Keywords: CTAB-based extraction, genomic DNA, plant genomics, lablab purpureus, genotyping-by-sequencing, next-generation sequencing, RNase A treatment, nanodrop, qubit fluorometry, SNP discovery.