An Improved Protocol for Genomic DNA Isolation from Bryophyte Species

Abstract

Genetic diversity studies of plants depend upon the quality and quantity of isolated DNA sample. For many lower groups of plants, apart from usual steps involved for DNA isolation, several additional steps are invariably required specific to the particular plant group. In bryophytes, nucleic acid isolation has always been a difficult task. The Bryophytes possess a substantial quantity of polyphenols and polysaccharides that are known to interfere with DNA isolation and Polymerase chain reaction amplification. In order to overcome these problems, an improved protocol is developed for DNA isolation by optimizing important components of the cetyltrimethylammonium bromide (CTAB) extraction protocol by varying concentrations of CTAB (2–4%), polyvinylpolypyrrolidone (PVP) (0–4%) and β-mercaptoethanol (0–1.5%). It was observed that CTAB (3%), PVP (2%), and β-mercaptoethanol (1%) is optimum with a DNA yield of 900–1582 µg/0.5 gm leaf sample. DNA concentration (25 ng/µl), MgCl2 (1.5 mM), primer (1 µm), annealing temperature (48–52 °C) and a number of cycle set at 35 is optimum for the reproducible and high-intensity Simple sequence repeats amplification. The efficacy of this protocol has been validated in 6 moss taxa and 2 liverwort taxa.