An improved Shorea robusta genomic DNA extraction protocol with high PCR fidelity.

Abstract

Shorea robusta (Dipterocarpaceae), commonly known as Sal, is an economically and culturally important timber species, known to contain a wide spectrum of polyphenols, polysaccharides, and other secondary metabolites in the tissues, which can interfere with the extraction of high-quality genomic DNA. In order to screen simple sequence repeat (SSR) markers and carry out other DNA-based analyses for this species in our laboratory, a high-throughput DNA extraction methodology was needed. Hence, we have optimized a simple, rapid, safe, and reliable high-throughput protocol for DNA extraction suitable for both fresh and dry leaves. The standardized protocol delivered good DNA yield of ∼1500 µg from 1 g of leaf tissue, with purity indicated by a 260 nm/280 nm absorbance ratio ranging from 1.70 to 1.91, which validated the suitability of extracted DNA and revealed reduced levels of contaminants. Additionally, the protocol that we developed was found to be suitable for polymerase chain reaction (PCR) amplification using microsatellite markers. Genome-wide characterization with SSR markers has been established in S. robusta, which further validates the protocol and its usefulness in DNA-based studies across the genus and/or family.