In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes

Abstract

Mature oak (Quercus spp.) leaves, although abundantly available during the plants’ developmental cycle, are rarely exploited as viable sources of genomic DNA. These leaves are rich in metabolites difficult to remove during standard DNA purification, interfering with downstream molecular genetics applications. The current work assessed whether in situ dark adaptation, to deplete sugar reserves and inhibit secondary metabolite synthesis could compensate for the difficulties encountered when isolating DNA from mature leaves rich in secondary metabolites. We optimized a rapid, commercial kit based method to extract genomic DNA from dark- and light-adapted leaves. We demonstrated that in situ dark adaptation increases the yield and quality of genomic DNA obtained from mature oak leaves, yielding templates of sufficiently high quality for direct downstream applications, such as PCR amplification and gene identification. The quality of templates isolated from dark-adapted pin oak leaves particularly improved the amplification of larger fragments in our experiments. From DNA extracts prepared with our optimized method, we identified for the first time partial segments of the genes encoding 18S rRNA and isoprene synthase (IspS) from pin oak (Quercus palustris), whose full genome has not yet been sequenced.

Polyphenol Profile and Pharmaceutical Potential of Quercus spp. Bark Extracts

“…The bark of Quercus species in particular is receiving increased attention because of its diverse traditional medicinal uses, its abundance and the low price of its wood residues, such as bark [4]. The genus Quercus, belonging to the Fagaceae family, contains trees that are distributed worldwide, with an estimated 450 species [5,6]. There are differences in their morphological appearance and chemical composition.…”

A novel, magnetic bead-based extraction method for the isolation of antimicrobial resistance genes with a case study in river water in Malawi

“…Commercially available kits are typically used for DNA extraction from environmental samples, as they offer standardized sets of reagents and are safer than phenol‐chloroform‐isoamyl alcohol (PCI) extraction methods (Hinlo et al, 2017 ). Adaptations of manufacturer's instructions are often reported (Barta et al, 2017 ; Renshaw et al, 2015 ), but novel methods are rarely incorporated in high‐throughput studies (Oberacker et al, 2019 ). This has led to inconsistent application amongst environmental researchers and often the kit used is determined by cost, accessibility of materials or personal preference (Hinlo et al, 2017 ).…”

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

“…To achieve reliable molecular data in oak studies, efficient DNA extraction protocols are required for different species and ecotypes. Therefore, isolating high quantities of contaminantfree genomic DNA for downstream applications from different tissues of species, especially those species rich in different contaminants, such as sugars, polyphenolics, and terpenoids, prompt the urgent need to revisit, adapt, and improve DNA extraction protocols (Barta et al 2017). Previously, many molecular studies have been performed on different species of oak trees using classical DNA extraction procedures, such as SDS or CTAB (e.g., Pandey and Tamta 2015;Toader et al 2009;Makela et al 2016) and different commercial kits (e.g., Hipp et al 2014;Barta et al 2017;Vranckx et al 2014).…”

“…Therefore, isolating high quantities of contaminantfree genomic DNA for downstream applications from different tissues of species, especially those species rich in different contaminants, such as sugars, polyphenolics, and terpenoids, prompt the urgent need to revisit, adapt, and improve DNA extraction protocols (Barta et al 2017). Previously, many molecular studies have been performed on different species of oak trees using classical DNA extraction procedures, such as SDS or CTAB (e.g., Pandey and Tamta 2015;Toader et al 2009;Makela et al 2016) and different commercial kits (e.g., Hipp et al 2014;Barta et al 2017;Vranckx et al 2014). However, oaks show large phenotypic and genotypic variations (e.g., physical size, leaf and stem type, metabolites, and genome size and structure), and DNA extraction from some species mainly from warmest climates is not as efficient as for temperate oaks or it is even deficient (Finkeldey et al 2010;Finch-Savage 1992;Sunderlíková et al 2009).…”