High throughput DNA extraction of legume root nodules for rhizobial metagenomics

Gabriel Johnson

doi:10.1186/s13568-019-0771-z

Abstract

A novel combination of tissue homogenization, cell lysis, and DNA purification techniques was developed for isolating total DNA from whole legume root nodules on an automated robotic system. Silica dehydrated root nodules from soy bean, Glycine max cv. Tara, and mung bean, Vigna radiata cv. Crystal, were homogenized in 96-well plates and were enzymatically and chemically lysed before being loaded into the AutoGenprep 965 for automated phenol–chloroform based DNA isolation. The resulting DNAs were of relatively high concentration, low fragmentation, and free of phenolic and polysaccharide contaminants. In contrast to manual methods, this adaptation of the Autogen Prep automated DNA extraction instrument allows for 100’s to 1000’s of samples to be prepared in a fraction of the time and cost.

Highlights

Nitrogen fixation by rhizobial endosymbionts occurs in specialized root structures called nodules and is essential for the integrity of both natural and agronomic ecosystems (Becker 2017; Beijerinck 1888; Hellriegel and Wilfarth 1888)
This highly developed methodology allows for even minute amounts of poor quality DNA to be analyzed; the primers used in these studies cannot be used to explore the vast diversity of nodule rhizobia found in non-crop legumes around the world (Zahran 2001; Weir et al 2004)
Molecular studies of non-agricultural legume root nodules are relatively few (Batzli et al 1992; Eardly et al 2017; Freitas et al 2014; Le Roux et al 2014; Ma et al 2012; Sarita et al 2005), they consistently uncover novel species of nitrogen fixating bacteria that cannot be fully characterized using primers developed for agricultural studies

Summary

Introduction

Nitrogen fixation by rhizobial endosymbionts occurs in specialized root structures called nodules and is essential for the integrity of both natural and agronomic ecosystems (Becker 2017; Beijerinck 1888; Hellriegel and Wilfarth 1888). Controlled inoculation of pulses has long been shown to improve agricultural productivity (Brooks 1901; Cotrell et al 1900; Hopkins 1904) Due to their great importance in crop studies, a diversity of techniques has been developed to allow for the rhizobial endosymbionts to be cultured and isolated for characterization and inoculation studies. Since the genera of agriculturally important rhizobia are relatively well known, primers targeting specific variable regions have been developed to profile these strains with amplicon-based methods (e.g. 16S ribosomal DNA, Multilocus Sequence Analysis) This highly developed methodology allows for even minute amounts of poor quality DNA to be analyzed; the primers used in these studies cannot be used to explore the vast diversity of nodule rhizobia found in non-crop legumes around the world (Zahran 2001; Weir et al 2004). Molecular studies of non-agricultural legume root nodules are relatively few (Batzli et al 1992; Eardly et al 2017; Freitas et al 2014; Le Roux et al 2014; Ma et al 2012; Sarita et al 2005), they consistently uncover novel species of nitrogen fixating bacteria that cannot be fully characterized using primers developed for agricultural studies

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