Abstract

BackgroundHigh throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers.ResultsWe have developed a high-throughput DNA extraction protocol for conifers using an automated liquid handler and modifying the Qiagen MagAttract Plant Kit protocol. The modifications involve change to the buffer system and improving the protocol so that it almost doubles the number of samples processed per kit, which significantly reduces the overall costs. We describe two versions of the protocol: one for medium-throughput (MTP) and another for high-throughput (HTP) DNA isolation. The HTP version works from start to end in the industry-standard 96-well format, while the MTP version provides higher DNA yields per sample processed. We have successfully used the protocol for DNA extraction and genotyping of thousands of individuals of several spruce and a pine species.ConclusionA high-throughput system for DNA extraction from conifer needles and seeds has been developed and validated. The quality of the isolated DNA was comparable with that obtained from two commonly used methods: the silica-spin column and the classic CTAB protocol. Our protocol provides a fully automatable and cost effective solution for processing large numbers of conifer samples.

Highlights

  • IntroductionMarker-assisted selection, population and conservation genetic studies require processing of a large number of samples

  • Most genetic linkage mapping, marker-assisted selection, population and conservation genetic studies require processing of a large number of samples

  • Recently-introduced magnetic fishing protocols allow for fully-automated, flexible throughput DNA isolation from certain samples, such as blood

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Summary

Introduction

Marker-assisted selection, population and conservation genetic studies require processing of a large number of samples. Cost-effective high-throughput DNA extraction is a major bottleneck for many of these applications because handling and quantification of non-liquid samples, such as plant tissues, are labour-intensive and difficult to automate. High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. Many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. We describe a procedure for high-throughput DNA isolation from conifers

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