Protocol: A versatile, inexpensive, high-throughput, sequencing-quality plant genomic DNA extraction method suitable for genotyping-by-sequencing

High-throughput, inexpensive methods for extracting high-quality plant genomic DNA (gDNA) underpin deployment of next-generation sequencing marker technologies such as genotyping-by-sequencing (GBS). Several high-throughput DNA extraction methods are available, but typically provide low yield or quality gDNA, or are costly (US$6-$9/sample). We modified a non-organic solvent protocol to extract microgram quantities (1–13 μg) of sequencing-quality gDNA inexpensively in 96-well plates from fresh, freeze-dried or silica gel-dried plant tissue. The method was effective for several difficult-to-extract forage, crop and horticultural species. Tissue ground in a 96-well plate format is mixed with a homogenisation buffer, followed by precipitation to remove impurities by centrifugation. The supernatant is applied to a 96-well silica filter plate, washed, then the gDNA eluted. This scalable, readily-automated protocol requires approximately three hours to process 192 samples (384-570 samples/day) at US$0.62/sample for consumables. Integral to our GBS and molecular breeding projects, this method produces sequencing-quality gDNA for multiple applications.