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

Abstract

BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or “orphan” crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6–$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1–13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384–576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

Breaking Free: The Genomics of Allopolyploidy-Facilitated Niche Expansion in White Clover

“…For the three Trifolium species in this study, nuclear DNA was extracted from young leaves as described in Anderson et al (2018) and sequenced by Macrogen. Using a range of platforms (Roche 454-GLX; Illumina HiSeq 2000), libraries (shotgun, paired-end; mate-paired) and insert sizes (180 bp to 8,000 bp), sequence data were generated to an approximate coverage of 4923, 1143, and 2303 for T. occidentale, T. pallescens, and T. repens, respectively (Supplemental Table 1).…”

“…Using a range of platforms (Roche 454-GLX; Illumina HiSeq 2000), libraries (shotgun, paired-end; mate-paired) and insert sizes (180 bp to 8,000 bp), sequence data were generated to an approximate coverage of 4923, 1143, and 2303 for T. occidentale, T. pallescens, and T. repens, respectively (Supplemental Table 1). Furthermore, high-molecular-weight DNA was extracted (Anderson et al, 2018) to create six white clover libraries of Illumina TSLRs generated by the Illumina FastTrack Sequencing Services Long Reads pipeline to provide a 33 coverage (Supplemental Table 1). Original sequence data are deposited in the National Center for Biotechnology Information (NCBI) under Bioprojects PRJNA523044, PRJNA521254, and PRJNA523043 for white clover, T. occidentale, and T. pallescens, respectively, as detailed in "Accession Numbers.…”

“…White clover scaffolds were assigned to subgenomes and ordered into pseudomolecules using pairwise LD (calculated as r 2 ) between microsatellite (SSR) markers and GBS-derived biallelic SNPs segregating in F 1 progeny (n 5 93) of a white clover biparental cross (PGen33PGen9). To enable linkage group (LG) identification and distinguish homeologs in this population, and to align the sequence assembly pseudomolecules with previously published white clover linkage groups, DNA extracted from the parent and progeny plants (Anderson et al, 2018) was screened with 16 SLHS SSR markers (Supplemental Table 2) as described in Griffiths et al (2013).…”

A sorghum Practical Haplotype Graph facilitates genome-wide imputation and cost-effective genomic prediction

“…Sequencing data took 48 hours to run through the founder PHG, giving us about a 4-week turnaround time from sample collection to genotype data and leaving two weeks to make selections and plan crosses. Additional time could potentially be saved with cheap, on-site DNA extractions and library preparations (Anderson et al, 2018;Zou et al, 2017). The extra time gained with on-site methods could make a second round of tissue collection and sequencing possible if samples are contaminated or if more data is needed for a particular breeding line.…”

Divergent Genomic Selection for Herbage Accumulation and Days-To-Heading in Perennial Ryegrass

“…For Pop III G3, pseudostem tissue was supplied directly by Grasslands Innovation Ltd. and provided for genotyping. DNA was isolated from 50-100 mg of harvested pseudostem tissue using a semi-automated method for the extraction of sequencing-quality high molecular weight DNA from plants [27]. Each seedling was also subject to an immuno-detection blot [28], to confirm the presence of fungal endophyte (Epichloë festucae var lolii).…”