Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications

Abstract

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

Fig 1. Gel images of nucleic acids extracted using the described protocol.

(A) Electrophoresis (1% agarose-TBE) of genomic DNA extracted from Eucalyptus grandis (lanes 3–8) and cashew (Anacardium occidentale) (lanes 9–14) leaves; lanes 1 and 2 contain 100 and 200 ng of uncut Lambda DNA (Thermo Scientific), respectively; (B) Pippin pulse (Sage Science, Beverly, MA, USA) gel electrophoresis (0.75% agarose-KBB buffer) image provided by McGill/Genome Quebec Innovation Centre with cashew high molecular weight genomic DNA samples; lane 1 is the 2.5 kb molecular ruler (BioRad Laboratories); (C) Electrophoresis (1% agarose-TAE) of total RNA from E. grandis leaf, fruit bud and cambium (lanes 2–4); lane 1 is kb Plus DNA ladder (Thermo Scientific).

Fig 2. Resuspended DNA samples extracted from Lafoensia spp.

The top row of tubes contains samples extracted using our standard CTAB protocol and the bottom row contains paired samples extracted using the same protocol with the addition of two sorbitol solution pre-washes.

Fig 3. ITS PCR of Lafoensia spp.

1.5% agarose gel containing PCR products from reactions performed using either standard CTAB extracted DNA (top row of wells) and paired samples extracted using the same protocol with the addition of two sorbitol solution pre-washes (bottom row). The molecular marker is the 100 bp ladder (Invitrogen).

Fig 4. Pellets formed following centrifugation of isopropanol-precipitated DNA extracts from Pereskia aculeata.

The top row of tubes contains samples extracted using our standard CTAB protocol and the bottom row contains paired samples extracted using the same protocol with the addition of two sorbitol solution pre-washes.