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.

Autor: Inglis PW; Plant Genetics Laboratory, Embrapa Genetic Resources and Biotechnology, Brasília, Brazil., Pappas MCR; Plant Genetics Laboratory, Embrapa Genetic Resources and Biotechnology, Brasília, Brazil., Resende LV; Plant Genetics Laboratory, Embrapa Genetic Resources and Biotechnology, Brasília, Brazil., Grattapaglia D; Plant Genetics Laboratory, Embrapa Genetic Resources and Biotechnology, Brasília, Brazil.; Genomic Sciences and Biotechnology Program, Universidade Católica de Brasília, Brasília, Brazil.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2018 Oct 18; Vol. 13 (10), pp. e0206085. Date of Electronic Publication: 2018 Oct 18 (Print Publication: 2018).
DOI: 10.1371/journal.pone.0206085
Abstrakt: 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.
Competing Interests: The authors have declared that no competing interests exist.
Databáze: MEDLINE
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