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      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

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          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.

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          Most cited references37

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          Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components

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            The genome of Eucalyptus grandis.

            Eucalypts are the world's most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.
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              A simple and efficient method for isolating RNA from pine trees

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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: Validation
                Role: Funding acquisitionRole: InvestigationRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                18 October 2018
                2018
                : 13
                : 10
                : e0206085
                Affiliations
                [1 ] Plant Genetics Laboratory, Embrapa Genetic Resources and Biotechnology, Brasília, Brazil
                [2 ] Genomic Sciences and Biotechnology Program, Universidade Católica de Brasília, Brasília, Brazil
                University of Helsinki, FINLAND
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-5513-8918
                Article
                PONE-D-18-11939
                10.1371/journal.pone.0206085
                6193717
                30335843
                ce9a0f19-c84e-435c-9938-f1402354eb91
                © 2018 Inglis et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 20 April 2018
                : 5 October 2018
                Page count
                Figures: 4, Tables: 3, Pages: 14
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award Recipient :
                This work was funded by a grant to D.G. from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq competitive grant 402659/2013-9). www.cnpq.br. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Research and analysis methods
                Extraction techniques
                DNA extraction
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Plant Tissues
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Leaves
                Biology and Life Sciences
                Biochemistry
                Glycobiology
                Polysaccharides
                Biology and life sciences
                Genetics
                DNA
                DNA electrophoresis
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA electrophoresis
                Research and analysis methods
                Electrophoretic techniques
                DNA electrophoresis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Custom metadata
                All relevant data are within the paper and its Supporting Information files. RNA sequencing data for Eucalyptus grandis are available at Phytozome: https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Egrandis.

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