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Abstract BackgroundThere is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications. ResultsIn a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant,Arabidopsis thaliana(L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastidrbcLand nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis. ConclusionsThe method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics.
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DNA barcoding of terrestrial invasive plant species in Southwest Michigan
Abstract Because of the detrimental effects of terrestrial invasive plant species (TIPS) on native species, ecosystems, public health, and the economy, many countries have been actively looking for strategies to prevent the introduction and minimize the spread of TIPS. Fast and accurate detection of TIPS is essential to achieving these goals. Conventionally, invasive species monitoring has relied on morphological attributes. Recently, DNA‐based species identification (i.e., DNA barcoding) has become more attractive. To investigate whether DNA barcoding can aid in the detection and management of TIPS, we visited multiple nature areas in Southwest Michigan and collected a small piece of leaf tissue from 91 representative terrestrial plant species, most of which are invasive. We extracted DNA from the leaf samples, amplified four genomic loci (ITS,rbcL,matK, andtrnH‐psbA) with PCR, and then purified and sequenced the PCR products. After careful examination of the sequencing data, we were able to identify reliable DNA barcode regions for most species and had an average PCR‐and‐sequencing success rate of 87.9%. We found that the species discrimination rate of a DNA barcode region is inversely related to the ease of PCR amplification and sequencing. Compared withrbcLandmatK, ITS andtrnH‐psbAhave better species discrimination rates (80.6% and 63.2%, respectively). When ITS andtrnH‐psbAare simultaneously used, the species discrimination rate increases to 97.1%. The high species/genus/family discrimination rates of DNA barcoding indicate that DNA barcoding can be successfully employed in TIPS identification. Further increases in the number of DNA barcode regions show little or no additional increases in the species discrimination rate, suggesting that dual‐barcode approaches (e.g., ITS + trnH‐psbA) might be the efficient and cost‐effective method in DNA‐based TIPS identification. Close inspection of nucleotide sequences at the four DNA barcode regions among related species demonstrates that DNA barcoding is especially useful in identifying TIPS that are morphologically similar to other species.
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- Award ID(s):
- 2146882
- PAR ID:
- 10581621
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Plant Direct
- Volume:
- 8
- Issue:
- 6
- ISSN:
- 2475-4455
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/pld3.615
