We thank Alan Tasker (USDA-APHIS, Riverdale, MD), Stephen Compton (Clemson), Sherry Aultman (Clemson), Craig Ramsey (USDA-APHIS, Fort Collins, CO), and Betty Marose (UMD) for assistance in obtaining specimens. We thank students Andrew Adrian (UA-Huntsville) and Derek Thacker (UA-Huntsville) for their assistance in testing this protocol, and Joseph Herdy for his work in the filming of the video. This work was funded by National Fish and Wildlife Foundation.

1. Specimen Collection and Preservation
This method was developed and tested using fresh, frozen, and recently dried leaf tissues.
<ol>
	<li>Identify cogongrass and/or JBG tissues with the help of a taxonomist that specializes in grass species identification. With its bright red leaves, ornamental JBG is easy to visually distinguish from wild-type cogongrass and JBG revert; however, cogongrass and JBG revert are almost indistinguishable from one another. Cogongrass and the JBG reverted phenotype have green, longer leaves, considerably larger and longer rhizomes, and more leaf area than JBG12 (Figure 1).</li>
	<li>Fresh leaf tissue provides the most abundant and highest quality DNA and can be collected from field or greenhouse grown I. cylindrica plants. If fresh tissue is to be used, extract DNA within 3 hours of collection to help prevent degradation. Otherwise, prepare tissue for storage, keeping the tissue cool and out of direct sunlight.</li>
	<li>To store tissues for DNA extraction at a later date, the most optimal method is to freeze and store the tissue at -80&deg;C immediately. Do not allow the frozen tissue to thaw prior to DNA extraction. Transfer the frozen tissue to liquid nitrogen prior to the grinding steps of DNA extraction to prevent thawing.</li>
	<li>If a -80&deg;C freezer is not available, dry the tissue immediately. For dry storage, place the tissue into a paper envelope and store the envelope in dehydrated silica gel or other active desiccants at room temperature. A small amount of indicator silica mixed in with non-indicating silica will ensure that silica is fully dehydrated and suitable for drying plant tissue.</li>
	<li>Use at least 10 times more silica gel than fresh leaf tissue by weight. Plant tissue should dry within 24 hours. The quality and quantity of DNA is reduced through dry storage over time (as in the case of herbarium specimens). </li>
</ol>
2. DNA Extraction
To extract DNA from plant tissue, follow the DNeasy Plant Mini Kit (Qiagen, Valencia, CA; Cat# 69104 or 69106) manufacturer's instructions with one minor modification. Instead of using the suggested less than 100 mg fresh tissue or less than 20 mg dry tissue for each column, grind greater than 100 mg, and then transfer 100 mg from fresh or frozen tissue (or &gt; 20 mg from dry tissue) to appropriate tubes for extraction. Nuclear and plastid DNA is extracted simultaneously.
<ol>
	<li>Before starting these procedures, verify that ethanol was added to buffers AP3/E and AW.</li>
	<li>Grind &gt;100 mg of fresh or frozen leaf tissue (or &gt;20 mg of dry leaf tissue) to a fine powder using three rounds of liquid nitrogen with grinding in a chilled mortar and pestle. Insufficient disruption of the starting material or insufficient lysis can also lead to lower yields of DNA. Carefully grind the tissue and do not overload the columns with too much tissue.</li>
	<li>Transfer 100 mg frozen powder from fresh or frozen tissue (or 20 mg of powder from dry tissue) to a 1.5 ml microcentrifuge tube containing 400 &mu;l of Buffer AP1 and 4 &mu;l of RNase A. Each tube can be placed in a small rack on a balance to monitor the correct weight of tissue per tube.</li>
	<li>Vortex or shake sample(s) to mix and incubate for 10 min at 65&deg;C, inverting the tube(s) 2-3 times during incubation.</li>
	<li>Add 130 &mu;l of Buffer AP2 to each sample. Mix by inverting the tube(s) several times, and incubate for 5 min on ice.</li>
	<li>Pipet each lysate into a separate QIAshredder Mini spin column, and place each column in a 2 ml collection tube (provided with the kit). Centrifuge column(s) for 2 min at 20,000 x g (~14,000 rpm), and transfer each flow-through fraction into a new tube (not supplied with the kit) without disrupting any formed pellet.</li>
	<li>Add 1.5 volumes of Buffer AP3/E, and mix by pipetting.</li>
	<li>Transfer 650 &mu;l of mixture into a DNeasy Mini spin column in a 2 ml collection tube. Centrifuge column(s) for 1 min at 6,000 x g (~8,000 rpm), and discard the flow-through. Repeat this step with the remaining mixture for each sample.</li>
	<li>Place the spin column(s) into a new 2 ml collection tube(s), and add 500 &mu;l of Buffer AW to the top of each column. Centrifuge column(s) for 1 min at 6,000 x g (~8,000 rpm), and discard flow-through.</li>
	<li>Add another 500 &mu;l of Buffer AW to the top of each column. Centrifuge for 2 min at 20,000 x g (~14,000 rpm). This step will dry the column, thus removing any residual ethanol contained in the buffers that can inhibit PCR.</li>
	<li>Transfer each spin column to a new 1.5 ml microcentrifuge sample tube. Add 100 &mu;l Buffer AE to the top of each column for elution, and incubate column(s) for 5 min at room temperature. Centrifuge column(s) for 1 min at 6,000 x g (~8,000 rpm) to collect the DNA.</li>
	<li>Repeat these elution steps one time, eluting the DNA into the same 1.5 ml microcentrifuge tube to yield 200 &mu;l of sample. Store DNA samples at -20&deg;C until use. DNA concentrations depend on tissue type and storage conditions. Optimal yields are obtained when eluting DNA with a total of 200 &mu;l of buffer AE; however, concentrations can be increased if elution volumes are reduced to as little as 50 &mu;l. </li>
</ol>
3. Verification of DNA Quality and Quantity
<ol>
	<li>Test the quality and quantity of extracted DNA prior to PCR setup using a spectrophotometer or fluorometer and gel electrophoresis. This will help ensure the success of subsequent steps.</li>
	<li>Using a spectrophotometer, test DNA quality and quantity. Good DNA yields should be between 50 and 150 ng/&mu;l with 260/280 and 230/280 ratios close to 2.0. As an example, Figure 2 shows good quality results using the NanoDrop spectrophotometer (ThermoScientific, Wilmington, DE).</li>
	<li>Conduct electrophoresis using a standard 1% agarose gel. Verify the presence of relatively large bands (&gt;10 Kb) with no to little streaking from RNA contamination (Figure 3).</li>
	<li>If the DNA concentration is high, dilute DNA samples to 70 ng/&mu;l for subsequent steps.</li>
</ol>
4. PCR Primers
The PCR primers used in this protocol are based on sequence differences between the plastid trnL-F spacer region of cogongrass and JBG genotypes. These differences come in the form of SNPs (Single Nucleotide Polymorphisms) and InDels (Insertions and Deletions) that allowed the development of variety-specific primers by locating the primers at the sites of unique sequences (Figure 4).
<ol>
	<li>The quality of the primers can have a significant impact on the PCR results. Order primers from a reputable company. We order our primers from Oblique Bio, Inc. (<a href="http://www.obliquebio.com/web/" target="_blank">http://www.obliquebio.com/web/</a>, Huntsville, AL), requesting only standard desalting procedures.</li>
	<li>trnL-F positive control primers: This primer set amplifies the plastid trnL-F spacer region of most grass taxa11-13 and serves as a good positive control (resulting in an 890 bp band). 
	<table border="1">
		<tbody>
			<tr>
				<td>Name</td>
				<td>Sequence</td>
			</tr>
			<tr>
				<td>trnF(GAA)-F</td>
				<td>5'-ATTTGAACTGGTGACACGAG-3'</td>
			</tr>
			<tr>
				<td>trnL(5' exon)-C</td>
				<td>5'-CGAAATCGGTAGACGCTACG-3'</td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>Wild-type cogongrass primers: This primer set is specific to cogongrass and does not amplify the trnL-F region of JBG genotypes. The set results in a band that is 595 bp. 
	<table border="1">
		<tbody>
			<tr>
				<td>Name</td>
				<td>Sequence</td>
			</tr>
			<tr>
				<td>trnLF-C-F1</td>
				<td>5'-TCCACTTTTTTGAAAAAACAAGTGCAA-3'</td>
			</tr>
			<tr>
				<td>trnLF-C-R1</td>
				<td>5'-GCCGATACTCTAATAAATAAAAAAAAAAAAGAAAT-3 '</td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>JBG and JBG Revert primers: This primer set is specific to JBG genotype and does not amplify the trnL-F region of cogongrass. The set results in a band that is 594 bp. 
	<table border="1">
		<tbody>
			<tr>
				<td>Name</td>
				<td>Sequence</td>
			</tr>
			<tr>
				<td>trnLF-R-F2</td>
				<td>5'-CCAAATCCACTTTTTTGAAAAAACAAGTGGTT-3'</td>
			</tr>
			<tr>
				<td>trnLF-R-R2</td>
				<td>5'-CGAGATTCCTTGCCGATACTCTAATAAAA-3'</td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>Resuspend each primer in enough volume of nuclease-free ddH2O to obtain a 100 mM stock solutions that can be stored at -20&deg;C, long term.</li>
	<li>Dilute each primer stock to 12 mM prior to the PCR setup steps.</li>
</ol>
5. PCR Setup
DNA extractions are amplified using each of the above primer sets in PCR reactions. Include a positive control to ensure that all PCR reagents are working well and can generate a band. Include a negative control to ensure that none of the reagents are contaminated with unwanted DNA. The negative control contains no-template and should result in no band production.
<ol>
	<li>Prepare all reactions in thin-walled PCR tubes to allow better heat transfer between the thermocycler block and the sample. We recommend using commercially available aerosol-free pipette tips to help avoid contamination.</li>
	<li>For each isolated DNA sample, set up 50 &mu;l PCR reactions using each of the above primer sets in 0.2-ml thin-walled PCR tubes by adding the following reagents on ICE in the order listed below. If multiple samples are being prepared, make a cocktail containing all reagents with the exception of the DNA template to establish uniform conditions between all reactions. 
	<table border="1">
		<tbody>
			<tr>
				<td>PCR Reagent</td>
				<td>Volume Used</td>
				<td>Final Concetration</td>
			</tr>
			<tr>
				<td>Nuclease-free ddH2O</td>
				<td>40.5 &mu;l</td>
				<td>&nbsp;</td>
			</tr>
			<tr>
				<td>10X Advantage 2 PCR Buffer (Clonetech, CA)</td>
				<td>5.0 &mu;l</td>
				<td>10% (v/v)</td>
			</tr>
			<tr>
				<td>Advantage UltraPure PCR dNTP Mix (10 mM each, Clonetech, CA)</td>
				<td>1.0 &mu;l</td>
				<td>0.2 mM</td>
			</tr>
			<tr>
				<td>Primer 1 (12&mu;M in ddH2O)</td>
				<td>1.0 &mu;l</td>
				<td>0.24 &mu;M</td>
			</tr>
			<tr>
				<td>Primer 2 (12&mu;M in ddH2O)</td>
				<td>1.0 &mu;l</td>
				<td>0.24 &mu;M</td>
			</tr>
			<tr>
				<td>Advantage 2 Polymerase Mix (Clonetech, CA)</td>
				<td>0.5 &mu;l</td>
				<td>1% (v/v)</td>
			</tr>
			<tr>
				<td>DNA extraction (70 ng/reaction; adjust ddH2O volume as needed)</td>
				<td>1.0 &mu;l</td>
				<td>1.4 ng/&mu;l</td>
			</tr>
			<tr>
				<td>&nbsp;</td>
				<td>Total: 50.0 &mu;l</td>
				<td>&nbsp;</td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>To ensure that all PCR reagents are working well, setup the positive control using the positive control primers. This primer set works equally well for cogongrass, JBG, JBG revert and other grasses and will result in a band that is 890 bp. </li>
	<li>Setup the negative control using the same control primer set as the positive control, using ddH2O instead of the DNA extraction. If all reagents are free of DNA contaminates, this reaction will result in no band. </li>
	<li>If the DNA concentration is low, more DNA can be added to each reaction, adjusting the amount of Nuclease-free ddH2O used to bring the total reaction volume to 50 &mu;l. Do not use more than 5 &mu;l of DNA (10% of the total volume) per reaction, as possible impurities contained in the DNA samples can inhibit PCR reactions.</li>
</ol>
6. PCR Cycling
<ol>
	<li>Carry out PCR amplifications in a thermocycler equipped with a heated lid using the following PCR cycling parameters. We use the Mastercycle pro S thermocycler (Eppendorf, Hauppauge, NY) set to operate with standard temperature ramping conditions. Any quality thermocycler should perform well. 
	<table border="1">
		<tbody>
			<tr>
				<td>Cycle</td>
				<td>Denaturation Annealing</td>
				<td>Polymerization</td>
			</tr>
			<tr>
				<td>1</td>
				<td>2 min at 95&deg;C</td>
				<td>&nbsp;</td>
			</tr>
			<tr>
				<td>2</td>
				<td>30 sec at 95&deg;C</td>
				<td>30 sec at 61&deg;C 90 sec at 68&deg;C 35 Cycles</td>
			</tr>
			<tr>
				<td>3</td>
				<td>&nbsp;</td>
				<td>5 min at 68&deg;C</td>
			</tr>
			<tr>
				<td colspan="3">Hold at 4&deg;C until the sample is removed</td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>Optimize the conditions for PCR (including primer annealing temperature, extension times, and number of cycles) as needed depending on the quality of the DNA, primers, Taq polymerase or type of thermocycler used. We recommend using a gradient capable thermocycler when determining the optimal annealing temperatures. </li>
	<li>If the thermocycler being used does not have a heated lid, add 1 drop of mineral oil to the top of each sample to prevent evaporation during PCR cycling.</li>
</ol>
7. Gel Electrophoresis of PCR Products
To visualize the results of the analysis, separate PCR products on a 1% agarose gel using standard electrophoresis.
<ol>
	<li>Combine 2 &mu;l of a standard DNA loading buffer (typically a 5x or 6x solution) with 5 &mu;l of each amplified PCR product.</li>
	<li>Load samples onto a 1% agarose gel containing EtBr (ethidium bromide for DNA staining) made with either TAE or SB (sodium borate) buffer systems16. We use 1 &mu;l of a 10 mg/ml EtBr stock solution per 100 ml 1% agarose (0.1 &mu;g/ml).</li>
	<li>Run samples at ~120V until the dye front reaches &frac34; of the total length of the gel.</li>
	<li>Under UV light (e.g. a short wave UV light box), inspect the resulting bands to see if an appropriate fragment was amplified.</li>
	<li>Document the gel and resulting bands using an available photo-documentation system or camera.</li>
</ol>
8. Representative Results
Upon visualization of the PCR products, cogongrass has a unique banding pattern compared to that of JBG or reverted JBG (Figure 5). For each DNA sample, the trnL-F positive control primer set should result in a single high-intensity band at ~890 bp. This verifies that all PCR reagents are working well. Similarly, the negative control (no template) reaction should contain no bands for any primer set used. This verifies that none of the reagents were contaminated.
If the DNA sample is derived from wild-type cogongrass, a PCR reaction using the cogongrass-specific primer set will result in a single band at ~595 bp while the JBG-specific primers will result in no band. Likewise, if the DNA sample is derived from JBG or reverted JBG, a PCR reaction using the JBG-specific primer set will result in a single band at ~594 bp while the cogongrass-specific primers will result in no band. Because JBG and reverted JBG have identical nucleic acid sequence, they will hence have identical banding patterns. If many samples are to be compared on a gel at the same time, we recommend running all samples derived from each primer set next to one another, thus making it easier to scan the samples for positive results.
Morphological differences between JBG and JBG reverts are fairly obvious (e.g. red color of the leaves and smaller stature of JBG vs. the green coloration, larger stature and aggressive growth of the JBG revert), so while PCR results will be the same, the JBG varieties are easy to distinguish using plant morphology.
<img alt="Figure 1" src="/files/ftp_upload/3265/3265fig1.jpg" /> 
Figure 1. Comparison of greenhouse grown Imperata cylindrica var. koenigii (Japanese blood grass), Reverted I. cylindrica var. koenigii (JBG Revert) and I. cylindrica (Wild-type cogongrass).
<img alt="Figure 2" src="/files/ftp_upload/3265/3265fig2.jpg" /> 
Figure 2. An example of DNA samples verified using a NanoDrop spectrophotometer. Note that, irrespective of the spectrophotometer used, the 260/280 ratio should be close to 1.8 and the 260/230 ratio should close to 2.0.
<img alt="Figure 3" src="/files/ftp_upload/3265/3265fig3.jpg" /> 
Figure 3. DNA samples verified using standard gel electrophoresis on a 1% agarose gel. A commercial DNA marker was used for size analysis. Lane #4 is an example of poor quality DNA sample, showing smearing and some RNA contamination.
<img alt="Figure 4" src="/files/ftp_upload/3265/3265fig4.jpg" /> 
Figure 4. Sequence alignments of the trnL-F regions of Imperata cylindrica var. koenigii (Japanese blood grass), Reverted I. cylindrica var. koenigii (JBG Revert) and I. cylindrica (Wild-type cogongrass). Vertical black arrows indicate differences in sequences resulting from SNPs and InDels. Horizontal green arrows indicate the positions of the Wild-type cogongrass primers used for cogongrass-specific PCR. Horizontal red arrows indicate the positions of the JBG and JBG Revert primers used for JBG-specific PCR. Please <a href="https://www.jove.com/files/ftp_upload/3265/3265fig4_lg.jpg">click here</a> to see a larger version of this figure.
<img alt="Figure 5" src="/files/ftp_upload/3265/3265fig5.jpg" /> 
Figure 5. Representative result of gel electrophoresis of PCR products derived from cogongrass, JBG and JBG revert DNA samples combined with cogongrass- and JBG-specific primers as well as the trnL-F positive control and a no template negative control.

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We have nothing to disclose.

The U.S. nursery and landscape industries thrive on cultivating and selling exotic and novel plant species. This, coupled with the growing globalization of trade, increases the chances that an invasive plant species will enter, establish, and spread in the U.S. The ability to federally regulate such plants relies on information that is often not available, including the potential to become invasive, correct taxonomy, and genetic distinctness from native and naturalized taxa. Because our knowledge of invasive plants is often limited, imported plants with hidden invasive characteristics have been willingly introduced only to learn later that they invade our agricultural and natural resources. This protocol aims to address such problems associated with I. cylindrica varieties by providing the first simplified molecular method that can accurately distinguish between wild-type cogongrass and the reverted form of its ornamental JBG counterpart.
For the development of this protocol, wild-type cogongrass was collected from naturalized populations at the Pond Creek Forestry Unit in Santa Rosa County near Jay, FL in June of 2008. JBG was procured from a commercial nursery (Bluebird Nursery, Inc.) in June 2008 as well as from a homeowner's collection in Columbia, MO. JBG reverts were obtained from the courtyard of the Campbell Geological Museum at Clemson University, SC in June 2008; from University Park in Riverdale, MA in June 2009; and from the front yard of a homeowner in Columbia, MO in 2009 (identified by Leland Cseke). All plants were maintained in a greenhouse located at the University of Alabama in Huntsville (Huntsville, AL).
Genetic sequencing of DNA collected from these plants included the in depth comparisons of 9 independent DNA regions commonly used to barcode plants2. In all cases, the sequences of JBG were a 100% match to those of the JBG revert, thus helping to verify that JBG does indeed revert to a green, invasive form. Only the nuclear ITS and the chloroplast trnL-F regions have differences that can be used to genetically distinguish between cogongrass and JBG. The ITS region has a total of 3 SNPs (single nucleotide polymorphisms) between cogongrass and JBG, while the trnL-F region has 2 SNPs and 2 InDels (insertions and deletions). These genetic differences allowed variety-specific PCR primers to be developed that can distinguish between wild-type cogongrass and JBG reverts. The most reliable results have come from primers derived from the plastid trnL-F region. Thus, this protocol is based on the sequence differences between the trnL-F regions of the chloroplast genome of cogongrass, JBG and reverted JBG (Figure 4).
To help generate primers that are more specific to the varieties in question and to help avoid false positives from closely-related species, all known trnL-F sequences of I. cylindrica varieties were compared with trnL-F sequences from related grass species (43 independent sequences from 29 species, e.g., Cymbopogon citratus, Sorghastrum incompletum, Coix lacryma-jobi, Miscanthus sinensis, Saccharum officinarum, Sorghum halepense). Although, we examined variety-specific primer sequences across 29 species of grass, the specificity of the variety-specific primers has not been comprehensively examined for its ability to amplify DNA from most other grass species. Consequently, tissue used for DNA extraction should be carefully identified as I. cylindrica prior to starting this protocol. If the grass cannot be identified as either cogongrass or JBG, then we suggest sequencing the PCR product to make sure that sequences are an exact match to cogongrass or JBG. Currently, the most accurate method to verify the identity of a given grass specimen is to perform PCR on both the trnL-F and ITS regions, followed by sequence verification of PCR products and comparison of the sequences to known sequences from accurately identified taxa. DNA can be amplified using control primers detailed in this protocol (for the trnL-F region) or other primers that available in other publications13-15. Sequencing is much more labor and cost intensive than using our simplified procedure.
The quality of the primers used for PCR is critical to the success of the procedure. We have made the primers for this procedure available from Oblique Bio, Inc. (<a href="http://www.obliquebio.com/web/" target="_blank">http://www.obliquebio.com/web/</a>, Huntsville, AL). The advantage to ordering the primers from Oblique Bio is that they store large numbers of aliquots of each primer from the identical sample production series as the primers we used for optimization in our laboratory. Consequently, primers not only have the same sequence, but they come from the exact same production lot that was used in this protocol. Using primers from the same lot, can help avoid extraneous variables in the procedure that may result from differences in the quality of PCR primers. Likewise, while other Taq polymerases should work fine for PCR, the quality of the Taq polymerase used will have an impact on the quality of PCR results. To allow for better consistency in PCR reagents, we have optimized the protocol using reagents from Clontech. The Advantage 2 Polymerase (Clontech, Mountain View, CA, Cat# 639201 or 639202) is a mixture of a robust, hot-start Taq polymerase and a proof-reading enzyme that helps to provide high specificity and more accurate amplifications.
Because this protocol relies on chloroplast DNA, which is maternally inherited in grasses, hybridization events between cogongrass and JBG genotypes may not be captured with our molecular identification procedure. In cases where hypridization is suspected, we recommend using nuclear regions that are inherited from both parents. The most commonly used non-plastid variable region to consider in plant genotyping is the nuclear ribosomal ITS region13-15,17. Currently, we are making progress towards multiplexing the amplification of the chloroplast trnL-F region with that of the nuclear ITS region in the same PCR tube. Multiplexing plastid with nuclear DNA regions would potentially circumvent the limitations of using either alone; however, such methods require optimization and additional evaluation to determine feasibility on a case by case basis. The use of quantitative real-time PCR (qPCR) and newer technologies, such as molecular beacons (fluorescent primer probes), are also being evaluated as fail-safe and accurate plant genotyping methods.
The protocol presented here provides a fast and reliable approach to distinguish the JBG revert from that of wild-type cogongrass. We encourage users of this protocol to contact us to report results derived from the use of this protocol. Such shared information will help provide information on the distribution of JBG reverts. This will also help regulators at the USDA make informed decisions on actions that may be need to circumvent the spread and potential hybridization of the highly invasive cogongrass varieties.

<table border="1">
 <tbody>
 <tr>
 <td>Name of 			the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>DNeasy Plant 			Mini Kit</td>
 <td>Qiagen, 			Valencia, CA</td>
 <td>69104 			or 69106</td>
 <td>Any reputable 			genomic plant DNA kit that yields good quality DNA should work 			fine for these procedures.</td>
 </tr>
 <tr>
 <td>trnF(GAA)-F</td>
 <td>Oblique Bio, 			Inc.</td>
 <td>3-0578</td>
 <td>Forward 			positive control primer</td>
 </tr>
 <tr>
 <td>trnL(5' exon)-C</td>
 <td>Oblique Bio, 			Inc.</td>
 <td>3-0579</td>
 <td>Reverse 			positive control primer</td>
 </tr>
 <tr>
 <td>trnLF-C-F1</td>
 <td>Oblique Bio, 			Inc.</td>
 <td>3-0864</td>
 <td>Forward 			wild-type cogongrass primer</td>
 </tr>
 <tr>
 <td>trnLF-C-R1</td>
 <td>Oblique Bio, 			Inc.</td>
 <td>3-0865</td>
 <td>Reverse 			wild-type cogongrass primer</td>
 </tr>
 <tr>
 <td>trnLF-R-F2</td>
 <td>Oblique Bio, 			Inc.</td>
 <td>3-0866</td>
 <td>Forward JBG 			and JBG revert primer</td>
 </tr>
 <tr>
 <td>trnLF-R-R2</td>
 <td>Oblique Bio, 			Inc.</td>
 <td>3-0867</td>
 <td>Reverse JBG 			and JBG revert primer</td>
 </tr>
 <tr>
 <td>Advantage 			UltraPure PCR dNTP Mix</td>
 <td>Clontech, 			Mountain View, CA</td>
 <td>639125</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Advantage 2 			Polymerase</td>
 <td>Clontech, 			Mountain View, CA</td>
 <td>639201 or 			639202</td>
 <td>A good 			proof-reading, hot-start Taq polymerase</td>
 </tr>
 </tbody>
</table>

a pcr-based genotyping method to distinguish between wild-type and ornamental varieties of imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

Watch this Scientific Journal Video about A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica at JoVE.com

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

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Research

JoVE Journal

Biology

31.5K visualizações.  The University of Alabama, Huntsville. Nós fornecer um protocolo de genotipagem custo-eficácia e rápida molecular que emprega iniciadores variedade-PCR específicos que as diferenças de sequência alvo de ADN dentro da região espaçadora cloroplasto trnL-F para diferenciar entre as variedades de Imperata cylindrica (Cogongrass) que não podem ser distinguidos pela morfologia sozinho. Estas variedades incluem a erva federal listado nocivo, cogongrass e estreitamente relacionada, a variedade ...

Vídeo: A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...