The main aim of this procedure is to provide a cost-effective and rapid molecular genotyping protocol that can differentiate between varieties of Retta cylindrica, typically called gon grass, that cannot be distinguished by morphology alone. This is accomplished by first identifying and isolating tissue from a gon grass variety of interest. The next step is to extract nuclear and plastered DNA from the collected tissue.
Next polymerase chain reaction is performed on the extracted DNA using variety specific primers that target sequence differences in the PLAs turn LF region, as well as appropriate positive and negative controls. The final step is to perform gel electrophoresis on each of the four PCR reactions to visualize the amplified PCR products. Ultimately, the variety of gon grass collected is determined through analysis of the resulting variety specific PCR product bans on the gel Invasive plans cost the United States about 3.4 billion annually, causing substantial harm to both agricultural and natural resources.
Wild type imparato Electrica, also known as Cogan, grass is one of the top 10 worst invasive weeds in the world. Cogan grass forms rapid spread in mono stands that displace native vegetation, and in turn, the animals that depend on that native vegetation GaN grass currently infest 490 million hectares worldwide and about 500, 000 hectares in the United States and ornamental variety of ti of tus centrica is widely marketed under the names of tus Centrica, Arbra, red Baren, and Japanese blood crass as we'll call it JBG. This variety is putatively sterile and non-invasive, and is considered desirable for its red colored leaves.
Under certain conditions, Japanese blood grass can produce viable seeds and revert to an all green form that is indistinguishable from wild type cogan grass. As you can see, they both possess green leaves and are larger than traditional ornamental variety with larger leaves. Additionally, the reverted and wild type plants have larger rhizomes and the ornamental variety increasing its asexual reproductive potential.
The reversion makes identification through morphology alone. A trying task. Molecular methods provide a means to accurately distinguish invasive plants and plant materials that cannot be identified through normal visual means.
Molecular methods are critical to safeguard against the spread and introduction of invasive plants. Application of this molecular protocol provides a means to accurately distinguish Japanese blood grass reverts from Kogan grass. This protocol can help prevent the co-occurrence with Kogan grass and the possible formation of viable hybrids.
The primary advantage to this technique over existing techniques such as amplified fragment length polymorphism, or A FLP analysis and things like DNA sequencing, is that it provides a very cost effective and rapid molecular genotyping technique that can easily distinguish the difference between wild type kogan grass and varieties of Japanese blood grass. Demonstrating the technique will be Joseph Hery, an undergraduate from our laboratory. First identify a specimen from a site where gon grass or Japanese blood grass are growing and need to be distinguished from one another.
Japanese blood grass is easily distinguished from wild type gon grass by its bright red leaves. However, the Japanese blood grass revert is almost indistinguishable from Kogan grass as they both have longer green leaves, more leaf area and larger rhizomes than the ornamental Japanese blood grass. This method can be used on fresh, frozen, and recently dried leaf tissues.
If storing tissues for DNA extraction at a later date, the best method is to freeze and store the tissue at minus 80 degrees Celsius immediately. If dry storage is necessary, place the tissue into a paper envelope and store the envelope in dehydrated silica gel or other active desiccants at room temperature. If fresh tissue is to be used, ensure that DNA is extracted within three hours of collection to prevent degradation.
This procedure uses the DNEZ plants mini kit from Kyogen according to the manufacturer's instructions with one minor modification.Here. The procedure has been adapted so that greater than 100 milligrams of fresh or frozen tissue or greater than 20 milligrams of dried tissue is used. First grind greater than 100 milligrams of fresh or frozen leaf tissue, or greater than 20 milligrams of dry leaf tissue to a fine powder using three rounds of liquid nitrogen with grinding in a chilled mortar and pestle.
Weigh the frozen powder from fresh or frozen tissue into 100 milligram aliquots. If using dry leaf tissue, then weigh the powder into 20 milligram aliquots. After this step, proceed with the DNA extraction according to the manufacturer's instructions using a spectrophotometer such as the NanoDrop spectrophotometer test, DNA, quality and quantity good DNA yields should be between 50 and 150 nanograms per microliter with two 60 over two 80 and 2 32 80 ratios close to 2.0 as seen.Here.
Conduct electrophoresis using a standard 1%AROS gel. Verify the presence of relatively large bands greater than 10 KB with no to little streaking from RNA contamination if the DNA concentration is high, dilute samples to 70 nanograms per microliter for subsequent steps. The PCR primers used in this protocol indicated by the red and green arrows are based on sequence differences between the plastered turn LF spacer region of Cogan grass and Japanese blood grass genotypes.
These differences come in the form of single nucleotide polymorphisms and insertions and deletions indicated by the vertical black arrows, which allow the development of variety specific primers. By locating the primers at the sites of unique sequences, the primer sequences are contained in the written portion of the protocol for each isolated DNA sample set up two 50 microliter PCR reactions in 0.2 milliliter thin walled PCR tubes on ICE using each primer set according to the written protocol. If the DNA concentration is low, more DNA can be added to each reaction while adjusting the amount of nuclease free double distilled water so that the total reaction volume remains 50 microliters.
Do not use more than five microliters of DNA or 10%of the total volume per reaction as impurities contained in the DNA samples may inhibit PCR reactions to ensure that all PCR reagents are working, well set up the positive control. Using the positive control primers set up the negative control using the same control primer set as the positive control, but with double distilled water instead of the extracted DNA at this point, there are a total of four PCR reactions for each sample being tested. Next, carry out PCR Amplifications in a thermocycler equipped with a heated lid using the PCR cycling parameters in the written protocol optimization for prime kneeling temperature, extension times and number of cycles may be needed depending on the quality of the DNA primers tack polymerase or the type of thermocycler used.
Once the PCR reactions are complete, separate the PCR products on a 1%gel using standard electrophoresis. Run the samples at around 120 volts until the D front reaches three quarters of the total length of the gel. Visualize the gel under UV light and inspect the resulting bands to see if an appropriate DNA fragment was amplified.
This representative gel shows the results of electrophoresis of PCR products derived from Kogan grass, Japanese blood grass, and Japanese blood grass revert DNA samples combined with Kogan grass and JBG specific primers, as well as the turn LF positive control and a no template negative control. The turn LF positive control primer set works equally well for Cogan grass, Japanese blood grass, Japanese blood grass revert and other grasses, and will result in a band that is 890 base pairs. If all PCR reagents are free of DNA contaminants, the negative control reaction will not result in a PCR product.
If the DNA sample is derived from wild type kogan grass, A PCR reaction using the Kogan grass specific primer set will result in a single band at around 595 base pairs, while the JBG specific primers will result in no band. Likewise, if the DNA sample is derived from JBG or reverted J-B-G-A-P-C-R reaction, using the JBG specific primer set will result in a single band at around 594 base pairs. While the Kogan grass specific primers will result in no band.
Because JBG and reverted JBG have identical nucleic acid sequence, they will hence have identical banding patterns. However, morphological differences between JBG and JBG reverts are fairly obvious, so while the PCR results will be the same, the JBG varieties are easy to distinguish at the plant level. DNA sequencing of the PCR products can identify possible changes in DNA sequences for different cogan grass ecotype.
The development of this technique provides the first simplified molecular method that can accurately distinguish between wild type K and grass and the reverted form of its ornamental variety Japanese blood grass. We encourage users of this protocol to contact us to report the results derived from the use of this protocol. Such shared information will help provide information on the distribution of Japanese blood grass.
This will also help regulators at the USDA make informed decisions on actions that might be needed to circumvent the spread and potential hybridization of the highly invasive Cogan grass varieties.
