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The DNA from four plants (Dill, Cilantro, Fennel, and Parsley) was extracted and their rDNA ITS region amplified using PCR and Sanger method to sequence the data and create UPGMA matrix and tree to show the phylogeny between the four plants and know which plants are more related than the other plant. The DNA was first extracted in eight steps using specific buffers and chemicals to bind, wash and elute it. It was amplified in three PCR steps: denaturation, primer annealing and extension at specific temperatures. It was sequenced using Sanger method by Exosap program and analyzed using data sequencing technique to create a UPGMA matrix and tree to calculate the genetic distance. According to the tree obtained using the data from BLAST program that was used to align the sequences, Dill and Fennel had the most common ancestor with Cilantro having the least common ancestor.
Key words: BLAST, Sanger method, Denaturation, Primer annealing, Extension, genetic distance
Internal Transcribed Spacers (ITS) region of rDNA genes when used and amplified correctly is the key of studying taxa and their phylogeny using the analysis of their sequences. The main parts to amplify in the ITS region are 18S, ITS1, 5.8S, ITS2 and 26S by using PCR program in the thermo cycler machine. Using four plants (Fennel, Cilantro, Parsley and Dill) the DNA was extracted, amplified and sequenced to study their phylogeny and genetic distance creating a UPGMA tree. Genetic distance shows the timeline between the taxa with the more recent separation and common ancestor having the least genetic distance and vice versa. Using a special program called BLAST, the sequences are aligned and the calculations are used to create a UPGMA tree and matrix. One of the advantages of using this tree is that the nucleotide has a one data point that is used in calculation of the distance between two organisms. In the other hand, this method has a disadvantage because sequences are changeable with time. (Dillon et.al 2009). The four plants are from the same subfamily of apiaceae, which is apioideae . This sub-family is distinguished by lobed or divided leaves and herbaceous stems while the flowers have pentamerous perianths. Scientists in taxonomy usually classified these plants according to their fruit appearance (Plunkett and Downie 1999). It is very difficult to differentiate between these four plants and other plants in this family so ITS region is used nowadays to place these plants in species and genera. For a long time these plants family, sub-family and tribe consensus was a mystery until the ITS method was used to analyze these plants. Anatomy and morphology is not enough to place plant in sub-families. ITS technology is more powerful and accurate based on an experiment done to study the sub-families of apiaceae (Downie et.al 1998). The UPGMA tree is useful to see if two plants have the same common ancestor and identifying that ancestor.(Soltis et.al 1997).The UPGMA tree created using the amplified rDNA ITS region was more useful and accurate in Taxonomy than a tree based on morphology
Methods and materials
This is the first step in this experiment. It was performed by eight steps to have the DNA that was necessary for the experiment. The first step was grinding in which a mechanical disruption is obtained by using liquid nitrogen to break the tissue. The second step was lysing in which an AP buffer was used to obtain biochemical disruption to destroy the cell wall at high temperature. The third step was combined with the second by adding RNase to destroy RNA. The fourth step was done by using an AP2 buffer in low temperatures to make unwanted components precipitate. In step Five, a filter was used to remove unwanted components. In step Six, a chaotic salt was added to make the H-bonds weaker to bind the DNA to the silica membrane. In step Seven, the DNA was washed with ethanol buffer AW. The last step was removing the DNA from the filter by applying a water based AE buffer.(Dillon et.al 2009).
After finishing from the extraction, a PCR was done using a thermo cycler machine. The PCR was performed using three main steps and 28 cycles. The first step was denaturation, which was done at 95 C0 . The second step, primer annealing was performed at 50 C0. The extension was performed at 70 C0. A negative and positive sample was used to get results that are more accurate. (Dillon et.al 2009).
The DNA was sequenced in Sanger method using Exosap reaction that removes any primers and residual dNTPs. Two different reactions (forward and reverse) were used after the Exosap program and the DNA was placed in thermo cycler while using OPTI-SEQUENCE program. (Ozaki 2009).
The genetic distance between the taxa was calculated using sequence data method while using BLAST program in computer to calculate the gaps between sequences. To organize the genetic distance calculations a UPGMA matrix and tree were used to show phylogeny between the four plants. (Dillon et.al 2009).
After eluting the DNA in extraction experiment, the DNA was ready for amplification using the PCR. A negative sample was used for checking any contamination. A positive sample was used to make sure that the sample is in working condition. The third step was sequencing using Sanger method with Exosap program for more pure DNA, with forward and reverse reactions. The reactions were analyzed and the data used in creating the UPGMA matrix (Table1) and UPGMA tree (Figure1). Genetic distance between each one of the four plants was used to fill the UPGMA matrix in which values are chosen one after another until there is no more values are available to choose. The smallest genetic distance was used to start the calculations. The gaps between the four plants sequences were calculated using BLAST program. (Dillon et.al 2009)
The data that was obtained from this experiment was not just useful for creating UPGMA tree but with further analysis it is useful to place plants in more precise species, genera and sub-families using the ITS region instead of appearance or morphology (Downie et.al 1998). Errors came from using the wrong pipette set up, different higher or lower temperatures than the ideal for successful experiment especially in the PCR steps. Another source of error is using data sequencing method because the sequences are changeable with time. A negative or positive sample was used to avoid errors in the PCR samples because this experiment is very complicated and sensitive to change in the components used. UPGMA tree was more useful than a tree based on morphology as shown in scientific articles used in this lab report. After creating the UPGMA tree, the dill and fennel were the most related with most recent common ancestor between them by five points. Cilantro was the least related with least recent common ancestor by 32.75 points. Scientists nowadays use the ITS region to differentiate between different groups of plants and divide them in a specific family, sub-family or specie. (Soltis et.al 1997) The UPGMA tree supported the hypothesis stating that the UPGMA tree is more useful and specific to classify plants than a tree based on morphology.
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L. Ozaki. (2009). DNA Sequencing Methods. Virginia Commonwealth University, Department of Biology.
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