Introduction
Modern biology is characterized by the development of instrumental methods of scientific analysis, which allow us to obtain qualitative answers to the questions posed by the discipline. Thus, a significant area of interest in the biological sciences is phylogeny, which aims at a profound study of the relationship between taxa in the light of the evolutionary transformations of life on Earth. For these studies, there is a set of developed instrumental methods of analysis, among which DNA barcoding deserves special attention. In general, such a procedure should be understood as a method of molecular biology that allows one to make accurate judgments about the membership of a species in a taxon, using short fragments of a DNA sample as evidence (DeSalle and Goldstein, 2019). To put it another way, DNA barcoding should be seen as a method that greatly accelerates species identification processes using a minimal sequence of genetic material. The efficiency of this instrumental method is built on the idea of close similarity in the structure of DNA molecules — to be more precise, the arrangement of nucleotides in it — between closely related species: the closer the classification relatedness, the higher the percentage of similarity in alignment. As a consequence, if one has the DNA sequence of some suspected species as a reference point, it becomes possible to determine, using DNA barcoding, how closely the sample under study is evolutionarily related to them.
The use of benchmarks of the genetic material of already studied species is a necessary measure for the present study. More specifically, to determine the classification position of an unknown specimen in a taxon sequenced DNA sequences for Corn poaceae, Pine pinaceae, Magnolia magnoliaceae, and Grape vitaceae were used. The gene of the Unknown specimen was selected for use in the present work, which turned out to be Hibiscus punaluuensis at the end of the study. The high confidence in the correspondence between the given unknown specimen and Hibiscus punaluuensis is justified by the use of digital phylogeny tools and the study of the Hibiscus punaluuensis genome separately. The working hypothesis of the experiment was that it was expected that plants with closer relatedness should be more strongly related to each other. In practice, this is expressed by the proximity of species on the phylogenetic tree, which means a higher percentage of DNA overlap in their alignment. The present study aims to qualitatively summarize the results obtained in order to establish the Unknown specimen.
Procedure
Carolina Biological Supply Company: Using DNA Barcodes to Identify and Classify Living Things (211385P) was used as the methodological guide for this report, so the test steps became largely a repeat of the laboratory measures described. The first phase was sample preparation, in which a DNA sequence was isolated from an Unknown specimen given by the instructor for academic purposes. This was done using standard PCR methods for DNA multiplication and gel electrophoresis for qualitative identification. These methods are widely used to isolate a fragment of DNA and quantify it to simplify subsequent manipulations, including the quality study of DNA composition through differences in the movement of molecules in the gel electrophoresis chamber. Upon receipt of the genetic material, the sample was delivered to Genewiz DNA Sequencing Service by mail.
Sequencing was performed through the use of a digital tool, namely the Basic Local Alignment Search Tool (BLAST), to collect the DNA sequences of an unknown plant into a single test database. Using the known number identifier of the unknown planet, a long nucleotide sequence was found that was stored and sent to the next stage of the test. As a result of using BLAST, a digital copy of the gene sequence for the plant being analyzed was obtained. Then, the DNA Subway tool was the second product that was used for the assay. Using DNA Subway, the previously obtained DNA copy of the unknown plant was loaded into the field for work, where the results were compared, aligned, and processed. Through analytical comparison of the order of nucleotides in the DNA sequence between the Unknown sample and the most similar species, the program determined the degree of their classification proximity. In other words, DNA Subway produced a phylogenetic tree that is used to make a descriptive comparison of the proximity of the species under study.
Results
Because the central result of the test is the construction of a phylogenetic tree to determine classification proximity between organisms, an unknown specimen was prepared for study. In more detail, identification numbers were given by the instructor for study. The unknown plant specimen obtained had the number #30-358425317 (AJ233121.1). This number was used to work with the BLAST and DNA Subway platforms. In more detail, a summary alignment statistic was obtained using DNA Subway, shown in Table 1. In addition, a phylogenetic tree model was constructed using the built-in DNA Subway functionality, as shown in Figure 2.
Table 1. Alignment results for the unknown specimen.
Based on the results obtained, it is possible to state with great confidence that the unknown plant was Hibiscus punaluuensis, the classification structure of which is shown in Table 2. More specifically, the Hibiscus punaluuensis chloroplast rbcL gene, partial, was most likely taken as the DNA sequence. Figure 1 and Figure 3 show the appearance of this plant: flower, leaves, and approximately reproductive organs.
Table 2. Classification structure of Hibiscus punaluuensis from clade to species name.
Discussion
In the present laboratory work, the critical research interest was to critically test the efficacy of the DNA barcoding method to identify the species name for an unknown specimen accurately. A DNA sequence apparently isolated from the Hibiscus punaluuensis chloroplast rbcL gene, partial, was given by the instructor. This gene is located in the chloroplasts inside the plant cell, which means that the DNA in which it is included should be considered semi-autonomous. This assumption is justified by the study of the Hibiscus punaluuensis genome in terms of searching for correspondence between the given sequence and the reference. Based on the data in Table 1, it is clear that with 99% probability, the obtained alignment has reliability or, in other words, the qualitative definition of Hibiscus punaluuensis is very accurate. At the same time, an error of 1% can be justified by the presence of mutational processes that distinguish species and genera from each other. This includes point mutations and single-nucleotide polymorphisms, as well as deletions and duplications that eventually led to polynucleotide chain changes (Mehmood et al., 2020). In turn, such changes caused the evolutionary divergence of plant organisms. At the same time, the phylogenetic tree constructed unambiguously identifies the studied specimen as related to those plants that were used for the alignment. The numbers on the tree show how closely related species are to each other in light of the comparison of their genetic material.
Conclusion
Since BLAST and DNA Subway digital tools were used to search for the DNA fragment, align it with the sequences already stored in the database, and model the phylogenetic tree, one can postulate with great confidence the high efficiency of the DNA barcoding method. In the context of assessing the validity of the working hypothesis, it should be labeled as confirmed since it was empirically proven that closely related plant organisms had a higher percentage of similarity. As a result, DNA barcoding has proven to be an excellent tool of analytical biology for phylogenetic studies.
References
DeSalle, Rob, and Paul Goldstein. 2019. Review and interpretation of trends in DNA barcoding. Frontiers in Ecology and Evolution 7: 302-310.
Eickhoff, D. 2009. Hibiscus arnottianus subsp. Punaluuensis. Flickr. Web.
Kolev, N. 2010. Macro of the flower of Hibiscus arnottianus subsp. Flickr. Web.
Mehmood, F., Shahzadi, I., Ali, Z., Islam, M., Naeem, M., Mirza, B., Lockhart, P.J., Ahmed, I. and Waheed, M.T., 2020. Correlations among oligonucleotide repeats, nucleotide substitutions, and insertion-deletion mutations in chloroplast genomes of plant family Malvaceae. Journal of Systematics and Evolution 59(2): 388-402.