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The purpose of the study is to estimate the genetic variation and species differentiation according to geographical differences of Hygrophila pogonocalyx by using RAPD (random amplified polymorphic DNA) fingerprinting, which is a PCR-based fingerprinting method. With the study of the plants genetic variations and phylogeography, we can get a better view on the plants evolutionary history and can find solutions to protect the endangered species.
H. pogonocalyx is an aquatic plant which is severely threatened due to human activities. In Taiwan, there are four populations remaining separated by geographical differences which is the mountain ridge. As a result, genetic variation between the populations is significant. From the above observations, population differentiation is influenced by phylogeography, and thus there are genetic drifts among the plant populations. Besides from geographical differences, pollinators such as insects will also influenced the genetic variability of a population. This is due to the fact that some insects will only be localized at a specific area. In addition, some seeds dispersal of plants will also limited to a certain area.
From the above observation, RAPD analysis can be used to study the differences and similarities between the populations of H. pogonocalyx. RAPD is a polymerase chain reaction (PCR)-based method which was used to amplify H. pogonocalyx DNA. By using the RAPD fingerprinting, we can determine that there is genetic diversity present as it functions as a genetic marker to detect genetic variations among strains of H. pogonocalyx.
MATERIALS AND METHODS
H. pogonocalyx was collected from northeastern and western Taiwan. As there are only limited populations available, only 10% is collected from each population and this will make sure that the same individual is not collected twice. The samples collected must be placed in a cool area and is cultured. Before proceeding to the grinding process, the leaves of young shoots are washed and the grinded leaf tissue powder was placed in liquid nitrogen to break down cell wall material and allow access to DNA while harmful cellular enzymes and chemicals remain inactivated. The genomic DNA of the plant was extracted by using the plant DNA extraction method. The DNA was resuspended in CTAB buffer. To purify the DNA, centrifugation was used to remove insoluble particulates and other materials were separated by mixing with chloroform and centrifuged. The supernatant was transferred and was centrifuged again and the pellet was washed by ethanol to remove contaminating salts. The pellet was then resuspended in TE buffer. The genomic DNA extracted was run on agarose gel, stained with ethidium bromide and observed under UV light.
The genomic DNA was amplified using PCR method. The amplified products were run again in gel electrophoresis, stained with ethidium bromide and photographed.
AMOVA-PREP was used to analyse the data between the populations studied. With AMOVA, we can estimate the molecular variance of the populations. The molecular variance was estimated in pairs.
From the RAPD data, the populationâ€™s distance matrix can be obtained. The distance matrix was used to draw a UPGMA dendrogram using software and the genetical variation was estimated by bootstrapping.
A formula was used to calculate the similarity index and certain software analyzes the coordinate using the similarity index. The first three coordinates was drawn in three-dimensional graph and the genetic relationship of the populations can be read from the graph.
From RAPD fingerprinting, the bands produced have similarities among the populations. The molecular variance also shows that it is also a factor that can be used to deduce that there are differences among the population studied with similar and different geographical area. Besides that, the pairing comparison of molecular variance also shows that there are differences between the populations.
The UPGMA dendrogram was analyzed and it shows that there are two geographically different groups. By sampling two of the population with same geographical region, the bootstrap value is 100%, confirming the similarities of population between same geographical areas. Moreover, there is no evidence shown that subpopulation structure is present.
The first three coordinate of the population used to draw the three-dimensional graph was found to be consistent with the UPGMA dendrogram. From this, we can conclude that there are two geographically different populations present.
The AMOVA analysis shows that genetic variation between populations of different geographical area is high compare to from the same geographical region. When all of the populations were analyzed regardless of geographical area, the genetic differences among populations are also high. From the data, the differences may be caused by insect pollination. Plants which adopt wind dispersal were found that it caused genetic differences just within the population.
As the insect pollinators were restricted to just a certain area, the gene flow between populations was not so significant. From this observation, it shows that the genetic variation is associated with geographical area.
The UPGMA dendrogram and coordinate analysis also shows that there are differences among population from different geographical area. Due to the fact that different genetic variation was present between populations of different geographical area, the loci of the population have attained their monophyly and this will caused genetic drift. Natural selection present at a particular area will also influence the gene flow of the population. This phenomenon of genetic variation shows that the population may have evolved to exhibit the similar phenotypic features but highly differentiated genetic composition.
Although H. pogonocalyx is an endangered species, but there are some genetically variate population present. Sexual reproduction is responsible for the genetic variation as from the study carried out; the progeny produced from sexual reproduction would have a higher living ability than the selfing ones. Somatic mutation is also responsible for this genetic variation. Human activities will cause the habitat of the plants to be destroyed and genetic variation would be lost. This will cause the population to extinct as times passed as they will lose their ability to adapt to harsh condition. So, to protect the plants from extinction and avoid genetic loss, conservation must be done to ensure that the plants can live and grow better in their habitats.
From the RAPD fingerprinting, genetic variation between the populations of H. pogonocalyx was still maintained although they are endangered species. Somatic mutation, insect pollination and selfing can also contribute to the genetic differences but inbreeding between the plants will lower the genetic differences. From the molecular variance data and UPGMA dendrogram, the population from different geographical area was shown to be separated genetically. As they are different genetically, they must be managed with different methods.