Masters Degrees (Genetics)

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Browsing Masters Degrees (Genetics) by Subject "Amaryllidaceae"

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    Genetic variation in the most primitive Clivia species
    (University of the Free State, 2010-11) Van der Westhuizen, Hester Maria; Spies, J. J.; Spies, P.
    English: The genus Clivia Lindl., which belongs to the family Amaryllidaceae J. St-Hil. (1805), is comprised out of seven different species. Clivia nobilis, C. caulescens, C. miniata, C. gardenii, C. mirabilis, C. robusta and the natural hybrid Clivia xnimbicola all forms part of this genus. Clivia mirabilis is found in the Northern Cape Province and is geographically isolated from the six other species which grows along the Eastern Coast and escarpment of South Africa. Conrad et al. (2003) proved that C. mirabilis and C. nobilis were the two most primitive species in the genus Clivia. During this study sequencing results were used to detect barcodes/SNPs for C. nobilis and C. mirabilis and to reveal genetic variation between the Clivia species. Clivia nobilis and C. mirabilis were tested with cross-species microsatellite makers to reveal intraspecific variation. Seven different gene regions were sequenced. Six were chloroplast regions, namely the atpH-I, matK, rpoB, rpoC1, rpl16, the trnL-F regions and one was a nuclear region, ITS1. The regions used for sequencing were evaluated as potential barcoding/SNP regions for future use. They were also used to infer the evolutionary development of C. nobilis and C. mirabilis. All seven Clivia species were analysed but this study focused mainly on morphologically different specimens of C. nobilis and C. mirabilis. The sequences were aligned and edited with Geneious Pro. A total of forty-seven polymorphic sites were observed between al seven species. Within the rpl16 region eleven parsimony informative sites were observed. The matK and trnL-F regions each had eight parsimony informative sites. ITS1 had three sites and rpoB and rpoC1, one parsimony informative site each. Within the atpHI region no parsimony informative sites were observed. The sequencing data obtained could be used for species identification and, therefore, showed great potential as barcoding regions. We propose that matK, rpl16 and trnL-F are used as a barcode in C. nobilis and C. mirabilis because they had the most parsimony informative sites. The cladogram obtained from the combined data set (atpH-I, rpoB, rpoC1, matK and trnL-F) confirmed that C. nobilis and C. mirabilis are two separate species. Clivia caulescens and C. xnimbicola forms a monophyletic group. Within the rpl16 chloroplast region intraspecific variation in C. mirabilis and interspecific variation between C. nobilis and C. mirabilis were observed. The phylogentic tree representing the sequencing results of the rpl16 region revealed three distinctive groups within the four different C. mirabilis populations. Two plants within one of the Donkerhoek populations showed more variation than the rest of the population. The rpl16 gene region proved to be ideal in order to test intraspecific variation in C. nobilis and C. mirabilis. To evaluate the use of cross-species markers, microsatellite makers designed for Phaedranassa tunguraguae, Hymenocallis coronia and Clivia miniata were tested on C. nobilis and C. mirabilis. Although amplification was obtained, in most cases the results could not be optimized in order to provide reliable analysis. In future species specific primers for C. nobilis and C. mirabilis will be developed. This study undoubtedly identified barcodes/SNPs for C. nobilis and C. mirabilis which can be used to eliminated mistaken identity. Gene regions specific for intra- and interspecific variation were identified and can be used in future for population studies.
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    Genetic variation of Clivia caulescens
    (University of the Free State, 2011-06) Stegmann, Suzanne; Spies, J. J.
    English: At present, the genus Clivia consists of six species, including Clivia nobilis Lindl., C. miniata (Lindl.) Regel, C. gardenii Hook., C. caulescens RA Dyer, C. mirabilis Rourke and C. robusta Murray, Ran, De Lange, Hemmet, Truter & Swanevelder. Many of the species and cultivars are extensively grown worldwide, making this group of considerable horticultural importance. This study mostly focused on Clivia caulescens with a natural habitat on the escarpment from Limpopo to Swaziland through Mpumalanga. The overlapping distribution between C. miniata and C. caulescens resulted in the formation of a natural hybrid between these species at the Bearded Man Mountain. The occurrences of natural hybrids between the various species are rarely recorded. In an attempt to find out if genetic erosion is currently a threat to the various C. caulescens populations and Bearded Man Mountain clivias, this study was conducted to establish if genetic variation is present. Genetic variation refers to the variation in the genetic material of a population, and includes the nuclear, mitochondrial, ribosomal DNA as well as the DNA of other organelles. The relative genetic diversity among individuals or populations can be determined using morphological and molecular markers. Five chloroplast DNA regions, i.e. atpH-I, matK, rpoB, rpoC and trnL-F, were used in an attempt to study the molecular diversity of C. caulecsens. This study concentrated on Single Nucleotide Polymorphisms (SNPs) from these regions and microsatellites to study genetic variation. The aim of this study was to determine the genetic variation between and within the different populations of C. caulescens, to determine whether gene flow occur between the different populations and to determine which of the DNA regions included in the study can contribute to the identification of plants from a specific geographical area. Regarding the study of Clivias situated at the Bearded Man Mountain, the main objectives were to estimate genetic diversity and determine the genetic relationship among the different species of Clivia (C. miniata, C. caulescens and C. xnimbicola) from this area. Of the initial five regions that were sequenced, trnL-F amplification failed repeatedly, and this region was therefore excluded from all analyses. The other four regions showed variation between the different populations of C. caulescens and for the Bearded Man Mountain clivias, except the rpoC1 region. When the results of the phylogenetics and statistical analysis (genetic distances) were combined, it was detected that most Bearded Man Mountain specimens and God‟s Window specimens clustered together in the cladograms and in the mean distances tables. Intraspecific variation was present in all the regions and combined dataset. All attempts during this study to amplify STRs and test allelic diversity in 13 microsatellite loci for 20 specimens failed. Cross-species amplification was not as effective as hoped. Microsatellites‟ species-specific nature could have a negative effect on obtaining results, although other researchers (as mentioned in the introduction of Chapter 4) could employ cross species markers successfully. Glen & Schabble (2005) reported that a given pair of microsatellite primers rarely works across broad taxonomic groups, so primers are usually developed anew for each species. The next step would therefore be to attempt the designing of specific primers for C. caulescens.
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    ItemOpen Access
    Identification and expression analysis of flavonoid biosynthetic genes in the genus Clivia
    (University of the Free State, 2010) Snyman, Marius Christian; Spies, J. J.; Viljoen, C. D.
    English: Anthocyanins belong to a large group of secondary plant metabolites, the flavonoids, and fulfil a range of biological functions that include the cyanic pigmentation they provide to flowers, fruits, vegetables and leaves. The anthocyanin biosynthetic pathway has been well elucidated and much effort has been made by researchers to modify some of the catalytic steps, thereby changing the colour of some ornamental and cut flower species. The genus, Clivia, is an ornamental monocot indigenous to South Africa and there has been a growing interest among local and international Clivia breeders to introduce novel flower colour varieties into the market. Transgene technology holds new possibilities to ensure modification of Clivia flower colour. However, the genetics and biochemistry of the Clivia anthocyanin biosynthetic pathway must first be investigated before any attempts regarding biotechnology can be made. The current study is the first to deal with the identification and expression analysis of flavonoid biosynthetic genes in the genus Clivia, specifically those involved in anthocyanin biosynthesis, thus identifying future prospects and motivating research in unexplored territory. A previous study concerning an HPLC analysis of Clivia anthocyanin content confirmed the presence of cyanidin and pelargonidin derivatives as the main pigments in the tepals and fruits. This enabled the establishment of a putative Clivia anthocyanin biosynthetic pathway illustrating each enzymatic event. Conventional PCR with degenerate primers and a tepal cDNA template was used to isolate four different target sequences. Consensus cDNA fragments of 586 bp, 326 bp, 510 bp and 225 bp confirmed the existence of Clivia orthologues for Chalcone synthase (CHS), Chalcone isomerase (CHI), Flavanone 3-hydroxylase (F3H), and Dihydroflavonol 4-reductase (DFR), respectively. The deduced amino acid sequences of CHS, DFR and F3H harboured important conserved residues that confirmed the existence of functional enzymes. Furthermore, nucleotide sequence analyses between each new Clivia cDNA fragment and the corresponding fragments of other higher plants, regarding similarity/identity and phylogeny demonstrated closer homologies and evolutionary relatedness to other monocot species. The identification of the Clivia flavonoid biosynthetic genes enabled the expression analyses of CHS and DFR. These structural genes encode enzymes responsible for two important controlling steps necessary to determine the nature of the final end-product(s) of the pathway. Real-time quantitative RT-PCR involving SYBR® Green chemistry was used to investigate the temporal expression of the two genes in the tepal, stamen and carpel tissues during five flower developmental stages of an orange and yellow variety of Clivia miniata. Statistical analyses were used to support any findings where possible. Each respective tissue type revealed its own trend in expression for both CHS (an early biosynthetic gene) and DFR (a late biosynthetic gene) throughout flower development except in the stamens of the yellow flowers. These findings suggested the co-ordinate regulation of the Clivia miniata anthocyanin biosynthetic genes as a single module, a model of transcriptional regulation that is often found in certain monocot species (Dooner et al., 1991; Meldgaard, 1992; Martin and Gerats, 1993). To understand the regulatory system that confers flowers colouration, genes that encode transcription factors should be isolated and their spatial and temporal expression investigated. The „parallelism‟ between anthocyanin biosynthetic gene expression and anthocyanin production in the tepals of the orange and yellow Clivia miniata varieties was also investigated. UV-visible spectrophotometry at A530nm was used to quantify total anthocyanins at each developmental stage after extraction. At full bloom the orange flowers had almost 16 times more anthocyanins, which support orange colour development, than the yellow flowers. It was confirmed by the outcomes of statistical analyses that the trends in expression of CHS and DFR and anthocyanin production were similar. Methods such as HPLC are recommended for more precise qualitative and quantitative determination of total monomeric anthocyanins.