Genetics

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'n Ontleding van die genetiese bydrae van KI-bulle tot die samestelling van die Friesras in Suid-Afrika.
(1978) Cilliers, Barend.; Weyers, W. H.
No abstract available.
The development of a white clover for use in the eastern high potential areas of South Africa.
(1988) Smith, Albert.; Reusch, J. Dieter H.
The problems associated with the use of white clover in pastures in the eastern high potential areas of South Africa i.e. high P requirements, low tolerance to high Al levels and low pH in the soil as well as a limited survival time of approximately 30 months, were identified and found to be related to the inadequate root system of white clover cultivars. During the improvement programme cultivars available on the world market were introduced and evaluated under dryland conditions. Selections were made from these introductions on the basis of root conformation in high AI, low pH soils, their response to grazing and induced moisture stress. A laboratory technique for the improvement of Al tolerance was developed and the tolerance of white clover plants to high levels of Al was improved but due to the complexity of pasture plant improvement it was decided that the selection for tolerance to Al could be more effectively carried out in the field. The effectiveness of vesicular arbuscular mycorrhizas as phosphate gatherers indicated that local strains of mycorrhizas combined as effectively with white clover as the imported strains. As no seed production of white clover is undertaken in South Africa guidelines for local seed production were also established. As a result of the improvement programme, Trifolium repens cv. DUSI was developed as an open pollinated synthetic variety, based on thirty eight selected mother lines. DUSI has a greater tolerance to high AI, low pH, low P in the soil and due to an improved root system with a high percentage of secondary taproots produces better under dryland conditions and has a longer stand life than any of the cultivars of white clover available on the local market. Plant Breeders Rights were obtained for cv. DUSI and the cultivar was inscribed on the South African variety list. Limited amounts of Breeders seed have been made available to the South African Forage Seed Association for commercial seed production.
Breeding for disease resistance to the major foliar pathogens of dry beans (Phaseolus vulgaris) in South Africa.
(1994) Edington, Brian Ross.
Resistances to bean common mosaic virus, halo, common and Ascochyta blight, angular leaf spot, anthracnose and rust pathogens of beans in South Africa were combined by reverse dichotomous crossing. Full resistance to Uromyces appendiculatus from Carioca 80 was conditioned by a single dominant gene. Partially dominant resistance to Phaeoisariopsis griseola was conditioned by a single gene in Carioca 80 and two genes in PAl 127. Differences in aggressiveness of isolates of Phoma exigua var. exigua were found. Different levels of Ascochyta blight resistance were found in the glasshouse, but field testing showed little difference after flowering. Inoculations of differential cultivars indicated the presence of at least eight races of U. appendiculatus and the a-Brazil race of Colletotrichum lindemuthianum . Inoculations of the old set of halo blight differential cultivars identified races 1 and 2. Forty-five lines with partial resistance to rust were obtained by recurrent selection. Very highly significant differences were noted between ratings of percentage leaf area affected by rust and yield of 23 cultivars planted in field trials. Significant genotype x environment interaction was noted for rust ratings. Ratings at different dates within a trial were correlated with one another, showing few ratings are required per trial, and a correlation of -0.678 between yield and rust rating was found. Inheritance of partial resistance and improved yield of eight cultivars crossed in a full diallel was mostly due to additive effects but non-additive effects were also very highly significant. Reciprocal effects were not significant for yield and rust ratings. Genotype x environment interactions were significant for rust ratings and yield. High estimates of narrow-sense heritability for rust resistance were obtained. No relationship between resistance and time to flowering, pustule size, leaf hairs and stomata was found. Latent periods in unifoliate leaves did not correlate with resistance but a closer match was found in the fourth trifoliate leaves. Inoculations with three additional single-pustule isolates of the 23 parent cultivars indicated the cultivars had similar levels of resistance. Ring necrosis was found in nine cultivars or crosses with them. The ring reaction was conditioned by a single dominant gene and possibly by the epistatic interaction of two dominant genes in Carioca 80. Differences in symptom severity in plants derived from Epicure indicated the possibility of additional gene interaction.
An investigation into the heritability of commercially important traits in a sugarcane population under dryland conditions.
(1995) O'Reilly, Kerry.; Shanahan, Paul Edward.; Hohls, Trevor.
Inheritance studies have previously been undertaken at the South African Sugar Association Experiment Station (SASEX) under irrigated conditions. Since most sugarcane is grown in South Africa under dryland (raingrown) conditions, heritability estimates were calculated under these conditions in this study and compared to those previously obtained under irrigated conditions. A sugarcane population consisting of 12 crosses, 32 offspring in each cross, and their parents were planted in the first two selection stages of the SASEX selection programme to ascertain which stage provided the most useful information when selecting parent cultivars. Data collected from Stage 2 was more reliable than data collected from Stage 1. Variance components, narrow and broad sense heritabilities, correlations among traits, and clonal repeatabilities between seasons were determined for 11 sugarcane traits at Stages 1 and 2. These traits studied included: stalk population; stalk diameter; stalk height; cane mass; dry matter % cane; fibre % cane; brix % cane; brix % dry matter; purity; pol % cane; and ers % cane. Narrow sense heritabilities of the sugarcane traits were estimated by mid-parent offspring regression . Alternative heritability estimates were obtained through restricted maximum likelihood (REML) analysis of the unbalanced North Carolina design II at Stage 2. Although narrow sense heritabilities determined by mid-parent-offspring regression were comparable with those previously determined at SASEX and by other workers, REML was more efficient than regression. Use of REML enabled additive and non-additive genetic variance components to be estimated by allocating degrees of freedom to treatments and the interactions between the different treatments. Heritability estimates varied for different traits and compared favourably with those obtained under irrigated conditions and by other workers. Additive genetic variance was more important than non-additive genetic variance for some characters, but not for stalk population, cane mass, and dry matter % cane, for which both variances were important. Selection of parent cultivars for all sucrose-related traits, fibre % cane, and stalk diameter should be as successful under raingrown as under irrigated conditions, provided that the environmental variation is determined efficiently under raingrown conditions. Environmental correlations were observed between some traits, particularly between the yield related traits, and may have influenced heritability estimates for those traits determined by mid-parent offspring regression. Stalk diameter, fibre % cane, and brix % dry matter were the most repeatable traits between seasons. Cane mass was the least repeatable trait between Stages 1 and 2 but was highly repeatable between plant (-P) and ratoon (-R) crops of Stage 2. Stalk diameter was positively correlated with brix % dry matter (0.457-P and 0.623-R) and strongly negatively correlated with stalk population (-0.790-P and -0.711-R) and fibre % cane (-0.628-P and -0.651-R). Cane mass was strongly positively correlated with brix % dry matter (0.638-P and 0.679-R). By selecting for brix % dry matter and stalk diameter, indirect selection for cane mass would be possible. Brix % dry matter was determined as the most reliable trait on which to base parental and commercial cultivar selection because it was highly heritable, highly repeatable and highly positively correlated with stalk diameter and cane mass.
The transformation of South African soya bean cultivars with a synthetic Basta resistance gene.
(1995) Van Huyssteen, Tracy.; Wallis, Frederick Michael.
The development of a genetic engineering system for soya bean (Glycine max L.) is described in this thesis. Routine tissue culture regeneration systems were developed for South African cultivars of soya bean despite the recalcitrant nature of this plant to in vitro manipulation. Regeneration of shoots was obtained when cotyledons were excised from seeds germinated for two days and cultured on B5 BA 20 medium containing 2 mg/I BA. The important problems of in vitro shoot elongation and rooting were overcome by culturing cotyledons in the dark for four weeks to produce shoots with unusually long stems. This was followed by one week of culture under conditions of high light intensity to obtain healthy green shoots which could be rooted , either in sorbarods or on solid Y2MS 30 medium. The use of a mist bed for the hardening off of rooted soya bean regenerants was essential for the recovery of fertile soya bean plants. Molecular techniques for the cloning of foreign genes into binary vectors suitable for plant genetic engineering were also studied and are described in the thesis. The Basta herbicide resistance gene, pat, was successfully cloned into the binary vector pBI121 which contains the [beta]-glucuronidase (GUS) reporter gene, uidA. The new construct, pB1121/Ac, was conjugated into various disarmed Agrobacterium tumefaciens strains and these strains, along with other binary vector-containing strains, were used to transform soya bean plant material. Although a protocol for the routine transformation of soya bean was not developed, transgenic soya bean material resistant to kanamycin and showing GUS activity was obtained. Transformation of wound sites on cotyledons was obtained in several experiments and transgenic shoots were regenerated from inoculated cotyledons. Only the A. tumefaciens strain C58C1 (pGV2260)(pJIT119) was able to transform cotyledonary cells of soya bean and, therefore, only kanamycin resistant soya bean shoots were produced. Transgenic soya bean plants resistant to the herbicide Basta were not produced due to the recalcitrant nature of the crop to genetic engineering. Transformation of the non-recalcitrant plant, tobacco, which is a model system for plant genetic engineering was achieved. The binary pat gene containing vector constructed in th is study, as well as vectors obtained from AgrEvo, were tested. The transgenic Basta resistant tobacco plants obtained were used to optimize assay systems for the analysis of transformed plant material containing the pat gene. These assay systems included the use of the polymerase chain reaction as well as digoxigenin-Iabelling of a DNA probe suitable for detection of the pat gene.
Isolation of an acetochlor detoxifying bacterium and cloning of an associated gene.
(1995) Martin, Darren Patrick.; Cress, William A.
A Pseudomonas strain, AI08, which was capable of detoxifying the herbicide acetochlor (2- chloro-N-ethoxymethyl-6'-ethylacet-o-toluide) was isolated from soils. The microbe was isolated using a combination of batch culture enrichment techniques, phenotypic agar plate based assays and a qualitative bioassay for detecting acetochlor detoxification. With the aid of a bioassay developed specifically for the quantification of acetochlor concentrations, it was determined that over a 21 day period Al 08 was capable of detoxifying 20 % of the acetochlor present in a medium containing no other organic carbon and 53 % of the herbicide in a medium containing glucose and yeast extract at concentrations of 0.02 g.l-l and 0.005 g.l-l respectively. A fragment of A108 DNA was cloned in Escherichia coli which produced recombinant cells with both elevated acetochlor resistance and the ability to detoxify 15 % of the acetochlor present in a minimal nutrient medium (containing 0.02 g.l-l glucose and 0.005 g.l-l yeast extract) over a 21 day period. Partial sequencing of the cloned A108 DNA revealed that it encoded an amino acid sequence with significant homology with the dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complexes of Azotobacter vinlandii, E. coli and Alcaligenes eutrophus. Theories are proposed as to the possible biochemical mechanisms whereby expression of the dihydrolipoyltransacetylase gene of Al 08 in recombinant E. coli cells may function in the detoxification of acetochlor.
Direct transformation of maize (Zea mays L.) tissue using electroporation and particle bombardment, and regeneration of plantlets.
(1996) Jenkins, Megan Joy.; Shanahan, Paul Edward.
Please open electronic version for Abstract.
The inhibition of Fusarium oxysporum f.sp. cubense race 4 by Burkholderia cepacia.
(1997) Pan, Manjing.; Hastings, John W.
Inhibition of Fusarium oxysporum f. sp. cubense race 4 by Burkholderia cepacia was evident when grown on various media (TSA, PDA, PSA, YM, KMB, PPM, NYGA, LA) with different carbon sources and under various pH and temperature conditions. In addition, B. cepacia was able to inhibit several fungal pathogens in vitro. Antagonism of B. cepacia against F. oxysporum f. sp. cubense occured at high levels of Fe³+, which may suggest that antagonism by B. cepacia did not involve siderophore production. Thin layer chromatogram (TLC) examination showed that B. cepacia produced several substances, one of which had similar R[f] value to that described for pyrrolnitrin. Cell-free supernatant of a 4-day culture of 6. cepacia was applied to an Amberlite XAD-2 column and inhibitory activity co-eluted with the 95% methanol (pH 9.5) fraction. The concentrated activated fractions showed inhibitory activity against F. oxysporum f. sp. cubense. A GC-MS chromatogram indicated numerous components in the antifungal extracts. The only compound identified in the Wiley 138 library, was 1,2- Benzenedicarboxylic acid, bis (2-Ethylhexyl) ester. Observations by light microscopy indicated that B. cepacia inhibited spore germination in F. oxysporum f. sp. cubense race 4 and retarded the mycelial growth. The interaction between the endophytic bacterium, B. cepacia and F. oxysporum f. sp. cubense race 4 was investigated with aid of scanning and transmission electron microscopy. This demonstrated that the bacterium was able to colonize the surface of hypha and macrospore of F. oxysporum f. sp. cubense. Mycelial deformation, terminal and/or intercalary swelling were evident. At later stages, hyphae of F. oxysporum f.sp. cubense, colonized by B. cepacia, were found to have collapsed. Further studies in vivo confirmed that B. cepacia colonized the hypha of F. oxysporum f. sp. cubense which had invaded banana roots. TEM observation showed that in the banana plant B. cepacia was closely associated with the healthy banana roots and a matrix was frequently found to be present between the bacterium and the plant surface. In addition, B. cepacia exists mainly in the intercellular space of the banana roots. UV irradiation treatment of B. cepacia resulted in a mutant that had lost inhibitory activity against F. oxysporum f. sp. cubense on TSA agar. Transposon mutagenesis of B. cepacia was performed by Tn5 insertion. Six mutants which had lost or had reduced inhibitory activity against F. oxysporum f. sp. cubense were generated. These mutants showed no inhibitory zones on TSA medium in the presence of the fungus. It was observed that one mutants. cepacia :: Tn5-188 appeared to lose the ability to colonize the fungal hypha, whilst a different mutant B. cepacia ::Tn5 - 217 was still able to colonize the fungal hyphae. TLC analyses showed that there was a decrease in antibiotic production in mutants B. cepacia :: Tn5 - 217 and B. cepacia - UV - 34, compared with the wild type. GC- MS analyses showed that there was no evidence of the peaks at 14.62 minutes, 20.0 minutes and 20.46 minutes in both chromatograms of mutants B. cepacia :: Tn5 -217 and 8. cepacia -UV - 34, compared with the wild type B. cepacia. No PCR products were detected using primers that were developed from sequences within the biosynthetic loci for Phi of P.fluorescens Q2-87(GenBank accession no. U41818) and PCA of P. fluorescens 2-79 (GeneBank no. L48616). Colony hybridization suggested that genomic DNA from B. cepacia could contain both Phi- and PCA probes. It was found that hybridization of genomic DNA digested with Cla-I of B. cepaca with Phl2a probe only occurred at low stringency. A hybridization signal was detected from a Cla-l fragment of approximately 2800bp.
Optimisation of the randomly amplified polymorphic DNA (RAPD) technique for the characterisation of selected South African maize (Zea mays L.) breeding material.
(2000) Edwards, Nicola Rachel.; Shanahan, Paul Edward.
Maize (Zea mays L.) is an important agronomic crop with the maize industry forming an important component of the South African economy. Considerable effort has been directed towards the genetic improvement of maize through both conventional breeding and biotechnology. Genotype identification by DNA fingerprinting is becoming an important activity in plant breeding. A widely used molecular based and relatively inexpensive method for DNA fingerprinting is the randomly amplified polymorphic DNA (RAPD) technique. The RAPD technique was tested in this study for its potential use in maize breeding programmes. Initial results using the technique showed a low degree of reproducibility, therefore both the DNA isolation and RAPD protocols were extensively optimised. DNA quality and quantity, and choice of Taq polymerase buffer were three of the variables found to be influential in ensuring reproducibility. The ability of the RAPD technique to characterise seven maize genotypes was evaluated. Sixty random oligonucleotide primers were screened. Forty two primers scored a total of 233 fragments (an average of 5.5 per primer), but not all primers gave reproducible profiles. Eighteen primers scored a total of 110 loci for the presence (1) and absence (0) of DNA fragments. RAPD markers were able to distinguish between all seven genotypes with five primers producing specific fragments for four genotypes. Genetic similarity matrices were calculated using two software programmes i.e. Genstat 5™ release 4.1 (1993) and PAUP (Phylogenetic Analysis Using Parsimony) 4.0 beta version (Swafford, 1998). Cluster analysis was used to generate dendrograms to visualise the genetic relationships of the seven maize genotypes (only minor differences were observed between the Genstat or PAUP method of analysis). Genetic diversity ranged from 0.62 to 0.96. The estimation of genetic relationship was in accordance with the presumed pedigree of the genotypes showing that the RAPD technique demonstrates potential for genome analysis of maize. The applicability of the technique for marker assisted selection was also evaluated. Near-isogenic lines (NILs) for leaf blight (Helminthosporium spp.) were screened for polymorphisms using a total of 120 primers. Ten primers identified polymorphisms between the NILs. Four primers produced five polymorphic fragments present in the resistant inbred K0315Y and absent in the susceptible inbred D0940Y. A small F2 population of 14 individuals was produced by selfing the F1 of a cross between K0315Y and D0940Y. To speed up the generation time, the F1 and F2 plants were cultured by embryo rescue from 18d old harvested seed. One fragment of 627 base pairs produced by primer OPB-01 (5' GTTTCGCTCC 3') showed a 3: 1 segregation in the small F2 population and was considered putatively linked to the HtN gene for leaf blight resistance. This study shows that the RAPD technique does have application in maize breeding programmes.
Phylogenetic and evolutionary analysis of the Borna disease virus.
(2002) Blank, Elena.; Fossey, Annabel.
The characteristic trait of the Borna disease virus is that it is a complicated single negative stranded RNA virus that is capable of infecting a wide array of mammalian species including human beings. It has been implicated in a diverse variety of human neuropsychiatric diseases. The infection capability, mechanism of infection and range of protein action of this virus remain to be identified. The purpose of the present study was to determine (1) whether the previous Bornaviridae family classification is indeed accurate as the action of BDV indicates that it is related to other viruses and (2) to estimate the number of synonymous (nucleotide substitution) and non-synonymous (amino acid change) evolutionary mutation rates of proteins (nucleoprotein, phosphoprotein, glycoprotein, matrix protein) exhibited by various Borna disease virus host species and the proteins (nucleoprotein, phosphoprotein, glycoprotein, matrix and X protein) of three Borna disease virus strains. The latter study would give an indication as to which proteins are subjected to positive selection. Phylogenetic methods were used to determine the accuracy of the Bornaviridae classification. Phylogenetic trees obtained through an alignment and analysis of the polymerase protein, which displays a uniquely conserved GDN motif, of various RNA negative single stranded viruses using neighbourhood and parsimony methods enabled comparison with other RNA virus families. A method adapted from Ina, (1995) for estimating the synonymous and non-synonymous evolutionary mutation rate was applied to various BDV proteins in order to provide more information on inter (host virus) and intra (virus) mutation rate. This information in turn was used to create an evolutionary model to clarify the positive and neutral evolutionary trend of the inter- and intra-virus proteins examined, which may help clarify and enhance the lack of current knowledge relating to species infection and the epidemiological nature of the virus. The results obtained by the polymerase alignment analysis indicates the presence of two newly discovered BDV motifs, v and vi, confirmed by three diverse alignment programmes. An analysis of the alignment of BDV proteins indicated that the BDV nucleoprotein nuclear localization signal aligns the BDV nucleoprotein between motifs IV and vi of the BDV polymerase. The results obtained by the phylogenetic analysis indicate that the Rabies virus and the Vesicular stomatitis virus are the most closely related animal viruses to BDV, whereas the Rice transitory yellowing (unclassified Rhabdovirus) and Sonchus yellow net plant virus are closet to BDV than other animal Rhabdoviridae raising intriguing questions on the evolutionary origins of the Borna disease virus. The phylogenetic analysis indicates that the Borna disease virus does not fall into a separate Bornaviridae family classification, and suggests that BDV may be more appropriately placed into a separate subfamily in the family Rhabdoviridae. The results of the evolutionary analysis indicate considerable diversity between BDV host virus (inter-species) and BDV virus (intra-species) protein sequences. In the host virus sequence comparison analysis all of the proteins examined displayed a high pattern of non random evolution, which is in contrast to the intra species comparison in which only three proteins; the BDV glycoprotein, nucleoprotein and X protein; displayed a non random pattern of evolution. The positive selection effect displayed by the inter-species (host) proteins may be attributed to antigenic variation displayed by the inter-species sequences and a super infection hypothesis, which indicates that positive selection on host variants could arise during the course of an infection as a result of specific immune responses. The positive and neutral selection trend of the proteins displayed by the intra-species (virus) sequences may be a result of a pattern of nucleotide substitution that is physio-chemically conservative. Conservation may be evident in volume, polarity, hydrophilicity, or molecular weight of amino acids of the proteins.
Near infrared analysis of sugarcane (Saccharum spp hybrid) bud scales to predict resistance to Eldana stalk borer (Eldana saccharina Walker).
(2003) Coetzee, N. A.; Shanahan, Paul Edward.; Greenfield, Peter L.
The eldana stalk borer (Eldana saccharina Walker) is the most serious pest of the Southern African sugarcane industry, and it is imperative that effective control measures are available to minimize economic damage. Because conventional control methods have had limited success, cultivar resistance is seen as the most viable method of controlling infestation. However, due to the space- and time-consuming nature of the present screening methods, only small numbers of cultivars can be tested relatively late in the Plant Breeding selection programme. Increased resistance in breeding and selection populations is therefore slow. Buds are a preferred entry point of eldana larvae as they are softer than the rind that is present on the rest of the stalk surface. Preliminary results by other workers suggested that near infrared spectroscopy (NIRS) could provide a rapid screening method for the chemical profile in bud scales, the outer coating of buds and therefore the first contact point of an invading larva. If feasible, analysis of samples using this method could be done in the South African Sugar Experiment Station's (SASEX) stage two selection trials, providing an early indication of eldana resistance on large numbers of cultivars, without the necessity of separate trials. However, knowledge of how environments, position of bud scales on the stalk and age affect NIRS is required in order to determine the feasibility of the method. Planting of a trial with an identical set of genotypes across a range of environments, sampled at a number of ages, would provide the necessary information on environmental effects, whilst simultaneously providing the necessary range of samples to develop a calibration between bud scale chemical profiles and eldana resistance ratings. Inheritance patterns of the characteristics being measured is also required if they are to be used in a breeding programme. The original work by Rutherford (1993) was carried out on only five calibration sets (a set of standard clones with relatively well-known eldana resistance ratings), and different sets were not comparable due to what was assumed to be environmental differences between calibration sets. One aspect of the current experiment was to examine more closely the effect of genotype x environment interaction (G x E) on the performance of the NIRS technique under a range of conditions. Two sites were chosen to represent the conditions encountered in trials carried out by SASEX. The crops were sampled at three ages, representing the range of ages at which sugarcane is harvested in South Africa. Two locations on the stalk were also examined, top and bottom, for removal of bud scales, based on the assumption that aging of bud scales may affect chemical composition. A new NIRSystems 6500 instrument was acquired during the course of this study. Data from the new instrument indicated that there were no longer differences between the different calibration sets, and therefore no longer differences between environments. Spectra for different samples were very close, the differences being of the same scale as those recorded with repeated measures of the same samples, or between the readings for the standard solvent solution. This led to the conclusion that the differences observed on the original NIRSystems 5000 instrument were due to instrument error, not environmental differences. More importantly, the different calibration sets were not comparable despite being similar to each other. Prediction from one calibration set to another was low. These observations led to the conclusion that NIRS was not a suitable method for determining chemical compounds associated with tolerance of sugarcane genotypes to eldana borer. The original NIRS instrument was subject to error, and the small number of calibration sets included in the study led to the erroneous conclusion that NIRS was suitable for the prediction of varietal tolerance to eldana. With the acquisition of the new instrument, the errors generated by the old instrument became apparent. With the increase in number of calibration sets included in the study, it also became apparent that a global calibration covering all environments was not possible. An analysis of the heritability of the chemical compounds associated with eldana resistance was also included in this study. A biparental progeny design of 24 crosses with 33 unselected offspring per cross was used. This trial would have been analysed once the calibration had been developed using the environmental trial, and it would have provided knowledge of the breeding behaviour of the chemical compounds associated with tolerance to eldana. Because the NIRS technique proved to be unsuitable for detection of chemical compounds associated with eldana resistance, the heritability of these chemical compounds could not be studied. As the NIRS study did not produce data, the G x E interaction analysis and determination of heritability was applied to the bud scale mass data set. This study showed a relatively low positive correlation between bud scale mass and resistance to eldana. The broad sense heritability estimate for bud scale mass from the G x E interaction analysis was 0.45, and the narrow sense heritability estimate from parent-offspring regression analysis was approximately 0.27, suggesting a low degree of genetic determination in bud scale mass. The G x E interaction analyses gave varying results depending on the method used. The ANOVA analysis suggested that ages, sites and years had an effect on bud scale mass, while deviation from maximum plot showed no significance for G x E interactions. The number and choice of genotypes selected as unstable also varied with the method used to determine the stability of individual genotypes. Regression analysis and rank order analysis revealed a number of unstable genotypes, whilst stability variance and ecovalence, which produced similar results, detected only two unstable genotypes. In the rank order analysis correction of data to remove genotype effect, reduced the number of unstable genotypes, suggesting that the G x E interaction effect was partially confounded with the bud scale mass of the genotypes. This was a more reliable method than the uncorrected rank order analysis, and would be the preferred analysis type of all those tried.
Cloning, expression and purification of the immunity factor associated with leucocin A production.
(2004) Pillay, Kovashni.; Beukes, Mervyn.
Leucocin A is a bacteriocin produced by Leucoconostoc gelidium UAL 187-22. Bacteriocin producer strains possess an immunity protein, which enables the strain to protect itself against its own bacteriocin. The immunity gene from Leucoconostoc gelidium was isolated via PCR from a recombinant clone pJF5.5. This fragment was cloned by amplifying the immunity gene from pJF5.5 and ligating it into pMALc2. The resulting recombinant plasmid pKP1 was then transformed into Escherichia coli strain JM103. The clone putative, was confirmed by DNA sequencing and southern blot hybridization using the primers EAL-2 and EAL-3. It was shown to contain an insert of 3.6 kb. Expression analysis showed the construct as an in frame malE fusion protein expressed within E. coli. The fusion construct was isolated by affinity chromatography. Leucocin A was purified to 90% purity, from the supernatant of Leucocnostoc gelidium UAL 187-22 by ion-exchange chromatography and HPLC. It was found to elute from a C18 reverse phase column at 55% actetonitrile, 0.1% TFA. Binding interaction and the stability of the immunity gene fusion protein were compared using a Biacore 2000. The supernatant and cytoplasmic extract isolated from Leucocnostoc gelidium UAL 187-22 were tested for interaction with the fusion construct. Surface Plasmon resonance studies indicated that there was no binding partner present in the supernatant which would influence the immunity process. However, a stable interaction was found between the immunity protein and an orphan ligand within the cytoplasm.
Estimation of genetic variation and marker identification in black wattle (Acacia mearnsii De Wild) with RAPD fingerprinting.
(2004) Sewpersad, Yaksha.; Fossey, Annabel.; Dunlop, Robert William.
No abstract available.
Characterization of streptococcal infections in KwaZulu-Natal Durban by random amplified polymorphic DNA anaylsis and DNA macrorestriction analysis.
(2004) Madlala, Paradise Zamokuhle.; Beukes, Mervyn.
A collection of 29 clinical streptococcal isolates obtained from the University of KwaZulu-Natal, Medical School, Durban Metro area (South Africa) were studied to establish their penicillin G susceptibility patterns often refered to as minimal inhibitory concentration (MIC) and to determine the genetic diversity among them using two genotyping methods, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) analysis. All isolates with MIC less than or equal to 0.12 µg/ml were considered susceptible, intermediate resistant if MIC was between 0.25 µg/ml and 4 µg/ml and resistant if greater than 4 µg/ml, The percentage of isolates with resistance was relatively high (75.9%), only 10.3% of isolates showed intermediate resistance and 13.8% of the isolates were completely susceptible to penicillin G. Some of the resistant isolates were highly resistant reaching penicillin G MIC levels of 5000 µ/ml. They were speculated to contain Path altered penicillin binding proteins and high level of crosslinking cell wall induced by the gene products of the MurMN operon. RAPD analysis was performed using three primers, MBPZ-1, MBPZ-2, and MBPZ-3, respectively. RAPD analysis allowed for the identification of 27 RAPD types with MBPZ-1 and MBPZ-3 and 26 RAPD types with MBPZ-2. Ninety-eight percent of these isolates were clustered into two groups, group I and group II, with 90% to 100% dissimilarity among them. Fifty two percent of the isolates of MBPZ-1 group I were in MBPZ-2 group I, 72% isolates of MBPZ-1 group I were in MBPZ-3 group I, and 72% of the isolates of MBPZ-2 group I were in MBPZ-3 group 1. This shows the discriminatory ability of the primers used in this study. Despite clustering of isolates, relatively high diversity was seen. PFGE analysis of macrorestriction fragments obtained after digestion of chromosomal DNA by restriction enzyme, SmaI showed 24 PFGE patterns. The 24 PFGE patterns were divided into three groups (I, II and III) of isolates, with an average of 85% dissimilarity (15% homology) among them. At 25% homology, four clusters, A (13 isolates), B (9 isolates), C (4 isolates), and D (4 isolates) were observed. Two pairs of isolates in group I, cluster A, showed 100% homology. This suggested that each represent the same strain. Four isolates of group I, cluster B, also exhibited 100% homology. This study showed that most of streptococcal isolates with the same penicillin G susceptibility patterns grouped together in a phylogenetic tree by both RAPD and PFGE analysis. There was also some similarity between the results obtained by RAPD analysis and PFGE analysis. Seventeen and nine of the 29 isolates grouped into group I and group II, respectively, two pairs of isolates were indistinguishable, and two pairs of islates were closely related by both RAPD (using MBPZ-3) and PFGE analysis. Although, RAPD analysis is sensitive, specific, faster and cost effective, the ease with which PFGE analysis can be performed, high discriminatory power, reproducibility of the results, and the polymorphism seen in the patterns, suggests that PFGE method has the potential to be very useful for epidemiological evaluations of nosocomial streptococcal infections in KwaZulu-Natal.
Individual identification and parentage analysis of Struthio camelus (ostrich) using microsatellite markers.
(2005) Essa, Fatima.; Fossey, Annabel.; Cloete, Schalk Willem P.
Ostrich (Struthio camelus) breeding is a well-developed industry in South Africa. However, successful genetic management has yet to be implemented. Parentage in colony breeding ostriches is unknown, where for a given offspring, a number of possible parents exist. Molecular markers have been extensively used in the livestock industry to resolve parentage issues and are only beginning to be utilized to address the issues of the ostrich industry. The aims of this investigation were to test known microsatellite markers developed for other ostrich subspecies in a South African Black ostrich population, and to further test these markers for their use in individual and parentage identification. DNA was extracted from venous blood obtained from two pair bred families and a colony of 97 individuals. Eleven polymorphic microsatellite markers were tested by PCR amplification of DNA samples followed by multiplexing on polyacrylamide gels to generate DNA fingerprints for each individual. Alleles were sized and quantified and used to create genotypes for each individual. Parentage analysis was performed using exclusion and likelihood methods. Pedigrees were constructed for the families by comparison of genotypes. Breeding statistics were calculated for the colony individuals. Three microsatellite markers did not amplify in this population and one marker was found to be monomorphic in this population. Four of the microsatellite markers that successfully amplified produced anonymous amplification products suggesting a second annealing site in the genome sequence of Blacks. All loci displayed low observed heterozygosities indicative of little genetic variation in this population. For the colony sample, four individuals were not assigned either parent and one female did not contribute any offspring. On average females produced 4.86 ± 2.71 fertile eggs during the sampling period with a coefficient of variation of 55.86%. A total of 79.2% of individuals were assigned paternity and 88.3% were assigned maternity. A greater number of loci are required to improve the power of parentage analysis within breeding flocks incorporating all eggs laid.
Fingerprinting of full and half-sib black wattle (Acacia mearnsii) progenies using Random Amplified Polymorphic DNA (RAPD).
(2005) Naguran, Riann.; Fossey, Annabel.
Black wattle (Acacia mearnsii), which belongs to the genus Acacia, is one of the many species of trees or hardwoods grown commercially in South Africa. Black wattle is a species indigenous to Australia and was introduced into South Africa by the van der Plank brothers in 1864. These trees are grown in South Africa because of its tannin-rich bark, the extract of which is used by the leather tanning industry. Black wattle is also grown for its timber, timber products and pulp. The introduction and cultivation history of black wattle suggests that the South African plantations contain limited genetic variation with relatedness amongst groups estimated to be high, thus implying a narrow genetic base in the South African black wattle population. In this investigation, Random Amplified Polymorphic DNA (RAPD) was used to estimate the genetic variation between seven different black wattle groups. A total number of 34 individuals obtained from different areas in South Africa were examined; Piet Retief (group 47 and 50: half-sibs), Kumbula (group 85: unrelated individuals), Howick (group 400: unrelated individuals) and an unknown area (groups 88, 89, 91: full-sibs). As this investigation was the first of its kind, a DNA isolation method as well as a PCR-RAPD protocol had to be modified. Total genomic DNA was successfully extracted using the CTAB DNA extraction method. This method removed large amounts of tannin present in the cells of the black wattle leaves and extracted high quality DNA to conduct between 50-100 RAPD reactions. The DNA purities ranged from 0.1 to 1.8, with an average of 1.46. A total of fourteen 10-mer RAPD primer sequences were randomly selected from the Operon Technologies primer list A, and tested in this investigation. Of the 14 primers used, only nine primers produced clear, single and repeatable bands. Therefore nine primers were selected for subsequent analyses. Ninety one loci that generated bands ranging from 300-3050 base pairs were produced. Seven to 13 loci per primer were generated. A total of 95.6 % of the loci were polymorphic. The overall expected mean heterozygosity (H = 0.3) obtained in this study was high in comparison to other studies conducted on acacias. The high levels of genetic variation were attributed to mating systems, dissortative mating and geographic distribution. The statistical packages POPGENE and ARLEQUIN were used to analyse the RAPD fingerprints. The genetic measures, Nei's diversity and Shannon's Information Index, showed that there was greater diversity exhibited (Nei's gene diversity = 32.09 % and Shannon's = 48.31 %), in the whole population than in each of the groups (with average of Nei's gene diversity = 20.33 % and Shannon's = 34.64 %). With regards to individual group analyses, low levels of genetic variation was obtained in group 400 (unrelated), from the Howick region, and group 85 (unrelated), from the Kumbula region, (mean 0.14 and 0.17 respectively). The low genetic values were attributed to limited gene exchange occurring in these two areas, bottlenecks and selection pressures. Groups 88, 89 and 91, from the unknown region (full-sib groups), were the most variable in comparison to the other groups, with means of (0.27,0.24 and 0.18 respectively). These high genetic variation values could be due to the fact that gene migration could have occurred between these groups and others in the area. It is thought that most acacias are insect-pollinated and this could have lead to gene migration between groups or populations, thereby explaining the high mean values. The gene flow obtained for the seven groups (FST = 0.174) indicated that great genetic differentiation existed in this population of black wattle studied. This value is higher in comparison to other woody species; however it is similar to other acacia species. UPGMA cluster analysis using Nei's unbiased genetic distance, revealed four distinct clusters of groups corresponding to the distribution areas represented in this study. The Howick (group 400: unrelated) and Kumbula (group 85: unrelated) were more closely related to each other than to the other groups, since both these groups are from Natal. The Piet Retief groups (groups 47 and 50: half-sibs), branched-off together, indicating that they are distinct from the other groups. The pairwise analysis of identity showed that the relationship between the group from Howick (group 400: unrelated) and all the other groups from the other regions was the lowest, ranging from 64 % to 79 %. The relationship between all the groups beside the group from Howick (group 400: unrelated) was reasonably high, ranging from 78 % to 90 %. This distance displayed by group 400 (unrelated) from Howick in relation to the groups, is attributed to the fact that it is frost resistant and the other groups not. Genetic variation was also detected and partitioned, between and within groups, by Analysis of Molecular Variance (AMQVA). Majority of the variation existed within groups (82.65 %) but significant differentiation was recorded between groups (17.44 %). This high level of within group differentiation may be explained by many aspects, such as the species breeding system, genetic drift or genetic isolation of groups or populations. The application of RAPD fingerprinting in black wattle has provided a more in depth understanding of the genetic variation residing in the South African population. The results achieved implementing this technique has shown that significant genetic variation exists within the black wattle population in South Africa. The results obtained in this study are also important since it is contrary to the expectation that the black wattle population in South Africa has low genetic variation. This knowledge is of great value to genetically discriminate between individuals or groups, to improve the selection of superior genotypes and allowing improved quality control in breeding programmes and seed orchard management.
The elucidation of the possible mechanism of vancomycin-resistance in selected streptococcal and enterococcal species.
(2005) Desai, Rizwana.; Beukes, Mervyn.
Three Streptococcal strains: S. milleri P213, S. milleri P35 and S. milleri B200 and three enterococcal strains: E. faecalis 123, E. faecalis 126 and E. faecium were used to test for vancomycin resistance. Two strains were used as reference strains that were already characterized as vancomycin resistant. E. faecium BM4147 was used as a VanA control and E. faecalis ATCC was used as a VanB control. Susceptibility of each strain to this antibiotic was tested by disk-diffusion assay and the MIC values for the strains were found to be between 5 - 10 ug/ml and for the VanA control, the MIC was > 64 ug/ml and for the VanB control was 32 ug/ml. These MIC values indicate that S. milleri P213, S. milleri P35, S. milleri B200, E. faecalis 123, E. faecalis 126, and E. faecium are all of the VanC phenotype. All strains were tested for lysis by means of addition of vancomycin (10 ug/ml) to the bacterial cultures. Lytic curves were constructed and the VanB control was found to be most autolytic upon addition of vancomycin and E. faecalis 123 was the least autolytic. However, under normal conditions in phosphate buffer, lytic curves showed that S. milleri P213 was the most autolytic and the VanA control, the least autolytic. PCR assays were performed to detect specific antibiotic resistant genes. Primers were selected from Dukta-Malen et al., 1995. The VanA primer yielded amplification of 732 bp for only the VanA control DNA and the VanB primer set yielded products for the VanB control DNA. S. milleri P213, P35, B200 and E. faecalis 123 and 126, and E. faecium DNA were amplified with the VanC primers. This supports the results obtained in MIC that these strains are possibly VanC resistant strains. Amplified VanA control and that of E. faecalis 126 were thereafter sequenced. VanA control amplicon was correctly amplified since it showed homology to E. faecium BM4147 as well as the VanB amplicons which was found to be homologous to the transposon Tn1549 found on the well-characterized E. faecalis strain which is known to harbour the VanB vancomycin-resistant genes. Whilst E. faecalis 126 which represented the VanC phenotype showed 96% homology to E. gallinarum BM4147 which is a well-characterized glycopeptide-resistant enterococci belonging to the VanC phenotype. Southern blots were performed using specific primers as a probe to verify whether the gene sequences for the specific genotype were present in these strains and results confirmed those found in the PCR assays and in DNA sequencing. The peptidoglycan precursors of each strain were arrested in vancomycin (20 ug/ml) to block transpeptidation and transglycosylation steps of peptidoglycan synthesis and bacitracin (100 ug/ml) was used to amplify precursors at the transglycosylation step. Precursors were extracted and analysed by reverse-phase HPLC. UDP-MurNAc-tetrapeptides cell wall precursors, which are found abundantly in vancomycin-resistant strains, were found in large proportions in all strains, except in E. faecalis 123 when arrested with vancomycin. This precursor has a noticeably decreased affinity for vancomycin, hence contributing to its resistance. The precursor accumulated when arrested with bacitracin, was, UDPMurNAc-tetrapeptide in all strains except in E. faecalis 126. UDP-MurNAc-pentapeptides were also found in moderate amounts in most strains. The molecular masses of the peptidoglycan precursors obtained from mass spectrometry correctly identified them. This confirmed that the bacterial strains investigated were in fact resistant to the antibiotic vancomycin and this study shows that results obtained from conventional phenotypical screening methods reliably correlated with the genotypes classified using more advanced techniques such as PCR, southern blot/hybridisation and DNA sequencing.
Investigation of leptin genotypes and economically important dairy traits in jersey cows.
(2005) Todd, Caryn Jayne.; Fossey, Annabel.
Dairy farming is one of the most important agricultural industries in South Africa, and thus improving the performance of dairy cows, with respect to economically important dairy traits, would be beneficial. Selection of dairy cows has traditionally been phenotypic, but new molecular techniques have made it possible to evaluate phenotypic dairy traits at the DNA level, providing the possibility of more accurate selection. The economically important dairy traits, milk production and reproductive performance, are quantitative traits, and are therefore controlled by many genes and the environment. A number of genes have been identified that have been shown to influence economically important dairy traits, including the lep gene. This gene encodes the hormone leptin, which has been proven to regulate feed intake, energy balance, fertility and immune function. A polymorphism has been identified in the lep gene, which may be associated with economically important dairy traits. This study on a South African Jersey herd investigated the possible association of the polymorphism, RFLP-Kpn 21, with milk production and reproductive performance. The lactation records of fifty Jersey cows that completed their first lactation between 1997 and 2004 were collected, and these cows were genotyped for the RFLP-Kpn 21 polymorphism, located at exon 2 of the lep gene. This involved the extraction of DNA from venous blood, using a salting out technique. The extracted DNA was amplified using PCR primers; the reverse primer included a purposeful mismatch. The role of the purposeful mismatch was to create a recognition site for a restriction enzyme (Kpn 21), thus allowing the alleles of the polymorphism to be identified through a restriction digestion protocol. Two alleles were identified, the C- and the Tallele. The genotype of each cow was identified using PAGE. The significance of the genotype effects on the milk production traits and the reproductive performance traits were estimated using the F-statistic provided by a GLM Univariate analysis. In conclusion, no significant effect of the RFLP-Kpn 21 polymorphism was found for milk yield, butterfat and protein percentage, ICP and SPC (p > 0.05), but a possible association with lactose percentage was suggested by the statistical analysis (p < 0.05). Further investigation of South African Jersey cows will be necessary in order for conclusive results to be obtained.
Characterization of the autolytic systems in selected streptococcal species.
(2005) Naidoo, Kershney.; Beukes, Mervyn.
Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified.
Characterisation of antibiotic resistance in Streptococcus, Enterococcus and Staphylococcus using a bioinformatics approach.
(2005) Ramsuran, Veron.; Beukes, Mervyn.
The rate at which bacterial pathogens are becoming resistant to antibiotics is quite alarming, and therefore much attention has been focussed on this area. The mechanism whereby the bacterial cells acquire resistance is studied in order to determine how this process works as well as to determine if any future resistance mechanisms can be circumvented. In this study three different genera and the antibiotics that are resistant to them were used, namely, penicillin resistant Streptococcus, vancomycin resistant Enterococcus and methicillin resistant Staphylococcus. The results prove that the active sites SXXK, SXN and KT(S) G in the penicillin resistance Streptococcus plays a major role in resistance. It is seen in this study that the SXXK active site is found in all the resistant and most of the intermediate strains, therefore proving to be an important component of the cell wall resistance. It was subsequently noticed the greater the number of mutations found in the sequences the higher the resistance. Three dimensional structures showed the actives sites and their binding pockets. The results also show the change in conformation with a mutation in the active site. The results also proved that the Penicillin Binding Protein (PBP) genes essential for resistance are PBP Ia, PBP 2b and PBP 2x. The results obtained, for the vancomycin resistance in Enterococcus study, proved that the VanC and VanE cluster are very much alike and VanE could have evolved from VanC. There is also close similarity between the different ligase genes. The VanX 3D structure shows the position of the critical amino acids responsible for the breakdown of the D-Ala-D-Ala precursors, and the VanA ligase 3D structure shows the amino acids responsible the ligation of the D-Ala-D-Lac precursors. The analysis performed on the methicillin resistance in Staphylococcus study showed that the genes used to confer resistance are very similar between different strains as well as different species.

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