No abstract available. / Thesis (Ph.D.)-University of KwaZulu-Natal, Piertermaritzburg, 2010.

The genus Enterococcus comprises of a group of commensal organisms of the human gut which has been associated with cases of endocarditis and urinary tract infections. In the present study, 12 Enterococcus isolates were obtained from clinical specimens and characterized using genotyping techniques that have become an integral part of clinical research. There were three different genotyping methods used to identify the enterococci to species level and to determine the level of genetic diversity among the selected strains. These techniques were, randomly amplified polymorphic DNA-PCR (RAPD-PCR), 16S rDNA ribotyping analysis and pulse field gel electrophoresis (PFGE) respectively. The minimum inhibitory concentration (MIC) to penicillin and vancomycin were also determined using a disc diffusion assay and a microtitre plate dilution assay. All twelve strains were found to be vancomycin resistant enterococci (VRE) at a MIC value greater than 100μg/ml. Penicillin growth inhibition based on MIC values were categorized into three groups, susceptible (< 0.25 μg/ml), intermediate (≤ 3μg/ml) and resistant (≥ 4μg/ml) respectively. RAPD-PCR was performed using four random primers. Primers yielding the highest discriminative power were used for phylogenetic analysis. The phylogenetic analysis indicated that all 12 strains yielded clonal dissemination, therefore a low genetic diversity between them. The 16S rDNA of all strains were used to identify the enterococci at species level. The rDNA were sequenced and analysed using the NCBI BLAST algorithm and found to belong to three species of Enterococcus. These were E.faecalis, E.faecium and E.durans. PFGE analysis was performed by restriction of all 12 strain’s genomic DNA with the restriction enzyme SmaI. The PFGE patterns were divided into two groups with low genetic diversity. Compared with the RAPD PCR patterns PFGE gives a higher discriminatory power as a higher dissimilarity between the strains was observed. Similar penicillin MICs for each of the strains in the three categories are grouped together in the phylogenetic trees for both PFGE and RAPD-PCR. RAPDPCR is a sensitive, faster, specific and cost effective technique, PFGE analysis has given a higher discriminatory power, higher reproducibility of the results and the polymorphism seen in the patterns suggest that PFGE has a potential of being an essential tool in clinical diagnostics. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / The Russian wheat aphid, Diuraphis noxia (Mordvilko), is a serious insect pest of wheat and barley. It affects the quality and yield of grain by sucking plant sap from the newest growth whilst toxic substances are injected that destroy plant tissue. The Russian wheat aphid also acts as a vector of plant viruses. The cultivation of aphid resistant cultivars is the preferred control strategy and nine resistance genes, designated Dn1 to Dn9, have been identified. Another undesignated gene, Dnx, was found in the wheat accession PI220127. Mapping of the resistance genes relative to known markers will improve their use in breeding programs. The dominant RWA resistance gene, Dn5, was identified in the accession PI294994 and mapped to chromosome arm 7DL. However, recent reports have placed Dn5 on ...

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The indigenous abalone, Haliotis midae has been a successfully cultured aquaculture species in South Africa since 1990. It has a slow growth rate and takes from two to five years to reach market size. Like for most other commercially important abalone species, the slow growth rate of H. midae is a cause of concern with regard to the profitability of farming and global competitiveness of the species. Ploidy manipulation of the maternal genome, a universally growing practice in shellfish culture, is considered a promising method to improve the growth rate of abalone - a desirable trait in aquaculture organisms from a commercial perspective. This manipulation technique is employed to achieve sterility, which results in limited gonad development. The consequent re-allocation of resources to somatic growth results in improved growth. The purpose of this study was to establish a viable method for the induction and validation of triploidy, on a commercial scale, in the South African abalone, H. midae. The focus was on hydrostatic pressure as a method of induction and flow cytometry as the method of validation. The results obtained confirm hydrostatic pressure as an effective method for the induction of triploidy in H. midae, delivering high percentages of triploidy (>80%) over a wide range of pressures and times, in 48 hour-old larvae. Hydrostatic pressure had a negative effect on survival in 20 hour-old larvae. Flow cytometry was validated as a reliable, fast and accurate, though expensive, method for identification of triploidy in H. midae. As an outcome of this study a manual of “Procedures for the Induction and Validation of Triploidy in the abalone” is presented (Appendix 1) together with recommendations for further studies on triploidy in the South African abalone, H. midae. / AFRIKAANSE OPSOMMING: Die inheemse perlemoen, Haliotis midae, is sedert 1990 ‘n suksesvol gekweekte akwakultuur spesie in Suid-Afrika. ‘n Kenmerk van die spesie is die stadige groeitempo van tussen twee en vyf jaar ten einde bemarkbare grootte te bereik. Soos vir die meerderheid perlemoen van kommersiële belang, is hierdie stadige groeitempo rede tot kommer met betrekking tot die winsgewende kweek en wêreldwye mededingendheid van die spesie. Die manipulasie van ploïdie van die moederlike genoom is ‘n toenemende praktyk in skulpvisboerdery en word gereken as ‘n belowende metode om die groeitempo van perlemoen te verbeter. Hierdie manipulasietegniek word gebruik om steriliteit te verkry wat manifesteer as onderdrukte ontwikkeling van die geslagsklier. Die gevolg is die herkanalisering van bronne na somatiese groei. Die doel van hierdie studie was om ‘n lewensvatbare metode vir die induksie van triploïdie op ‘n kommersiële skaal in die Suid-Afrikaanse perlemoen, H. midae, te vestig. Daar is op hidrostatiese druk as metode vir die induksie en vloei-sitometrie as metode vir die geldigverklaring van triploïdie gefokus. Die resultate van hierdie studie bevestig dat hidrostatiese druk ‘n effektiewe metode vir die induksie van triploïdie in H. midae is. Hoë persentasies van triploïdie (>80%) is oor ‘n wye reeks van drukke en tye in 48 uur oue larwes verkry. Daar is gevind dat hidrostatiese drukbehandeling ‘n negatiewe effek op die oorlewing van 20 uur oue larwes het. Vloei-sitometrie is bevestig as ‘n betroubare, vinnig en akkurate, maar duur metode vir die identifikasie van triploïdie in H. midae. As ‘n uitvloeisel van die studie word ‘n handleiding “Procedures for the Induction and Validation of Triploidy in the abalone” (Appendix 1) aangebied tesame met aanbevelings vir verdere studies rakende triploïdie in die Suid-Afrikaanse perlemoen, H. midae.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Oesophageal cancer (OC) is the sixth leading cause of cancer related deaths in the world with approximately 300 000 new cases reported each year. OC may be characterized into two forms with 90% of cases presenting as squamous-cell carcinoma (SCC) and the remaining 10% as adenocarcinoma (ADC). Several factors have been attributed to the development of OC, including oesphageal injury and/or irritation, chronic inflammation and excess iron associated with enhanced tumour growth. The HAMP gene codes for a 25 amino-acid protein found to be primarily expressed in the liver and crucial to regulation of bodily iron status. Defects occurring in the HAMP gene could therefore lead to the dysregulation of the gene, resulting in an iron overload status. Iron overload is a previously described risk factor in the development of various cancers, including OC, and therefore the aim of this study was to investigate whether dysregulation of the HAMP gene may be involved in the cancer phenotype exhibition. The study cohort comprised of 48 unrelated patients presenting with SCC and a control group of 51 healthy, unrelated population-matched individuals. Mutation detection techniques included polymerase chain reaction (PCR) amplification, heteroduplex single-stranded conformation polymorphism (HEX-SSCP) analysis and bi-directional semi-automated DNA sequencing analysis. Screening of the 5’ regulatory region (5’UTR) of the HAMP gene revealed one known (-582A/G) and two novel (-188C/T and -429G/T) variants with the -429G/T variant showing statistically significant reduction in expression in patients relative to controls. Iron parameters were correlated between patient and control cohorts, as well as for variant presence and absence within individuals. Luciferase reporter constructs were used to investigate the functional implications of the presence of a variant on HAMP gene expression, and how these results correlated to the iron parameter statistics obtained. Luciferase reporter assay results indicated the -188C/T and -429G/T variants to result in under-, and the -582A/G variant to result in over-expression at the basal level, relative to the respective wild-type sequence constructs. Correlation of the luciferase data with the iron parameter statistics, indicate the -429G/T variant to be coupled to significantly higher levels of ferritin and C-reactive protein (CRP) and significantly lower levels of serum-iron and transferrin when compared to individuals without the variant. Considering only the patient group, the presence of the -188C/T and -429G/T variants were coupled to significantly lower levels of transferrin in patients with either variant, compared to patients without. The variants found within the HAMP promoter region are therefore able to alter gene regulation to an extent where iron parameters deviate between healthy and OC afflicted individuals, and also between patients with and without a variant. This dysregulation in iron homeostasis may play a role in the development and/ or progression of OC. Characterisation of the 5’ UTR of the HAMP gene may contribute to linking iron regulation to the establishment of an effective screening program, facilitating the early detection of OC. / AFRIKAANSE OPSOMMING: Slukdermkanker (SK) is die sesde grootste oorsaak van kanker-verwante sterftes in die wêreld, met sowat 300 000 nuwe gevalle wat aangemeld word elke jaar. SK kan geklassifiseer word in twee vorme, waar 90% van die gevalle plaveisel-selkarsinoom (SSC) vorm en die oorblywende 10%, adenokarsinoom (ADC). Verskeie faktore word toegeskryf aan die ontwikkeling van SK, insluitend slukderm beserings en/ of irritasie, chroniese inflammasie en oormatige ystervlakke wat geassosieer word met verhoogde gewasgroei. Die HAMP geen kodeer vir 'n 25 aminosuur proteïen wat hoofsaaklik in die lewer uitgedruk word en noodsaaklik is vir die regulering van ystervlakke in die liggaam. Defekte wat in die HAMP geen voorkom kan dus die onreëlmatige regulering van die geen tot gevolg hê, wat lei tot yster-oorlading. Yster-oorlading is voorheen beskryf as ‘n risiko faktor in die ontwikkeling van verskillende vorme van kanker, insluitend SK en gevolglik was die doel van hierdie studie om te bepaal of die wanregulering van die HAMP geen betrokke mag wees by die uitdrukking van die kanker fenotipe. Die studiepopulasie het bestaan uit 48 onverwante pasiënte met SSC en ‘n kontrole-groep van 51 gesonde, onverwante soortgelyke individue. Die mutasie opsporingstegnieke wat gebruik is, het polimerase kettingreaksie (PKR) amplifisering, heterodupleks enkelstring-konformasie polimorfisme (HEX-SSCP) analise en bidireksionele semi-outomatiese DNS volgordebepaling-analise van die geïdentifiseerde variante ingesluit. Sifting van die 5’ regulerende area (5'UTR) van die HAMP geen het een bekende (-582A/G) en twee nuwe (-188C/T en -429G/T) variante opgelewer, met die -429G/T variant wat statisties beduidend onderdruk is in pasiënt uitdrukkings vlakke relatief tot 'n gesonde kontole-groep. Yster-parameters van alle pasiënt en kontole individue is gekorreleerd tussen pasiënt en kontrole groepe, sowel as vir teenwoordigheid of afwesigheid van variante in elke individu. Luciferase verklikker konstrukte is gebruik om die funksionele implikasies van die teenwoordigheid van ‘n variant op HAMP geenuitdrukking te ondersoek, en hierdie resultate te korreleer met yster-parameter statistieke wat verkry is. Luciferase verklikkertoetse dui aan dat die -188C/T en -429G/T variante tot verminderde, en die -582A/G variant lei tot die verhoogte uitdrukking op die basale vlak lei, relatief tot die onderskeie wilde-tipe konstukte. Korrelasie van die luciferase data met die yster-parameter statistieke, dui aan dat die -429G/T-variant gekoppel is aan aansienlik hoër vlakke van feritien en C-reaktiewe proteïen (CRP) en beduidend laer vlakke van serum-yster en transferrien in vergelyking is met individue sonder die variant. Met oorweging van slegs die pasiënt-groep, is die teenwoordigheid van die -188C/T en -429G/T variante beduidend gekoppel aan laer vlakke van transferrien in pasiënte met die variant, in vergelyking met pasiënte daarsonder. Variante binne die HAMP promotor is dus in staat om geenregulasie te verander tot so 'n mate dat die yster-parameters afwyk tussen gesonde en SK geaffekteerde individue, sowel as tussen pasiënte met en sonder ’n variant. Hierdie wanregulering in yster homeostase kan 'n rol speel in die ontwikkeling en/ of die progressie van SK. Karakterisering van die 5’ regulerende area van die HAMP geen kan grootliks bydra om ysterregulasie te verbind met die implementering van ‘n effektiewe siftingsprogram, en sodoende die vroeë opsporing van SK fasiliteer.

Thesis (MScAgric)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Research into markers for the detection of genetic diversity and cultivar identification has become an important component of the genetic improvement of crops. However, the incorporation of marker assisted selection (MAS) as a tool for the identification and characterization of breeding material has not been fully implemented in the breeding of spring triticale at Stellenbosch University’s Plant Breeding laboratory (SU-PBL). The present study served as a case study in order to achieve this. The first part of the study concerned the detection of genetic diversity in 101 newly sourced triticale cultivars, from a USDA germplasm bank, together with five local control cultivars, in order to identify possible crossing parents. Eight SSR markers, including five derived from rye and three from wheat, and five agronomic characteristics were used to assess diversity. In seedling screening the foreign cultivars showed resistance towards the stem rust isolate used, but were mostly susceptible to the leaf rust isolate. Out of the 8 SSR markers tested, 7 markers were polymorphic and revealed 140 alleles varying from 12 to 26 with an average of 17.5 alleles per locus. The observed polymorphic information content (PIC) value ranged from 0.39 to 0.88 with an average of 0.70, indicative of the good discriminatory ability of the SSR markers. The data revealed that the South African cultivars were genetically closely related to cultivars from the USA and Canada. The second part of the study focused on the introgression of a blue aleurone layer gene (Ba), carried by a wheat cultivar, ‘Cltr1202STR’, and purple pericarp genes (Pp1; Pp3) also carried by a wheat cultivar, ‘Amethyst’, into a triticale background. Unfortunately the introgression of the purple pericarp genes failed. Two lines containing the blue aleurone layer, 11T023 and 11T028, were however successfully created. Molecular typing of these lines with SSR markers were able to show that BC4F1 line 11T023 (Ba) B was genetically similar to the recurrent parent ‘Agbeacon’; and that the BC4F1 11T028 line (Ba) A was closest to the ‘US2007’ recurrent parent. The study illustrated that MAS was a reliable tool for detecting genetic diversity in newly sourced germplasm, and assisted in making a backcross breeding effort more effective. The data generated from MAS could therefore clearly assist in making the SU-PBL breeding program more effective by moving, better informed, decision making toward data based partly on the genotype, thereby minimizing the risks associated with purely phenotypic based decisions. / AFRIKAANSE OPSOMMING: Navorsing rondom die gebruik van merkers vir die bepaling van genetiese-diversiteit en kultivar identifikasie is ‘n toenemend belangriker komponent vir die genetiese verbetering van gewasse. Die inkorporering van merker-bemiddelde-seleksie (MBS) as gereedskap vir die identifikasie en karaktarisering van telingsmateriaal is nog nie ten volle geïmplimenteer in die lente korogtelingsprogram van die Stellenbosch Universiteit Planteteeltlaboratorium (SU-PTL). Die studie het gedien as gevallestudie ten einde dit te bereik. Die eerste gedeelte van die studie het gehandel oor die tipering van die genetiese diversiteit van ‘n 101 kultivars verkry vanaf ‘n USDA kiemplasmabank saam met 5 plaaslike kontroles. Dit was gedien ten einde moontlike kruisings-ouers te kon identifiseer. Agt SSR merkers, insluitend vyf afkomstig van rog en drie vanaf koring, asook vyf agronomiese kenmerke is aangewend om die materiaal se diversiteit te tipeer. Saailingtoetsing is ook gedoen en het aangetoon dat die meeste kultivars weerstandig was vir die stamroes-isolaat, maar nie die blaarroes-isolaat nie. Van die agt SSR merkers getoets het sewe getoon om polimorfies te wees en het ‘n 140 allele gegee wat gewissel het vanaf 12 tot 26 per lokus met ‘n gemiddeld van 17.5. Die waargenome polimorfiese inligtings inhoud (PII) waarde het gewissel vanaf 0.39 tot 0.88 met ‘n gemiddeld van 0.70. Die merkers kon dus suksesvol diskrimineer. Die data het aangetoon dat die Suid-Afrikaanse kultivars genetiese die naaste verwant was aan die kultivars afkomstig vanaf die VSA en Kanada. Die tweede gedeelte van die studie het gefokus op die introgressie van ‘n blou aleuron-laag geen (Ba), afkomstig vanaf die koringkultivar ‘Cltr1202STR’, en twee pers-perikarp gene (Pp1; Pp3), afkomstig vanaf die koringkultivar ‘Amethyst’, na ‘n korog agtergrond. Ongelukkig het die oordrag van die pers-perikarp gene gefaal. Twee lyne wat die blou aleuron- laag bevat, 11T023 en 11T028, is egter suksesvol geskep. Tipering van die lyne met die SSR merkers het aangetoon dat die BC4F1 lyn 11T023 (Ba) B genetiese baie na aan die herhalende ouers ‘Agbeacon’ is en dat die BC4F1 11T028 lyn (Ba) A nader is aan die herhalende ouer ‘US2007’. Die studie het dus geïllustreer dat MBS gebruik kan word as ‘n betroubare manier om genetiese diversiteit te bepaal en by te dra tot die sukses van ‘n terugkruisingsprogram. Die data wat dus voortspruit uit MBS kan dus help om die SU-PTL se telingsprogram te assisteer in die besluitnemingsproses tydens teling deur beter genotipe gebaseerde besluite te neem wat die riskio van fenotipe gebaseerde besluite kan help verminder.

Thesis (MScAgric)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Abalone production is the largest financial contributor to aquaculture in South Africa and practically all of the abalone produced is exported to Asia. This means that the product must be globally competitive and many technologies have been applied to this cause. One that specifically shows great promise for bivalve mollusc production is triploidy; more precisely, sterility due to the induction of aneuploidy. Under normal maturation, energy is diverted from somatic growth through sexual maturation, therefore inhibiting or retarding gametogenesis through a process such as aneuploidy is expected to increase growth and decrease the time to marketing. Two studies preceding this one investigated the induction of triploidy through hydrostatic shock (De Beer, 2004) and the comparative growth rate of triploid genotypes from 8 to 24 months, prior to the onset of sexual maturation (Schoonbee, 2008). During this comparative growth stage, no convincing statistical evidence of faster growth or of seasonal environmental effects could be obtained. It was recommended that growth between triploid and diploid variants be compared during the age period when sexual maturity becomes a factor to determine whether triploidy in Haliotis midae is a useful biotechnological tool to improve biological productivity and global competiveness of the abalone industry. The growth measured as shell length and wet weight in the period from 29 to 62 months showed a statistically significant difference in mean weight and mean length with diploids showing a superior growth rate compared to their triploid siblings. This difference of 1.99 mm and 5.13 g was however not perceived as being commercially significant. Important production parameters including canning yield percentage and gonadosomatic index were also measured during this trial. For both these parameters, the triploid genotype showed statistically and commercially significant improvement of 10.68% increased canning yield and 28.42% reduction in gonadosomatic index when compared to their diploid counterparts. Triploid abalone was found to be not completely sterile; gametes and even mature gonads were observed in some instances. Even though complete sterility was not achieved there appeared to be a retarded gonadosomatic development in triploid variants. The delay in sexual maturation, together with the improvement in canning yield, may justify triploidy’s commercial application, despite its reduced growth rate. / AFRIKAANSE OPSOMMING: Perlemoen produksie lewer die grootste finansiële bydra tot akwakultuur in Suid Afrika en feitlik al die Perlemoen word uitgevoer na Asië. Dit beteken dat die produk moet kompeteer op die wêreld mark en verskeie tegnologieë word reeds aangewend vir die spesifieke doel. Een so tegnologie wat potensiaal toon ten opsigte van akwakultuur produksie is triploïedie; meer spesifiek, sterieliteit veroorsaak deur aneuploïedie induksie. Onder normale volwassewording, word energie weggeneem van somatiese groei wanneer geslagsrypheid intree en daarom kan groeitempo verhoog word deur gametogenese te inhibeer of te vertraag deur ‘n proses soos aneuploïedie en, word korter tydperk benodig om bemarkingsgrootte te bereik. Twee voorafgaande studies het gehandel oor die induksie van triploïedie deur hidrostatiese druk skok (De Beer, 2004) en die vergelykende groeitempo van triploïede genotipes vanaf ouderdom 8 tot 24 maande (Schoonbee, 2008) alvorens geslagsrypheid intree. Tydens hierdie vergelykende groeifase kon geen statisties betekenisvolle aanduidings van vinniger groei of seisoenale omgewingseffekte aangetoon word nie. Die studie handel vervolgens oor die uitbreiding van die vergelykende groeistudies tussen triploïede en diploïede genotipes tot ouderdom van 62 maande wat die intrede van geslagsrypheid insluit, ten einde te bepaal of die induksie van triploïedie in Haliotis midae voordele inhou ten opsigte van produksiedoeltreffendheid en mededingendheid op wêreldmarkte. Groei gemeet in terme van skulplengte en lewende massa oor die tydperk van 29 tot 62 maande het statisties betekenisvolle verskille getoon in gemiddelde massa en lengte van diploïede genotipes bo die van triploïede verwante individue. Die verskille van 1.99 mm en 5.13 g kan egter nie as kommersieel betekenisvol beskou word nie. Belangrike produksie eienskappe waaronder persentasie opbrengs van eindproduk en die gonadosomatiese indeks is ook bepaal. Vir beide die produksie eienskappe het die triploïede genotipe statisties sowel as kommersieel betekenisvolle verbetering van 10.68% getoon vir opbrengs en 28.42% verlaging in gonadosomatiese indeks in vergelyking met die diploïede genotipe. Triploïede genotipes was nie volledig steriel nie, gegewe die aanwesigheid van gonades en gamete in sommige individue. Selfs al is totale steriliteit nie bereik nie, het dit wel voorgekom asof daar vertraging in gonadosomatiese ontwikkeling plaasgevind het in triploïede genotipes. Die vertraging in geslagsrypheid tesame met die verhoogde persentasie opbrengs van die eindproduk hou voordele in bo die andersins effense stadiger groei van triploïede genotipes.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Gene transfer from wild gras species to wheat is complicated by the simultaneous integration of large amounts of alien chromatin. The alien chromatin containing the target gene is inherited as a linkage block and the phenomenon is known as linkage drag. The degree of linkage drag depends on whether, and how readily, recombination occurs between the foreign and wheat chromatin. The S13 translocation line was developed by the department of Genetics, US. A cross was made between Chinese Spring and a leaf rust resistant Aegilops speltoides accession. Resistant backcross F1 was backcrossed to Chinese Spring and W84-17. S13 was selected from the backcross progeny and found to carry three rust resistance genes temporarily named LrS13, SrS13 and YrS13. Unfortunately, the resistance genes were completely linked to gametocidal (Gc) genes that were co-transferred from the wild parent. In wheat Gc genes cause reduced fertility, poor plant phenotype and hybrid necrosis. In order to use employ the rust resistance genes commercially they need to be separated from the Gc genes. At the onset of this study four putative shortened forms of the S13 translocation were provided. The four lines were identified in a homoeologous paring induction experiment (involving the test cross 04M127). This study aimed to achieve the following: (i) characterize the four recombinants with the use of molecular markers, (ii) use the knowledge gained to identify further recombinants in the 04M127 cross, (iii) identify the shortest (most useful) recombinant, and (iv) attempt to shorten the shortest recombinant form still further and thereby remove as many of the Gc genes as possible. In total, seven recombinants of the S13 translocation (04M127-1, -2, -3, -4, -7, -11 and -12; referred to as recombinant group A) were identified and characterised with microsatellite and SCAR markers. These recombinants have exchanged different amounts of foreign chromatin for wheat chromatin, but were still associated with Gc genes, showing hybrid necrosis and seed shrivelling. Some of the recombinants have lost the undesirable „brittle rachis‟ phenotype which occurs in Ae. speltoides and the S13 translocation line. In plants VII having this trait, the rachis spontaneously disarticulates after the third spikelet upon ripening of the ear. Recombinant 3 appeared to be least affected by Gc genes and was therefore used in further attempts to shorten the translocation. Recombinant 3 was crossed with wheat (W84-17) and resistant F1 (heterozygous for the translocation) were test crossed with Chinese Spring nullisomic 3A tetrasomic 3B/D plants. Thirty five resistant testcross F1 plants were identified (named recombinant group B). The resistant group B recombinants as well as nine susceptible test cross F1 (which also appeared to be recombinant) were characterised making use of microsatellites and a SCAR marker. From the results it appeared that each of the 35 resistant plants exchanged substantial amounts of Ae. speltoides chromatin for wheat chromatin. The species chromatin that remained (and which contains LrS13) is probably located either close to the 3AS telomere or within the proximal regions of 3AS and 3AL. A SCAR marker that has been developed specifically for the S13 translocation provided useful confirmation of the presence of Ae. speltoides chromatin in the 35 recombinants. If the SCAR marker proves to be tightly linked to LrS13 it may eventually be used for marker assisted selection of the resistance or it may be employed in continued attempts to reduce the amount of foreign chromatin. Seedling rust resistance tests showed that the recombinants have lost SrS13 and YrS1 during recombination. An attempt was also made to develop additional markers that specifically detect the translocation in order to further characterise the group B recombinants. Published information on Ae. speltoides specific repeated and transposon sequences were obtained and used for primer design. Unfortunately, no suitable markers could be found and the primers that were designed tended to amplify the same fragments in both the wheat and species genomes. DArT markers were also employed in an attempt to characterise the 35 group B recombinants and controls. The DArT results provided an independent verification of the results obtained with the microsatellite markers. The DArT results confirmed that the group B recombinants exchanged large amounts of species chromatin for wheat chromatin. Even though the 35 resistant group B recombinants have undergone extensive recombination they still show signs of residual Gc effects. It is believed these effects can be removed by continued backcrossing to wheat accompanied by selection against Gc symptoms. While the effects of Gc genes per se were not studied, their properties were reminiscent of those of transposable elements. Indications were that complex interactions involving the Gc genes themselves as well as genetic factors in the wheat genome may have a drastic effect on the selective survival of recombinant gametes.

MScAgric / Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: In the Western Cape small grain cereals, triticale (×Triticosecale Wittmack ex A. Camus) in particular, appear to be among the most promising starch-carrying raw materials for the production of bio-ethanol. A core group of cultivars and lines from the Stellenbosch University Plant Breeding Laboratory spring triticale breeding programme were subjected to initial testing for the purpose of ethanol production. They underwent multi-location field-testing across six (season 2006–2007) and nine (season 2007–2008) locations representing the Western Cape cereal production area. Climatic conditions during the study were characterised as generally favourable, especially in the 2007 season. During the season, trials were visited in order to make in situ observations. Disease susceptibility was given specific attention. After harvesting, grain yield (kg.ha-1), test weight (kg.HL-1), total starch content in whole grain (%), amylose/amylopectin ratio, protein content (%), ethanol output (L.tonne-1) and ethanol yield (L.ha-1) were analysed. Near infra-red reflectance spectroscopy calibration models were developed for moisture and starch contents. The best calibration based on whole grain spectra for moisture content had RPD = 1.691, R2 = 0.657 and SEP = 0.271%, and for starch content RPD = 1.646, R2 = 0.634 and SEP = 1.356%. Calibrations developed from milled grain showed better results for moisture content RPD = 2.526, R2 = 0.843, SEP = 0.182%, and for starch content RPD = 1.741, R2 = 0.673, SEP = 1.277%. These calibrations are suitable for rough screening of samples. In the 2006 season, starch yield was highly positively correlated with grain yield (R2 = 0.988, P <0.001). Both starch yield and grain yield were positively correlated with days to heading (R2 = 0.533 and R2 = 0.556, respectively; P <0.001). The 2007 season was characterised by a generally higher starch yield (2952– 3142kg.ha-1, 95%CI) compared to the 2006 season (2077–2315kg.ha-1, 95%CI). Starch yield was strongly positively correlated with grain yield (R2 = 0.975, P <0.001). Test weight demonstrated weak positive correlation with ethanol yield (R2 = 0.238, P <0.01) and grain yield (R2 = 0.279, P <0.001). Mean ethanol output ranged between 466–477L.tonne-1 at the 95%CI. Ethanol output was demonstrated to be more dependent on starch and other polysaccharides accessibility to enzymatic digestion than on the total starch content as such. The best lines for ethanol output in the 2007 season were G2, D3 and H2 for the Swartland region, and D3, G2 and D1 for the Overberg region. The best triticale lines under investigation showed their potential from a biological point of view to be a suitable crop for ethanol production in the Western Cape, with the achieved ethanol yield ranging between 2446–2625L.ha-1 at the 95%CI. For the Swartland region the best genotypes for ethanol yield were D1, H1 and D2, and for the Overberg H1 and G2. The 23 best lines were selected from the elite and senior blocks, and then used for the establishment of a recurrent massselection pre-breeding block. / AFRIKAANSE OPSOMMING: In die Wes-Kaap is kleingrane, meer spesifiek korog (×Triticosecale Wittmack ex A. Camus), van die mees belowende styseldraende rou-materiale vir die produksie van bio-etanol. ‘n Kern versameling van kultivars en telerslyne van die Universiteit van Stellenbosch se Planteteeltlaboratorium se lente korogteeltprogram is blootgestel aan aanvanklike toetsing met die doel om etanol produksie te meet. Die materiaal het veldtoetsing ondergaan oor verskeie lokaliteite gedurende die 2006–2007 (ses lokaliteite) en 2007–2008 (nege lokaliteite) seisoene wat verteenwoordigend was van die Wes-Kaapse produksie gebied. Klimaatstoestande gedurende die studie kan beskryf word as gunstig, veral gedurende die 2007 seisoen. Gedurende die groeiseisoen is proeflokaliteite gereeld besoek ten einde in situ observasies te kon maak, siektevatbaarheid het veral aandag geniet. Na die oes van proewe was graanopbrengs (kg.ha-1), hektolitermassa (kg.HL-1), totale-styselinhoud in heelgraan (%), amilose/amilopektien-verhouding, proteïeninhoud (%), etanolopbrengs (L.ton-1) en etanolopbrengs per hektaar (L.ha-1) gemeet. Naby-infrarooispektroskopie kalibrasies was ontwikkel vir vog- en styselinhoud. Die beste kalibrasies vir heelgraan voginhoud het ‘n RDP = 1.691, R2 = 0.657 en SEP = 0.271% en vir styselinhoud RPD = 1.646, R2 = 0.634 en SEP = 1.356% opgelewer. Die kalibrasies gebaseer op meel was aansienlik beter vir voginhoud RPD = 2.526, R2 = 0.843 en SEP = 0.182%, sowel as vir styselinhoud RPD = 1.741, R2 = 0.673 en SEP = 1.277%. Die kalibrasies is bruikbaar vir aanvanklike sifting van monsters. 5 Gedurende die 2006 seisoen het styselinhoud en graanopbrangs ‘n baie hoë korrelasie (R2 = 0.988, P <0.001) getoon. Beide stysel- en graanopbrengs was positief gekorreleerd met dae tot aar (R2 = 0.533 en R2 = 0.556; P <0.001). Die 2007 seisoen is gekenmerk deur ‘n hoër styselopbrengs (2952– 3142kg.ha-1, 95%VI) teenoor die 2006 seisoen (2077–2315kg.ha-1, 95%VI). Styselopbrengs was positief gekorreleerd met graanopbrengs (R2 = 0.975, P <0.001). Hektolitermassa het swak korrelasie getoon met etanolopbrengs (R2 = 0.238, P <0.01) en graanopbrengs (R2 = 0.279, P <0.01). Gemiddelde etanolopbrengs het gewissel tussen 466–477L.ton-1 by 95%VI. Data het aangedui dat etanolopbrengs meer aangewese is op stysel en ander polisakkariedverbindings se ensiematiese toeganklikheid eerder as totale stysel aanwesig. Die beste lyne wat etanolopbrangs betref in 2007 was G2, D3 en H2 vir die Swartland en D3, G2 en D1 vir die Overberg. Van die koroglyne wat deel was van die ondersoek het goeie potensiaal getoon, uit ‘n suiwer biologiese oogpunt, as gewas vir die produksie van etanol in die Wes-Kaap met ‘n gerealiseerde etanolopbrengs in die omgewing van 2446-2625L.ha-1 by 95%VI. In die Swartland was die beste genotipes D1, H1 en D2 en in die Overberg H1 en G2. Die beste 23 lyne is geselekteer uit die elite en senior telingsblokke en aangewend in die vestiging van ‘n herhalende-seleksie voortelingsblok.

The aim of this study was to analyse testcross-material that was generated during a homoeologous pairing-induction experiment. Absence of the homoeologous pairing suppressor gene, Ph1, was employed to induce meiotic pairing between the Lr59 translocation (Aegilops peregrina) and 1AL of normal wheat. The study aimed to characterize the test-cross plants derived from this experiment and to identify recombinants which retained the least amount of species chromatin but which still contained the Lr59 gene. The test-cross F1 population, 07M5 (total 635 plants), was screened for Lr59 resistance by inoculating seedlings with the leaf rust pathotype, UVPrt8. The 168 resistant plants were characterized with molecular markers in order to identify recombinants. The data were used to construct a physical map which showed the relative sizes of the recombinants and which could be used to identify those recombinants which contained the least amount of residual species chromatin. Microsatellite (Xcfa2219, Xbarc83 and Xgwm164) and SCAR (S15T3) analysis was used for the initial identification of recombinants. The results showed that 152 of the 168 resistant plants were recombinants for the four loci; that eight of the remaining 16 plants represented non-recombinant, wild species-types and that the last eight plants represented the wheat parental-types which were resistant (and thus, also recombinants). This extremely high recombination frequency can largely be attributed to strong segregation distortion that was evident in the cross. It is also possible that the translocation segment could derive from the S genome rather than the U genome of Ae. peregrina. The S genome is closer related to the wheat genomes than the U genome and may be more prone to recombination. With the use of the microsatellite and SCAR data, a physical map was constructed which showed the relative location of the Lr59 gene on the translocation. It appeared that the eight shortest recombinants retained terminal species chromatin. In an attempt to characterize the eight recombinants, additional marker loci had to be identified within that region. RAPD, iv AFLP and DArT markers were investigated for this purpose. RAPD analyses did not produce any useful markers. AFLP and DArT analyses did identify useful markers with which the eight recombinants could be screened. The data showed which recombinants probably retained the least amount of species chromatin. Seeing that AFLP and DArT markers are anonymous and that the distances between marker loci are unknown, it is not possible to say which recombinant is the shortest and consequently it will be nessecary to also evaluate the group of eight recombinants agronomically in order to identify the most useful ones. The results showed that multiple cross-overs apparently occured on both sides of Lr59. Multiple cross-overs are higly unlikely in material of this nature, therefore it was speculated that the observation resulted from incomplete synteny between the telomeric areas of the translocation and 1AL. A structural difference between the two chromosome regions might have given rise to abnormal meiotic pairing structures and thus unexpected gamete genotypes. Each of the eight recombinants did express one or more of the Ae. peregrina derived AFLP loci which can in future be verified for use as a marker for marker assisted selection. The study succeeded in identifying a number of potentially useful recombinants which contain the Lr59 resistance. It would, however, be risky to select only one of the shortest recombinants for further development on the basis of the present knowledge as some recombinants may contain genetic abnormalities which resulted from reduced synteny in the Lr59 region. It would therefore be wise to further evaluate all eight recombinants before the best one is selected for agronomic use.