Teses / dissertações sobre o tema "Wheat Genetics"

Thesis ( MScAgric) -- Stellenbosch University, 1997.<br>ENGLISH ABSTRACT: Stem rust is considered the most damaging of the wheat rusts causing yield losses of more than 50% in epidemic years. Similarly, Russian wheat aphids (RWA) can be regarded as one ofthe most devastating insect pests of wheat. Yield losses due to R W A primarily result from a reduction in plant resources (sucking plant sap). Secondary losses are incurred by viruses transmitted during feeding. Mapping disease and insect resistance genes that are effective against prevailing pathotypes and biotypes of South Africa will optimize their utilization in breeding programmes. The wheat line, 87M66-2-l, is homozygous for a single dominant stem rust resistance gene located on chromosome lD. This stem rust resistance gene has been derived from Triticum tauschii accession RL5289 and is here referred to as Srtau. The aim of this study was to determine the chromosome arm involved. Following the chromosome arm allocation of Srtau, its possible linkage with the genes Rg2, Lr 21 , Sr X and Sr 33 was studied. A telosomic analysis has shown that Srtau is located on chromosome arm 1 DS and is linked to the centromere with a recombination frequency of 21 ± 3 .40%. Glume blotch and a heavy mildew infection of segregating families planted in the field in 1996 made the linkage study between Lr 21 (leaf rust resistance) and Rg2 (glume colour) impossible. However, estimated linkages of 9 ± 1.9 map units between Sr33 (stem rust resistance) and Srtau, ± 6 map units between Sr X (stem rust resistance) and Sr 3 3 and ± 1 0 map units between Sr X and Srtau suggested that SrX, Sr33 and Srtau are closely linked on I DS. Taking existing map data into consideration, it seems that the most likely order of the genes is: centromere - Srtau - Sr 3 3 - Sr X. A single dominant R W A resistance gene, Dn5, was identified in the T aestivum accession 'SA 463' and is located on chromosome 7D. The aim ofthis study was to determine the chromosome arm involved. The possible linkage of Dn5 with the endopeptidase locus, Ep-D1 b. and chlorina mutant gene, cn-D1, was then studied. Endopeptidase zymograms of 'SA 463' revealed two unknown polymorphisms. F 2 monosomic analyses involving the chromosomes 7 A, 7B and 7D were performed in an attempt to identify the loci associated with these polymorphisms. Dn5 was mapped on chromosome arm 7DL. A recombination frequency of60 ± 4.53% between Dn5 and the centromere suggested the absence of linkage. Linkage between Ep-Dl and cn-Dl could not be calculated as a result of similar isoelectric points of the 7DL encoded endopeptidases of the parental material studied. Recombination frequencies of32 ± 4.97% between Dn5 and EpDl and 37 ± 6.30% between Dn5 and cn-Dl were, however, encountered. The two novel endopeptidase alleles encountered in 'SA 463' were designated as Ep-Dle and Ep-Ald. A RWA resistance gene was transferred from the rye accession ' Turkey 77' to wheat and in the process the RWA resistant wheat lines 91M37-7 and 91M37-51 were derived. No rye chromatin could be detected in these plants following C-banding. The aim of this study was to determine (i) on which chromosome the gene(s) is located, and (ii) whether the resistance can be the result of a small intercalary translocation of rye chromatin. A monosomic analysis of the RWA resistance gene in 91M37-51 has shown that a single dominant resistance gene occurs on chromosome 7D. The use of rye-specific dispersed probes did not reveal any polymorphisms between the negative controls and RW A resistant lines 91M3 7- 7 and 91M37-51 which would suggest that it is unlikely that the resistance was derived from rye.<br>AFRIKAANSE OPSOMMING: Stamroes word as die mees vemietigende graanroessiekte beskou en het in epidemiese jare oesverliese van meer as 50% tot gevolg. Russiese koringluise is eweneens een van die emstigste insekplae van koring. Russiese koringluise veroorsaak oesverliese deurdat dit plantsap uitsuig en die plant van voedingstowwe beroof. Dit tree egter ook as 'n virusvektor op en kan so indirekte oesverliese veroorsaak. Kartering van siekte- en insekweerstandsgene wat effektief is teen die Suid-Afrikaanse patotipes en biotipes, sal hulle gebruik in teelprogramme optimiseer. Die koringlyn, 87M66-2-l , is homosigoties vir 'n dominante stamroes-weerstandsgeen wat op chromosoom ID voorkom. Hierdie weerstandsgeen is uit die Triticum tauschii aanwins, RL5289, afkomstig en word hiema verwys as Srtau. Daar is gepoog om te bepaal op watter chromosoomarm Srtau voorkom, waama sy koppeling met betrekking tot die gene Rg2, Lr21 , SrX en Sr33 bepaal is. 'n Telosoomanalise het getoon dat Srtau op chromosoom-arm 1 DS voorkom en gekoppel is aan die sentromeer met 'n rekombinasie-frekwensie van 21 ± 3.40%. Segregerende populasies wat in 1996 in die land geplant is, is hewig deur aarvlek en poeieragtige meeldou besmet en dit het die moontlike bepaling van koppeling tussen Lr21 (blaarroesweerstand) en Rg2 (aarkaffie kleur) belemmer. Koppelingsafstande van 9 ± 1. 9 kaart-eenhede tussen Sr 33 (stamroesweerstand) en Srt au, ± 6 kaart -eenhede tussen Sr X ( stamroesweerstand) en Sr 3 3 en ± 1 0 kaart -eenhede tussen SrX en Srtau is geraam en toon dat SrX, Sr33 en Srtau nou gekoppel is. Die waarskynlikste volgorde van die gene op lDS is: sentromeer- Srtau- Sr33- SrX. 'n Enkele dominante Russiese koringluis-weerstandsgeen, Dn5, is in dieT aestivum aanwins 'SA 463 ' ge"identifiseer en kom op chromosoom 7D voor. Die studie het ten doel gehad om te bepaal op watter chromosoom-arm Dn5 voorkom, asook wat die koppeling van Dn5 met die endopeptidase lokus, Ep-Dl, en die chlorina mutante geen, cn-Dl , is. Endopeptidase simograrnme van 'SA 463' het twee onbekende polimorfismes getoon. Die gene wat kodeer vir hierdie twee polimorfismes is met behulp van F2 monosoom-analises wat die chromosome 7 A, 7B en 7D betrek, gei:dentifiseer. Dn5 is op chromosoom 7DL gekarteer. 'n Rekombinasie-frekwensie van 60 ± 4.53% is gevind vir die sentromeer en Dn5 en dui op die afwesigheid van koppeling. Koppeling tussen Ep-Dl en cn-Dl kon nie bepaal word nie omdat die endopeptidase bande geproduseer deur die ouerlike materiaal wat in die studie gebruik is, nie met sekerheid in die nageslag onderskei kon word nie. Rekombinasie-frekwensies van 32 ± 4.97% tussen Dn5 en Ep-Dl en 37 ± 6.30% tussen Dn5 en cn-Dl is egter bereken. Dit word voorgestel dat daar na die twee onbekende endopeptidase-allele wat in 'SA 463 ' voorkom, verwys word as Ep-Dle en Ep-Ald. 'n Russiese koringluis-weerstandsgeen is uit die rog-aanwins, 'Turkey 77', oorgedra na koring en in die proses is die Russies koringluis weerstandbiedende lyne, 91M37-7 en 91M37-51 , geproduseer. Geen rog-chromatien kon egter met behulp van C-bande in hierdie lyne waargeneem word nie. Die doel van die studie was om te bepaal (i) op watter chromosoom die geen(e) voorkom, en (ii), of die Russiese koringluis weerstandsgeen die gevolg kan wees van 'n klein interkalere translokasie van rog- chromatien. 'n Monosoom-analise van die Russiese koringluis-weerstandsgeen in 91M37-51 het getoon dat 'n enkele dominante weerstandsgeen op chromosoom 7D voorkom. Rog-spesifieke herhalende peilers het geen polimorfismes tussen negatiewe kontroles en die Russiese koringluis weerstandbiedende lyne 91M37-7 en 91M37-51 getoon nie. Dit is dus onwaarskynlik dat die weerstand in die lyne uit rog verhaal is.

Bibliography: leaves 137-163. his is a comprehensive study of induced homoeologous recombination along most of the complete genetic length of two homoeologous chromosomes in the Triticeae (7A of common wheat and 7Ai of Agropyron intermedium), using co-dominant DNA markers. Chromosome 7Ai was chosen as a model alien chromosome because is has been reported to carry agronomically important genes conferring resistance to stem rust and barley yellow dwarf virus on its short and long arms, respectively.

Thesis (PhD (Agric)) -- Stellenbosch University, 2001.<br>ENGLISH ABSTRACT: Chromosome arm 7DL of common wheat carries genes for agronomically important traits such as leaf rust, stem rust, Russian wheat aphid and eye spot resistance. Some of these genes occur on introgressed foreign chromatin, which restricts their utility in breeding. The 7DL genetic maps are poorly resolved, which seriously hampers attempts to manipulate the genes and introgressed regions in breeding. This dissertation represents an attempt to improve our knowledge of the relative map positions of three resistance genes that have significant potential for use in local breeding programmes. The leaf rust resistance gene, Lr19, is located on a Thinopyrum ponticum-derived translocation which occupies a large part of the terminal end of 7DL. The translocation also carries genes for less favourable traits such as yellow flour colour. Attempts have been made to reduce the size of the translocation through allosyndetic pairing induction; the primary aims being to remove deleterious genes and to minimise the amount of foreign chromatin associated with Lr19 so it can be recombined with other useful 7DL genes. Twenty-nine 'Indis'-derived Lr 19 deletion mutants were previously produced by gamma irradiation and a physical map was constructed. In this study, the set of mutant lines were further analysed using 144 Sse8387I/Msei and 32 EcoRI/Msel amplified fragment length polymorphism (AFLP) primer combinations. The previous physical map, which was based on five restriction fragment length polymorphism (RFLP) markers and five structural gene loci, was extended and now includes 95 novel AFLP markers (86 Sse8387I/Msei and 9EcoRI!Msel markers), of which seven map close to Lr 19. Most of the deletions could be ordered according to size and the improved map has already been used to characterise shortened recombinant forms of the Lr 19 translocation. An unsuccessful attempt was made to convert one of the seven markers closest to Lr 19 into a sequence-specific marker. However, an AFLP marker located distally from Lr 19 was successfully converted into a sequence-specific marker in collaboration with other researchers. An attempt was also made to map and tag the Russian wheat aphid (RWA) resistance gene, Dn5. A doubled haploid mapping population consisting of 94 lines was created and typed for Dn5, four microsatellite loci and the endopeptidase locus, Ep-Dl. The Dn5 locus mapped 25.4 cM and 28.6 cM distally from Xg.vm111 and Xg.vm437, respectively, but was not linked to Xgwm428, Xgwm3 7 or Ep-Dl. Tagging of Dn5 was attempted by screening twelve homozygous resistant and seven homozygous susceptible F2 lines from a cross between 'Chinese Spring' and 'PI 294994' with 70 Sse8387IIi\1sei AFLP primer combinations. Only two potentially useful polymorphisms (one in coupling and one in repulsion phase) were identified. Conversion of the coupling phase marker to a sequence-specific marker was not successful. The eyespot resistance gene, Pchl , was derived from Triticum ventricosum and is present in the wheat VPM-1. Close association between Pchl and the endopeptidase Ep-Dlb allele has been reported previously. Pchl/Ep-Dl was tagged by screening ten wheat genotypes (each homozygous for the confirmed presence or absence of Pchl and/or Ep-Dl b) with 36 Sse83 87I/ Msei AFLP primer combinations. Three AFLP markers were closely associated with Pchl I Ep-D 1, one of which was targeted for conversion into a sequence-specific marker. The sequence-specific marker contained a microsatellite core motif and was found to be useful for tagging Pchl!Ep-Dl. A genetic distance of 2 cM was calculated between the novel microsatellite marker and Ep-Dl. The microsatellite marker was also polymorphic for the Lr 19 translocation and it was possible to map it between the Wsp-Dl and Sr25 loci. In this dissertation, mapping and/or tagging of three important resistance genes were achieved. Due to the fact that all markers used in these studies were not polymorphic between all of the targeted regions, it was not possible to fully integrate the data obtained for the three regions.<br>AFRIKAANSE OPSOMMING: Chromosoom arm 7DL van broodkoring dra gene vir agronomies-belangrike kenrnerke soos blaarroes, stamroes, Russiese koringluis en oogvlek weerstand. Sommige van hierdie gene kom voor in blokke spesie-verhaalde chromatien wat hul bruikbaarheid in teling beperk. Die genetiese kaarte van 7DL is swak ontwikkel en dit maak dit baie moeilik om hierdie gene en spesie-verhaalde streke tydens teling te manipuleer. Hierdie proefskrif verteenwoordig 'n paging om kennis van die relatiewe kaart liggings van drie weerstandsgene, met betekenisvolle potensiaal in plaaslike tee! programme, te verbreed. Die blaarroes weerstandsgeen, Lr 19, kom voor op 'n Thinopyrum ponticum-verhaalde translokasie wat 'n groot terminale gedeelte van 7DL beslaan. Die translokasie dra ook gene vir minder gewensde kenrnerke soos gee! meelkleur. Pogings is aangewend om die translokasie deur homoeoloe parings-induksie te verkort. Die doe! was om nadelige gene te verplaas en die hoeveelheid vreemde chromatien geassosieer met Lr 19 te minimiseer sodat dit met ander nuttige gene op 7DL gerekombineer kan word. Nege-en-twintig 'Indis'-verhaalde Lr 19 delesie mutante is vroeer met gamma bestraling geproduseer en gebruik om 'n fisiese kaart op te stel. Teenswoordig is die stel mutante verder ontleed met behulp van 144 Sse8387I!Msei en 32 EcoRII Msel amplifikasie-fragment-lengte-polimorfisme (AFLP) inleier kombinasies. Die bestaande fisiese kaart, wat gebaseer was op vyf restriksie-fragment-lengte-polimorfisme (RFLP) merkers en vyf strukturele geen loki, is uitgebrei en sluit nou 95 unieke AFLP merkers (86 Sse8387I/Msel en 9EcoRI/Msel merkers) in, waarvan sewe naby aan Lr19 karteer. Die meeste van die delesies kon op grond van hulle grootte gegroepeer word en die verbeterde fisiese kaart is alreeds gebruik om verkorte rekombinante vorms van die Lr 19 translokasie te karakteriseer. 'n Onsuksesvolle paging is aangewend om een van die sewe merkers naaste aan Lr 19 om te skakel na 'n volgorde-spesifieke merker. 'n AFLP merker wat distaal van Lr 19 karteer is egter wel suksesvol in samewerking met ander navorsers omgeskakel na 'n volgordespesifieke merker. 'n Paging is ook aangewend om die Russiese koringluis (RKL) weerstandsgeen, Dn5, te karteer en merkers gekoppel aan die geen te identifiseer. 'n Verdubbelde-haplo!ede karteringspopulasie van 94 lyne is geskep en getipeer vir Dn5, vier mikrosatelliet loki en die endopeptidase lokus, Ep-D1. Die Dn5 lokus karteer 25.4 cM en 28.6 cM distaal van Xgwml11 en Xgwm437, respektiewelik, maar was me gekoppel met Xgwm428, Xgwm37 of Ep-D1 me. Twaalf homosigoties weerstandbiedende en sewe homosigoties vatbare F2 lyne uit die kruising: 'Chinese Spring' I 'PI 294994' is met 70 Sse8387VMsel AFLP inleier kombinasies getoets in 'n poging om merkers vir Dn5 te identifiseer. Slegs twee moontlik bruikbare polimorfismes (een in koppelings- en een in repulsie fase ), is ge'identifiseer. Omskakeling van die koppelingsfase merker na 'n volgorde-spesifieke merker was onsuksesvol. Die oogvlek weerstandsgeen, Pch1, is uit Triticum ventricosum oorgedra en kom voor in die koringlyn, VPM-1. Noue koppeling van Pch1 en die endopeptidase alleel, Ep-D1 b, is vantevore gerapporteer. Merkers is vir P chl I Ep-D 1 gevind deur tien koring genoti pes ( elkeen homosigoties vir die bevestigde teenwoordigheid of afwesigheid van Pch1 en/of Ep-D1 b) te toets met 36 Sse83871/kfsel AFLP inleier kombinasies. Drie AFLP merkers is gevind wat nou koppel met Pchl!Ep-D1 , waarvan een gekies is vir omskakeling na 'n volgorde-spesifieke merker. Die volgorde-spesifieke merker het 'n mikrosatelliet kernmotief bevat en was nuttig as merker vir Pch1/Ep-D1. 'n Genetiese afstand van 2 cM is tussen die unieke mikrosatelliet merker en Ep-D1 bereken. Die mikrosatelliet merker was ook polimorfies vir die Lr 19 translokasie en dit is tussen die Wsp-D1 en Sr25 loki gekarteer. Kartering en/of identifikasie van merkers vir drie belangrike weerstandsgene was suksesvol in hierdie studie. Omdat al die merkers wat gebruik is, nie polimorf was tussen al die streke van belang nie, was dit nie moontlik om die data vir elk van die drie streke ten volle te integreer nie.

1 v.<br>Title page, contents and abstract only. The complete thesis in print form is available from the University Library.<br>Thesis (Ph.D.)--University of Adelaide, Dept. of Agronomy, Waite Agricultural Institute, 1986

Bibliography: leaves 234-256. Genetic studies of tolerance of durum wheat (Triticum turgidum L. var durum) to high concentrations of boron (B) were undertaken to identify genetic variation in response to B, the mode of gene action, number of genes and chromosomal locations of genes controlling tolerance. Results demonstrated that tolerance to B is under simple genetic control as observed in bread wheat. High levels of tolerance can be transferred into sensitive commercial varieties via backcrossing and selection can be performed during seedling growth at early generations.

Wheat improvement most often has been accompanied by a narrowing germplasm base, as newer cultivars have been derived from intercrosses between elite germplasm. However, there is a concern that narrow germplasm may restrict breeding improvement for important traits such as resistances to new biotic and abiotic stresses. In addition to germplasm base, the wheat kernel is a major component of wheat grain yield and an important factor for milling characteristics. Focusing on wheat kernel characteristics might be a key element to improve wheat genotypes for agronomic and quality traits. With the intention to broaden the wheat germplasm, and to explore the associations between kernel traits and agronomic as well as quality traits, a two-year study was initiated in 2009 to examine the influence of the kernel traits on the agronomic and quality attributes of a 160 Recombinant Inbred Lines (RIL) population developed from an adapted (ND 705) and a non-adapted genotype (PI 414566). The experiment was conducted at Prosper and Carrington, North Dakota, during 2009 and 2010. The RIL population had a better performance at Carrington than Prosper due to favorable climatic conditions at this location, in 2009 and 2010. The results in this study showed that kernel traits had a high correlation among them and they exhibited continuous variations suggesting a polygenic inheritance. Grain yield, kernel volume weight (KVW), and flour extraction were highly correlated with kernel width, length/width ratio, weight, and area. Eight RIL yielded better than the adapted parent ND 705 and two of the RIL along with three checks were significantly superior for gram yield compared with the other genotypes across all environments. Although the non-adapted parent has a facultative grown habit, several RIL required fewer days to flower compared to the adapted parent. Two RIL had better flour extraction compared to the other genotypes included in this study. These results indicated that kernel traits can play a significant role in improving agronomic and quality traits. Higher values for grain yield, KVW, and flour extraction were significantly associated with spheroid or round shape (short and plump), large, and heavy kernels. The high agronomic and quality attributes showed by some RIL demonstrated that the use of a non-adapted parent can broaden the genetic variability while increasing the genetic gain for certain traits. Also, breeders should pay attention to kernel size and shape during the parental selection for the development of populations with improved agronomic and quality traits.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.<br>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.

With increasing demands on the quality and quantity of food required now and in the future, improvements to current agriculture practices are required. Increased food production requires utilisation of more agricultural land, pushing crops into non- traditional areas. The need for advances in agricultural technologies are not only required for current crop varieties, but for new varieties with increased tolerance to environmental stresses. Technological improvement means better crop yields and reduced land, water, fertilizer and pesticide use. Diversity Arrays Technology (DArT) was used to study wheat diversity, specifically to identify polymorphic markers between various wheat cultivars for use in marker- assisted breeding programs. The hybridisation based technology was used to analyse various bread and durum wheat cultivars for increased understanding of genomic diversity. Analysis shows that DArT is able to discriminate between tissue samples from wheat cultivars grown under various environmental stresses with polymorphic markers identified between samples treated with differing salt, light and temperature conditions. Epigenetic diversity was analysed through methylation detection using DArT to identify a list of candidate polymorphic markers. Markers were identified using the methylation sensitive restriction enzyme McrBC to generate control and treated targets. Diversity through cultivar exploration, looking at breeding experiments between cultivars with phenotypic extremes to examine salt tolerance versus in-tolerance using DArT produced a recombinant inbred line genetic linkage map. Bulk segregant analysis was also used to group phenotypic samples. Candidate markers were identified between cultivars that can be used to genotyping tetraploid and hexaploid wheat cultivars for germplasm identification. In addition, the identification of trait-linked molecular markers, such as salt resistance, plant breeders can genotype individual plants and populations of cultivars to determine the most suitable cultivar to plant that best complements to its local environment. This eliminates the need for multiple planting cycles to optimize crop selections, and gives the plant breeder the highest possible chance for crop success (yield, quality, performance and cost).

Grain hardness is an important quality and end use determinant of wheat grain. The precise factor controlling grain hardness is not known but it is thought to be controlled at the starch granules-protein matrix interface in the endosperm tissue. The work described in this thesis consists of three broad approaches. The first approach involves isolation of candidate hardness regulating gene sequences from Triticum tauschii, a soft grained diploid relative of wheat. Sequences of the candidate genes and surrounding areas were obtained. A D genome specific GSP-1 sequence was identified in these studies. Sequence differences between A genome and D genome GSP-1 genes were observed. This is an important aspect of the study because grain hardness is regulated by chromosome 5D in wheat. wGSPlD-13, a sequence downstream of the GSP-1 coding region from Triticum tauschii, is not associated with A genome GSP-1 sequences. This was used successfully in mapping studies involving the hardness locus. The coding region sequences may later be used in transformation studies to clarify their involvement in the control of grain hardness. A second approach was concerned with characterisation of members of the GSP-1/ puroindoline family ( candidate hardness controlling genes) that are expressed in developing wheat endosperm. An aim of this approach was to ascertain whether new members of the family could be identified that may be responsible for grain hardness control or could assist in future high resolution mapping applied to the region surrounding the grain hardness locus. A novel clone was identified that contained an entire GSP-1 coding region and upstream sequence, this upstream sequence showed homology to an element in a high molecular weight glutenin subunit gene. Further work is required to confirm this result. If the sequence is expressed in typical wheat endosperm it may have an involvement in regulating grain hardness. The third approach made no assumption about the mode of grain hardness control and assumed only that there is a genetic difference responsible for variation in the character. RAPD analysis was performed on DNA bulks generated from material differing only in grain hardness that is controlled by chromosome SD. Variations to the RAPD protocol were assessed and polymorphic bands were cloned. An attempt was made to create STS markers from sequence information obtained from the cloned polymorphic bands. One primer pair produced a polymorphism when used on soft and hard DNA bulks, but difficulty reproducing the polymorphism was experienced with new primer aliquots. DNA sequence information and cloned sequences generated in these studies will provide a valuable contribution to clarifying an involvement of candidate genes in grain hardness control and to high resolution mapping studies that will precisely define the grain hardness locus.

Thesis (PhD (Agric)) -- Stellenbosch University, 2003.<br>ENGLISH ABSTRACT: Stripe (yellow) rust of wheat, caused by Puccinia striiformis f.sp. tritici, was first detected as a single introduction into South Africa in 1996. Two additional pathotypes have since been identified. Control of the disease may be achieved by use of genetic adult plant resistance (APR) as is present in the local cultivar 'Kariega'. The aim of this project was to understand the genetic basis of the APR in 'Kariega' to facilitate breeding of new varieties with genetic resistance to stripe rust. A partial linkage map of a 'Kariega X Avocet S' doubled haploid population covering all 21 wheat chromosomes was generated using 208 DNA markers, viz, 62 SSR, 133 AFLP, 3 RGA and 10 SRAP markers, and 4 alternative loci. The different marker techniques detected varying polymorphism, viz, overall SSR: 46%, AFLP: 7%, SRAP: 6% and RGA: 9%, and the markers produced low levels of missing data (4%) and segregation distortion (5%). A significant feature of the linkage map was the low polymorphism found in the D genome, viz, 19% of all mapped DNA markers, 11% of all AFLP markers and 30% of the total genome map distance. A region exhibiting significant segregation distortion was mapped to chromosome 4A and a seedling resistance gene for stem rust (Puccinia graminis f.sp . tritici), Sr26, mapped to chromosome 6A close to three SSR markers. The leaf tip necrosis gene, Ltn, which was also segregating in the population, mapped to chromosome 7D. Protocols for SRAP and RGA were optimised, and SRAP marker use in wheat genetic linkage studies is reported for the first time. The linkage map was used together with growth chamber and replicated field disease scores for QTL mapping. Chromosomes showing statistically significant QTL effects were then targeted with supplementary SSR markers for higher resolution mapping. The quality of disease resistance phenotypic data was confirmed by correlation analysis between the different scorers for reaction type (0.799±0.023) and for transformed percentage leaf area infected (0.942±0.007). Major QTL were consistently identified on chromosome 7D (explaining some 25-48% of the variation) and on chromosome 2B (21-46%) using transformed percentage leaf area infected and transformed reaction type scores (early and final) with interval mapping and modified interval mapping techniques. Both chromosomal regions have previously been identified in other studies and the 7D QTL is thought likely to be the previously mapped APR gene Yr 18. Minor QTL were identified on chromosomes lA and 4A with the QTL on 4A being more prominent at the early field scoring for both score types. A QTL evidently originating from 'Avocet S' was detected under growth chamber conditions but was not detected in the field, suggesting genotype-environment interaction and highlighting the need for modifications of growth chamber conditions to better simulate conditions in the field. The genetic basis of the APR to stripe rust exhibited by 'Kariega' was established by mapping of QTL controlling this trait. The linkage map constructed will be a valuable resource for future genetic studies and provides a facility for mapping other polymorphic traits in the parents of this population with a considerable saving in costs.<br>AFRIKAANSE OPSOMMING: Streep of geelroes van koring word veroorsaak deur Puccinia striiformis f. sp tritici, en is die eerste keer in 1996 in Suid-Afrika na introduksie van 'n enkele patotipe waargeneem. Twee verdere patotipes is sedertdien in Suid-Afrika gei"dentifiseer. Beheer van die siekte word veral moontlik gemaak deur die gebruik van genetiese volwasseplantweerstand soos gei"dentifiseer in die plaaslike kultivar 'Kariega'. Die doel van hierdie studie was om die genetiese grondslag van die streeproesweerstand te ontrafel ten einde die teling van nuwe bestande kultivars moontlik te maak. 'n Verdubbelde haplo1ede populasie uit die kruising 'Kariega X Avocet S' is aangewend om 'n gedeeltelike koppelingskaart vir die volle stel van 21 koring chromosome saam te stel. Die kaart het uit 208 DNA merkers, nl., 62 SSR, 133 AFLP, 3 RGA, 10 SRAP merkers en 4 ander lokusse bestaan. Totale polimorfisme wat deur die verskillende merkersisteme opgespoor is, was as volg: SSR: 46%, RGA: 9%, AFLP: 7% en SRAP: 6%. Die mate van ontbrekende data was gering (4%) asook die mate van segregasie distorsie (5%) van 'n enkele geval wat op chromosoom 4A gekarteer is. 'n Prominente kenmerk van die koppelingskaart is die relatiewe gebrek aan polimorfiese merkers op die D-genoom, nl., slegs 19% van alle DNA merkers en 11% van alle AFLP merkers wat slegs 30% van die totale genoom kaartafstand bestaan het. Die stamroes (Puccinia graminis f. sp. tritici) saailingweerstandsgeen, Sr26, karteer op chromosoom 6A naby drie SSR merkers. Die geen vir blaartipnekrose, Ltn, karteer op chromosoom 7D. Protokolle vir SRAP en RGA merkers is ge-optimiseer en gebruik van SRAP merkers in koppelings-analise word vir die eerste keer in koring gerapporteer. Die koppelingskaart is in kombinasie met groeikamerdata en gerepliseerde veldproefdata gebruik om die gene (QTL) vir volwasseplant streeproesweerstand te karteer. Chromosome met statisties betekenisvolle QTL is met aanvullende SSR merkers geteiken om die resolusie van kartering verder te verhoog. Die kwaliteit van fenotipiese data, soos in die proewe aangeteken, is bevestig deur korrelasies te bereken tussen lesings geneem deur onafhanklike plantpataloe (0.799 ± 0.023 vir reaksietipe en 0.942 ± 0.007 vir getransformeerde persentasie blaaroppervlakte besmet). Hoofeffek QTL vir die twee maatstawwe van weerstand is deur middel van die metodes van interval QTL kartering en gemodifiseerde interval QTL kartering konsekwent op chromosome 7D (25-48% van variasie verklaar) en 2B (21-46% van variasie verklaar) ge"identifiseer. In vorige studies is aangetoon dat beide chromosome 7D en 2B QTL vir volwasseplant streeproesweerstand dra. Die 7D QTL is waarskynlik die weerstandsgeen, Yr 18. QTL met klein effekte op weerstand is op chromosome lA en 4A ge"identifiseer. Die effek van laasgenoemde geen was meer prominent in die velddata in die vroee datum van weerstandsbeoordeling. Een QTL, afkomstig van 'Avocet S', is slegs onder groeikamertoestande identifiseerbaar. Dit dui op moontlike genotipe-omgewing wisselwerking en beklemtoon die noodsaaklikheid om aanpassings te maak in groeikamertoestande vir beter simulasie van veldproeftoestande. Die genetiese grondslag van volwasseplantweerstand teen streeproes in die kultivar 'Kariega' is deur QTL kartering bepaal. Die 'Kariega X Avocet S' koppelingskaart kan as 'n waardevolle basis dien vir toekomstige genetiese ontledings van ander polimorfiese kenmerke in die populasie.

Wheat (Triticum aestivum) is the world's most widely grown and economically important crop. It is both a staple food for humans and a raw material for many industrial processes. World trade in wheat is important for economic stability and an ability to grow wheat is a valuable national resource. Wheat is Australia's major crop with an annual production of about 23 million tonnes. One-quarter of this is used domestically and meets all of Australia's requirements; the remaining three-quarters is exported. Therefore, Australia's wheat industry provides both the national staple food source and the basis of an export industry worth almost 2 billion dollars. There is great potential for further genetic improvement of wheat, not only by increasing grain yields by improved resistance to pathogens and tolerance to adverse environmental conditions, but also by improving functional quality. For example, one can change the physical properties of the storage components, starch and protein, to increase their usefulness in conventional applications and for novel uses. Some examples of the physical properties of starch affecting its uses are, the large starch granules from wheat that are suitable to make carbonless copy paper (Bligh, 1999), the small starch granules from rice that are used as a fat substitute because the a comparable mouthfeel, the high-amylose starches that have film-forming properties desirable for fried food coating batters and some forms of plastics, and the low-amylose starch that swells more in water and can be used for soft foods such as Asian noodles. Continued improvement of wheat is vital to meet the quantity and quality demands of the local and international wheat markets. One specialty market for Western Australian wheat, is export to Japan and South Korea for the production of Japanese white salted Udon noodles, an export market worth more than $200M pa (Garlinge, 1996). Udon noodles have specific eating qualities including a light, creamy, uniform colour, a 'bright' appearance to the noodle, a soft but elastic texture to the noodle, and a smooth 'mouthfeel', all of which result from the quality of the wheat flour starch they are made from(Crosbie, 1991; Batey et al., 1997; Zeng et al., 1997). The Australian Standard White Noodle (ASWN) wheat that Australia exports to produce Udon noodles is soft-grained, white coloured, contains between 9.5% and 11.5% protein, and produces flour of fine particle size with little starch damage (V. Reck, DAWA, pers. comm.). The flour also has good starch-swelling characteristics, moderate dough strength and good dough extensibility. The good starch-swelling characteristics of the flour result, for the most-part, from containing relatively less of the starch amylose than other varieties (22-23% compared to 25%), a property controlled by the GBSS genes (Nelson and Rines, 1962; Garlinge, 1996). When less amylose is present in the starch granule as it is heated in water, the amylopectin matrix inside the granule can swell, causing the finished Udon noodle to be soft. When more amylose is present in the starch granule, the amylopectin matrix cannot swell as much, and the finished noodle is too hard to have the desired 'mouthfeel' of an Udon noodle. The amylose fraction of starch is produced by the granule-bound starch synthase (GBSS) enzymes, encoded by the GBSS genes. The overall aim of the research described in this thesis was to investigate the genomic organization of the GBSS genes of wheat. Since the GBSS genes influence wheat starch quality, an understanding of the action of these genes is needed for future improvement of starch quality in noodle-wheats. There are three loci for GBSS genes in wheat, and these are located on chromosomes 4A, 7A and 7D. Both wild-type alleles and non-functional 'null' alleles exist at each locus. At the start of the project, these alleles had not been sequenced and the molecular differences between the alleles were not known. Other GBSS alleles were also thought to exist in Australian varieties that had yet to be identified and characterised. GBSS genes from a selection of wheat varieties, and from all three GBSS loci, were sequenced searching for DNA polymorphisms that were different between the different alleles. If any DNA polymorphisms were found to result in GBSS protein sequence differences, or differences in GBSS enzyme expression, they could influence the functional characteristics of the starch. Identifying GBSS allelic variants would enable molecular markers to be developed to detect the alleles and investigate their potential effects upon starch quality. Different PCR-based methods and one non-PCR-based method were used to investigate the genomic organization of the GBSS genes in a selection of genetically diverse wheat varieties. The 31 wheat varieties studied included noodle-wheat varieties from the ASWN classification, varieties with similar genetic background to ASWN wheat varieties but of unsuitable quality for noodle production, unrelated varieties of Australian Standard White wheat, and were compared with those 'Chinese Spring' varieties described in the literature. Most of the varieties are grown in the Western Australian wheatbelt and southern regions, either for export and the production of Asian noodles, or for the production of domestic baked-goods. A 500bp section from the middle of the GBSS genes was amplified, from a selection of wheat varieties, and sequenced to search for polymorphisms. Twenty-one single nucleotide differences were found between genes at the three loci and two PCR-based tests were designed to validate these differences as Single Nucleotide Polymorphisms (SNPs). A novel microsatellite was also discovered in intron 4 of the GBSS 7A genes. This (TGCCG)n microsatellite was variable between wheat varieties and so defines a novel allele in the Australian germplasm present at a frequency of 40%. A PCR-based test was developed to identify this variable locus. However, the new GBSS allele was not linked to Flour Swelling Volume (FSV) quality properties. The variable microsatellite locus Xsun1 (Shariflou and Sharp, 1999) in the 3' untranslated region of the GBSS genes and linked to GBSS allelic variation was used to genotype a wheat breeding population for its GBSS status. The population (n=69) contained combinations of wild-type and null alleles at the 7A and 7D loci. Once genotyped using this marker, the GBSS alleles were assessed for possible likage to starch variation. Although the trend suggested that the presence ofnull alleles increased the FSV, the size of the population tested was too small for the differences in FSV between wild-type and partially-waxy wheats to be statistically significant. The linkage between the Xsun1 microsatellite variation and the (TGCCG)n microsatellite variation from intron 4 of the GBSS 7A genes was studied. By combining these two microsatellite loci, which are closely linked to the GBSS coding regions, GBSS genes at the 7A locus could be separated into 12 allelic groups. Although none of these groups could be linked to specific changes in starch qualities, they can be analysed further for functional differences. In order to access a larger section of the GBSS genes using PCR, new PCR primers were designed and optimized to amplify segments of the GBSS genes. Primers for GBSS genes tend to generate many PCR products, but many of these were shown to be non-specific. These artifacts could be reduced by increasing the annealing temperatures, and non-specific priming was repressed by the presence of the second primer in the PCR reaction. Using one primer set, a nearly 2000bp segment of the GBSS 7A genes from wheat varieties 'Kulin' and 'Eradu' was amplified and sequenced. These sequences indicated the presence of single nucleotide differences that resulted in changed amino acids in the protein when compared to published GBSS sequences. The sequencing should be repeated to validate this result, which indicates that these are novel alleles, but it does suggest that allelic variation for GBSS exists in Australian wheat varieties and that these alleles are different from those described internationally. The EcoR1, HindIII and BamH1 restriction enzyme sites surrounding the GBSS genes were identified using Southern hybridisation. This provided the potential to access the entire GBSS gene, including the promoter and untranscribed regions, by restriction enzyme mediated cloning of genomic DNA. However, attempts to clone the genomic GBSS genes into both plasmid and viral vectors were not successful.The potential existence of pseudogene copies of the GBSS genes in the wheat genome was investigated using both PCR and Southern hybridisation techniques. No evidence of GBSS pseudogenes was found, and this suggests that the wheat genome does not contain them. This result was unexpected since organisms with large genomes, such as wheat, normally contain repeated sequences and pseudogenes. However, the absence of repeated sequences and pseudogenes should be beneficial in molecular wheat breeding because it suggests that there will not be interference from non-coding GBSS sequences in identifying molecular markers to GBSS genes. The GBSS genes present in Australian wheat varieties were similar enough to those described internationally that Australian breeders can make full use of research and molecular tests for GBSS genes developed elsewhere. However, enough variation exists between overseas and domestic varieties to warrant further investigation of novel GBSS alleles in domestic wheat, which may relate to differences in functionality.

Wheat (Triticum aestivum) is the world’s most widely grown and economically important crop. It is both a staple food for humans and a raw material for many industrial processes. World trade in wheat is important for economic stability and an ability to grow wheat is a valuable national resource. Wheat is Australia’s major crop with an annual production of about 23 million tonnes. One-quarter of this is used domestically and meets all of Australia’s requirements; the remaining three-quarters is exported. Therefore, Australia’s wheat industry provides both the national staple food source and the basis of an export industry worth almost 2 billion dollars. There is great potential for further genetic improvement of wheat, not only by increasing grain yields by improved resistance to pathogens and tolerance to adverse environmental conditions, but also by improving functional quality. For example, one can change the physical properties of the storage components, starch and protein, to increase their usefulness in conventional applications and for novel uses. Some examples of the physical properties of starch affecting its uses are, the large starch granules from wheat that are suitable to make carbonless copy paper (Bligh, 1999), the small starch granules from rice that are used as a fat substitute because the a comparable mouthfeel, the high-amylose starches that have film-forming properties desirable for fried food coating batters and some forms of plastics, and the low-amylose starch that swells more in water and can be used for soft foods such as Asian noodles. Continued improvement of wheat is vital to meet the quantity and quality demands of the local and international wheat markets. One specialty market for Western Australian wheat, is export to Japan and South Korea for the production of Japanese white salted Udon noodles, an export market worth more than $200M pa (Garlinge, 1996). Udon noodles have specific eating qualities including a light, creamy, uniform colour, a ‘bright’ appearance to the noodle, a soft but elastic texture to the noodle, and a smooth ‘mouthfeel’, all of which result from the quality of the wheat flour starch they are made from(Crosbie, 1991; Batey et al., 1997; Zeng et al., 1997). The Australian Standard White Noodle (ASWN) wheat that Australia exports to produce Udon noodles is soft-grained, white coloured, contains between 9.5% and 11.5% protein, and produces flour of fine particle size with little starch damage (V. Reck, DAWA, pers. comm.). The flour also has good starch-swelling characteristics, moderate dough strength and good dough extensibility. The good starch-swelling characteristics of the flour result, for the most-part, from containing relatively less of the starch amylose than other varieties (22-23% compared to 25%), a property controlled by the GBSS genes (Nelson and Rines, 1962; Garlinge, 1996). When less amylose is present in the starch granule as it is heated in water, the amylopectin matrix inside the granule can swell, causing the finished Udon noodle to be soft. When more amylose is present in the starch granule, the amylopectin matrix cannot swell as much, and the finished noodle is too hard to have the desired ‘mouthfeel’ of an Udon noodle. The amylose fraction of starch is produced by the granule-bound starch synthase (GBSS) enzymes, encoded by the GBSS genes. The overall aim of the research described in this thesis was to investigate the genomic organization of the GBSS genes of wheat. Since the GBSS genes influence wheat starch quality, an understanding of the action of these genes is needed for future improvement of starch quality in noodle-wheats. There are three loci for GBSS genes in wheat, and these are located on chromosomes 4A, 7A and 7D. Both wild-type alleles and non-functional ‘null’ alleles exist at each locus. At the start of the project, these alleles had not been sequenced and the molecular differences between the alleles were not known. Other GBSS alleles were also thought to exist in Australian varieties that had yet to be identified and characterised. GBSS genes from a selection of wheat varieties, and from all three GBSS loci, were sequenced searching for DNA polymorphisms that were different between the different alleles. If any DNA polymorphisms were found to result in GBSS protein sequence differences, or differences in GBSS enzyme expression, they could influence the functional characteristics of the starch. Identifying GBSS allelic variants would enable molecular markers to be developed to detect the alleles and investigate their potential effects upon starch quality. Different PCR-based methods and one non-PCR-based method were used to investigate the genomic organization of the GBSS genes in a selection of genetically diverse wheat varieties. The 31 wheat varieties studied included noodle-wheat varieties from the ASWN classification, varieties with similar genetic background to ASWN wheat varieties but of unsuitable quality for noodle production, unrelated varieties of Australian Standard White wheat, and were compared with those ‘Chinese Spring’ varieties described in the literature. Most of the varieties are grown in the Western Australian wheatbelt and southern regions, either for export and the production of Asian noodles, or for the production of domestic baked-goods. A 500bp section from the middle of the GBSS genes was amplified, from a selection of wheat varieties, and sequenced to search for polymorphisms. Twenty-one single nucleotide differences were found between genes at the three loci and two PCR-based tests were designed to validate these differences as Single Nucleotide Polymorphisms (SNPs). A novel microsatellite was also discovered in intron 4 of the GBSS 7A genes. This (TGCCG)n microsatellite was variable between wheat varieties and so defines a novel allele in the Australian germplasm present at a frequency of 40%. A PCR-based test was developed to identify this variable locus. However, the new GBSS allele was not linked to Flour Swelling Volume (FSV) quality properties. The variable microsatellite locus Xsun1 (Shariflou and Sharp, 1999) in the 3’ untranslated region of the GBSS genes and linked to GBSS allelic variation was used to genotype a wheat breeding population for its GBSS status. The population (n=69) contained combinations of wild-type and null alleles at the 7A and 7D loci. Once genotyped using this marker, the GBSS alleles were assessed for possible likage to starch variation. Although the trend suggested that the presence ofnull alleles increased the FSV, the size of the population tested was too small for the differences in FSV between wild-type and partially-waxy wheats to be statistically significant. The linkage between the Xsun1 microsatellite variation and the (TGCCG)n microsatellite variation from intron 4 of the GBSS 7A genes was studied. By combining these two microsatellite loci, which are closely linked to the GBSS coding regions, GBSS genes at the 7A locus could be separated into 12 allelic groups. Although none of these groups could be linked to specific changes in starch qualities, they can be analysed further for functional differences. In order to access a larger section of the GBSS genes using PCR, new PCR primers were designed and optimized to amplify segments of the GBSS genes. Primers for GBSS genes tend to generate many PCR products, but many of these were shown to be non-specific. These artifacts could be reduced by increasing the annealing temperatures, and non-specific priming was repressed by the presence of the second primer in the PCR reaction. Using one primer set, a nearly 2000bp segment of the GBSS 7A genes from wheat varieties ‘Kulin’ and ‘Eradu’ was amplified and sequenced. These sequences indicated the presence of single nucleotide differences that resulted in changed amino acids in the protein when compared to published GBSS sequences. The sequencing should be repeated to validate this result, which indicates that these are novel alleles, but it does suggest that allelic variation for GBSS exists in Australian wheat varieties and that these alleles are different from those described internationally. The EcoR1, HindIII and BamH1 restriction enzyme sites surrounding the GBSS genes were identified using Southern hybridisation. This provided the potential to access the entire GBSS gene, including the promoter and untranscribed regions, by restriction enzyme mediated cloning of genomic DNA. However, attempts to clone the genomic GBSS genes into both plasmid and viral vectors were not successful.The potential existence of pseudogene copies of the GBSS genes in the wheat genome was investigated using both PCR and Southern hybridisation techniques. No evidence of GBSS pseudogenes was found, and this suggests that the wheat genome does not contain them. This result was unexpected since organisms with large genomes, such as wheat, normally contain repeated sequences and pseudogenes. However, the absence of repeated sequences and pseudogenes should be beneficial in molecular wheat breeding because it suggests that there will not be interference from non-coding GBSS sequences in identifying molecular markers to GBSS genes. The GBSS genes present in Australian wheat varieties were similar enough to those described internationally that Australian breeders can make full use of research and molecular tests for GBSS genes developed elsewhere. However, enough variation exists between overseas and domestic varieties to warrant further investigation of novel GBSS alleles in domestic wheat, which may relate to differences in functionality.

Studies were conducted to evaluate the effect of glaucousness, number of tillers and plant height on response to water stress using near-isogenic wheat lines under two water regimes. The effect of glaucousness and number of tillers was studied under both field and glasshouse conditions while that of plant height was studied only under field conditions. In addition 28 wheat cultivars were evaluated for epicuticular wax content and its relationship with spectral reflectance. Highly significant differences (1.51 to 2.8 mg/dm2) were found in the amount of epicuticular wax (Ew) among the cultivars. Water stress conditions promoted the development of Ew content significantly. Ew content under control conditions and that under stress conditions were positively correlated (r = 0.85, p <0.01) suggesting that selection for this trait could be practised in either of the environments. Surface reflectance was reduced when the waxy layer from the leaf was removed with chloroform. The reduction for the abaxial surface was twice that for the adaxial surface indicating that the abaxial surface was more waxy than the adaxial one. The mean reduction (both surfaces) termed '5' was positively correlated with the amount of Ew (r = 0.59, p <0.01). The effect of glaucousness was studied using six pairs of nearisogenic lines (four pairs in case of glasshouse experiment). The lines differed significantly in all the characters studied except one. However, significant difference between the mean of glaucous and that of non—glaucous lines was found only for epicuticular wax content and water consumption per g of grain. 0n the average, glaucous lines consumed 4.8% less water per g of grain under well-watered conditions and 17.8% less under stress conditions than the non-glaucous lines.

Thesis (PhD)--Stellenbosch University, 2014.<br>ENGLISH ABSTRACT: The Russian wheat aphid (Diuraphis noxia, Kurdj., Hemipetra, Aphididae, RWA) is an important pest of wheat, causing large-scale damage and yield losses. Various studies have been done at a transcriptomics level, including complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), suppressive subtractive hybridization (SSH) and micro-array, which have identified genes putatively involved in RWA resistance. Even though these candidate genes have been identified, their role in host defence still needs to be verified using a functional genetics approach. In this study virus induced gene silencing (VIGS) using a barley stripe mosaic virus (BSMV) vector, has been utilized to knock-down candidate genes of interest in a wheat cultivar with the Dn1-resistance gene (TugelaDN). In this study it was hypothesized that genes involved in the hypersensitive response (HR) may contribute towards resistance and were thus targeted for silencing. These include glutathione-S-transferase (GST), superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX). However, since aphid feeding also results in wounding, the genes were also analyzed under wounding only. Aphid fecundity is considered an indicator of involvement in RWA resistance, as susceptible plants result in higher aphid fertility. Findings in the study suggest that with wounding only, that Dn1 containing plants produce a greater hypersensitive response than susceptible controls. Ascorbate peroxidase was found to be important for wounding-induced resistance in Dn1 wheat plants. Under infestation conditions, silencing of superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX) was found not to have an effect on aphid fertility and thus are not directly involved in resistance signaling. Knock-down of a phi-class glutathione-S-transferase F6 (TaGSTF6) transcripts however, had a large effect on aphid nymph numbers and thus may contribute to Dn1-resistance. Putative resistance genes silenced under aphid infestation conditions were a nucleotide binding protein (NBP) and resistance gene analogue 2 (RGA2). Analysis of NBP revealed its identity as a part of the iron homeostasis machinery in the cytosol, responsible for Fe-cluster assembly. Silencing of both NBP and RGA2 resulted in the expression of a susceptible phenotype. T10rga2-1A is an NBS-LRR protein known to be required for rust resistance in concert with resistance gene Lr10. T10rga2-1D silenced treatments resulted in susceptibility and plant death after aphid infestation, suggesting that T10rga2-1D may be a good up-stream candidate in Dn1-resistance.<br>AFRIKAANSE OPSOMMING: Die Russiese-koringluis (RWA) is ‘n pes wat ‘n belangrike ekonomiese invloed op koring opbrengste het en infestasie kan tot grootskaalse skade en oes verlies lei. Verskeie studies, onder andere komplimentêre DNA amplifiseerde fragment polimorfismes (cDNA-AFLPs), onderdrukkende onderskeidende hibridisaie (SSH) en mikro-reekse wat voorheen op transkriptomiese vlak gedoen is, het moontlike gene wat by RWA weerstand betrokke is, geïdentifiseer. Alhoewel hierdie gene reeds geidentifiseer was, hulle rol is nogtans onbekend. Dié gene moet nog getoets word, duur funksionele genetiese benaderingste maak. In hierdie studie is ‘n gars streep mosaïek virus vektor (BSMV) gebruik om kandidaat-gene van belang in ‘n Dn1-weerstandige geen-bevattende kultivar (TugelaDN) te onderdruk. Ondrukking van gene het deur middel van virus geïnduseerde geen onderdrukking (VIGS) plaasgevind. In hierdie studie is die hipotese gestel dat die gene betrokke by die hipersensitiewe reaksie (HR) ‘n invloed op plantweerstand kan hê en is dus geteiken vir geen-onderdrukking-studies. Hierdie gene het die volgende ingesluit: glutatioon-S-transferase (GST), superoksied dismutase Cu/Zn (SOD) en askorbien peroksidase (APX). Egter, omdat luisinfestasie ook tot verwonding aanleiding gee, is die onderdrukte gene ook onder alleenlik verwondingstoestande getoets. Luis vrugbaarheid is gebruik as indikator van betrokkenheid omdat meer vatbare plante ‘n hoër luis vrugbaarheid tot gevolg het. In die studie is gevind dat onder alleenlik verwondingkondisies, plante wat Dn1 bevat, ‘n groter hipersensitiewe respons vertoon, as vatbare kontroles. Daar is verder gevind dat askorbien peroksidase ‘n belangrike rol tydens verwondings-geïnduseerde weerstand in Dn1-plante speel. Daar is verder bevind dat die onderdrukking van superoksied dismutase Cu/Zn (SOD) en ‘n tilakoïed-geassosïeerde askorbien peroksidase (tAPX). Onder luis-infestasie kondisies, geen effek op luisvrugbaarheid gehad het nie en dus nie direk by die weerstandsrespons betrokke is nie. Die onderdrukking van ‘n phi-klas glutatioon-S-transferase F6 (TaGSTF6) het egter ‘n groot invloed op luis-vrugbaarheid gehad en kan dus ‘n rol in Dn1-weerstand speel. Die moontlike weerstands gene, geïdentifiseer as nukleotied bindings proteïen (NBP) en weestandsgeen anoloog 2 (T10rga2-1D), is getoets onder luis-infestasie kondisies. Die analise van NBP het getoon dat dit ‘n integrale deel van die yster homeostase meganisme in die sitosol, wat vir Fe-kluster samestelling verantwoordelik is, vorm. Onderdrukking van beide die NBP en T10rga2-1D het tot die uitdrukking van ‘n vatbare fenotipe aanleiding gegee. T10rga2-1A is ‘n NBS-LRR proteïen wat bekend is om noodsaaklik te wees tydens roes weerstandigheid in teenwoordigheid van die weerstandsgeen Lr10. T10rga2-1D-onderdrukte behandelings het tot vatbaarheid aangeiding gegee en daartoe gelei dat plante na luis-infestasies doodgaan. Hierdie resultate dui dus ‘n rol vir T10rga2-1D in Dn1-weerstandigheid aan, en suggereer verder dat hierdie geen ‘n goeie stroom-op kandidaat in Dn1-weerstandigheid is.

Thesis (MSc (Genetics))--University of Stellenbosch, 2005.<br>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)--Stellenbosch University, 2014.<br>ENGLISH ABSTRACT: Global food security is hampered by a variety of insects/pest and plant diseases. In wheat, the Russian wheat aphid (RWA) is a significant pest problem in many areas of the world. Wheat has developed defensive mechanisms against the RWA over time which are activated upon feeding. One such mechanism is the hypersensitive response (HR) which is effective against phloem-feeding insects i.e. D. noxia (Diuraphis noxia, Kurdjumov, RWA). In this study, two genes associated with the hypersensitive response i.e. ascorbate peroxidase (APX) and glutathione S transferase (GSTF6b) were investigated to elucidate their function in the defensive mechanism of wheat using a reverse genetic approach i.e. particle bombardment. This study has succeeded in the established of a tissue culture and transformation system which generated three genetically modified wheat plants with decreased resistance to RWA feeding due to gene silencing. The establishment of this system enabled to test the association of defensive related genes in wheat to RWA resistance. Expression analysis performed on obtained transgenics before and after RWA infestation reavealed that the silenced plants were more susceptible to RWA feeding. Chlorosis was observed in the Gamtoos-S-APX transgenic plant which is an indicator of oxidative damage to the photosynthetic machinery of the plant. Decreased GSTF6b transcripts was found in the transgenic Gamtoos-S-GSTF6b and transgenic Gamtoos-R-GSTF6b transgenic plants but no visible symptoms of infestation was observed in these two plants. Resistance breeding could be strengthened by developing broad spectrum resistance plants by incorporating wheat defensive related genes with known function into the breeding programs. The use of this transformation system will allow rapid identification and introduction of agronomically important genes by upregulating these genes to enhance bread wheat against aphid infestation.

This study covered three aspects, namely; assessment of genetic diversity for rust resistance genes among released cultivars, inheritance of adult plant resistance (APR) in two North American wheat cultivars and genetic association between the stern rust resistance gene Sr3 9 and the stripe rust resistance gene YrSp. Queensland and northern New South Wales cultivars carried more diverse gene combinations in comparison to rest of the nation. Genetic diversity for all three rust diseases is a concern in Victoria and South Australia. There is need to release cultivars carrying combinations of resistance genes against three rust diseases. This will ensure environmentally safe control of rust diseases. Cultivars Jagger carried three genes for APR to stripe rust. Combinations of these genes provide high levels of resistance. Isolation of these genes individually was initiated and molecular mapping will determine identities of these genes. American workers detected the presence of Yr] 7 in at least one source of Jagger. Based on marker genotyping, the Jagger source used in this study did not carry Yr] 7. Cultivar Katepwa carried a single APR gene for stripe rust resistance. It is likely to be located on chromosome 2D. Repulsion linkage between the Triticum speltoides-derived stem rust and leaf rust genes Sr39/Lr35 and the stripe rust resistance gene YrSp was demonstrated. A recombination faction of 7.7:tl.5 cM between these genes was computed. These results refuted the previous location of YrSp in the short arm of chromosome 2B. Comparative results from other sources suggested the location of YrSp in the long arm of chromosome 2B. Progenies from three recombinant genotypes (Sr39Sr39YrSpyrSp) would enable the isolation of Sr39, Lr35 and YrSp in a single genotype. Such triple rust resistant genotype will be useful in improving rust resistance in future wheat cultivars.

Thesis (PhD)--Stellenbosch University, 2015<br>ENGLISH ABSTRACT : Diuraphis noxia Kurdjumov (Russian Wheat Aphid; RWA) is a pest of wheat and barley that has spread from its home range in the fertile crescent to most wheat producing countries except Australia. Since its first introduction to South Africa and the USA in the late 20th century, breeding programs for wheat phenotypes resistant to the aphid were put in place. Conventional breeding practices rely on phenotypic screening to verify traits carried by offspring and genetic tools such as marker assisted selection (MAS) have greatly aided this process in speed and accuracy. The size and complexity of the wheat genome, its allopolyploid nature and repetitive elements have, however, posed a challenge to studies on the genetics of this cereal crop. Many studies have focused on chromosome 3B which is the largest of the wheat chromosomes and easily separated from the redundant genomic background by techniques such as flow cytometry. The similarity in size of the remaining chromosomes however, limits the application of flow cytometry to their isolation. Databases such as Grain-Genes (http://wheat.pw.usda.gov/GG2/index.shtml) house marker data from various mapping studies for all wheat chromosomes and in 2014 the International Wheat Genome Sequencing Consortium (IWGSC) completed the draft genome sequence of wheat categorized by chromosome. Sources of resistance (Dn resistance genes) against RWA are located on chromosome 7D. but despite the marker and sequence data available currently, mapping studies specific for the Dn resistance genes are few. Additionally, sequence data available is derived from cultivars susceptible to RWA and is not comprehensively annotated and assembled in many cases. In this study, we demonstrate a novel, combined approach to isolate and characterize the Dn resistance genes through the use of a genetic map constructed from Amplified Fragment Length Polymorphism (AFLP), Expressed Sequence Tag (EST) and microsatellite markers and a physical map constructed from Next Generation Sequencing (NGS) data of ditelosomic chromosomes (7DS and 7DL) isolated by microdissection on the PALM microbeam system. A 122.8 cM genetic map was produced from 38 polymorphic AFLP markers and two ESTs with the microsatellite Xgwm111 as anchor to related genetic maps. Through comparison to maps available on GrainGenes the location of the Dn1 resistance gene was narrowed down to a deletion bin (7DS5-0.36-0.62) on the short arm of chromosome 7D with an AFLP marker (E-ACT/M-CTG_0270.84) mapping closely at 3.5 cM and two ESTs mapping at 15.3 cM and 15.9 cM from Dn1. Isolation of individual chromosome arms 7DS and 7DL using the PALM Microbeam system allowed sequencing of the chromosome without the redundancy of the remainder of the hexaploid genome. Through isolating the chromosome arms in this way, a >80-fold reduction in genome size was achieved as well as a major reduction in repetitive elements. Analysis of the sequencing data confirmed that 7DL is the physically shorter arm of the chromosome though it contains the majority of protein coding sequences.<br>AFRIKAANSE OPSOMMING : Diuraphis noxia Kurdjumov (Russiese koring-luis; RWA) is « pes wat op koring en gars voorkom. Die pes het vanaf sy tuiste in die midde Ooste na meeste koringproduserende lande behalwe Australië versprei. Sedert die eerste bekendstelling van RWA in Suid Afrika en die VSA in die vroeë 20ste eeu is teelprogramme ten gunste van koring lyne met weerstand teen RWA begin. Tradisionele teelprogramme maak op fisieise observasie van die fenotipe staat om te verifieer of plante in die nageslag die gewenste eienskap dra. Genetiese metodes soos merkerondersteunde seleksie (MAS) versnel hierdie selekteringsproses grootliks. Die grootte en kompleksiteit van die koring genoom asook die polyploïde en herhalende natuur daarvan is « groot hindernis vir genetiese studies van hierdie graangewas. Baie studies het op chromosoom 3B gefokus wat die grootste van die koring chromosome is en dus maklik vanaf die res van die oorbodige genomiese agtergond deur tegnieke soos vloeisitometrie geskei word. Die ooreenkoms in grootte tussen die res van die chromosome bemoeilik die toepassing van vloeisitometrie om hulle te isoleer. Databasisse soos GrainGenes (http://wheat.pw.usda.gov/GG2/index.shtml) bevat merker data vanaf verskeie karterings-studies vir al die chromosome en in 2014 het die "International Wheat Genome Sequencing Consortium"(IWGSC) die voorlopige basispaarvolgorde van die koring genoom bekendgestel, gekategoriseer volgens chromosoom. Weerstandsbronne (Dn weerstandsgene) teen RWA kom meestal op chromosoom 7D voor. Ten spyte van merker en basispaarvolgorde data tans beskikbaar is karterings-studies spesifiek tot die Dn gene skaars en basispaarvolgorde data is vanaf kultivars afkomstig wat nie weerstandbiedend teen RWA is nie en waarvan die annotasie en samestelling baie keer nie goed is nie. In hierdie studie demonstreer ons « nuwe, gekombineerde aanslag om die Dn weerstandsgene te isoleer en karakteriseer deur van « genetiese kaart opgestel met "Amplified Fragment Length Polymorphism"(AFLP), "Expressed Sequence Tag"(EST) en mikrosatelliet merkers asook « fisiese kaart saamgestel deur die volgende-generasiebasispaarvolgordebepaling van ditelosomiese chromosome (7DS en 7DL) geïsoleer deur mikrodisseksie met die "PALM Microbeam"sisteem gebruik te maak. « Genetiese kaart van 122.8 cM was met 38 polimorfiese AFLP merkers en twee EST merkers geskep. Die mikrosatelliet, Xgwm111, is ook ingesluit en het as anker vir verwante genetiese-kaarte gedien. Deur vergelyking met genetiese-kaarte op GrainGenes is die posisie van die Dn1 weerstandsgeen vernou na « delesie bin (7DS5-0.36-0.62) op die kort arm van chromosoom 7D met « AFLP merker (EACT/ M-CTG_0270.84) wat ongeveer 3.5 cM vanaf die geen karteer. Die twee EST merkers is 15.3 cM en 15.9 cM vanaf die geen gekarteer. Isolering van die individuele chromosoom arms, 7DS en 7DL, deur van die "PALM Microbeam"sisteem gebruik te maak het basispaarvolgordebepaling van die chromosoom toegelaat sonder die oortolligheid van die res van die hexaploïde genoom. Deur die chromosoom so te isoleer is « >80-maal verkleining in genoom grootte bereik insluitend « groot reduksie in herhalende elemente. Analise van die data vanaf basispaarvolgordebepaling het bevestig dat chromosoom 7D die fisiese kleiner chromosoom is maar dat dit die meerderheid van proteïn koderende basispaarvolgordes bevat.

Periodic evaluation of improvements in yield and disease resistance is necessary to assess breeding progress over time, and the elucidation of underlying traits responsible for yield gains can help direct future breeding. Objectives of this study were: 1) to determine the rate and magnitude of yield progress in eastern soft red winter (SRW) wheat (Triticum aestivum, L.) cultivars released from 1950 to 2009 relative to a historical cultivar Red May (1919) and; 2) to determine effects of leaf rust (Puccinia triticina f. sp. tritici) and powdery mildew [Blumeria graminis (DC.) E.O. Speer f. sp. tritici Em. Marchal] on grain yield components and agronomic traits. Replicated yield trials were grown at Warsaw, VA in 2010 and 2011, and at Holland and Blacksburg, VA in 2011. For objective 1, the genetic progress experiment: flag leaf angle, kernel weight, spikes m-2, lodging, flowering date and harvest index collectively explained the most yield variation in multiple environments on the basis of linear regression analysis. Rate of genetic yield improvement ranged from 0.56% yr-1 at Holland in 2011 to 1.4% yr-1 at Blacksburg in 2011. For objective 2, the disease loss experiment: yield losses ranged from 1% at Holland in 2011 to 21% at Warsaw in 2011. Losses primarily due to powdery mildew and leaf rust were as high as 14% and 33%, respectively. Powdery mildew had the largest negative correlation with harvest index and seeds spike-1, while leaf rust had the largest negative correlation with plant biomass and harvest index.<br>Master of Science

Thesis (MSc)--Stellenbosch University, 2012.<br>ENGLISH ABSTRACT: Plant diseases are among the major causes of food insecurity. In South Africa the wheat fungal diseases including stem rust caused by Puccinia graminis f. sp. tritici, leaf rust caused by P. triticina and stripe rust caused by P. striiformis f. sp. tritici are the most important. Genetic resistance is a viable way of protecting wheat crops against the wheat rusts, especially cultivars carrying multiple genes that confer durable resistance. In order to breed for multi-gene resistance an effective breeding strategy that allows for selecting multiple resistance genes and other desirable traits needs to be devised. The aim of this study was to identify a number of genotypes with combinations of different rust resistance genes, good grain yield and end-use quality out of an existing pre-breeding population and thereby identify superior parents. In order to achieve the stated aim the following objectives have been identified: identify wheat lines through marker-assisted selection (MAS) carrying the gene complexes, Sr31/Lr26/Yr9, Lr24/Sr24, Lr37/Sr38/Yr17, Lr34/Yr18 and Sr2; to develop inbred lines to evaluate selected lines under field trials. From the initial subset of 64 lines, 60 were chosen and advanced to the doubled haploid (DH) phase and seed multiplication. The 60 lines either carried one or more of the three rust resistance gene complexes. The genes that were the most prominent were Sr31/Lr26/Yr9 and Lr24/Sr24. The selected lines were incorporated into a DH seed multiplication phase. After 4 cycles of seed increases and preliminary field evaluation during multiplication, 15 lines were chosen and subjected to multi-location field trails. The extensive multi-location field trails carried out in this study aided in identifying genotypes from the 15 MS-MARS lines with good adaptability and stability in regards to yield and baking quality. An important observation was that the molecular markers employed to indentify quality loci correlated well with the genes encoding the HMW-GS 5, 10 and 12 as observed with the Agilent© 2100 Bioanalyzer. In future studies the lines which performed the best could be re-introduced into the existing MSMARS pre-breeding programme of the Stellenbosch University’s Plant Breeding Laboratory (SUPBL). The frequencies of desired alleles could be increased in this manner. Since the majority of these characteristics are influenced by quantitatively inherited alleles, using these lines as recurrent parents will increase the frequencies of these alleles in the existing SU-PBL pre-breeding population.<br>AFRIKAANSE OPSOMMING: Plantsiektes is van die belangrikste oorsake van voedselonsekerheid ter wêreld. In Suid-Afrika is die roesswamme van die belangrikste plantsiektes wat koring produksie beïnvloed. Hierdie siektes sluit in, stamroes wat veroorsaak word deur Puccinia graminis f. sp. tritici, blaarroes wat veroorsaak word deur P. triticina en streeproes wat veroorsaak word deur P. striiformis f. sp. tritici. Genetiese weerstand is ‘n uitstekende manier om koring te beskerm teen hierdie swamsiektes. Weerstand wat gebasseer is op veelvuldige weerstandsgene is veral ‘n goeie middel om genetieseweerstand op ‘n volhoubare basis in koringteling toe te pas. Om veelvuldige weerstandsgene in koringkultivars in te teel word ‘n effektiewe telingstrategie benodig. Die doel van die studie was om genotipes te identifiseer met kombinasies van veelvuldige weerstandsgene vir roes, sowel as goeie eienskappe belangrik vir graanopbrengs en bakkwaliteit. Lyne is geïdentifiseer uit ‘n bestaande voortelingspopulasie van Stellenbosch Universiteit se Planteteelt Laboratorium (SU-PTL) wat geteel was met spesifiek weerstand en opbrengs potensiaal in gedagte. Om die doel van die studie te bereik is sekere doelwitte daar gestel. Hierdie doelwitte sluit in om lyne uit die populasie te selekteer deur middel van merker bemiddelde seleksie (MBS) vir gene naamlik Sr31/Lr26/Yr9, Lr24/Sr24, Lr37/Sr38/Yr17, Lr34/Yr18 en Sr2; om die geselekteerde lyne suiwertelend te maak; sowel as om die suiwertelende lyne in veld proewe in te sluit. Van die oorspronklike stel van 64 lyne, is 60 gekies vir verdere studie. Deur middel van die verdubbelde haploïed (VH) tegniek is die lyne suiwertelend gemaak. Die 60 lyne het een of meer van die geselekteerde gene bevat. Die mees prominente gene was die twee geen komplekse Sr31/Lr26/Yr9 en Lr24/Sr24. Na vier siklusse van saadvermeerdering en voorloppige seleksies is 15 lyne ingesluit by ‘n multi-omgewing veldproef. Hierdie uitgebreide multi-omgewing veldproewe het gehelp om individue uit die 15 lyne te identifiseer wat oor goeie aanpasbaarheid en stabiliteit beskik met betrekking tot opbrengs en bak kwaliteit. Die molekulêre merkers gebruik om die gene verantwoordelik vir die kodering van HMGGS 5, 10 en 12 op te spoor het goed gekorreleer met die HMG-GS bande bepaal met behulp van die Agilent© 2100 Bioanalyzer. Toekomstige studies kan moontlik insluit die gebruik van die lyne wat geïdentifiseer was met goeie kenmerke in die bestaande MS-MARS teelprogram van die SU-PTL. Die frekwensies van die verlangde allele kan op hierdie manier in die populasie verhoog word.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.<br>Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were v identified: S8-introgression (Triticum dicoccoides) = one RAPD and two SCARs; S13-translocation (Aegilops speltoides) = four RAPDs, three RGAPs and five SCARs; S15-translocation (Ae. peregrina) = one RAPD and two SCARs; S20-translocation (Ae. neglecta) = two RAPDs, two RGAPs and one SCAR. The markers are already being employed in current projects aiming to map and shorten these translocations. Some of the markers can be combined in multiplex reactions for more effective mass screening. No repeatable markers could be identified for the four remaining translocations (S12 from Ae. sharonensis; S14 from Ae. kotschyi; Smac from Ae. biuncialis and Lr19-149-299 from Thinopyrum ponticum).

The research described in this týesis was focused on achieving a better understanding of the genetics of resistance of wheat to septoria tritici blotch (Mycosphaerella graminicola). Firstly, a detached leaf technique that could be a useful complement to field trials and an alternative to whole seedling assays in assessing cultivar resistance and investigating the genetics of the host-pathogen interaction was developed. Sets of inter-varietal substitution lines, developed at the John Innes Centre, involving known and possible sources of resistance to septoria tritici blotch, were tested with several single-pycnidium isolates in both the seedling and the adult plant stage. Two specific resistance genes were identified on chromosomes of `Synthetic 6x' and `Bezostaya 1'. A resistance gene, named Stb5, was identified using the M. graminicola isolate IP094269 and mapped on the short arm of chromosome 7D of `Synthetic 6x'. `Bezostaya I's specific resistance gene to IP0323 seems to be located in the same region as Stb6, and is indeed likely to be the same gene. `Bezostaya 1' and `Cappelle Desprez' also seemed to carry components for partial resistance. Triticum macha resistance to septoria tritici blotch on the other hand was both of a specific and isolatenon- specific nature. The specific components carried by T. macha seemed to be the Stb6 gene and an additional resistance gene, but it was not possible to identify their chromosomal location. To evaluate the relationship of heading date and plant height components to severity of septoria tritici blotch, an F6 single seed population of `Apollo' x `Thesee' was studied in natural conditions. Septoria tritici blotch levels were substantially lower in later-heading than in earlier-heading lines. Total plant height had comparatively little effect on disease severity, but increased distance between the two upper leaves increased disease levels. `Apollo' seems to carry partial resistance involving more than one QTL.

The development of wheat cultivars with unique quality traits is of major importance to the Australian wheat grower. Varietal diversity and flour quality of wheat are determined by genes and their products - proteins. Therefore, characterisation of the grain proteins will lead to discovery of potential protein markers for varietal identification and desirable quality attributes such as hardness, dough strength and extensibility. In order to screen the identified protein markers among thousands of breeder’s lines, specific high throughput detection methods are needed. In this study, a proteomics approach was applied to search for polymorphic grain proteins, and specific and high throughput antibody—based immunoassays were then developed. In order to increase resolution of inter-varietal polymorphic proteins, sub-fractionation (referred to as “de-complexing”) on the basis of protein solubility and multiple comparative analyses of hundreds of wheat varieties were carried out. A series of protein analytical techniques were used including native and denaturing one-dimensional polyacrylamide gel electrophoresis (PAGE), twodimensional gel electrophoresis (2-DE) and electrospray ionisation tandem mass spectrometry (ESl-MS/MS).

Thesis (MSc)--Stellenbosch University, 2014.<br>ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) commonly known as the Russian wheat aphid (RWA), is a small phloem-feeding pest of wheat (Triticum aestivum L). Virulent D. noxia biotypes that are able to feed on previously resistant wheat cultivars continue to develop and therefor the identification of factors contributing to virulence is vital. Since energy metabolism plays a key role in the survival of organisms, genes and processes involved in the production and regulation of energy may be key contributors to virulence: such as mitochondria and the NAD+/NADH that reflects the health and metabolic activities of a cell. The involvement of carotenoids in the generation of energy through a photosynthesis-like process may be an important factor, as well as its contribution to aphid immunity through mediation of oxidative stress. The complete mitochondrial genome of global Diuraphis noxia populations was characterised using Next Generation sequencing, and was found to be 15 721bp in size and consisting of 38 genes typically found within most insects. Single nucleotide polymorphism (SNP) analyses of the genomes of nine populations revealed 125 SNPs in the protein coding genes with the majority of the SNPs occurring in the ND genes, and the least in the ND4L gene. Low SNP variant frequency was found for the atp6 and atp8 genes, which differed from other reports in the Hemiptera. Variable ND5 expression levels were observed among the biotypes, although no correlation was apparent between ND5 expression and the virulence associated with each biotype. Whereas atp6 transcription was higher in the highly virulent biotype (SAM) under normal and stressful conditions in comparison to the least virulent biotype (SA1). A significantly higher NAD+/NADH ratio was also observed for the SAM biotype under stressful conditions in comparison to the lesser virulent biotypes. UPLC-MS analysis did not reveal any lycopene or β-carotene due to low compound concentrations in the extracted samples but various hydrophobic compounds were present in different concentrations among the biotypes. The carotene desaturase expression profile revealed that SA1 had the lowest relative expression of the gene involved in carotenoid products, while SAM had the highest, under normal and stressful conditions. The results indicate that sequence conservation in mitochondrial genes are associated with key energy processes to maintain a state of homeostasis under variable conditions and that the generation of energy is a contributing factor to the virulence development of D. noxia. The results also show that carotenoids may possibly contribute to fitness of D. noxia through reactive oxygen species scavenging or the production of additional energy, but further investigation is needed for confirmation.<br>AFRIKAANSE OPSOMMING: Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) algemeen bekend as die Russiese koringluis (RWA), is ‘n klein floëem-voedende pes van koring (Triticum aestivum L). Virulente D. noxia biotipes wat instaat is om op voorheen bestande koring kultivars te voed gaan ontwikkel voortdurend, en daarom is die identifisering van faktore wat kan bydrae tot virulensie so belangrik. Omdat energie-metabolisme ‘n sleutelrol in die oorlewing van organismes speel, kan gene en prosesse wat by die produksie en regulering van energie betrokke is belangrike bydraers tot virulensie lewer: soos onder andere mitokondria en die NAD+/NADH-verhouding wat die gesondheid en metaboliese aktiwiteit van ‘n sel reflekteer. Die betrokkenheid van karotenoïede in die produksie van energie deur 'n fotosintese-verwante proses kan 'n belangrike faktor bydraend tot luis fiksheid wees, asook die bydra daarvan tot plantluis-immuniteit deur bemiddeling van oksidatiewe stres. Die volledige mitochondriale genoom van globale Diuraphis noxia populasies is met behulp van volgende generasie DNA volgordebepaling gekarrakteriseer, en daar is bevind dat dit 15 721 bp in grootte is en uit 38 gene bestaan wat tipies binne insekte voorkom. Enkelnukleotied- polimorfisme (SNP) ontleding van die genome van nege populasies het onthul dat daar 125 SNPs in die proteïen-koderende gene voorkom, met die meerderheid van die SNPs in die ND-gene, en die minste in die ND4L-geen. Lae SNP-frekwensies is gevind vir die atp6- en atp8- gene, wat verskil van verslae oor ander Hemiptera. Veranderlike ND5-uitdrukkingsvlakke onder die biotipes is waargeneem, alhoewel geen korrelasie duidelik was tussen ND5-uitdrukking en die virulensie geassosieer met elke biotipe nie. Die transkripsie van atp6 was hoër in die hoogs virulente biotipe (SAM) onder normale en stresvolle toestande in vergelyking met die minste virulente biotipe (SA1). ‘n Aansienlike hoër NAD+/NADH-verhouding is ook waargeneem vir die SAM-biotipe onder spanningsvolle omstandighede in vergelyking met die minder virulente biotipes. UPLC-MS-analise het geen likopeen of β-karoteen geïdentifiseer nie as gevolg van lae verbinding konsentrasies in die onttrekte monsters, maar verskeie hidrofobiese verbindings was in verskillende konsentrasies tussen die biotipes teenwoordig. Die karoteen desaturase-uitdrukkingsprofiel het aangetoon dat SA1 die laagste relatiewe uitdrukking van gene betrokke by karotenoïed produksie het, terwyl SAM die hoogste relatiewe uitdrukking onder normale en spanningsvolle omstandighede het. Die resultate van die studie dui daarop dat die volgorde bewaring in mitochondriale gene verband hou met die sleutel energie prosesse om 'n toestand van homeostase onder wisselende omstandighede te handhaaf en dat die produksie van energie 'n bydraende faktor tot die ontwikkeling van virulensie in D. noxia is. Die resultate toon ook aan dat karotenoïede moontlik kan bydra tot fiksheid van D. noxia deur reaktiewe suurstofspesies te aas of deur die produksie van addisionele energie, maar verdere ondersoeke word benodig ter bevestiging.