Call number: LD2668 .T4 1933 B41 / Master of Science

Call number: LD2668 .T4 1985 B68 / Master of Science

Thesis (PhDAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Maize is undoubtedly South Africa's most important field crop. The identification of markers and genes for traits of interest is important to sustain the improvement of maize cultivation. Northern corn leaf blight (NClB) is a disease that occurs worldwide and can dramatically reduce yield. A number of single dominant resistance genes have been identified for NClB and some have been mapped. Currently there are no simple PCR markers for any of these resistance genes, making markerassisted selection (MAS) difficult. The aim of this study was to develop PCR markers for the NClB resistance genes Ht1, Ht2, Ht3 and Htn1 in maize. To accomplish this, the AFlP (amplified fragment length polymorphism) technique was first optimised. The results indicated that the Mlul/Msel restriction enzyme combination produces a higher percentage of polymorph isms when compared to the PstllMsel enzyme combination. It was also shown that the enzyme combination plays an important role in the percentage of polymorphic fragments observed, whereas the number of restriction enzymes used in AFlP analysis only significantly affects the total number of fragments scored. Populations segregating for the different resistance genes were not available for this study. Nearly-isogenic lines (Nils) were used in combination with AFlP technology to identify markers that map close to the genes. AFlP markers common in at least two resistant or susceptible lines were cloned and converted to PCR markers. Two commercially available recombinant inbred line (Ril) populations were then used to map the identified markers. For Htn1 fifteen polymorphic fragments were present in both resistant lines. They were selected for sequence specific marker conversion. Seven of the fifteen sequence characterized amplified region (SCAR) markers were polymorphic on the Nil pairs and five mapped to one region of maize chromosome 8.05/06. Twenty-one AFlP markers were identified for Ht1 and four SCAR markers were polymorphic In the Ht1 Nils. Three of these were mapped to chromosome 2.07. Three AFlP markers were identified for Ht2 of which two were converted to SCAR markers. Both SCAR markers were polymorphic on the Ht2 Nils and mapped to chromosome 8.05/06. On the Ht3 NILs, four AFLP markers were identified and two converted SCAR markers and one microsatellite marker (bnlg1666) were polymorphic. One of the SCAR markers and the microsatellite marker were mapped to chromosome 7.04 using a RIL population. This reports the first tentative mapping position for the Ht3 locus. The next step was to determine if a set of marker alleles could be used in a number of Htn 1 resistance lines to identify a common donor region selected by the breeders. Nine markers consisting of five SCAR markers, three converted RFLP markers and one microsatellite marker were used on 16 Htn1 resistant lines. The marker allele of us3 was in 12 of the 16 lines in coupling with Htn1 resistance. Second was the marker us5 in 11 of the 16 lines. Using this data 14 of the 16 lines shared a common introgressed region between the markers us3 and us5. A further common introgressed region between 11 of the inbred lines was found between the markers us14 and asg17. The last aim of this study was to propose a new marker technique that might be more successful than the AFLP technique in the identification of markers closely linked to genes. A new marker approach was identified where a MITE (Hbr) primer was used as an anchor primer in combination with resistance gene analog primers. This was found to be a highly polymorphic marker technique that could be used to identify markers and possibly candidate genes. It is a robust technique, which is affordable since amplifications occur from undigested genomic DNA and the primers mainly amplify fragments from genic regions. / AFRIKAANSE OPSOMMING: Mielies (Zea mays) is ongetwyfeld Suid Afrika se belangrikste lanbou gewas. Vir volgehoue opbrengs verbetering is die identifisering van merkers en gene vir belangrike eienskappe noodsaaklik. Noordelike blaarskroei (NBS) kan opbrengs wesenlik kan beïnvloed. Tans is daar reeds "n aantal enkel weerstandsgene geïdentifiseer, maar geen PKR-merkers is beskikbaar vir merker gebaseerde seleksie nie. Die doelwit van hierdie studie was om PKR-merkers te ontwikkel vir vier enkel weerstands gene (Ht1, Ht2, Ht3 en Htn1) teen NBS in mielies. Om die doelstelling te bereik is die AFLP-tegniek eers geoptimiseer. Op grond van waargenome aantal polimorfismes, was Mlul/Mse/"n beter restriksie ensiem kombinasie as Pstl/Msel. In die studie is ook bewys dat die aantal (meer as twee) restriksie ensieme wat gebruik word slegs die aantal fragmente, en nie die persentasie polimorfismes, wesenlik beïnvloed nie. Geen segregerende populasie was vir die verskillende gene beskikbaar nie. Naby isogeniese lyne (NILe) is daarom in kombinasie met die AFLP-tegniek gebruik om merkers te identifiseer wat naby die gene karteer. Alleenlik polimorfiese merkers wat in ten minste twee weerstand biedende of vatbare lyne voorgekom het, is gekloneer en omgeskakel na PKR-merkers. Daarna is twee kommersiële rekombinante ingeteelde lyn populasies gebruik om die gene te karteer. Vyftien fragmente is gevind wat gekoppel was met die Htn1 weerstand. Sewe van hierdie merkers is omgeskakel in polimorfiese SCAR-merkers waarvan vyf gekarteer is in een gebied op chromosoom 8.05/06. Een-en-twintig AFLP-merkers is geïndentifiseer vir Ht1 en vier is omgeskakel na polimorfiese SCAR-merkers. Drie hiervan is gekarteer op chromosoom 2.07. Drie AFLP-merkers is geïndetifiseer vir Ht2 waarvan 2 omgeskakel is na polimorfiese SCAR-merkers. Altwee hierdie merkers is gekarteer op chromosoom 8.05/06. Op die Ht3 lyne is vier AFLP-merkers geïdentifiseer waarvan twee omgeskakel is na polimorfiese SCAR-merkers. Een mikrosatelliet merker (bnlg1666) is ook gevind wat die selfde polimorfiese patroon wys op die Ht3 lyne. Die mikrosateliet en een van die SCAR-merkers het gekarteer op chromosomale posisie 7.04. Hierdie is die eerste tentatiewe posisie vir die Ht3 lokus. Die volgende stap was om te bepaal of "n stel polimorfiese merker-allele gebruik kan word om die donor DNA-segment te identifiseer wat die plantteiers geselekteer het. Nege PKR-merkers wat bestaan het uit vyf SCAR-merkers, 3 omgeskakelde RFLP merkers en een mikrosateliet is gebruik op 16 Hnt1 weerstandslyne. Us3 was die merker alleel wat in die meeste gevalle gekoppel was met die Htn1 weerstandslyne (12/16). Tweede was die merker us5 (in 11 van die 16 lyne). Uit die data blyk dit dat 14 van die 16 lyne "n donor segment het wat beide merkers us3 en us5 bevat. Merkers us14 en asg17 het in 11 van die 16 bestande lyne saam voorgekom. Die laaste doelstelling van hierdie studie was om "n nuwe tegniek te ontwikkel wat dalk meer suksesvol as AFLPs kan wees om merkers te identifiseer nabyaan gene. "n Nuwe tegniek word voorgestel waar "n MITE (Hbr) inleier gebruik kan word in kombinasie met weerstandgeen-analoog inleiers. Dit is gevind dat hierdie kombinasie van inleiers "n hoogs polimorfiese band patroon gee en dat die merkers ook dalk kandidaat-gene kan wees. Die tegniek is maklik uitvoerbaar, relatief goedkoop en maak gebruik van onverteerde genomiese DNA. Die fragmente wat geamplifiseer word is hoofsaaklik vanaf geenryke areas.

Call number: LD2668 .T4 1986 K84 / Master of Science / Grain Science and Industry

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.

Includes bibliographical references (l5 leaves) The aim of the work presented in this thesis was to develop molecular genetic markers for YD2 (the gene in barley which provides protection against barley yellow dwarf luteovirus) which could be used for the marker assisted selection of the gene in breeding programs and enable the gene to be cloned via a map-based approach.

Graduation date: 2002

Molecular tools, coupled with unique germplasm stocks and rigorous phenotyping, are useful for developing a better understanding of qualitative and quantitative disease resistance genes in plants. The identification of molecular markers linked to all types of resistance genes provides opportunities for implementing a range of resistance breeding strategies, ranging from gene pyramiding to gene deployment. This thesis consists of two chapters. The first describes a disease resistance gene mapping effort and the second describes a disease resistance gene introgression effort. The number, location, and effects of genes determining resistance to stripe rust, leaf rust and Barley Yellow Dwarf Virus were determined using a population of doubled haploid (DH) lines from the cross of Shyri x Galena. Resistance to leaf rust was qualitatively inherited, and the locus was mapped to the long arm of chromosome 1. Resistance to stripe rust and BYDV was quantitatively inherited. Multiple QTLs were detected for each type of resistance. The principal stripe rust resistance QTL was on the short arm of chromosome 5 and the principal BYDV resistance QTL was on the long arm of chromosome 1, linked in repulsion phase with the leaf rust resistance gene. Additional QTLs and QTL x QTL interactions were detected. The majority of the qualitative and quantitative resistance loci detected in the Shyri x Galena population coincided with Resistance Gene Analog Polymorphisms (RGAPs) mapped in the same population. These RGAPs were based on degenerate primers derived from cloned resistance gene sequence motifs. These associations should be useful for efficient resistance gene mapping and provide an approach for ultimately isolating and describing quantitative and qualitative resistance genes. The second chapter describes a molecular marker assisted selection (MMAS) effort to introgress stripe rust resistance QTLs on chromosomes 4 and 7 into susceptible germplasm. DH lines were derived form a MMAS backcross-one (BC-1) population, extensively phenotyped for stripe rust resistance, and genotyped for the introgressed QTLs and background genome. The resistance QTLs that were introgressed were significant determinants of resistance in the new genetic background. Additional resistance QTLs were also detected. Together, these chapters describe an integrated approach to disease resistance gene characterization and utilization. / Graduation date: 1999

The level of polymorphisms of many biochemical and DNA markers are low in cultivated sunflower (Helianthus annuus L.). The number of mapped public DNA markers is limited. Molecular markers have not been developed for the most important diseases of sunflower, such as downy mildew. The objectives of this study were (i) to help alleviate the problem of low DNA marker polymorphisms by developing simple sequence repeat (SSR) markers, (ii) to build an integrated AFLP-RFLP linkage map by using previously described probes and newly developed AFLPs, and (iii) to clone and characterize candidate disease resistance genes. Forty-four polymorphic SSR markers were developed from a genomic DNA library. Diversity analysis of these SSRs for variability among 10 public inbred lines produced an average of 1.86 alleles per locus and mean heterozygosity of 0.21. The number of alleles ranged from 1 to 5. Trinucleotide SSRs were less polymorphic than dinucleotide and mononucleotide SSRs. Cluster analysis and multidimensional scaling separated elite inbred lines from wild species. There was more polymorphism in wild species than in elite lines. Three hundred and six AFLP markers were developed using 18 primer combinations. Two sets of previously mapped RFLP markers were tested for segregation in an F��� mapping population. A total of 401 markers were assigned to 17 linkage groups covering 1326 cM with a mean spacing of 3.3 cM between adjacent markers. The RFLP markers were well spaced and well distributed throughout the genome. Some linkage groups are sparsely populated with common markers. There were two gaps of 30 or more cM in two linkage groups. We cloned candidate disease resistance genes for downy mildew resistance based on sequence homology among resistance genes in other species. Eleven unique nucleotide binding sequence (NBS) containing clones were isolated and showed similarity to the corresponding domains of cloned disease resistance genes in other plant species. Seven clones mapped to four linkage groups and identified nine loci. A cleaved amplified polymorphic sequence (CAPS) marker that was 3.7 cM from the Pl1 resistance gene was developed by analysis of NILs. This CAPS marker should facilitate marker-assisted selection in sunflower. / Graduation date: 1999

Strawbreaker foot-rot is a major limiting factor to cost efficient winter wheat production in the Pacific Northwest. Development of resistant cultivars has been hindered by the lack of adequate levels of genetic resistance and screening techniques which can consistently detect desired genotypes. Studies were conducted to determine if the reported strawbreaker foot-rot resistance of the cultivar "Rendezvous" is effective on isolates of Pseudocercosporella herpotrichoides found in the Pacific Northwest. Protected, naturally infected and artificially inoculated treatments were employed to determine the level of resistance of 10 cultivars including Rendezvous. Different concentrations of inoculum and stages of development were also used to determine if observations on leaf sheath penetration of seedlings obtained in the greenhouse were related to disease severity index readings taken in the field for selected cultivars. In addition, the nature of inheritance of strawbreaker foot-rot was studied in two crosses involving Rendezvous. Experiments were conducted at three locations and over two years at one location. Despite cultivar x treatment interaction, consistent levels of infection were observed in all experiments at each location. Significant differences were found for treatments and cultivars for most attributes. Yield losses, including the components of yield spikes per square meter, 1000 kernel weight, and kernel number per spike were proportional to the severity of the disease. Losses were greater when lodging occurred, which was also associated with disease severity. However, even in the absence of lodging losses were recorded in the naturally and artificially inoculated plots. Traits measured involving Rendezvous and Vpm/Mos 95//*2Hill were only slightly influenced by the treatments. Under greenhouse conditions, it was possible to distinguish the level of resistance of Rendezvous from susceptible cultivars at concentrations of 100 spores/ml, two weeks after inoculation at the seedling stage. Leaf sheath penetration of seedlings was found to be closely associated with the disease severity index obtained under field conditions. Generation means analysis performed in crosses involving Rendezvous indicated that additive and additive x additive gene action were responsible for most of the genetic variability associated with resistance. Narrow-sense heritability estimates also confirmed these fmdings. It would appear that Rendezvous has at least two major genes for resistance to strawbreaker foot-rot. / Graduation date: 1992