Rhizoctonia blight (sensu lato) is a common and serious disease of many turfgrass species. The most widespread causal agent R. solani consists of several genetically different anastomosis groups (AGs) and subgroups. Though anastomosis or hyphal fusion reactions have been used to group Rhizoctonia species, they are time consuming and sometimes difficult to interpret. Anastomosis reactions are incapable of identifying isolates belonging to different AG subgroups within an AG. This study evaluated molecular techniques in comparison with traditional anastomosis grouping (AG) to identify and group isolates of Rhizoctonia. More than 400 Rhizoctonia isolates were collected from diseased turfgrass leaves from eight geographic areas in Virginia and Maryland. A random sample of 86 isolates was selected and initially characterized by colony morphology, nuclei staining and anastomosis grouping. Molecular identification was performed by analysis of rDNA-ITS region and DNA fingerprinting techniques universally primed PCR (UP-PCR) and amplified fragment length polymorphism (AFLP). The cladistic analysis of ITS sequences and UP-PCR fragments supported seven clusters. Isolates of R. solani AG 1-IB (n=18), AG 2-2IIIB (n=30) and AG 5 (n=1) clustered separately. Waitea circinata var. zeae (n=11), and var. circinata (n=4) grouped separately. A cluster of six isolates (UWC) did not fall into any known Waitea group. Most of the binucleate Rhizoctonia-like fungi (BNR) (n=16) grouped separately. AFLP grouping also largely agreed with the above results. However, UWC isolates clustered into two groups. Molecular analyses corresponded well with traditional anastomosis grouping by clustering isolates within an AG or AG subgroup together. UP-PCR cross-hybridization could distinguish closely related Rhizoctonia isolates to their infraspecies level. Genetically related isolates belonging to the same AG subgroups cross-hybridized strongly, while isolates of different AGs did not cross-hybridize or did so weakly. Sequence-characterized amplified region (SCAR) markers were generated from UP-PCR products to identify isolates of major pathogenic groups AG 1-IB and AG 2-2IIIB. Specific primer pairs successfully distinguished isolates of AG 1-IB and AG 2-2IIIB from isolates of other AGs. Sensitivity of Rhizoctonia species and AGs was tested in vitro to commercial formulations of iprodione, triticonazole and pyraclostrobin. W. circinata isolates were moderately sensitive to iprodione while isolates of R. solani and BNR were extremely sensitive. Isolates of AG 2-2IIIB showed less sensitivity to triticonazole than other Rhizoctonia isolates. W. circinata var. zeae isolates were moderately sensitive to pyraclostrobin while most of the other isolates were extremely sensitive. / Ph. D.

Resumo: A seleção assistida por marcadores moleculares têm contribuído com os estudos para o desenvolvimento de cultivares resistentes. Os ganhos mais evidentes podem ocorrer em doenças como a ferrugem asiática da soja, onde a alta variabilidade do patógeno e a busca por novas fontes de resistência têm dificultado o sucesso dos melhoristas. Assim, os objetivos deste trabalho foram estudar a herança da resistência à ferrugem em diferentes fontes e desenvolver marcadores SCAR ligados a um loco de resistência para seleção assistida. Populações F2 oriundas dos cruzamentos PI 459025 x CD 208 (1), PI 200526 x CD 205 (2), PI 200456 x Conquista (3) e GC 84058-21-4 x IAC Foscarin 31 (4) foram submetidas à inoculação com a ferrugem e avaliadas quanto ao tipo de lesão (RB - resistente ou TAN - suscetível). O teste de Qui-quadrado indicou a presença de um gene dominante para os cruzamentos 1 e 2, enquanto no 3 e 4 observou-se a presença de um gene recessivo. A análise multivariada agrupou os genótipos mais similares, com base nos caracteres severidade da doença, número de vagens, número de sementes e ciclo, avaliados nos cruzamentos 2 a 4. Diferenças significativas foram encontradas em número de lesões e esporulação para genótipos RB e TAN. Dois bulks de DNA foram obtidos de plantas homozigotas resistentes e suscetíveis no cruzamento 1, para a análise de BSA. Dos 600 iniciadores RAPD testados, três foram discriminativos e localizam-se a 4,5, 6,2 e 10,2 cM do loco de resistência, em fase de repulsão. Um desses marcadores foi convertido a SCAR e se manteve discriminativo, podendo ser indicado para seleção assistida de genótipos resistentes com a mesma fonte. / Abstract: Assisted selection by molecular markers has contributed to development studies of resistant cultivars. The most evident gains may occur for diseases like the Asian soybean rust, where the high variability of pathogen and the search for new resistance sources has difficult the breeder success. Thus, the aims of this work were to study the rust resistance inheritance in different sources and develop SCAR markers linked to a rust resistance locus for assisted selection. F2 populations from crosses PI 459025 x CD 208 (1), PI 200526 x CD 205 (2), PI 200456 x Conquista (3) and GC 84058-21-4 x IAC Foscarin 31 (4) were submitted to rust inoculation and evaluated by the lesion type reaction (RB - resistant or TAN - susceptible). Chi-square test indicated the presence of a single dominant gene for crosses 1 and 2, whereas the 3 and 4 crosses showed a single recessive gene. Multivariate analysis has grouped the most similar genotypes based on disease severity, number of pods, number of seeds and cycle for 2 to 4 crosses. Significant differences were observed in lesions number and sporulation rate for RB and TAN genotypes. Two DNA bulks were obtained, for BSA analysis, on cross 1, from resistant and susceptible homozygous plants. From the 600 tested RAPD primers, three of them were discriminative and located in repulsion phase at 4.5, 6.2 and 10.2 cM from the resistance locus. One of the RAPD markers was converted to SCAR and confirmed its discriminative nature. So it can be indicated for assisted selection of genotypes with the same resistance source. / Orientador: Antonio Orlando Di Mauro / Coorientador: José Baldin Pinheiro / Coorientadora: Sandra Helena Unêda Trevisoli / Banca: Eduardo Antonio Gavioli / Banca: Natal Antonio Vello / Banca: Janete Apparecida Desidério Sena / Banca: Rinaldo César de Paula / Doutor

Made available in DSpace on 2014-06-11T19:32:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-08Bitstream added on 2014-06-13T19:42:47Z : No. of bitstreams: 1 costa_mm_dr_jabo.pdf: 539143 bytes, checksum: 38e4165c8bd5de4a8cf35184bd7ea3a7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A seleção assistida por marcadores moleculares têm contribuído com os estudos para o desenvolvimento de cultivares resistentes. Os ganhos mais evidentes podem ocorrer em doenças como a ferrugem asiática da soja, onde a alta variabilidade do patógeno e a busca por novas fontes de resistência têm dificultado o sucesso dos melhoristas. Assim, os objetivos deste trabalho foram estudar a herança da resistência à ferrugem em diferentes fontes e desenvolver marcadores SCAR ligados a um loco de resistência para seleção assistida. Populações F2 oriundas dos cruzamentos PI 459025 x CD 208 (1), PI 200526 x CD 205 (2), PI 200456 x Conquista (3) e GC 84058-21-4 x IAC Foscarin 31 (4) foram submetidas à inoculação com a ferrugem e avaliadas quanto ao tipo de lesão (RB – resistente ou TAN – suscetível). O teste de Qui-quadrado indicou a presença de um gene dominante para os cruzamentos 1 e 2, enquanto no 3 e 4 observou-se a presença de um gene recessivo. A análise multivariada agrupou os genótipos mais similares, com base nos caracteres severidade da doença, número de vagens, número de sementes e ciclo, avaliados nos cruzamentos 2 a 4. Diferenças significativas foram encontradas em número de lesões e esporulação para genótipos RB e TAN. Dois bulks de DNA foram obtidos de plantas homozigotas resistentes e suscetíveis no cruzamento 1, para a análise de BSA. Dos 600 iniciadores RAPD testados, três foram discriminativos e localizam-se a 4,5, 6,2 e 10,2 cM do loco de resistência, em fase de repulsão. Um desses marcadores foi convertido a SCAR e se manteve discriminativo, podendo ser indicado para seleção assistida de genótipos resistentes com a mesma fonte. / Assisted selection by molecular markers has contributed to development studies of resistant cultivars. The most evident gains may occur for diseases like the Asian soybean rust, where the high variability of pathogen and the search for new resistance sources has difficult the breeder success. Thus, the aims of this work were to study the rust resistance inheritance in different sources and develop SCAR markers linked to a rust resistance locus for assisted selection. F2 populations from crosses PI 459025 x CD 208 (1), PI 200526 x CD 205 (2), PI 200456 x Conquista (3) and GC 84058-21-4 x IAC Foscarin 31 (4) were submitted to rust inoculation and evaluated by the lesion type reaction (RB – resistant or TAN – susceptible). Chi-square test indicated the presence of a single dominant gene for crosses 1 and 2, whereas the 3 and 4 crosses showed a single recessive gene. Multivariate analysis has grouped the most similar genotypes based on disease severity, number of pods, number of seeds and cycle for 2 to 4 crosses. Significant differences were observed in lesions number and sporulation rate for RB and TAN genotypes. Two DNA bulks were obtained, for BSA analysis, on cross 1, from resistant and susceptible homozygous plants. From the 600 tested RAPD primers, three of them were discriminative and located in repulsion phase at 4.5, 6.2 and 10.2 cM from the resistance locus. One of the RAPD markers was converted to SCAR and confirmed its discriminative nature. So it can be indicated for assisted selection of genotypes with the same resistance source.

Sunflower (Helianthus annuus L.) high oleic acid content lines differ from conventional sunflower by an increase in oleic acid (C18:1) content of more than 60%. The current sunflower cultivars under production in South Africa are standard sunflower with high levels of linoleic acid (C18:2). The aim of this study was to improve the quality of oil produced by local sunflower germplasm with respect to oleic acid through employing a marker-assisted breeding technique to facilitate and speed up the recovery of the high oleic acid allele into the background of the recurrent parent genome. Eleven sunflower breeding genotypes with high and low oleic acid traits were obtained from the Agricultural Research Council-Grain Crops Institute (ARC-GCI) in Potchefstroom. The breeding genotypes were phenotypically characterised based on their oleic and linoleic acid levels using gas chromatography. Results demonstrated that the average mean of oleic and linoleic acid contents in high oleic acid genotypes were 72% and 17% respectively, while the average mean of oleic acid and linoleic acid contents in wild type lines were 33.5 % and 54 % respectively. These results indicated a perfect negative correlation between the amount of oleic and linoleic acids possessed in high and low oleic acid genotypes (R2 = -99.16%). Sequence characterised amplified region (SCAR) markers were tested to ascertain if any of the ten available dominant FAD2-1 markers was segregating with the high oleic acid allele. Four dominant SCAR markers (FAD2-1F4/R1; FAD2-1F4/R2; FAD2-1F13/R1; FAD2-1F14/R2) were strongly associated with the high oleic acid trait (P< 0.001). With regard to the inheritance of the high oleic acid trait, 143 plants of the F2 segregating population derived from a cross between the high oleic acid parent (AP901-95-3-4-1) and low oleic acid parent (H55-9-2-1-1) were genotyped with the four SCAR markers to determine the genetic state concerning the high oleic acid gene (Ol). Results from a Chi square analysis of the observed frequencies of each dominant FAD2-1 marker locus in 143 F2 individuals indicated that the deviation from the expected ratio of 3:1 (high to low oleic acid) was not statistically significant (P< 0.95) from the observed segregation ratio. These results were consistent with the previous finding that an incomplete dominant gene governs sunflower high oleic acid. A multiplex assay of 78 Simple sequence repeat (SSR) markers was optimised and evaluated on 143 plants of the F2 population to determine suitable SSR markers that can be used in a marker-assisted background selection. Only 14 markers were suitable for marker-assisted background selection based on their high polymorphic information content, allele frequency and maximum allele numbers. In conclusion, this study demonstrated the potential of using SSR and SCAR marker systems as a breeding tool to characterise and speed up the selection process in marker-assisted backcross breeding. / Thesis (MSc (Botany))--North-West University, Potchefstroom Campus, 2013.

Sunflower (Helianthus annuus L.) high oleic acid content lines differ from conventional sunflower by an increase in oleic acid (C18:1) content of more than 60%. The current sunflower cultivars under production in South Africa are standard sunflower with high levels of linoleic acid (C18:2). The aim of this study was to improve the quality of oil produced by local sunflower germplasm with respect to oleic acid through employing a marker-assisted breeding technique to facilitate and speed up the recovery of the high oleic acid allele into the background of the recurrent parent genome. Eleven sunflower breeding genotypes with high and low oleic acid traits were obtained from the Agricultural Research Council-Grain Crops Institute (ARC-GCI) in Potchefstroom. The breeding genotypes were phenotypically characterised based on their oleic and linoleic acid levels using gas chromatography. Results demonstrated that the average mean of oleic and linoleic acid contents in high oleic acid genotypes were 72% and 17% respectively, while the average mean of oleic acid and linoleic acid contents in wild type lines were 33.5 % and 54 % respectively. These results indicated a perfect negative correlation between the amount of oleic and linoleic acids possessed in high and low oleic acid genotypes (R2 = -99.16%). Sequence characterised amplified region (SCAR) markers were tested to ascertain if any of the ten available dominant FAD2-1 markers was segregating with the high oleic acid allele. Four dominant SCAR markers (FAD2-1F4/R1; FAD2-1F4/R2; FAD2-1F13/R1; FAD2-1F14/R2) were strongly associated with the high oleic acid trait (P< 0.001). With regard to the inheritance of the high oleic acid trait, 143 plants of the F2 segregating population derived from a cross between the high oleic acid parent (AP901-95-3-4-1) and low oleic acid parent (H55-9-2-1-1) were genotyped with the four SCAR markers to determine the genetic state concerning the high oleic acid gene (Ol). Results from a Chi square analysis of the observed frequencies of each dominant FAD2-1 marker locus in 143 F2 individuals indicated that the deviation from the expected ratio of 3:1 (high to low oleic acid) was not statistically significant (P< 0.95) from the observed segregation ratio. These results were consistent with the previous finding that an incomplete dominant gene governs sunflower high oleic acid. A multiplex assay of 78 Simple sequence repeat (SSR) markers was optimised and evaluated on 143 plants of the F2 population to determine suitable SSR markers that can be used in a marker-assisted background selection. Only 14 markers were suitable for marker-assisted background selection based on their high polymorphic information content, allele frequency and maximum allele numbers. In conclusion, this study demonstrated the potential of using SSR and SCAR marker systems as a breeding tool to characterise and speed up the selection process in marker-assisted backcross breeding. / Thesis (MSc (Botany))--North-West University, Potchefstroom Campus, 2013.