Wheat grain quality and consumption is influenced by its constituents structure and concentrations. In the first part of the dissertation, six Canadian bread wheat cultivars; four (CDC Teal, AC Superb, AC Barrie, AC Splendor) belonging to the Canada Western Red Spring (CWRS), and two (AC Foremost, and AC Crystal) to the Canada Prairie Spring Red (CPSR) market classes were characterized for the relationship between their starch constituents and starch in vitro enzymatic hydrolysis. CPSR cultivars with relatively longer amylopectin chains of DP 37-45, reduced chain lengths of DP 15-18, and a low volume percent of small C-type starch granules, had reduced starch in vitro enzymatic hydrolysis rates. In the second part of the dissertation, near-isogenic wheat lines differing at the Waxy locus were analyzed for the influence of genome-specific granule-bound starch synthase I (GBSSI/Waxy; Wx-A, Wx-B, Wx-D) on starch composition, structure and starch in vitro enzymatic hydrolysis. Amylose concentration was more severely affected in genotypes with GBSSI missing from two genomes (double nulls) than from one genome (single nulls) of wheat, indicating dosage dependent amylose synthesis. Subtle differences in amylopectin chain length distribution were observed among non-waxy, partial and completely waxy starches, suggesting a non-limiting role of genome-specific GBSSI for amylopectin synthesis. A suppressive role of Wx-D on the short chain phenotype of wheat amylopectin was observed. In addition, Wx-D increased the volume percentage of large A-type starch granules and reduced starch hydrolysis index. Thus, among the waxy isoproteins, Wx-D might be the major contributor for reducing the rate of in vitro starch enzymatic hydrolysis in wheat. In the third part of the dissertation, endosperm starch’s physicochemical properties and structure during grain development in wheat waxy-null genotypes were analyzed. The study was conducted with pure starch isolated from wheat grains at 3-30 days post anthesis (DPA), at three day intervals. Changes in amylopectin structure were observed until 12 DPA, suggesting the formation of a basic amylopectin skeleton by this stage. A differential influence of waxy isoproteins on amylopectin structure formation has been suggested, with Wx-B and Wx-D affecting short glucan chains of DP 6-8 at 3 and 6 DPA, Wx-A being effective at 9 and 12 DPA, and Wx-D affecting DP 18-25 chains from 18-30 DPA.

Soybean (Glycine max [L.] Merr.) cultivars differ in their resistance to sudden death syndrome (SDS). The syndrome is caused by root colonization by Fusarium virguliforme (ex. F. solani f. sp. glycines). Breeding for improve SDS response has proven challenging, possible due to interactions among the 18 known loci for resistance. Four loci for resistance to SDS (cqRfs to cqRfs3) were found clustered within 20 cM of the rhg1 locus underlying resistance to soybean cyst nematode (SCN) on chromosome 18. Another locus on chromosome 20 (cqRfs5) was reported to interact with this cluster. The aims of this study were to compare the inheritance of resistance to SDS in a near isogenic line (NIL) population that was fixed for resistance to SCN but still segregated at 2 of the 4 loci (cqRfs1 and cqRfs) for resistance to SDS on chromosome 18; to examine the interaction with the locus on chromosome 20; and to identify candidate regions underlying quantitative trait loci (QTL). Used were a near isogenic line population derived from residual heterozygosity in an F5:7 recombinant inbred line EF60 1-40; SDS response data from 2 locations and years; four microsatellite markers and six thousand SNP markers. Polymorphic regions were found from 2,788 to 8,938 Kbp on chromosome 18 and 33,100 to 34,943 Kbp on chromosome 20. Both regions were significantly (0.005 < P > 0.0001) associated with resistance to SDS. A fine map was constructed that Mendelized the three loci. Substitution maps suggested the two loci on chromosome 18 were actually 3 loci (cqRfs, cqRfs1 and cqRfs19). Candidate genes for cqRfs19 were identified in a small region of the genome sequence of soybean. An epistatic interaction was inferred where the allele of loci on chromosome 18 determined the value of the locus on chromosome 20. It was concluded that SDS loci are both complex and interacting which may explain the slow progress in breeding for resistance to SDS.

Maize, Zea mays L., is the largest cereal grain crop grown in United States. Its yield and grain quality are adversely impacted by diseases every year. Aspergillus ear rot, caused by the fungus Aspergillus flavus, received little interest until its carcinogenic secondary metabolites, aflatoxins, were discovered. The objectives of this study were to introgress the quantitative trait loci (QTL) 2.04 from Mp313E and 5.03 from Mp715 into two commercial inbred lines, MonF and MonM; and evaluate their near isogenic lines (NILs) and testcrosses for preharvest aflatoxin accumulation and secondary agronomic traits. Marker assisted selection to create NILs and the testcross production was conducted by Bayer Company between 2015 and 2018. Field trials were conducted in summer 2019 as randomized complete block trials at three locations. The entry list of inbred trials included two donor parents (DP), two recurrent parents (RP), and their 58 NILs, and that of hybrid trials included 114 NIL testcrosses and 8 parental testcrosses. The top ear of each plant in every plot was inoculated with a 3.4 ml of A.flavus conidial suspension 13 days after mid-silk. All the inoculated ears were harvested at maturity, dried, machine shelled, ground, and aflatoxin concentration was determined by plot. Separate hybrid yield trials were conducted in four locations to measure the grain yield including an additional commercial check. Data on aflatoxin and other secondary traits was analyzed using SAS software. Overall, MonF NILs improved significantly more than MonM NILs in terms of their resistance to aflatoxin accumulation with the introgression of QTL 2.04 from Mp313E, but there were no differences with the introgression of QTL 5.03 from Mp715. Overall, Mp313E NILs improved more than Mp715 NILs when the recurrent parent was MonF, but the response was opposite when the recurrent parent was MonM. Compared to their respective recurrent parents, there were at least two NILs from each of the three out of four RP x DP crosses that significantly improved their resistance to aflatoxin accumulation with a minimal loss of their agronomic performance and testcross grain yields. These NILs could be considered as parents in future introgression projects.

No description available.

Master of Science / Department of Grain Science and Industry / Jon M. Faubion / Endosperm texture is an important characteristic in determining wheat processing and end-use. The presence of puroindoline proteins on the starch surface is the biochemical marker for wheat hardness. Near-isogenic samples over expressing puroindolines have been used to assess the effect of wheat hardness on final product characteristics. The objective of this study was to determine differences among starch isolated from near-isogenic samples and to investigate the role starch surface components play in pasting. The use of near-isogenic samples over expressing puroindolines combined with the use of two methods of starch isolation (batter and dough) was an effective means to create samples with varied amounts of surface components. Starch thermal properties were characterized and surface proteins and lipids were quantified. Starch isolated from hard wheat cultivars presented more similarities with starch isolated from its soft near-isogenic line when a dough method was used than when a batter method was used. Starch from soft experimental lines isolated using a batter method showed increased MVA peak viscosity, breakdown and swelling power. Increased levels of LysoPC in starch isolated from hard wheat cultivars or soft experimental lines by dough method could have complexed with amylose and restricted granule swelling. Thereby, decreasing peak viscosity, breakdown and swelling power.

Carotenoides são pigmentos encontrados em grande abundancia em frutos do tomateiro. A capacidade de sequestro de radicais destes pigmentos faz do tomate uma excelente opção para inclusão de compostos nutricionais pela dieta. Embora a síntese de carotenoides seja reconhecida como uma das respostas da cascata de eventos que levam ao amadurecimento, pouco se sabe a respeito de sua influência neste processo. Deste modo, o objetivo deste trabalho foi determinar se o conteúdo e a composição de carotenóide nos frutos acarretam alterações na conservação e na suscetibilidade dos frutos ao fungo B. cinerea. Foram utilizadas linhagens quase isogênicas (NILs) ao Micro Tom (MT) carregando as mutações, Beta carotene (B), tangerine (t), yellow flesh (r) e Delta carotene (Del). A caracterização dessas variações alélicas revelou, não apenas alterações no conteúdo de carotenoides, mas também outros efeitos pleiotrópicos relacionados ao amadurecimento, parâmetros de qualidade, espessura da cutícula e propriedades nutricionais dos frutos. Em face disso, subsequentemente investigamos se o diferencial conteúdo e composição de carotenóides nas referidas NILs altera a suscetibilidade dos frutos ao fungo Botrytis cinerea. A caracterização do amadurecimento demonstrou que o pico climatérico dos frutos do mutante r foi antecipado, assim como a transição para o estádio breaker. De modo contrário, a alteração na coloração da epiderme do mutante t foi atrasada, reafirmando suspeitas de outros autores que sugeriram que a enzima carotenoide-isomerase é necessária para a síntese de carotenoides no escuro. O conteúdo de ácido ascórbico também foi contrastante entre estes genótipos, sendo que no mutante r a sua concentração foi elevada, enquanto em t sua concentração foi reduzida. A perda de massa dos frutos foi menor no mutante Del, no entanto, esta característica não esteve relacionada a espessura da cutícula, que foi maior em B. A inoculação com B. cinerea por meio de ferimentos na pele dos frutos revelou menor suscetibilidade do mutante t, e em menor amplitude, do mutante Del. Ainda, o mutante t também se mostrou menos suscetível à inoculação do patógeno sobre frutos intactos. A maior espessura da cutícula de B não reduziu sua suscetibilidade ao fungo, que infectou os tecidos preferencialmente através da cicatriz do pedúnculo. A interação com B. cinerea desencadeou a produção de etileno e de espécies reativas de oxigênio (EROs), assim, é provável que a capacidade de sequestro de radicais livres tenha contribuído para a redução da suscetibilidade em t e Del, que mostraram menor produção de superóxido. Juntos, estes resultados sugerem que a modulação na atividade de enzimas da carotenogênese pode servir como ferramenta para obtenção de frutos com diferente vida útil durante o armazenamento pós-colheita de tomate. / Carotenoids are pigments found in great abundance in tomato fruits. The radical sequestration capacity of these pigments makes tomato an excellent option for inclusion of nutritional compounds in diet. Although the synthesis of carotenoids is recognized as one of the cascade responses that trigger ripening, little is known about its influence in this process. Thus, the aim of this study was to determine if the content and composition of carotenoids in the fruits alters the conservation and the susceptibility of the fruits to the B. cinerea fungus. Micro Tom (MT) near-isogenic lines (NILs) harboring the mutations Beta carotene (B), tangerine (t), yellow flesh (r) and Delta carotene (Del) has been used. The characterization of these allelic variations revealed not only changes in the carotenoid content but also other pleiotropic effects related to maturation, quality parameters, cuticle thickness and nutritional properties of fruit. In this way, we subsequently investigated whether the differential content and composition of carotenoids in NILs alters fruit susceptibility to the fungus Botrytis cinerea. The ripening characterization showed that the climacteric peak of the fruits of the r mutant was anticipated, as well as the transition to the breaker stage. Conversely, the change in epidermal coloration of mutant t was delayed, reaffirming suspicions of other authors who suggested that the carotenoid-isomerase enzyme is required for the synthesis of carotenoids in the dark. The ascorbic acid content was also contrasting among these genotypes, and in the r mutant its concentration was high, while at t its concentration was reduced. The fruit mass loss was lower in Del mutant, however, this characteristic was not related to cuticle thickness, which was higher in B. Wound-inoculation with B. cinerea revealed less susceptibility of the mutant t, and in a lesser extent, the mutant Del. Further, mutant t was also less susceptible to pathogen inoculation on intact fruits. The greater thickness of the cuticle of B did not reduce its susceptibility to the fungus, which infected the tissues preferentially through the scar of the peduncle. The interaction with B. cinerea triggered the production of ethylene and reactive oxygen species (ROS), so the free radical scavenging capacity probably contributed to the reduction of t and Del susceptibility, which showed lower production of superoxide. Together, these results suggest that modulation in carotenogenesis enzyme activity may serve as a tool to obtain fruit with different shelf-life during post-harvest tomato storage.

Carotenoides são pigmentos encontrados em grande abundancia em frutos do tomateiro. A capacidade de sequestro de radicais destes pigmentos faz do tomate uma excelente opção para inclusão de compostos nutricionais pela dieta. Embora a síntese de carotenoides seja reconhecida como uma das respostas da cascata de eventos que levam ao amadurecimento, pouco se sabe a respeito de sua influência neste processo. Deste modo, o objetivo deste trabalho foi determinar se o conteúdo e a composição de carotenóide nos frutos acarretam alterações na conservação e na suscetibilidade dos frutos ao fungo B. cinerea. Foram utilizadas linhagens quase isogênicas (NILs) ao Micro Tom (MT) carregando as mutações, Beta carotene (B), tangerine (t), yellow flesh (r) e Delta carotene (Del). A caracterização dessas variações alélicas revelou, não apenas alterações no conteúdo de carotenoides, mas também outros efeitos pleiotrópicos relacionados ao amadurecimento, parâmetros de qualidade, espessura da cutícula e propriedades nutricionais dos frutos. Em face disso, subsequentemente investigamos se o diferencial conteúdo e composição de carotenóides nas referidas NILs altera a suscetibilidade dos frutos ao fungo Botrytis cinerea. A caracterização do amadurecimento demonstrou que o pico climatérico dos frutos do mutante r foi antecipado, assim como a transição para o estádio breaker. De modo contrário, a alteração na coloração da epiderme do mutante t foi atrasada, reafirmando suspeitas de outros autores que sugeriram que a enzima carotenoide-isomerase é necessária para a síntese de carotenoides no escuro. O conteúdo de ácido ascórbico também foi contrastante entre estes genótipos, sendo que no mutante r a sua concentração foi elevada, enquanto em t sua concentração foi reduzida. A perda de massa dos frutos foi menor no mutante Del, no entanto, esta característica não esteve relacionada a espessura da cutícula, que foi maior em B. A inoculação com B. cinerea por meio de ferimentos na pele dos frutos revelou menor suscetibilidade do mutante t, e em menor amplitude, do mutante Del. Ainda, o mutante t também se mostrou menos suscetível à inoculação do patógeno sobre frutos intactos. A maior espessura da cutícula de B não reduziu sua suscetibilidade ao fungo, que infectou os tecidos preferencialmente através da cicatriz do pedúnculo. A interação com B. cinerea desencadeou a produção de etileno e de espécies reativas de oxigênio (EROs), assim, é provável que a capacidade de sequestro de radicais livres tenha contribuído para a redução da suscetibilidade em t e Del, que mostraram menor produção de superóxido. Juntos, estes resultados sugerem que a modulação na atividade de enzimas da carotenogênese pode servir como ferramenta para obtenção de frutos com diferente vida útil durante o armazenamento pós-colheita de tomate. / Carotenoids are pigments found in great abundance in tomato fruits. The radical sequestration capacity of these pigments makes tomato an excellent option for inclusion of nutritional compounds in diet. Although the synthesis of carotenoids is recognized as one of the cascade responses that trigger ripening, little is known about its influence in this process. Thus, the aim of this study was to determine if the content and composition of carotenoids in the fruits alters the conservation and the susceptibility of the fruits to the B. cinerea fungus. Micro Tom (MT) near-isogenic lines (NILs) harboring the mutations Beta carotene (B), tangerine (t), yellow flesh (r) and Delta carotene (Del) has been used. The characterization of these allelic variations revealed not only changes in the carotenoid content but also other pleiotropic effects related to maturation, quality parameters, cuticle thickness and nutritional properties of fruit. In this way, we subsequently investigated whether the differential content and composition of carotenoids in NILs alters fruit susceptibility to the fungus Botrytis cinerea. The ripening characterization showed that the climacteric peak of the fruits of the r mutant was anticipated, as well as the transition to the breaker stage. Conversely, the change in epidermal coloration of mutant t was delayed, reaffirming suspicions of other authors who suggested that the carotenoid-isomerase enzyme is required for the synthesis of carotenoids in the dark. The ascorbic acid content was also contrasting among these genotypes, and in the r mutant its concentration was high, while at t its concentration was reduced. The fruit mass loss was lower in Del mutant, however, this characteristic was not related to cuticle thickness, which was higher in B. Wound-inoculation with B. cinerea revealed less susceptibility of the mutant t, and in a lesser extent, the mutant Del. Further, mutant t was also less susceptible to pathogen inoculation on intact fruits. The greater thickness of the cuticle of B did not reduce its susceptibility to the fungus, which infected the tissues preferentially through the scar of the peduncle. The interaction with B. cinerea triggered the production of ethylene and reactive oxygen species (ROS), so the free radical scavenging capacity probably contributed to the reduction of t and Del susceptibility, which showed lower production of superoxide. Together, these results suggest that modulation in carotenogenesis enzyme activity may serve as a tool to obtain fruit with different shelf-life during post-harvest tomato storage.

No description available.

Doctor of Philosophy / Department of Grain Science and Industry / Finlay I. MacRitchie / Novel genetic materials were used to deduce gluten protein composition-functionality relationships. The Pegaso bread wheat near-isogenic lines (NILs) included addition, variation and/or deletion of major loci coding for HMW-GS, LMW-GS and gliadins. The waxy wheat lines (Svevo and N11 set) included wild, partial and complete waxy lines. Triticale translocations include 1R.1D and 1A.1D lines (GDS7, Trim, Rhino and Rigel sets) with HMW-GS 5+10 and 2+12. The main goal of the study was to establish the usefulness of NILs as appropriate materials to investigate the structure-function relationships of wheat proteins and to evaluate the performance of unique triticale translocations and waxy wheat lines. Effect of genetic variation on phytochemical (phenolic acid and policosanol) contents was also studied. Innovative methods like MALLS, Lab-on-a-chip and micro (10 g) baking were utilized along with traditional analytical methods. Results confirmed the potential of using NILs in understanding the effects of certain proteins coded at specific loci that might often be targeted in breeding programs. Removal of expected chain terminators at Gli-1/Gli-2 loci causes a shift in MWD to higher values, reflected in higher UPP and dough strength. Lines with HMW-GS 5+10 were clearly separated from 2+12 lines in terms of dough strength and UPP. The present study obtained evidence that modified ω-gliadins acts as chain terminators and cause reduction of protein polymer size and thus shifts in MWD. Marked differences in terms of milling characteristics, protein composition and ultimately in end-use functionality were observed with various waxy wheat null lines. Loaf volumes with waxy wheat flour alone were higher than a 50% blend with commercial wheat; however, breads were unacceptable to consumers in all aspects. Poor milling quality, very low mixing times with low bread loaf volumes were typical of all the triticales studied. However, translocation of the HMW-GS from wheat chromosome 1D increased dough strength, particularly the HMW-GS 5+10. Among the phytochemicals studied, double nulls at Gli-1 loci of Pegaso NILs had the highest total policosanols and total phenolic acid contents.Slight variation to wheat phenolic acid composition and contents were observed with waxy wheat and triticale lines.

This study represents the first comprehensive analysis of gene regulation underlying the distinct categories of resistance afforded to wheat (Triticum aestivum, L.) by different Dn genes. Russian wheat aphid (Diuraphis noxia, Mordv.) feeding on susceptible wheat cultivars causes leaf rolling, chlorosis and the eventual death of the plant. Plants expressing Dn genes are resistant to D. noxia infestation, but different Dn genes afford phenotypically distinct modes of resistance: the Dn1 gene confers an antibiotic effect to lower aphid fecundity; Dn2 confers tolerance to high aphid pressure; and Dn5 confers antixenosis, and aphids do not prefer such plants as hosts. Little is known about the components involved in establishing a successful defence response against D. noxia attack and how these differ between the distinct resistance categories. It is assumed that the Dn genes function as classic R genes in plant defence, being receptors for elicitors in aphid saliva. Upon recognition, defence response signalling is initiated, but the exact mechanics of subsequent cellular events in aphid resistance have only recently come under investigation. Evidence from cDNA microarray and subtractive hybridization experiments indicated the involvement of kinase signalling cascades and photosynthetic proteins in the response against D. noxia. However, expression analysis describing how these processes differ between plants carrying different Dn genes and how these differences account for antibiosis, antixenosis or tolerance had not been conducted. We consequently investigated the downstream components involved in or affected by the generation of these resistance mechanisms by comparing the responses in transcript regulation of Tugela near-isogenic lines with different Dn genes to D. noxia infestation. cDNA-AFLP analysis was selected as an appropriate functional genomics tool, since it is semi-quantitative, does not require prior sequence information and allows for the discovery of novel genes. cDNA-AFLP analysis yielded 121 differentially regulated transcript-derived fragments (TDFs) grouped into eight expression clusters. We cloned and sequenced 49 representative TDFs, which were further classified into five broad functional categories based on inferred similarity to database sequences. Transcripts involved in such diverse processes as stress, signal transduction, photosynthesis, metabolism and gene regulation were found to be differentially regulated during D. noxia feeding. Many TDFs demonstrated homology to proteins with unknown function and several novel transcripts with no similarity to previously published sequences were also discovered. Detailed expression analysis using quantitative RT-PCR and RNA hybridization provided evidence that the time and intensity of induction of specific pathways is critical for the development of a particular mode of resistance. This includes: the generation of kinase signalling cascades and the induction of several ancillary processes such as ubiquitination, leading to a sustained oxidative burst and the hypersensitive response during antibiosis; tolerance as a passive resistance mechanism countering aphid-induced symptoms through the repair or de novo synthesis of photosystem proteins; and the possible involvement of ethylene-mediated wounding pathways in generating volatile organic compounds during antixenosis. This is the first report on the involvement of KCO1, a vacuolar K+ channel, in assisting cytosolic Ca2+-influx and preventing leaf rolling, as well as on the role of iron homeostasis as a gene regulatory mechanism for sustaining the oxidative burst during the antibiotic defence response. This study opens up several areas of investigation heretofore unexplored in cereal-aphid interaction research. Of particular interest is the induction of genes involved in photosynthetic compensation during Dn2 tolerance responses, since these constitute a novel, passive resistance mechanism exclusive to aphid defence as opposed to the active resistance triggered in the presence of the Dn1 gene in the form of a general hypersensitive response. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted