Doctor of Philosophy / Department of Agronomy / Guihua Bai / Allan K. Fritz / Wheat leaf rust, caused by Puccinia triticina, is an important fungal disease worldwide. Growing resistant cultivars is an effective practice to reduce the losses caused by the disease, and using slow-rusting resistance genes can improve the durability of rust resistance in the cultivars. CI13227 is a winter wheat line that shows a high level of slow-rusting resistance to leaf rust and has been studied extensively. In this research, two recombinant inbreed line (RIL) populations derived from CI13227 x Suwon (104 RILs) and CI13227 x Everest (184 RILs) and one doubled haploid (DH) population derived from CI13227 x Lakin with 181 lines were used to identify quantitative trait loci (QTLs) for slow leaf rusting resistance. Each population and its parents were evaluated for slow-rusting traits in two greenhouse experiments. A selected set of 384 simple sequence repeat markers (SSRs), single nucleotide polymorphism markers (SNPs) derived from genotyping-by-sequencing (GBS-SNPs) or 90K-SNP chip (90K-SNPs) were analyzed in the three populations. Six QTLs for slow-rusting resistance, QLr.hwwgru-2DS, QLr.hwwgru-7BL, QLr.hwwgru-7AL, QLr.hwwgru-3B_1, QLr.hwwgru-3B_2, and QLr.hwwgru-1D were detected in the three populations with three stable QTLs, QLr.hwwgru-2DS, QLr.hwwgru-7BL and QLr.hwwgru-7AL. These were detected and validated by Kompetitive Allele-Specific PCR (KASP) markers converted from GBS-SNPs and 90K-SNPs in at least two populations. Another three QTLs were detected only in a single population, and either showed a minor effect or came from the susceptible parents. The KASP markers tightly linked to QLr.hwwgru-2DS (IWB34642, IWB8545 and GBS_snpj2228), QLr.hwwgru-7BL (GBS_snp1637 and IWB24039) and QLr.hwwgru-7AL (IWB73053 and IWB42182) are ready to be used in marker-assisted selection (MAS) to transfer these QTLs into wheat varieties to improve slow-rusting resistance in wheat.

Doctor of Philosophy / Department of Agronomy / Guihua Bai / Guorong Zhang / Fusarium head blight (FHB) is a devastating fungal disease in wheat, reducing not only grain yield but also quality. The pathogen produces the mycotoxin deoxynivalenol (DON) that induces severe toxicological problems in human and animals. Using host resistance has been the most efficient way to control the disease. To identify quantitative trait loci (QTLs) for FHB resistance in Chinese landrace Haiyanzhong (HYZ), a recombinant inbred lines (RILs) population derived from a cross between HYZ and Wheaton was developed. The RILs were evaluated for percentage of symptomatic spikelets (PSS) in three greenhouse experiments, and genotyped using simple sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) developed from genotyping-by-sequencing (GBS). Eight QTLs were identified for type II (PSS) resistance on chromosomes 5A, 6B, 7D, 2B (2), 3B, 4B, and 4D, with 5A as the major QTL. Ten SNPs closely linked to 5A, 6B, and 2B QTLs were successfully converted to Kompetitave allelic specific PCR (KASP) assays. To identify common QTLs across different populations, we constructed high-density GBS-SNP maps in an additional four RIL populations derived from the Chinese landraces, Wangshuibai (WSB), Baishanyuehuang (BSYH), Huangfangzhu (HFZ), and Huangchandou (HCD) and conducted meta-analysis of the QTLs for FHB resistance using a consensus map developed from the five populations. We identified six MQTLs on chromosomes 3BS (2), 3A, 3D, 2D, and 4D and 23 tightly linked GBS-SNPs to the MQTLs. These GBS-SNPs were successfully converted to KASPs. The KASPs linked to MQTLs can be used for pyramiding these QTL in breeding programs. To quickly reduce FHB damage in U.S. hard winter wheat (HWW), we transferred Fhb1, a major QTL with stable effects on FHB resistance, from Ning7840 into three adapted HWW cultivars Overland, Jagger, and Overley, by marker-assisted backcross (MAB), and assessed the effect of Fhb1 on FHB resistance in these different backgrounds. The results showed that Fhb1 can significantly lower FHB severity, Fusarium-damaged kernel (FDK), and DON accumulation in the all the three HWW backgrounds. Some of the selected lines showed high levels of FHB resistance, but agronomically similar traits as recurrent parents, can be used as resistant parents to improve HWW FHB resistance.

Master of Science / Department of Plant Pathology / John P. Fellers / Allan K. Fritz / Leaf rust is caused by Puccinia triticina and is one of the most widespread diseases of wheat worldwide. Breeding for resistance is one of the most effective methods of control. Lr16 is a leaf rust resistance gene that provides partial resistance at the seedling stage. One objective of this study was to use RNA-seq and in silico subtraction to develop new resistance gene analog (RGA) markers linked to Lr16. RNA was isolated from the susceptible wheat cultivar Thatcher (Tc) and the resistant Thatcher isolines TcLr10, TcLr16, and TcLr21. Using in silico subtraction, Tc isoline ESTs that did not align to the Tc reference were assembled into contigs and analyzed using BLAST. Primers were designed from 137 resistance gene analog sequences not found in Tc. A population of 260 F[subscript]2 lines derived from a cross between the rust-susceptible cultivar Chinese Spring (CS) and a Thatcher isoline containing Lr16 (TcLr16) was developed for mapping these markers. Two RGA markers XRGA266585 and XRGA22128 were identified that mapped 1.1 cM and 23.8 cM from Lr16, respectively. Three SSR markers Xwmc764, Xwmc661, and Xbarc35 mapped between these two RGA markers at distances of 4.1 cM, 10.7 cM, and 16.1 cM from Lr16, respectively. Another objective of this study was to use genotyping-by-sequencing (GBS) to develop single nucleotide polymorphism (SNP) markers closely linked to Lr16. DNA from 22 resistant and 22 susceptible F[subscript]2 plants from a cross between CS and TcLr16 was used for GBS analysis. A total of 39 Kompetitive Allele Specific PCR (KASP) markers were designed from SNPs identified using the UNEAK and Tassel pipelines. The KASP marker XSNP16_TP1456 mapped 0.7 cM proximal to Lr16 in a TcxTcLr16 population consisting of 129 F[subscript]2 plants. These results indicate that both techniques are viable methods to develop new molecular markers. RNA-seq and in silico subtraction were successfully used to develop two new RGA markers linked to Lr16, one of which was more closely linked than known SSR markers. GBS was also successfully used on an F[subscript]2 population to develop a KASP marker that is the most closely linked marker to Lr16 to date.

Borage (Borago officinalis) is an annual herb plant for culinary and medicinal uses. Due to a high level of gamma-linolenic acid (GLA) in its seed oil and the health-related benefits of GLA, borage is commercially cultivated. However, a herbicide-resistant variety has not yet been developed for effective weed management in borage farming. Thus, this thesis aimed to create, identify and characterize ethyl methanesulfonate (EMS) induced borage mutants for herbicide imidazolinone resistance. An EMS-mutagenized borage population was generated by using a series of concentrations of EMS to treat M1 seeds. After screening M2 borage plants with the herbicide, tolerant plants were selected, self-pollinated and grown to their maturity. The offsprings were subjected to herbicide screening again to confirm the phenotype, resulting in identification of two genetically stable imidazolinone-resistant lines. Two acetohydroxyacid synthase (AHAS) genes, AHAS1 and AHAS2, involved in the imidazolinone resistance were isolated and sequenced from both mutant (resistant) and wild type (susceptible) borage plants. Comparison of these AHAS sequences revealed that a single nucleotide substitution occurred in the AHAS1 resulting in an amino acid change from serine (S) in the susceptible plant to asparagine (N) in the first resistant line. The similar substitution was later found in the AHAS2 of the second resistant line. A KASP marker was developed for the AHAS1 mutation to differentiate the homozygous susceptible, homozygous and heterozygous resistant borage plants for the breeding purpose. The in vitro assay showed homozygous resistant borage containing the AHAS1 mutation could retain significantly higher AHAS activity than susceptible borage across different imazamox concentrations. The herbicide dose response test showed that the resistant line with the AHAS1 mutation was tolerant to four times the field applied concentration of the “Solo” herbicide.

The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.

The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.

The Russian wheat aphid (RWA) (Diuraphis noxia Kurdjumov) is one of the important insect pests of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and other grasses. To date, there are four RWA biotypes identified in South Africa. The virulent biotypes emerged, partly due to climate change and new genetic variations within populations of RWA; hence there is a need to improve host-plant resistance, as an effective control measure. Bread wheat (Triticum aestivum L.) accession Cereal Introduction (CItr) 2401 is known to be resistant to all RWA biotypes worldwide. The goal of this study was to use a backcrossed near-isogenic line (NIL) BC5F5 mapping population, developed from a cross between CItr 2401 and susceptible Kavkaz, to identify and validate single nucleotide polymorphism (SNP) markers linked to the resistance phenotype in CItr 2401. This was achieved by (i) conducting a preliminary study that evaluated the suitability of simple sequence repeat (SSR) markers previously reported in literature for discriminating stacked RWA resistance genes and, (ii) employing SNP markers for the first time in a RWA resistance study as a future alternative to the widely used SSR markers. None of the tested SSR markers showed potential use in marker-assisted selection (MAS). The mapping population was phenotypically evaluated for RWA resistance using the four South African biotypes, viz. RWASA1, RWASA2, RWASA3 and RWASA4. Analysis of variance (ANOVA) showed significant (P<0.001) differences of genotypes after confirming the normality of residuals and homogeneity of variance. The Illumina iSelect 9,000 wheat SNP platform was used to genotype the two crossing parents and a selection of 24 NIL genotypes from the mapping population. Eight SNP markers found to be linked to the phenotype were converted to breeder-friendly and high-throughput Kompetitive allele-specific polymerase chain reaction (KASP) markers. The designed KASP markers were validated on the two crossing parents, the 24 NIL sent for SNP genotyping, on the mapping population and on the preliminary study genotypes for their effectiveness. The KASP assays developed in this study will be useful for stacking the RWA resistance from CItr 2401 with other Dn genes effective against the RWA. / Life and Consumer Sciences / M. Sc. (Life Sciences)