Pre-harvest contamination of maize grain with aflatoxin is a chronic problem worldwide and particularly in the southeastern U.S. Aflatoxin is a mycotoxin produced by the fungus Aspergillus flavus, an opportunistic ear-rot pathogen of maize (Zea mays). Resistance to aflatoxin accumulation is heritable, and resistant germplasm-lines are available. These lines are derived from “exotic” genetic backgrounds and were released as sources of resistance, not parental inbreds. However, all current sources of resistance are quantitative, which complicates conventional efforts to introgress resistance alleles from unadapted but resistant donor lines to adapted but susceptible recipient lines. Mapping quantitative trait loci (QTL) and their linked markers enables targeted introgression of the desired alleles via marker-assisted selection. Quantitative trait loci were identified in two F2:3 mapping populations, derived from crossing resistant inbreds Mp715 and Mp717 to a common susceptible parent (Va35). The Mp715 x Va35 population was phenotyped for aflatoxin accumulation under artificial inoculation in replicated field trials at Mississippi State (MSU) in 2015 and 2016. The Mp717 x Va35 population was phenotyped at MSU and Lubbock, TX in 2016. Populations were genotyped using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers and linkage maps created in JoinMap4. To locate QTL, linkage maps, genotypes, and phenotypes were analyzed jointly in QTL Cartographer 2.5 using composite interval mapping (CIM) and multiple interval mapping (MIM) procedures. Five QTL with the beneficial allele contributed by Mp715 were identified during CIM in bins 5.01, 6.06, 7.03 10.04 and 10.05. Three QTL with the beneficial allele contributed by Mp717 were identified during CIM in bins 3.07/3.08, 7.02/7.03, and 10.05. In both populations, QTL were identified with the beneficial allele contributed by Va35. Those QTL did not co-locate across populations but four of the six were on chromosome 1. Significant QTL effects from CIM were used as the initial model terms in MIM, where all QTL effects were fit simultaneously and their gene-action and epistatic interactions estimated.

The main goal of soybean breeding is to increase yield with improved seed quality characters. The objectives of this thesis were: i) to identify yield quantitative trait loci (QTL) across different mega-environments (ME); ii) to evaluate the relationship between yield and yield components and the co-localization of QTL; iii) to analyze environmental factors that affect QTL expression and identify different ME. Two Canadian x Chinese soybean populations were tested across Canada, northern United States and China. Yield QTL were identified at one, two and three hypothetical ME. Most of them were co-localized with agronomic trait QTL. These results give strong evidence that Canada, northern United States and China are different ME. Novel alleles from plant introductions can favorably contribute directly or indirectly to seed yield and the use of QTL would facilitate their introgression into breeding populations in both North America and China. / National Science and Engineering Research Council of Canada (NSERC) and the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA)

Soybean [Glycine max (L.) Merr.] seed is a major source of edible oil in the world and the main renewable raw material for biodiesel production in North America. Oil, which on average accounts for 20% of the soybean seed weight, is a complex quantitative trait controlled by many genes with mostly minor effects and influenced by environmental conditions. Because of its quantitative nature, the seed oil concentration may have an indirect effect on other economically important and agronomic traits such as seed yield and protein concentration. Increasing the oil concentration in soybean has been given more attention in recent years due to increasing demand for both edible oil and feedstock. To achieve this objective, it is important to understand the genetic control of the oil accumulation and its relationship with other traits. The main objectives of this thesis were to identify quantitative trait loci (QTL) and genes involved in oil biosynthesis in soybean. Two recombinant inbred line (RIL) populations were developed from crosses between moderately high oil soybean cultivars with high seed yield and protein concentration. In a population of 203 F3:6 RILs from a cross of ‘OAC Wallace’ and ‘OAC Glencoe’, a total of 11 genomic regions located on nine different chromosomes were identified as associated with oil concentration using multiple QTL mapping (MQM) and single-factor ANOVA. Among the 11 oil-associated QTL, four QTL were also validated in a population of 211 F3:5 RILs from a cross of ‘RCAT Angora’ and ‘OAC Wallace’. There were six oil QTL identified in this study that were co-localized with seed protein QTL and four for seed yield QTL. The oil-beneficial allele of the QTL tagged by marker Sat_020, on Chromosome 9, was positively associated with seed protein concentration. The oil-enhancing alleles at markers Satt001 and GmDGAT2B were positively correlated with seed yield. In this study, three sequence mutations were also discovered in either the coding or non-coding regions of three DGAT soybean genes (GmDGAT2B, GmDGAT2C, and GmDGAT1B) between ‘OAC Wallace’ and ‘OAC Glencoe’ that showed significant effects on some of the traits evaluated. GmDGAT2B showed significant association with seed oil and yield across different environments. The oil-favorable allele of the gene GmDGAT2B from ‘OAC Glencoe’ was also positively correlated with seed yield. GmDGAT2C was associated with seed yield, whereas GmDGAT1B showed significant effects on seed yield and protein concentration. However, neither of these two genes showed any association with seed oil. The yield-enhancing allele of GmDGAT1B showed negative association with protein concentration. The identification of oil QTL that were either positively associated with seed yield and protein or neutral to both traits and the development of new gene-based markers will facilitate marker-assisted breeding to develop high oil soybean cultivars with high yield and minimal effect on protein concentration. / Generous funding to conduct this research was provided by the Alternative Renewable Fuels II Program of the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) and by the Grain Farmers of Ontario.

Les allèles en ségrégation des deux génomes Elaeis ont été tracés dans un pseudo backcross interspécifique Elaeis (E. oleifera x E. guineensis) x E. guineensis. Dix-neuf QTL de proportion d'acides gras de l'huile de palme par rapport à 16 QTL des mêmes caractères d'un croisement intraspécifique E. guineensis ont mis en évidence des QTL communs ou spécifiques aux deux espèces. Aucune corrélation n'existe entre proportions d'acides gras et le rendement en huile de palme. Le déterminisme génétique et les principaux gènes associés aux acides gras du mésocarpe ont été étudiés, à l'aide de banques d'ADNc pleine longueur et des extraits d'ARNm de mésocarpe de fruits en développement des espèces Elaeis chacune représentée par quatre fonds génétiques. L'expression de 113 gènes Elaeis clés ou de facteurs putatifs de régulation des voies de synthèse de novo des acides gras et des triacylglycérols fut caractérisée au niveau de l'espèce par analyse in silico de profils de transcription et par analyse Real Time RT-qPCR. Les gènes ont été cartographiés sur le pseudo backcross avec 180 marqueurs SNP intra-géniques. Quarante-huit gènes étaient exprimés différemment entre espèces dont 94% plus exprimés chez E. guineensis. Le facteur de transcription EgAP2-2 (WRI1-like) influence le début de la synthèsede novo des acide gras par action principale sur les gènes MAT et KAS III, parait indépendant de l'expression d'autres gènes et non différentiel entre espèces. Les variations en C16:0 et C18:0 entre espèces s'expliquent par niveau d'expression allélique et l'activité enzymatique relative des isoformes présentes dans le génome de FATB (2), KAS II (1) et SAD (3). / An Elaeis interspecific pseudo-backcross of first generation (E. oleifera x E. guineensis) x E. guineensis allowed tracing segregating alleles from both Elaeis genomes. Nineteen quantitative trait loci (QTLs) associated to palm oil fatty acid proportions compared to 16 QTLs of same traits in an intra-specific oil palm cross evidenced common or specific QTLs in E. guineensis and E. oleifera. No correlation was found in oil palm between mesocarp fatty acid proportions and yieldtraits. The genetic determinism of main fatty acid proportions was confirmed. Genes related to palm oil fatty acids were investigated using full-length cDNA libraries and mRNA extracts from the mesocarp of developing fruits in each Elaeis species represented by four genetic pools. Expression of 113 key Elaeis genes or putative regulation factors of de novo fatty acid and triacylglycerol pathways were characterized at species level by in silico transcript profiling and Real Time RT-qPCR analysis. Genes were mapped on the pseudo-backcross using 180 intra-gene SNP markers. Forty-eight genes were differentially expressed between Elaeis species, with 94% over expressed in E. guineensis. The EgAP2-2 (WRI1-like) transcription factor might influence the start of the de novo fatty acid pathway by main action only on the MAT and KAS III genes, as independent in expression from other genes and not differential between species. Between species variations in C16:0 and C18:0 can be deciphered by relative expression levels and enzyme activities of the isoforms in the genome of FATB (2), KAS II (1) and SAD (3). An oil palm producing more oleic acid in proportion than pure E. oleifera is feasible.

Growth rate and flowering time are agriculturally important traits that are linked to fitness, productivity and reproductive success of plants. To study the genetic basis for natural variation in growth rate and flowering time between local accessions of Arabidopsis thaliana, hybrids were produced between fast growing / late flowering and slow growing / early flowering parents. F3 and F5 hybrid families were grown under a range of conditions – under a constant controlled environment, outside over the winter and outside in spring and early summer. Growth rates were estimated from repeated images of rosettes. Flowering time, as number of leaves to flower, was also recorded both in control and natural conditions for F5 lines. Damage by slugs and stress-induced production of anthocyanin pigments were also recorded for plants grown outside. Broad-sense heritability estimates were higher for F5 families than F3, in which more loci will segregate, and ranged from 48% to 89%. No significant correlation between growth rates under different environments was observed in most cases for F3 populations, however significant correlations were detected for F5 families outside and under controlled conditions, suggesting that same genes can affect growth rate in more than one environment. The genotypes of F3 families were determined at thirty-nine SSLP (simple sequence length polymorphism) loci and used in regression with phenotype data to search for quantitative trait loci (QTL). Significant QTLs were detected in F3 families for growth rate, flowering time and anthocyanin production, but not for herbivore damage. To confirm QTL detected in the F3 and to detect additional loci, bulk segregant analysis was carried out in F5 families grown under different conditions. Potentially linked markers were tested further in individual F5 plants and QTL mapped on a finer scale in F5 families that remained heterozygous for candidate regions. VIP5 and LDL1 were selected as potential candidate genes for flowering time variation. These genes were sequenced for two parental alleles. A transposon insertion and 5’ UTR deletion were found in the LDL1 allele from the late flowering parent and SNPs (single nucleotide polymorphisms) were observed throughout the gene. However both alleles appeared to be expressed at similar levels. Transgenic lines have been produced carrying the LDL1 allele from the early flowering parent (4D1) in the background of the later flowering parent (11C1). This work is on-going and will hopefully reveal whether LDL1 underlies differences in flowering behaviour seen between 11C1 and 4D1.

Thesis (PhD (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The South African abalone, Haliotis midae, is a commercially valuable mollusc and is mostly exported to the Far East. Genetics research on H. midae has increased substantially since a genetic improvement programme was introduced in 2006 by collaboration between Stellenbosch University, government and industry partners. The development of molecular markers, QTL-mapping, gene-expression and genome manipulations are the main focuses of the research currently being conducted. The end goal is to create high quality and fast growing animals for the industry. The present study focused on the development of microsatellite markers and the detection of quantitative trait loci (QTL) affecting growth traits (shell length, shell width, wet weight) in this species. A combination of three methods, namely selective genotyping and bulked segregant analysis (pooling analysis), single marker regression and interval mapping were used to identify putative QTL in two full-sib families from two different farmed locations. Additional methods and protocols were developed that can assist the industry in other molecular research aspects. A total of 125 microsatellite loci were characterised. A total of 82 of these loci were isolated using second generation sequencing, a first for any abalone species. A preliminary, low-density framework linkage map was constructed containing 50 loci that mapped to 18 linkage groups. The observed genome length was 148.72cm with coverage of ±47%. QTL analyses revealed two putative QTL for shell width and wet weight, with 17% and 15% variance explained, that mapped on one linkage group in the first family and three putative QTL, for shell length, shell width and wet weight, with 33%, 28.5% and 31.5% variance explained, that mapped on one linkage group in the second family. Additional methods and protocols developed include an automated high-throughput DNA isolation protocol, a real-time PCR assay for H. midae x H. spadicea hybrid verification, a triploid verification microsatellite assay and a pre- and post-PCR multiplex setup and optimisation protocol. Future studies focussing on QTL and marker assisted selection (MAS) should verify the QTL found in this study and also utilise additional family structures and determine QTL-marker phase within the commercial populations. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is ’n kommersieel waardevolle weekdier en word hoofsaaklik na die Verre-Ooste uitgevoer. Genetiese navorsing op H. midae het aansienlik toegeneem sedert ’n genetiese verbeteringsprogram in 2006 deur samewerking tussen die Universiteit van Stellenbosch, die regering en industrievennote ingebring is. Die ontwikkeling van molekulêre merkers, KEL-kartering, geen-uitdrukking en genoom manipulasies is die hooffokusse van die navorsing wat tans uitgevoer word. Die einddoel is om hoë kwaliteit en snelgroeiende diere vir die industrie te skep. Die huidige studie het op die ontwikkeling van mikrosatelliet merkers en die opsporing van groeiverwante (skulplengte, -breedte en nat gewig) kwantitatiewe eienskap lokusse (KEL) in hierdie spesie gefokus. ’n Kombinasie van drie metodes, naamlik selektiewe genotipering en versamelde segregaat analise (samevoegingsanalise), enkel merker regressie en intervalkartering is gebruik om waarskynlike KEL in twee vol-sibbe families van twee verskillende produksiegebiede te identifiseer. Aanvullende metodes en protokolle is ontwikkel wat die industrie in ander molekulêre navorsingsaspekte kan ondersteun. ’n Totaal van 125 mikrosatelliet lokusse is beskryf. ’n Totaal van 82 van hierdie lokusse is deur die gebruik van derde generasie volgordebepaling geïsoleer, ’n eerste vir enige perlemoen spesie. ’n Voorlopige, laedigtheid raamwerkkoppelingskaart is saamgestel met 50 lokusse wat op 18 koppelingsgroepe gekarteer is. Die waarneembare genoomlengte was 148.72cm met ’n dekking van ±47%. KEL-analises het twee waarskynlike KEL vir skulpbreedte en nat gewig blootgelê wat 17% en 15% variasie verduidelik en is op een koppelingsgroep in die eerste familie gekarteer asook drie waarskynlike KEL, vir skulplengte, -breedte en nat gewig wat 33%, 28.5% en 31.5% variasie verduidelik en is op een koppelingsgroep in die tweede familie gekarteer. Aanvullende metodes en protokolle wat ontwikkel is, sluit ’n geoutomatiseerde hoë-deurgang DNS-isolasieprotokol, ’n intydse PKR-proef vir H. midae x H. spadicea hibried verifikasie, ’n triploïed verifikasie mikrosatellietproef en veelsoortige pre- en post-PKR opstelling en optimaliseringsprotokol in. Toekomstige studies wat fokus op KEL en merker ondersteunde seleksie (MOS) behoort die KEL wat in hierdie studie gevind is te verifieer en ook bykomende familie strukture te benut om KEL-merker fases binne die kommersiële populasie te bepaal.

This thesis addresses the dissection and characterisation of quantitative trait loci (QTL) affecting production traits in sheep. Firstly, the association between specific genetic polymorphisms and complex variation in weight, muscle and fat depositions was investigated. Research concentrated on assessing the presence, correspondence and significance of two single nucleotide polymorphisms (SNPs) in the GDF8 region of ovine chromosome 2, reportedly affecting muscle production. Commercial populations of British Texel, Suffolk and Charollais sheep were studied. The SNPs were absent in Suffolk and almost fixed in Texel breeds. In the Charollais population, the SNPs segregated at intermediate frequencies and a significant association was found between these polymorphisms and muscle depth. The previously proposed causative allele at one of the loci resulted in increased muscle depth and, at allele frequency of 0.5, this locus would explain one third of the additive genetic variance for the trait. Partial recessive allelic expression is proposed by genotypic value predictions and is consistent with the previously postulated molecular mechanism by which it gives rise to muscle changes. Secondly, the thesis focused on detection of QTL associated with growth. Live weight is a composite of growth rates over time, with inter-age genetic correlations for live weight decreasing as time between weight measurements increases. To explore whether observed genetic correlation patterns translate into distinct loci acting on weight at different growth stages, a novel method was developed and the applicability of a second proposed method was explored. Both methods allowed simultaneous analysis of multiple live weights per animal, while accounting differently for the correlation among measurements ordered in time. In the first approach, a growth curve technique was developed and employed to map growth QTL for curve parameters and predicted growth descriptors. A study of actual live weights identified significant QTL at different ages on distinct chromosomes, with QTL significance and variance changing over time. Further application of this technique on a simulated dataset validated its effectiveness in detecting age-dependent QTL. An extension of the procedure resulted in a novel technique for genomic evaluation of longitudinal traits. In the second method examined, random regression (RR) models were applied for dissection of growth QTL. Systematic model selection and inclusion of relevant random effects resulted in apparently significant QTL, but the method was computationally demanding, model choice proved challenging and the results were questioned. To further explore the method, RR models were applied to various simulated growth phenotypes composed of time-dependent QTL trajectories, polygenic and environmental effects. Statistically optimal RR models succeeded in identifying significant QTL and predicting the simulated time-dependence for most scenarios. However, the issue of model choice was again prominent, as suboptimal models resulted in unreliable QTL variance trajectories and pronounced confounding between different time-dependent effects. Thus, the growth curve approach appeared to be the more flexible and robust process for analysing longitudinal data to map agedependent QTL.

Breeding programmes that use elite sires with the best estimated breeding values for muscling traits have achieved significant improvement in lamb production in the UK. Further acceleration of the rate of genetic gain for the desirable production traits could be achieved using DNA marker-assisted selection (MAS) breeding strategies. The underlying causal genetic variants associated with improved muscling may be unknown and lying between a cluster of genes known as quantitative trait loci (QTL) or could be single nucleotide polymorphisms (SNP). LoinMAXTM, Texel muscling QTL (TM-QTL) and c.*1232G > A myostatin mutation were genetic variants that reported to be associated with improved muscling characteristics and hence subjected to further analysis in this project. It is essential before incorporating segregating genetic variants in any breeding scheme to comprehensively evaluate their effects on carcass traits. In-vivo scanning (ultrasound scanning (US) and computed tomography scanning (CT)), and carcass video image analyses (VIA) were used in the current studies. Objective VIAprediction weights of the carcass primal cuts could be the backbone of a value-based marketing system that is suggested to replace the current Meat and Livestock Commission (MLC) carcass grades for conformation scores (MLC-C) and fat class (MLC-F). The effect of a single copy of LoinMAXTM QTL (LM-QTL) compared to noncarriers was evaluated in UK crossbred lambs out of Scottish Mule ewes. M. longissimus lumborum (MLL) width, depth and area, as measured by CT scanning, were significantly greater in lambs heterozygous for LM-QTL compared to noncarriers. VIA detected a significant effect of the LM-QTL on the predicted weight of saleable meat yield in the loin primal cut (+2.2%; P < 0.05). The effects of the ovine c.*1232G > A myostatin mutation (MM), found on sheep chromosome 2, on carcass traits in heterozygous crossbred lambs sired by Texel and Poll Dorset rams were studied. Texel crossbred lambs carrying MM had increased loin depth and area. In both crossbred lambs, MM-carriers had significantly higher CT-estimated lean weight and proportion (2 to 4%) and muscle to bone ratios (by ~3%). Poll Dorset heterozygous crossbred animals had higher muscle to fat ratio (28%) and significantly lower fat-related measurements. The c.*1232G > A (MM) mutation as well as TM-QTL effects were evaluated in a different genetic background of Texel x Welsh Mountain crossbreed lambs. Carrying two copies of MM was associated with a significant positive effect on 8 week weight, a negative effect on ultrasound fat depth, a substantial decrease in MLC-fat score, positive impact on VIA-estimated weight of the hind leg, chump and loin primal cuts, as well as the muscularity of the hind leg and loin regions with greater loin muscle width, depth and area. Two copies of MM altered lambs‟ morphological traits with significantly wider carcasses across the shoulders, breast and hind legs and greater areas of the back view of the carcass when measured by VIA. TM-QTL significantly increased US-muscle depth and TM-QTL carriers had significantly greater loin muscle width and area measurements. Comparing TM-QTL genetic groups (homozygote allele carriers (TM/TM), heterozygote carriers of paternal and maternal origin of allele (TM/+ and +/TM, respectively) and homozygote non-carriers (+/+)) and TM-QTL mode of action were then studied. TM/TM carcasses were significantly heavier than non-carriers by 1.6 kg and scored higher conformation values when compared to heterozygote groups only. TM/+ lambs had significantly higher VIA-predicted weight and muscularity in the hind leg and loin, and higher loin dimensions relative to some other genotypic groups. The effect of TM-QTL on some carcass shape measurements was significant. TM-QTL mode of action results on the loin muscling traits supports the earlier reports of polar over dominance. In the light of growing calls to replace the current subjective carcass payment system with the objective VIA system that values the carcass according to the superiority of its cuts, I investigated the ability of US and CT measurements to predict the VIAestimated weights of the carcass primal cuts. Several prediction equations were examined but the best could be achieved when ultrasound measurement, CT linear measurements and live weight were fitted in the model. Since CT scanning information of elite sires is now being used for genetic selection for carcass merit, genetic parameters and genetic relationships between CT scanning measurements and post mortem traits (VIA and MLC-FC) were estimated. However, results were not sufficiently accurate to be of practical use due to lack of data.

The seed size and shape of lentil are important traits because they determine the market class, cooking time, and can influence quality and yield of milled lentils. Understanding the genetic control of seed size and shape can help breeders develop varieties with improved seed size and shape characteristics such as seed diameter, seed thickness and seed plumpness. The objectives were to determine the heritability of seed size and shape and identify the genomic regions controlling these traits. This involved i) developing a linkage map for the LR-18 population (CDC Robin x 964a-46) using a recently developed single nucleotide polymorphism (SNP) assay; ii) analyzing the LR-18 population for seed size and shape QTLs; iii) analyzing an association mapping panel for seed size and shape QTLs. Phenotyping trials were grown at two different locations in Saskatchewan, Canada. The mapping population was grown in two different years while the association panel was only grown in one. Seed diameter and thickness were measured using sieves and this data were used to calculate seed plumpness. Days to flowering was also recorded to determine if it had any effect on seed size or shape. A linkage map consisting of 537 SNPs, 10 SSRs and 4 morphological markers on seven linkage groups was constructed and used for the QTL analysis. The heritability estimates were high for seed diameter and seed plumpness (0.92 and 0.94, respectively) while for seed thickness and days to flowering they were more moderate (0.60 and 0.45, respectively). QTL analysis revealed QTLs on five of the seven linkage groups. The association mapping study revealed similar heritability estimates of 0.97, 0.62, 0.94, and 0.62 for seed diameter, seed thickness, seed plumpness and flowering time, respectively. There were 31 different significant marker trait associations, however only 5 of those were significant for both locations. Four of those five markers did not map in the LR-18 linkage map so their genomic locations are still to be determined. Results showed that there are key regions in the genome that control seed size and shape and flowering time in lentil. These markers could be used for marker-assisted selection or for further candidate gene analysis.

Fusarium Head Blight (FHB) is a destructive disease caused by Fusarium graminearum that affects wheat (Triticum aestivum L.) worldwide. Breeding for resistance to FHB is arguably the best way to combat this disease. However, FHB resistance is highly complex and phenotypic screening is difficult. Molecular markers are a promising tool but breeding programs face the challenge of allocating resources in such a way that the optimum balance between phenotypic and genotypic selection is reached. An F2:3 population derived from a resistant x susceptible cross was subjected to phenotypic and genotypic selection. For phenotyping, a novel air separation method was used to measure percentage of damaged kernels (FDK). Heritability estimates were remarkably high, which was attributed to the type of cross and the quality of phenotyping. Genotypic selection was done by selecting resistance alleles at quantitative trait loci (QTL) on the 3BS (Fhb1) and the 2DL chromosomes. Fhb1 conferred a moderate but stable FHB resistance while the 2DL QTL conferred a surprisingly high level of resistance but with significant interaction with the environment. Phenotypic selection conferred higher or lower genetic gains than genotypic selection, depending on the selection intensity. Based on these results, different selection strategies are discussed.