Doctor of Philosophy / Department of Agronomy / Tesfaye Tesso / The approach used to identify inbred lines that can produce superior hybrids is costly and time-consuming. It requires creation of all possible crosses and evaluation of the crosses to estimate combining abilities for the desired traits. Predicting heterosis or hybrid performance in any way possible may help to reduce the number of crosses to be made and evaluated. In this study, four sets of experiments were conducted to determine whether heterosis can be predicted based on inbred line performance, genetic distance between parents and genomic prediction model. The first experiment was aimed at assessing the levels of genetic diversity, population structure and linkage disequilibrium (LD) in 279 public sorghum inbred lines, based on 66,265 SNPs generated using the genotyping-by-sequencing (GBS) platform. The inbred lines were developed at different times over the last two decades and harbor robust diversity in pedigree and agronomic characteristics. Some of the inbreds are resistant to Acetolactate synthase (ALS) and Acetyl co-enzyme-A carboxylase (ACC) inhibitor herbicides. The mean polymorphic information content (PIC) and gene diversity across the entire inbreds were 0.35 and 0.46, respectively with non-herbicide resistant inbreds harboring more diversity than the herbicide resistant ones. The population structure analysis clustered the inbred lines into three major subgroups according to pedigree and fertility-reaction with the maintainer lines (B-lines) distinctly forming a separate cluster. Analysis of molecular variance (AMOVA) revealed more variation within subgroups than among subgroups. Substantial linkage disequilibrium (LD) was detected between the markers in the population with marked variation between chromosomes. This information may facilitate the use of the inbreds in sorghum breeding programs and provide perspectives for optimizing marker density for gene mapping and marker-assisted breeding. The second experiment, based on 102 F1 hybrids developed by intercrossing closely and distantly related inbreds, was conducted to investigate the relationship of genetic distance between parents with hybrid vigor or heterosis. The F1 hybrids alongside their parents were evaluated at two environments in a randomized complete block design with three replications. The results show that correlations of genetic distance between parents with hybrid performance and heterosis were variable and dependent on the trait. Though most were statistically non-significant and not strong to be used as predictor for heterosis, the results tend to show that certain level of genetic distance between parents is needed to capture maximum heterosis and hybrid performance. The objective of the third research study was to determine whether traits measured on parents can be used to predict hybrid performance in sorghum and to assess the combining ability of selected inbreds. Forty-six parental inbred lines and 75 F1 hybrids generated from intercrossing the inbreds were evaluated in four environments in a randomized complete block design with three replications. The average performance of the parents (mid-parent) was significantly correlated with hybrid performance for thousand kernel weight, days to flowering and plant height. Significant general (GCA) and specific (SCA) combining abilities were observed for most traits, with highly significant GCA effects observed for most traits as compared to SCA indicating that additive genetic effects are more important in affecting the inheritance of the traits measured. Results show that studying parental inbred line performance could generate important information for predicting hybrid performance in sorghum. The fourth experiment was aimed at assessing the efficacy of genomic prediction of hybrid performance in sorghum. Genomic prediction was performed with five-fold cross-validation procedure on 204 F1 hybrids developed using 102 inbred lines. A total of 66,265 SNP markers generated using genotyping-by-sequencing were used in this study. Results showed that increasing training population size increased prediction accuracies for all traits with the effect being different for different traits. Also, considering additive effects alone versus additive and dominance effects in the model showed similar trend of prediction accuracy but the full model (considering both additive and dominance effects of the markers) provided better prediction at least for some of the traits. The results suggest that genomic prediction could become an effective tool for predicting the performance of untested sorghum hybrids thus adding efficiency to hybrid selection.

Doctor of Philosophy / Genetics Interdepartmental Program / Jesse Poland / The Green Revolution is credited with saving billions of lives by effectively harnessing new genetic resources and breeding strategies to create high-yielding varieties for countries lacking adequate food security. To keep the next billion people in a state of food security, plant breeders will need to rapidly incorporate novel approaches and technologies into their breeding programs. The work presented here describes new genomic and phenomic strategies and tools aimed at accelerating genetic gain in plant breeding. Plant breeders have long relied on regional testing networks to evaluate new breeding lines across many locations. These are an attractive resource for both retrospective and contemporary analysis due to the vast amount of data available. To characterize genetic progress of plant breeding programs in the Central Plains, entries from the Southern Regional Performance Nursery dating back to 1992 were evaluated in field trials. The trend for annual improvement was 1.1% yr⁻¹, matching similar reports for genetic gain. During the same time period, growth of on-farm yields stagnated. Genomic selection, a promising method to increase genetic gain, was tested using historical data from the SRPN. A temporal-based model showed that, on average, yield predictions outperformed a year-to-year phenotypic correlation. A program-based model found that the predictability of a breeding program was similar when using either data from a single program or from the entire regional collection. Modern DNA marker platforms either characterize a small number of loci or profile an entire genome. Spiked genotyping-by-sequencing (sGBS) was developed to address the need in breeding programs for both targeted loci and whole-genome selection. sGBS uses a low-cost, integrated approach that combines targeted amplicons with reduced representation genotyping-by-sequencing. This approach was validated using converted and newly-designed markers targeting known polymorphisms in the leaf rust resistance gene Lr34. Plant breeding programs generate vast quantities of data during evaluation and selection of superior genotypes. Many programs still rely on manual, error-prone methods to collect data. To make this process more robust, we have developed several open-source phenotyping apps with simple, intuitive interfaces. A contemporary Green Revolution will rely on integrating many of these innovative technologies into modern breeding programs.

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.

Brassica napus L. is an economically important oilseed species cultivated across Western Canada. Hybrid B. napus cultivars compose the majority of the market due to their seed yield and agronomic quality. It is important to attempt to predict high-yielding parental combinations in order to conserve resources during experimental hybrid evaluation. Genetic distance between parents has been implicated in producing high-yielding hybrids and is used as one criteria for determining parental combinations.In the current study, the genetic distance between high erucic acid rapeseed (HEAR) genotypes of B. napus was established using both phenotypic and genotypic criteria. Phenotypic criteria took the form of nine agronomic and seed quality traits gathered from 318 distinct B. napus genotypes over the 2013 and 2014 field seasons in Southern Manitoba. Genotypic criteria took the form of either 291,782 SNP markers identified in 231 distinct B. napus genotypes using genotyping-by-sequencing (GBS) or 230 polymorphic sequence-related amplified polymoprhism (SRAP) markers identified in 160 B. napus genotypes. The genetic distance between available pollinators and a single male-sterile female was established using each set of criteria in an attempt to correlate genetic distance with hybrid yield. Regression analysis was conducted with yield data from hybrid genotypes gathered from 37 field sites from 2011-2014. Using the phenotypic-derived genetic distance, a significant correlation between genetic distance and hybrid yield was uncovered explaining either 22 % or 42 % of the variation in hybrid yield depending upon whether hybrids were grown at three or more, or five or more sites in the analysis, respectively. No significant link was found between GBS or SRAP-derived genetic distance and hybrid yield. These results provide evidence that that phenotypic criteria can be used to establish genetic distance with utility in the selection of high-yielding hybrid genotypes. / February 2017

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.

Species of Valerianaceae in South America represent one of the best examples of rapid diversification on a continental scale. The phylogeny of Valerianaceae has received a lot of attention within the last 10 years, but relationships among the South American species are fairly unresolved. Results from previous studies have not been well resolved with traditional genetic markers, most likely due to its recent and rapid radiation. Species in this clade exhibit a variety mating systems and inflorescence types. For the first part of this research I used several traditional plastid markers, and 3 new low copy nuclear markers to better resolve the phylogeny and then explore mating system evolution within the clade. For the second part of this research I collected high-throughput “next-generation” genomic sequence data from reduced representation libraries obtained using genotyping-by-sequencing (GBS) protocols, along with several phylogenetic methods, to try to further resolve the phylogeny of this group.

Doctor of Philosophy / Department of Agronomy / Guihua Bai / Guorong Zhang / Wheat yield and quality is influenced by many abiotic and biotic environmental factors. Pre-harvest sprouting (PHS) occurs when physiologically matured spikes are exposed to wet field conditions before harvest, which results in seed germination and causes significant losses in yield and end-use quality. Wheat stripe rust is one of the most important biotic factors reducing grain yield and quality. To investigate the genetic basis of the resistance to PHS and stripe rust in hard white winter wheat cultivars Danby and Tiger and develop molecular markers for marker- assisted breeding, a double haploid (DH) population, derived from those two cultivars, was genotyped with simple sequence repeats (SSR) markers and simple nucleotide polymorphism (SNP) markers. This DH population was assessed for resistance to PHS and stripe rust in both greenhouse and field experiments. For PHS, one major resistant quantitative trait locus (QTL) was consistently detected on the short arm of chromosome 3A in all three experiments conducted and explained 21.6% to 41.0% of the phenotypic variation (PVE). This QTL is corresponding to a previously cloned gene, TaPHS1. A SNP in the promoter of TaPHS1 co- segregated with PHS resistance in this mapping population. Meanwhile, two other QTLs, Qphs.hwwg-3B.1 and Qphs.hwwg-5A.1, were consistently detected on the chromosome arms 3BS and 5AL in two experiments. These two QTLs showed significant additive effects with TaPHS1 in improving PHS resistance. For stripe rust, three major QTLs were consistently detected in four out of six environments for infection type (IT) or disease severity (DS). Two of them, QYr.hwwg-2AS1 and QYr.hwwg-4BL1, contributed by the Danby allele explained up to 28.4% of PVE for IT and 60.5% of PVE for DS. The third QTL, QYr.hwwg-3BS1, contributed by the Tiger allele, had PVE values up to 14.7% for IT and 22.9% for DS. QYr.hwwg-2AS1 and QYr.hwwg- 4BL1 are likely the same resistance genes reported previously on chromosome arms 2AS and 4BL. However, QYr.hwwg-3BS1 might be different from the reported gene cluster near the distal end of 3BS where Yr57, Yr4, Yr30 and Sr2 were located. Significant additive effects on reducing IT and DS were observed among these three major QTLs. In order to pyramid multiple QTLs in breeding, user-friendly Kompetitive allele specific PCR (KASP) markers were successfully developed for several QTLs identified in this study. The QTLs and their interactions found in this study together with those novel flanking KASP markers developed will be useful not only for understanding genetic mechanisms of PHS and stripe rust resistance but also for marker- assisted breeding to improve wheat resistance to PHS and stripe rust by gene pyramiding.

Norway spruce (Picea abies (L.) Karst.) together with the sister species Siberian spruce (P. obovata Ledeb.) form a vast continuous distribution over Eurasia. The present distribution of P. abies in Europe was formed recently, after the last glacial maximum. Theories about the colonization routes and history of this species differ depending on the datasets examined to date. This thesis aims to investigate the genetic structure and diversity of P. abies and establish its glacial refugia and postglacial migration. A range-wide sampling was performed of both P. abies and P. obovata, and a genotyping-by-sequencing approach was used to obtain whole-genome single nucleotide polymorphism (SNP) data. Two major genetic lineages of P. abies were found; northern and central Europe. The northern lineage was further divided into a Scandinavian and a north-east European cluster; the Scandinavian cluster being more closely related to P. obovata and the north-east European cluster to the central European lineage. Introgression from P. obovata was detected far into northern Fennoscandia. The central European lineage was divided into an Alpine and a Carpathian cluster, originating from different glacial refugia. Genetic diversity was higher in the northern part of the range, which can be attributed to the relatively large refugium that recolonized this area, as well as introgression from P. obovata. Genetic diversity was also somewhat elevated where the two central European clusters meet, as is expected in areas where two previously isolated lineages admix. This study is the first range-wide investigation of P. abies using whole-genome SNP information, and shows how the genetic structure of the species has been shaped by the last glacial maximum and postglacial recolonization.

Next-generation sequencing (NGS) has revolutionized the field of genetics by providing a means for fast and relatively affordable sequencing. With the advancement of NGS, whole- genome sequencing (WGS) has become more commonplace. However, sequencing an entire genome is still not cost effective or even beneficial in all cases. In studies that do not require a whole-genome survey, WGS yields lower sequencing depth and sequencing of uninformative loci. Targeted sequencing utilizes the speed and low cost of NGS while providing deeper coverage for desired loci. This thesis applies targeted sequencing to the genomes of two different, non-model plants, Artemisia tridentate (sagebrush) and Lupinus luteus (yellow lupine). We first targeted the transcriptomes of three species of sagebrush (Artemisia) using RNA-seq. By targeting the transcriptome of sagebrush we have built a resource of transcripts previously unmatched in sagebrush and identify transcripts related to terpenes. Terpenes are of growing interest in sagebrush because of their ability to identify certain species of sagebrush and because they play a role in the feeding habits of the threatened sage-grouse. Lastly, using paralogs with synonymous mutations we reconstructed an evolutionary time line of ancient genome duplications. Second, we targeted the flanking loci of recognition sites of two endorestriction enzymes in genome of L. luteus genome through genotyping-by-sequencing (GBS). GBS of yellow lupine provided enough single-nucleotide polymorphic loci for the construction of a genetic map of yellow lupine. Additionally we compare GBS strategies for plant species without a reference genome sequence.

Doctor of Philosophy / Department of Entomology / James F. Campbell / Thomas W. Phillips / Movement is a fundamental feature of animals that impacts processes across multiple scales in space and time. Due to the heterogeneous and fragmented nature of habitats that make up landscapes, movement is not expected to be random in all instances, and an increase in fitness is an expected consequence for those that can optimize movement to find valuable and scarce recourses. I studied the movement of Rhyzopertha dominica (Coleoptera: Bostrichidae), one of the most important pests of stored grain worldwide, within and between resource patches. At a fine spatial scale, I identified factors that contribute to overall and upward movement in the grain mass. Three-week-old insects tented to stay closer to the surface than one or two-week-old insects. Females tended to be more active and to explore more than males. I also found that males tended to stay closer to the surface than females and that might be related to the ability to attract females from outside the patch since there was no significant difference regarding female’s attraction within the grain patch. Interaction with feeding sites or other individuals of the same sex creates positive feedback and a more clumped spatial pattern of feeding and foraging behavior. On the other hand, interaction with individuals of different sex creates negative feedback and a more random or overdispersed pattern. At a broad spatial scale, I studied the long-term consequence of R. dominica movement on the development of population structure within the U.S. To evaluate population structure, I used reduced representation of the genome followed by direct sequencing of beetles collected from different locations across the U.S where wheat or rice is produced and stored. Ecoregions were more important in explaining structure of R. dominica populations than crop type. I also found significant isolation by distance; however, model selection primarily elected grain production and movement variables to explain population differentiation and diversity. Understanding animal movement is essential to establishing relationships between distribution and surrounding landscape, and this knowledge can improve conservation and management strategies.