netgwas: An R Package for Network-Based Genome-Wide Association Studies

Graphical models are powerful tools for modeling and making statistical inferences regarding complex associations among variables in multivariate data. In this paper we introduce the R package netgwas, which is designed based on undirected graphical models to accomplish three important and interrelated goals in genetics: constructing linkage map, reconstructing linkage disequilibrium (LD) networks from multi-loci genotype data, and detecting high-dimensional genotype-phenotype networks. The netgwas package deals with species with any chromosome copy number in a unified way, unlike other software. It implements recent improvements in both linkage map construction (Behrouzi and Wit, 2018), and reconstructing conditional independence network for non-Gaussian continuous data, discrete data, and mixed discrete-and-continuous data (Behrouzi and Wit, 2017). Such datasets routinely occur in genetics and genomics such as genotype data, and genotype-phenotype data. We demonstrate the value of our package functionality by applying it to various multivariate example datasets taken from the literature. We show, in particular, that our package allows a more realistic analysis of data, as it adjusts for the effect of all other variables while performing pairwise associations. This feature controls for spurious associations between variables that can arise from classical multiple testing approach. This paper includes a brief overview of the statistical methods which have been implemented in the package. The main body of the paper explains how to use the package. The package uses a parallelization strategy on multi-core processors to speed-up computations for large datasets. In addition, it contains several functions for simulation and visualization. The netgwas package is freely available at https://cran.r-project.org/web/packages/netgwasComment: 32 pages, 9 figures; due to the limitation "The abstract field cannot be longer than 1,920 characters", the abstract appearing here is slightly shorter than that in the PDF fil

Disentangling hexaploid genetics : towards DNA-informed breeding for postharvest performance in chrysanthemum

DNA-informed selection can strongly improve the process of plant breeding. It requires the detection of DNA polymorphisms, calculation of genetic linkage, access to reliable phenotypes and methods to detect genetic loci associated with phenotypic traits of interest. Cultivated chrysanthemum is an outcrossing hexaploid with an unknown mode of inheritance. This complicates the development of resources and methods that enable the detection of trait loci. Postharvest performance is an essential trait in chrysanthemum, but is difficult to measure. This makes it an interesting but challenging trait to phenotype and detect associated genetic loci. In this thesis I describe the development of resources and methods to enable phenotyping for postharvest performance, genetic linkage map construction and detection of quantitative trait loci in hexaploid chrysanthemum. Postharvest performance is a complicated trait because it is related to many different disorders that reduce quality. One of these disorders in chrysanthemum is disk floret degreening, which occurs after long storage. In chapter 2, we show that degreening can be prevented by feeding the flower heads with sucrose, suggesting carbohydrate starvation plays a role in the degreening process. To investigate the response to carbohydrate starvation of genotypes with different sensitivity to disk floret degreening, we investigated the metabolome of sugar-fed and carbohydrate-starved disk florets by 1H-NMR and HPAEC. We show that the metabolome is severely altered at carbohydrate starvation. In general, starvation results in an upregulation of amino acid and secondary metabolism. Underlying causes of genotypic differences explaining variation in disk floret degreening in the three investigated genotypes remained to be elucidated, but roles of regulation of respiration rate and camphor metabolism were posed as possible candidates. In chapter 3, disk floret degreening was found to be the most important postharvest disorder after 3 weeks of storage among 44 white chrysanthemum cultivars. To investigate the inheritance of disk floret degreening, we crossed two genotypes with opposite phenotypic values of both disk floret degreening and carbohydrate content to obtain a population segregating for disk floret degreening. To phenotype the cultivar panel and the bi-parental population precisely and in a high throughput manner, we developed a method that quantified colour of detached capitula over time. This method was validated with visual observations of disk floret degreening during vase life tests. In a subset of the bi-parental population we measured carbohydrate content of the disk florets at harvest. The amount of total carbohydrates co-segregated with sensitivity to degreening, which shows that the difference in disk floret degreening sensitivity between the parents could be explained by their difference in carbohydrate content. However, the correlation was rather weak, indicating carbohydrate content is not the only factor playing a role. In order to develop resources for DNA-informed breeding, one needs to be able to characterize DNA polymorphisms. In chapter 4, we describe the development of a genotyping array containing 183,000 single nucleotide polymorphisms (SNPs). These SNPs were acquired by sequencing the transcriptome of 13 chrysanthemum cultivars. By comparing the genomic dosage based on the SNP assay and the dosage as estimated by the read depth from the transcriptome sequencing data, we show that alleles are expressed conform the genomic dosage, which contradicts to what is often found in disomic polyploids. In line with this finding, we conclusively show that cultivated chrysanthemum exhibits genome-wide hexasomic inheritance, based on the segregation ratios of large numbers of different types of markers in two different populations. Tools for genetic analysis in diploids are widely available, but these have limited use for polyploids. In chapter 5, we present a modular software package that enables genetic linkage map construction in tetraploids and hexaploids. Because of the modularity, functionality for other ploidy levels can be easily added. The software is written in the programming language R and we named it polymapR. It can generate genetic linkage maps from marker dosage scores in an F1 population, while taking the following steps: data inspection and filtering, linkage analysis, linkage group assignment and marker ordering. It is the first software package that can handle polysomic hexaploid and partial polysomic tetraploid data, and has advantages over other polyploid mapping software because of its scalability and cross-platform applicability. With the marker dosage scores of the bi-parental F1 population from the genotyping array and the developed methods to perform linkage analysis we constructed an integrated genetic linkage map for the hexaploid bi-parental population described in chapter 3 and 4. We describe this process in chapter 6. With this integrated linkage map, we reconstructed the inheritance of parental haplotypes for each individual, and expressed this as identity-by-descent (IBD) probabilities. The phenotypic data on disk floret degreening sensitivity that was acquired as described in chapter 3, was used in addition to three other traits to detect quantitative trait loci (QTL). These QTL were detected based on the IBD probabilities of 1 centiMorgan intervals of each parental homologue. This enabled us to study genetic architecture by estimating the effects of each separate allele within a QTL on the trait. We showed that for many QTL the trait was affected by more than two alleles. In chapter 7, the findings in this thesis are discussed in the context of breeding for heterogeneous traits, the implications of the mode of inheritance for breeding and the advantages and disadvantages of polyploidy in crop breeding. In conclusion, this thesis provides in general a significant step for DNA-informed breeding in polysomic hexaploids, and for postharvest performance in chrysanthemum in particular.</p

Genetic studies on self-fertility in perennial ryegrass (Lolium perenne L.) with implications for hybrid breeding in allogamous grasses

Perennial grasses have diverse uses and are relevant from the agronomic and economic point of view, with main uses as forage, turf and bioenergy. In the grass family polyploidy is prevalent and both autopolyploids and allopolyploids are present. Also, within grasses there are a range of breeding systems, but hermaphrodite flower is the most frequent floral condition. Cross-pollination in species with hermaphrodite flowers is imposed by a gametophytic genetic self-incompatibility (SI). SI is controlled by two multiallelic and independent loci, S and Z. The incompatibility phenotype of the pollen grain is determined by its haploid genome. A pollen grain is incompatible when the same S and Z alleles carried by pollen are present in the pistil. This SI mechanism keeps its functionality at higher ploidy levels. Understanding the mechanisms involved in the breakdown of SI are crucial for implementing novel breeding practices. The aim of this work is to increase the knowledge of SF in outcrossing grasses for the purpose of inbred line development and making hybrid breeding possible. Mutations at S, Z and at a third locus are known to cause self-fertility (SF). In perennial ryegrass (Lolium perenne), a locus conferring SF is located in linkage group 5. Using segregation and linkage analysis, the SF locus region was reduced to 1.6 cM. This locus explained 94% of the observed variability. By aligning the flanking marker sequences to the Brachypodium distachium reference genome, it was found that it corresponds to an 807 Kbp region in B. distachium. This locus was studied at the tetraploid level and it was found that SF remained functional, the SF locus genotype was the main determinant of pollen compatibility explaining 54% of the variation, and there is incomplete dominance between alleles at this locus in the diploid pollen grain. The prospects of migrating the SF locus from perennial ryegrass to other related self-incompatible were discussed. Based on the available information on hybridization between Lolium and Festuca species, different types of crosses were propose according to the particular species involved. The results and observations presented here contribute to a better understanding of the trait at both diploid and tetraploid levels and are promising as SF may readily be incorporated into breeding programs.Agencia Nacional de Investigación e InnovaciónUSDA - National Institute of Food and AgricultureIowa State University - Plant Sciences Institute, RF Baker Center for Plant Breeding and K.J. Frey Chair in AgronomyINIA- Instituto Nacional de Investigación Agropecuari

Genetic studies on self-fertility in perennial ryegrass (Lolium perenne L.) with implications for hybrid breeding in allogamous grasses

Perennial grasses have diverse uses and are relevant from the agronomic and economic point of view, with main uses as forage, turf and bioenergy. In the grass family polyploidy is prevalent and both autopolyploids and allopolyploids are present. Also, within grasses there are a range of breeding systems, but hermaphrodite flower is the most frequent floral condition. Cross-pollination in species with hermaphrodite flowers is imposed by a gametophytic genetic self-incompatibility (SI). SI is controlled by two multiallelic and independent loci, S and Z. The incompatibility phenotype of the pollen grain is determined by its haploid genome. A pollen grain is incompatible when the same S and Z alleles carried by pollen are present in the pistil. This SI mechanism keeps its functionality at higher ploidy levels. Understanding the mechanisms involved in the breakdown of SI are crucial for implementing novel breeding practices. The aim of this work is to increase the knowledge of SF in outcrossing grasses for the purpose of inbred line development and making hybrid breeding possible. Mutations at S, Z and at a third locus are known to cause self-fertility (SF). In perennial ryegrass (Lolium perenne), a locus conferring SF is located in linkage group 5. Using segregation and linkage analysis, the SF locus region was reduced to 1.6 cM. This locus explained 94% of the observed variability. By aligning the flanking marker sequences to the Brachypodium distachium reference genome, it was found that it corresponds to an 807 Kbp region in B. distachium. This locus was studied at the tetraploid level and it was found that SF remained functional, the SF locus genotype was the main determinant of pollen compatibility explaining 54% of the variation, and there is incomplete dominance between alleles at this locus in the diploid pollen grain. The prospects of migrating the SF locus from perennial ryegrass to other related self-incompatible were discussed. Based on the available information on hybridization between Lolium and Festuca species, different types of crosses were propose according to the particular species involved. The results and observations presented here contribute to a better understanding of the trait at both diploid and tetraploid levels and are promising as SF may readily be incorporated into breeding programs.Agencia Nacional de Investigación e InnovaciónUSDA - National Institute of Food and AgricultureIowa State University - Plant Sciences Institute, RF Baker Center for Plant Breeding and K.J. Frey Chair in AgronomyINIA- Instituto Nacional de Investigación Agropecuari

Linkage mapping in prairie cordgrass (Spartina pectinata Link) using genotyping-by-sequencing markers

Global climate change from anthropogenic carbon emissions can be mitigated through the development of bioenergy systems. Bioenergy feedstocks tolerant of marginal land can reduce the tension between bioenergy production and food production. Prairie cordgrass (Spartina pectinata Link) is tolerant of poorly drained soils, saline soils, and low temperature. Prairie cordgrass (Spartina pectinata) is a polyploid Chloridoid grass with tetraploid (2n=4X=40), hexaploid (2n=6X=60), and octaploid (2n=8X=80) cytotypes, and is a potential dedicated energy crop with promising yields in marginal environments. Breeding efforts in prairie cordgrass are currently hampered by the lack of a linkage map, the lack of a Chloridoid reference genome, and by lack of information on inheritance patterns (disomic versus polysomic). Genotyping-by-sequencing (GBS) was applied to a population of 85 progeny from a reciprocal cross of heterozygous tetraploid parents. A total of 26,418 SNPs were discovered, with a distribution of allele frequencies suggesting disomic inheritance. A filtered set of 3,034 single-dose, high-coverage SNPs was used for pseudo-testcross mapping, resulting in two parental maps of 20 linkage groups containing 1,522 and 1,016 SNPs and a nearly 1:1 ratio of coupling to repulsion phase linkages, again suggesting disomic inheritance. Genomic contigs from tef (Eragrostis tef (Zucc.) Trotter), another Chloridoid grass, were used as a bridge to associate short prairie cordgrass reads with unique positions in the sorghum genome, providing a first glimpse into synteny between Chloridoids and other grasses. Genotyping-by-sequencing enabled rapid generation of a linkage map that will aid in future breeding and genomics efforts in prairie cordgrass

Geographical parthenogenesis

Diese Dissertation hat zum Ziel, das Phänomen der geographische Parthenogenese (GP) zu verstehen. GP bedeutet, dass sexuelle und asexuelle Populationen desselben Artkomplexes nicht die gleiche geographische Verbreitung aufweisen. Im allgemeinen besetzen asexuelle Populationen höhere Lagen und Breitengrade, und sind weiter verbreitet. Geographische Parthenogenese kombiniert verschiedene Aspekte, die für das Verständnis der Verbreiungsmuster betrachtet werden müssen: das Repoduktion-System, Polyploidisierung, genetische Diversität und die Fähigkeit zur Kolonisierung. Die alpine Art Ranunculus kuepferi ist als Modell zum Studium der geographischen Parthenogenese geeignet, da polyploide, asexuelle Populationen im gesamten Alpengebiet weit verbreitet sind, während die diploiden sexuellen Populationen auf glaziale Refugien beschränkt sind. Ich habe verschiedene Formen der Reproduktion und die Stabilität der Zytotypen mittels Flow Cytometry (FC) und Durchflusszytometrie an Samen (FCSS) untersucht. Die Herkunft der Polyploidisierung und die genetische Vielfalt der Populationen wurde mit molekular-biologischen Methoden, mit Amplified Fragment Length Polymorphism (AFLPs; dominante Marker) und Mikrosatelliten (SSR; kodominanten Marker) analysiert. Diese Studien wurden an 60 Populationen aus dem ganzen Verbreitungsgebiet dieser Art durchgeführt. All diese Marker haben dazu beigetragen, Aufschluss über die genetische Struktur und den Einfluss der Reproduktion auf die Entstehung von geographischer Parthenogenese zu erhalten. Die drei Kapitel der Arbeit behandeln die verschiedenen Aspekte wie folgt: Die Stabilität der Zytotypen wurde mittels FC, der Fortpflanzungsmodus mittels FCSS bestimmt. Diese Analyse ergab, dass die diploiden Populationen stabil und voll sexuell bleiben. Triploide individuen kommen in der Kontaktzone vor und sind ein Produkt von Rückkreuzungen zwischen diploiden und tetraploiden Populationen. Außerhalb der Kontaktzone weisen in tetraploiden Populationen c. 30% der Samen triploide Embryos auf, aber triploide erwachsene Pflanzen treten nur einmal im ganzen tetraploiden Verbreitungsgebiet auf. Die Mehrheit der Tetraploiden behält eine stabile Ploidiestufe mittels gametophytischer Apomixis, wobei die Bildung des Endosperms entweder pseudogam oder autonom erfolgt. Einige wenige Prozent der Samen der tetraploiden Pflanzen sind auf sexuellem Weg entstanden. Polyploidisierung kann eine Erklärung geben, warum Apomixis in der Art Ranunculus kuepferi entstanden ist. Die autopolyploide Herkunft der Tetraploiden wurde mittels Bayesian-Analyse der Populations-Struktur (BAPS) Analyse der SSRs nachgewiesen. Damit konnte gezeigt werden, dass die tetraploiden aus diploiden Populationen ohne den Beitrag eines anderen Genoms entstanden sind. Die tetraploiden entstanden mehrfach von diploiden Populationen, da sie fast keine genetischen Unterschiede und nur wenige neue SSRs-Allele zeigen. AFLPs bestätigen in einer Neighbor Joining Analyse, dass diploide und tetraploide Populationen den gleichen Gen-Pool aufweisen. Daher ist der geographische Erfolg der Apomikten nicht auf den Beitrag eines neuen Genoms zurückzuführen, wie es in Allopolyploiden anzunehmen ist. Apospory scheint die Probleme in der Meiose, die durch Multivalentbildung verursacht wird, zu überwinden, was vom Vorkommen von Genotypen mit multiple Allelen in den SSR Loci angenommen werden kann. Diese ungewöhnliche Kombination von Autopolyploidie und Aposporie stabilisiert das Reproduktionssystem und damit die Ploidiestufe. Die Auswirkung von Reproduktionssystem und der genetischen Diversität der Populationen wurden durch die Verwendung von AFLPs und SSRs untersucht. Tetraploide Populationen von Ranunculus kuepferi haben die Selbst-Inkompatibilität verloren, wodurch pseudogame Selbstbestäubung ermöglicht wird, während die diploiden selbst-inkompatibel bleiben. Daher können Apomikten leichter neue Populationen, potentiell sogar mit einem einzigen Samen gründen, und zeigen damit eine bessere Fähigkeit zur Kolonisierung. Eine BAPS Analyse der AFLP Daten bestätigt, dass die Apomikten populationsspezifische Gen-Pools aufweisen, die wahrscheinlich auf mehrfache Gründer-Effekte zurückzuführen sind. Diploide sexuelle Populationen hätten den Vorteil einer höheren genetischen Vielfalt durch Rekombination. Mein Ergebnis zeigt eine genetische Diversität, die typisch für sexuelle auskreuzende Arten ist. Die tetraploiden Apomikten weisen jedoch ein gleiches Ausmaß genetischer Diversität hinsichtlich Heterozygotie und der Fst Werte auf; außerdem zeigen sie keine Klonalität. Genetische Vielfalt wird über fakultative Apomixis beibehalten und ermöglicht die weite Verbreitung der Tetraploiden. Die tetraploiden Populationen profitieren von Vorteilen beider Reproduktions-Systeme (besser Kolonisierungsfähigkeit und genetische Vielfalt), und sind damit effizienter als die diploiden, ein großes Verbreitungsgebiet zu besiedeln.This doctoral thesis aims at understanding geographical parthenogenesis, which means that sexual and asexual populations of the same species complex do not share the same distribution area. In general, asexuals occupy higher altitudes and latitudes and are more widespread. Geographical parthenogenesis combines different aspects that need to be considered for understanding the pattern: the reproduction system, poly-ploidisation events, genetic diversity, and colonization ability. The alpine species Ranunculus kuepferi is a model for studying geographical parthenogenesis, since polyploid, putative asexual populations are widespread throughout the Alps, while diploids sexual are confined to small refugial areas. I investigated different modes of reproduction and stability cytotypes via Flow Cytometry (FC) and Flow Cytometric Seed Screening (FCSS) in seeds. The origin of polyploidisation and genetic diversity of populations was analyzed with molecular methods, by using Amplified Fragment Length Polymorphism (AFLPs; dominant marker) and microsatellites (SSRs; codominant marker). These studies were conducted on 60 populations out of the whole distributional range of the species. All these markers helped to reveal the population genetic structure and the importance of modes of reproduction for the evolution of geographical parthenogenesis. The three chapters of the thesis treat the different aspects as follows: Cytotype stability for each ploidy level was assessed over the distribution area with FC, and the mode of reproduction was determined via FCSS. This analysis revealed that diploids remain stable and fully sexual. Triploids in the contact zone are a product of backcrossing between diploids and tetraploids. Outside the contact zone, 30% of the seed display triploid embryos in tetraploid populations, but triploid adult plants occur only once in the whole tetraploid area. The majority of tetraploids maintain a stable ploidy level via gametophytic apomixis with either pseudogamous or autonomous endosperm formation. However, a few percent of seeds of tetraploids are formed in the sexual way. Polyploidization events may give an explanation why apomixis originated in the species Ranunculus kuepferi. Evidence for an autopolyploid origin of the tetraploids was presented by Bayesian Analysis of Populations Structure (BAPS) analysis of SSRs, which showed that tetraploids originated from diploids without the contribution of another genome. Tetraploids obviously originated several time from diploids since they show almost no divergence and only few new alleles in SSRs are present in tetraploids. AFLPs show in a Neighbor Joining analysis that diploids and tetraploids share the same gene pool. Therefore, geographical success of apomicts does not result from genomic novelty, which could be predicted in allopolyploids. Apospory seems to help to overcome the problems occurring in unbalanced meiosis caused by multivalent formation, which can be assumed from genotypes exhibiting multiple allelism in SSR loci. This uncommon combination of autopolyploidy and apospory stabilizes the reproductive system and hence the ploidy level. Effects of breeding system and genetic diversity of populations were further studied by using AFLPs and SSRs. Tetraploids of Ranunculus kuepferi have a breakdown of the self-incompatibility system, allowing for pseudogamous selfing, whereas diploids remain self-incompatible. Therefore, apomicts can easily establish new populations, potentially with a single seed, and thus they have superior colonization ability. A BAPS analysis of AFLP data confirms populations-specific gene pools in apomicts which have probably resulted from multiple founder events. Diploids ought to have the advantage of higher genetic diversity via recombination, as our result show a genetic diversity typical for sexual outcrossers. However, tetraploids show the same level of diversity with respect to heterozygosity and Fst values, as well as they exhibit no clonality. Genetic diversity is maintained via facultative apomixis and allows maintenance of the widespread distribution pattern of tetraploids. Tetraploids profit from the best of both reproductive systems (better colonization ability and genetic diversity), and therefore they are more efficient than the diploids to spread over the major distribution area

Genetic mapping in polyploids

Many of our most important crop species are polyploid – an unusual phenomenon whereby each chromosome is present in multiple copies (more than the usual two copies). The most common such arrangement is tetraploidy, where each chromosome is present four times. Plant species can tolerate this condition quite well (the same cannot be said of animals or humans). In fact, polyploidy can confer certain advantages such as larger fruits and flowers, seedless fruits (useful for fruit growers) or improved tolerance to environmental stresses. However, carrying multiple copies of each chromosome complicates things, particularly when crop breeders would like to use DNA information to help inform selection decisions. This PhD project looked at how DNA information of polyploids should be best analysed, developing methods and new software tools to achieve this. We analysed DNA information from polyploid crops such as potato, rose and chrysanthemum, yielding many novel insights and important results.</p

Breeding and genetics of lilacs and hardy hibiscus

Lilacs are a group of ornamental trees and shrubs in the Oleaceae family consisting of 22 to 30 species. There are six series within genus Syringa: Pubescentes, Villosae, Ligustrae, Ligustrina, Pinnatifoliae, and Syringa. Fertility and cross-compatibility among cultivars, species, and series have yet to be formally investigated. Over three years, a cross-compatibility study was performed using elite cultivars and species of shrub-form lilacs in series Syringa, Pubescentes, and Villosae. We report the success of each of these combinations and the fertility estimates of viable crosses. This study is a comprehensive investigation of lilac hybridization, and the knowledge gained on cross-compatibility will aid future efforts in lilac cultivar development.Genome size variation can be used to investigate biodiversity, genome evolution, and taxonomic relationships among related taxa. In addition, plant breeders use genome size variation to identify parents useful for breeding sterile or improved ornamentals. Reports conflict on genome evolution, base chromosome number, and polyploidy in lilac. Flow cytometry was used to estimate holoploid (2C) genome sizes in series, species, cultivars, and seedlings from parents with three ploidy combinations: 2x x 2x, 2x x 3x, and 3x x 2x. Monoploid (1Cx) genome sizes were calculated by dividing 2C genome size by ploidy, which was confirmed in a subset of taxa using root tip microscopy. Pollen diameter was measured toinvestigate the frequency of unreduced gametes in diploids and triploids. Interploid crosses between ‘Blue Skies’ (2x) and ‘President Grévy’ (3x) produced an aneuploid population with variable 2C genome sizes. One viable seedling was recovered from a cross between ‘President Grévy’ (3x) and ‘Sensation’ (2x). This near pentaploid (5x) seedling had a larger 2C genome size than either parent, and the largest 2C genome size currently reported in lilac. Pollen diameter measurements revealed that ‘Sensation’ produced 8.5% unreduced pollen. Increased ploidy may provide a mechanism for recovering seedlings from incompatible taxa in lilac breeding.Common lilac, Syringa vulgaris, is an important flowering shrub that accounts for a large share of spring sales in the U.S. nursery industry. However, little research has focused on shortening generation time for lilac breeders. In a previous cross-compatibility study, observations revealed that first-year hybrid seedlings undergo a quiescent phase of growth, producing few leaves but an extensive root system. This study investigated the effects of six germination and post-germination treatments of green seed and dry, dehisced seed on seed germination and subsequent growth in lilacs. Green seed extracted 20 weeks after pollination had the highest germination rate and an increase in vegetative growth compared to controls. Our results indicate that green seed sowing may provide a new tool for shortening juvenility and reduced breeding time in common lilac.Remontancy (reblooming) and disease resistance are two important traits in the dwarf lilacs (Syringa pubescens). Marker-assisted selection could prove useful at producing more disease-resistant, floriforous lilacs for future breeders. To aid future efforts at at marker discovery, genotyping-by-sequencing was applied to a bi-parental mapping population from S. meyeri ‘Palibin’ x S. pubescens Bloomerang® which varies for remontancy and resistance to bacterial blight. SNP-based genetic linkage maps were created for each parent, and maps will continue to be improved with further sequence data. Future efforts to phenotype the mapping population will be combined with these findings for marker-trait association.Althea (Hibiscus syriacus) is an ornamental shrub prized for its winter hardiness and large colorful summer flowers. Althea are primarily tetraploids (2n = 4x = 80) with higher level polyploids reported from experiments with spindle-fiberinhibitors. Previous studies report anatomical variation among althea polyploids, including changes in stomata size. The purpose of this study was four-fold. The first was to identify genome size and ploidy variation in althea cultivars via flow cytometry and root tip chromosome counts. The second was to create a ploidy series consisting of 4x, 5x, 6x, and 8x cytotypes using a combination of interploid hybridization and autopolyploid induction via colchicine and oryzalin. The third was to investigate the ploidy series for variation in stomatal guard cell length, stomatal density, and copy number of fluorescent rDNA signals. The fourth was to investigate segregation patterns in rDNA signals in a subset of pentaploid seedlings. Results of this study revealed ploidy differences among available cultivars. Polyploid induction and interploid hybridation were successful for producing a ploidy series that varied in stomata size, stomata density, and number of 5S and 45S rDNA signals. The rDNA loci confirmed ploidy levels in each cytotype of our ploidy series, and random segregation of rDNA loci provides evidence of random chromosome segregation in interploid hybrids of althea.Despite its attractive, ornamental flowers, althea produces capsules with numerous, fertile seeds that germinate and cause a nuisance in production and the home landscape. Breeding for sterile forms of althea has long been a goal for Hibiscus breeders, yet many popular “sterile” cultivars have been reported as weedy. The purpose of this study was to evaluate female and male fertility for tetraploid and hexaploid cultivars, and to evaluate the female fertility of pentaploid seedlings resulting from 4x x 6x and 6x x 4x crosses. Self- and cross-incompatibilities were discovered, as was variation in seeds per capsule and seeds per pollination. In addition, significant differences were found among flower forms (single, semi-double, and double) for fertility estimates. Double-flowered forms had reduced female fertility, which may indicate that breeding for increased petaloid stamen may result in a reduction in female fertility. Previously reported sterile taxa were also found to be fertile, including ‘Aphrodite’, ‘Diana’, ‘Helene’ and ‘Minerva’. Two hexaploids, ‘Pink Giant’ and Raspberry Smoothie™, were found to have reduced female fertility compared to tetraploids. Fertility testcrosses of pentaploid seedlings revealed a reduction in fertility compared to controls. The reduction in fertility ofpentaploids will likely lead to new, near sterile cultivars for the nursery industry. The combination of double flowers with pentaploid cytotypes will likely lead to completely sterile cultivars of althea.Although floral traits are most important for breeders of althea, little is known about their segregation patterns. The objectives of this study were to determine segregation patterns in eyespot presence, flower color, and flower form. Over four years, thousands of flowering seedlings were observed representing F1, F2, and backcross families. Based on our results, we propose that eyespot presence is controlled by a single locus and that a recessive allele called spotless results in a complete elimination of color. The gene controlling spotless is likely located upstream in the flavonoid biosynthetic pathway. We also propose that flowers with white to blush-pink petal body color and a red eyespot are controlled by a single recessive allele called geisha. This trait exhibits incomplete dominance and is under epistatic control by spotless. It is likely located downstream in the delphinidin biosynthetic pathway, responsible for lavender, dark pink, and blue pigments. In addition to color segregation, depth of color irrespective of hue (CIE L*) was also investigated (spotless and geisha seedlings removed). The deepest pigments were measured in crosses among hexaploid ‘Pink Giant’, taxa homozygous dominant for geisha, and taxa heterozygous for geisha. Conversely, the lightest pigments were observed in crosses between taxa homozygous recessive for geisha and taxa heterozygous for geisha. Future efforts at eliminating the geisha allele from a breeding population may allow for quantitative improvement in total anthocyanin production. Observations on petal number inheritance revealed that seedlings produced a continuous distribution of petal numbers between the petal numbers of the two parents, with occasional transgressive segregants. The highest average petal numbers were found in seedlings resulting from the cross of double-flowered taxa. Flower size (petal area), varied significantly among cross combinations and flower forms. The largest petals were observed in the seedlings of single-flowered by double-flowered crosses. Concomitant upregulation or expression of genes controlling laminar growth in stamen may not only result in petaloid stamen, but may also result in increased laminar growth in the true petals, resulting in wider,overlapping petals. However, further work must be undertaken to eliminate environmental effects on flower size estimates.Keywords: segregation, fertility, ploidy, hybridization, cross-compatibilit