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

EM Algorithm for Mapping Quantitative Trait Loci in Multivalent Tetraploids

Multivalent tetraploids that include many plant species, such as potato, sugarcane, and rose, are of paramount importance to agricultural production and biological research. Quantitative trait locus (QTL) mapping in multivalent tetraploids is challenged by their unique cytogenetic properties, such as double reduction. We develop a statistical method for mapping multivalent tetraploid QTLs by considering these cytogenetic properties. This method is built in the mixture model-based framework and implemented with the EM algorithm. The method allows the simultaneous estimation of QTL positions, QTL effects, the chromosomal pairing factor, and the degree of double reduction as well as the assessment of the estimation precision of these parameters. We used simulated data to examine the statistical properties of the method and validate its utilization. The new method and its software will provide a useful tool for QTL mapping in multivalent tetraploids that undergo double reduction

Development of novel theory and methods for QTL analysis and inferring crossover interference in autotetraploids

Polyploidization widely occurs in the evolutionary history of eukaryotes, especially for flowering plants. Exploring the evolutionary and agricultural important role by polyploidy is mystery and challenging job. In the first chapter of this thesis, I developed a quantitative genetics model based on orthogonal contrast scales, which would provide theoretical basis for any further bi-allelic QTL analysis in autotetraploid species. In the next chapter, I developed an interval mapping method for QTL mapping in outbred population for autotetraploid, considering both bivalent and quadrivalent pairing during meiosis. Extensive simulation work has been implemented to demonstrate the reliability of this method. This work would provide practical tools for breeding the world’s third most important crop, the cultivated potato. To give some insight into the evolutionary important role played by polyploidization, I developed a statistical method for inferring crossover interference based on three-locus analysis in autotetraploids in the third chapter and a method to predict genome-wide crossover rate for autotetraploid yeast in the final chapter

Mapping of the apomixis region and QTLs in "Megathyrsus maximu"s : an autotetraploid tropical forage grass

Orientadores: Anete Pereira de Souza, Liana JankTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O Brasil é destaque no mercado internacional quanto à produção e exportação de carne, visto que possui o maior rebanho bovino comercial do mundo. Dentre as principais gramíneas forrageiras de clima tropical responsáveis por parte da engorda animal a pasto, "Megathyrsus maximus" (Jacq.) B. K. Simon & S. W. L. Jacobs (sinonímia "Panicum maximum" Jacq.) é uma das mais produtivas, estando envolvida na alimentação de caprinos, equinos, ovinos, gado de corte e de leite. A autotetraploidia, a apomixia e o recente processo de domesticação indicam uma maior complexidade envolvida no melhoramento genético dessa espécie, que torna indispensável o esclarecimento dos mecanismos genéticos que podem estar envolvidos. Dessa forma, o melhoramento molecular poderá proporcionar o aumento da eficiência do programa de melhoramento e a redução do tempo nas etapas envolvidas para o lançamento de cultivares. Neste contexto, mapas de ligação têm sido utilizados como base para a compreensão da organização genômica das espécies, principalmente as não-modelo e complexas que não possuem o genoma sequenciado, tais como "M. maximus". Com isto, este trabalho teve como objetivo a construção de um mapa de ligação para a gramínea forrageira "M. maximus" a partir de marcadores SNPs com informação de dosagem alélica, visando detectar a região do locus da aposporia, bem como loci de caracteres quantitativos (QTLs) envolvidos com a produtividade e qualidade nutricional da forragem. A progênie de mapeamento deste trabalho foi obtida pelo programa de melhoramento genético da Embrapa Gado de Corte (Campo Grande, Mato Grosso do Sul), sendo composta por 136 híbridos F1 originados a partir do cruzamento de genitores autotetraploides (2n = 4x = 32), um sexual (S10) e o outro apomítico (cv. Mombaça). A partir do DNA extraído das plantas, bibliotecas de GBS (genotyping-by-sequencing) foram construídas com o uso de duas enzimas de restrição (PstI-MspI). Os dados obtidos com o sequenciamento foram analisados no software Tassel4-poly. O alinhamento das sequências foi realizado com cinco genomas de espécies filogeneticamente próximas: Panicum hallii, P. virgatum, Setaria italica, S. viridis e Urochloa ruziziensis, além de três transcriptomas de "M. maximus". A dosagem alélica dos marcadores SNPs identificados foi estimada por meio dos programas SuperMASSA e Updog, visando incluir no mapa todas as configurações de SNPs para um tetraploide. Marcadores monomórficos e SNPs com mais de 25% de dados perdidos foram removidos manualmente usando o software R. Para a construção do mapa de ligação foram utilizados os programas polymapR e TetraploidSNPMap, os quais possibilitam o uso de SNPs com informação de dosagem alélica. O genoma de P. virgatum apresentou melhor alinhamento aos dados de GBS, sendo utilizado como referência. O mapa de ligação consenso e integrado construído contém 8 grupos de homologia (GH) e 858 marcadores SNPs com todas as doses esperadas para uma progênie F1 tetraploide. A densidade média do mapa genético foi de aproximadamente 1,13 SNPs/cM, sendo o GH 7 o maior (108, 6 cM) e mais denso (159 SNPs), além de ter sido o único GH com todas as doses presentes. As análises de ligação permitiram detectar o evento da redução dupla, um tipo de distorção de segregação que ocorre principalmente em autopoliploides. Com este mapa e por meio de uma análise qualitativa, o locus ligado à aposporia foi mapeado no GH 2 a 65 cM. A partir da análise de similaridade da região aos marcadores adjacentes a esse locus, utilizando como base o genoma de P. virgatum, foi encontrado um gene relacionado à embriogênese somática. Por meio do método de mapeamento por intervalo, foram detectados 10 QTLs para caracteres agronômicos, como capacidade de rebrota e matéria seca total, e 36 QTLs relacionados a qualidade da forragem, como digestibilidade in vitro da matéria orgânica, lignina e celulose. Um total de 55 famílias gênicas foram identificadas por meio da análise de similaridade, com destaque para genes potencialmente envolvidos na regulação de hormônios de crescimento da planta e de síntese de lignina. Esta tese fornece contribuições inéditas para a espécie M. maximus, tais como a identificação do evento de redução dupla, um mapa de ligação de alta resolução construído a partir da informação de dosagem alélica dos SNPs e o mapeamento da região do locus da aposporia e de caracteres agronômicos e nutricionais. Além do avanço sobre o conhecimento da genética, esses resultados promovem informações práticas ao programa de melhoramento genético da espécie. Igualmente, são um incentivo a estudos adicionais sobre a apomixia e a evolução da herança polissômica nesta e em outras gramíneas tropicaisAbstract: Brazil has a prominent role in the international meat market for its production and exportation, once it has the largest commercial cattle herd in the world. Among the main forage grasses of tropical climate that are present in ruminant feed on pasture, "Megathyrsus maximus" (Jacq.) B. K. Simon & S. W. L. Jacobs (syn. "Panicum maximum" Jacq.) is one of the most productive, being involved in the feeding of goats, horses, sheep, beef and dairy cattle. Autotetraploidy, apomixis and the recent domestication process indicate a greater complexity involved in the genetic improvement of this species, which makes it essential to clarify the genetic mechanisms that may be involved. Thus, molecular improvement will be able to increase the efficiency of the breeding program and also reduce the time involved in steps on launching cultivars. In this context, linkage maps have been used as basis for understanding the genomic organization of species, mainly non-model and complex ones that do not have sequenced genomes, such as M. maximus. Thus, this work aimed to construct a linkage map for the forage grass M. maximus from SNP markers with allele dosage information. This was intended to detect the genomic region of the apospory locus and quantitative trait loci (QTLs) involved in forage yield and nutritional quality. The mapping progeny of this work was obtained from the breeding program of Embrapa Gado de Corte (Campo Grande, Mato Grosso do Sul), being composed of 136 F1 hybrids originated from the cross between autotetraploid parents (2n = 4x = 32), one sexual (S10) and the other apomictic (cv. Mombaça). The individuals were sampled in genotyping-by-sequencing (GBS) libraries constructed using two restriction enzymes (PstI-MspI). The data obtained with these sequencing were analysed using Tassel4-poly. Sequence alignment was performed with five genomes of phylogenetically close species: Panicum hallii, P. virgatum, Setaria italica, S. viridis and Urochloa ruziziensis, as well as three transcriptomes of M. maximus. Allele dosage for SNPs markers identified was estimated using the SuperMASSA and Updog softwares, aiming to include SNP higher doses (AAaa, AAAa and AAAA) in the map. Monomorphic markers and SNPs with over 25% missing data were manually removed using the R software. For the construction of the linkage map, polymapR and TetraploidSNPMap softwares, were used which enabled the usage of SNPs with allele dosage information. The P. virgatum genome presented better alignment with GBS data, wherein the linkage map obtained with TetraploidSNPMap was based on. The consensus integrated linkage map contains 8 homology groups (GH) and 858 SNP markers with all doses expected in a tetraploid progeny. The average density was about 1.13 SNPs/cM, with GH 7 presenting the largest size (108.6 cM) and the most markers (159 SNPs); only this group contained markers in all doses. Linkage analysis allowed the detection of a double reduction event, a type of segregation distortion that occurs mainly in autopolyploids. One locus in the apomixis region was mapped to HG II at 65 cM, using our linkage map. From similarity analysis to the markers, which are adjacent to the locus linked to apospory, using as base on the P. virgatum genome resulted in a gene related to somatic embryogenesis. Using interval mapping, ten QTLs for agronomic traits, such as regrowth capacity and total dry matter were detected, as well as 36 QTLs related to forage quality, such as in vitro digestibility of organic matter, lignin, and cellulose. A total of 55 gene families were identified by similarity analysis, highlighting putative genes involved in the regulation of plant growth hormones and lignin synthesis. This thesis provides unprecedented contributions to M. maximus, such as the identification of a double reduction event, a high-resolution linkage map constructed from SNPs with allele dosage information, and the mapping of apospory region, agronomic and nutritional traits. In addition to the advance on the knowledge of genetics, these results provide practical information for the breeding program of this species. They are also an incentive for further studies on apomixis and the evolution of polysomic inheritance in this and other tropical grassesDoutoradoGenetica Vegetal e MelhoramentoDoutora em Genética e Biologia Molecular88887.335258/2019-002017/17969-5CAPESFAPES

FitTetra 2.0-improved genotype calling for tetraploids with multiple population and parental data support

BackgroundGenetic studies in tetraploids are lagging behind in comparison with studies of diploids as the complex genetics of tetraploids require much more elaborated computational methodologies. Recent advancements in development of molecular techniques and computational tools facilitate new methods for automated, high-throughput genotype calling in tetraploid species. We report on the upgrade of the widely-used fitTetra software aiming to improve its accuracy, which to date is hampered by technical artefacts in the data.ResultsOur upgrade of the fitTetra package is designed for a more accurate modelling of complex collections of samples. The package fits a mixture model where some parameters of the model are estimated separately for each sub-collection. When a full-sib family is analyzed, we use parental genotypes to predict the expected segregation in terms of allele dosages in the offspring. More accurate modelling and use of parental data increases the accuracy of dosage calling. We tested the package on data obtained with an Affymetrix Axiom 60k array and compared its performance with the original version and the recently published ClusterCall tool, showing that at least 20% more SNPs could be called with our updated.ConclusionOur updated software package shows clearly improved performance in genotype calling accuracy. Estimation of mixing proportions of the underlying dosage distributions is separated for full-sib families (where mixture proportions can be estimated from the parental dosages and inheritance model) and unstructured populations (where they are based on the assumption of Hardy-Weinberg equilibrium). Additionally, as the distributions of signal ratios of the dosage classes can be assumed to be the same for all populations, including parental data for some subpopulations helps to improve fitting other populations as well. The R package fitTetra 2.0 is freely available under the GNU Public License as Additional file with this article.</p

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

Phenotypic and meiotic differences between diploid and polyploid plants

Polyploidy is present in a large number of crop plants and is considered as one of the driving forces in the evolution of angiosperms. Unlocking genetic variation in various autopolyploid crop plants is highly relevant to crop breeders. Homologous recombination, a tightly controlled cell process during the production of gametes in meiosis, is responsible for creation of genetic variation. Owing to the presence of more than two homologous chromosomes, polyploid meiosis faces a variety of challenges, such as multivalent formation and mis-segregation. Using a plant trial with more than 300 diploid and tetraploid Arabidopsis thaliana F2 individuals, significant differences were found in various traits between the two populations. Cytological analysis using FISH on diploid and tetraploid plants revealed an overall increase in meiotic recombination in tetraploids, although the per bivalent frequency was reduced. The process of meiotic recombination was further explored in potato (Solanum tuberosum), a globally important autotetraploid crop. Chiasma frequency and multivalent frequency for chromosomes 1 and 2 varied according to variety, where the diploid variety showed a reduced chiasma frequency compared with tetraploid varieties. Immunolocalisation of the axis and synaptonemal complex proteins, ASY1 and ZYP1, demonstrated the complexities that may arise during meiosis in an autotetraploid plant