Knowledge of the genetic variation of crop collections is essential for their efficient use in plant breeding programs. The Ethiopian Highland Maize Germplasm Collection Mission was launched throughout the highlands of Ethiopia in 1998 and 287 traditional maize accessions were collected from farmers’ fields. To date, no information was available on the morphological and genetic diversity in this important collection. Various molecular marker techniques and quantitative genetics approaches were applied to accurately unravel the extent of phenotypic and genetic diversity, to study patterns of morphological and molecular variation and to determine association of molecular markers with quantitative trait variation, with the view of designing a sound breeding program and management strategy for maize in the highlands of Ethiopia. The morphological study confirmed that traditional Ethiopian highland maize accessions contain large amounts of variation for agro-morphological traits. The broad trait diversity observed among the accessions suggested ample opportunities for the genetic improvement of the crop through selection directly from the accessions and/ or the development of inbred lines for a future hybrid program. Selection practices followed by local farmers are mostly consistent within agroecology and gave rise to morphologically distinct maize accessions in different agroecologies. This underscores the importance of considering farmers’ knowledge of diversity in the collection and evaluation of local accessions. The results of amplified fragment length polymorphism (AFLP) and microsatellite or simple sequence repeat (SSR) marker analyses showed that bulking leaf samples from 15 individual plants per out-bred accession is an effective means of producing representative profiles of individual plants, thereby reducing the cost of DNA extraction and subsequent marker analysis of open-pollinated varieties. Cluster analyses based on AFLP and SSR data showed that most of the accessions collected from the Northern agroecology were genetically distinct from the Western and Southern accessions suggesting that differentiation for adaptive traits for drought conditions may have occurred in the Northern accessions. However, there was very little genetic differentiation between the Western and Southern accessions suggesting gene flow between the two agroecologies and recent introduction of similar improved varieties in these agroecoogies . In both marker systems, high mean genetic diversity was observed among the traditional Ethiopian highland maize accessions. This is possibly due to (i) the continuous introduction of maize from abroad by different organizations; (ii) genetic variation generated through farmers management practices; and (iii) the presence of different environmental conditions in the highlands of Ethiopia to which local landraces may have been adapted. The correlation between the morphological dissimilarity matrix and the matrices of genetic dissimilarity based on SSR and AFLP markers were 0.43 and 0.39, respectively (p = 0.001 in both cases). The correlation between SSR and AFLP dissimilarity matrices was 0.67 (p = 0.001). These significant correlations indicate that the three independent sets of data likely reflect the same pattern of genetic diversity, and validate the use of the data to calculate the different diversity statistics for Ethiopian highland maize accessions. From this study, three groups of maize accessions with distinctive genetic profiles and morphological traits were identified that will be useful for future collection, conservation and breeding programs of maize for the highlands of Ethiopia. A pilot association study using SSR markers and quantitative trait variation indicated that molecular markers could be useful to identify genetic factors controlling earliness, tallness, grain yield and associated traits, which could be exploited by various breeding schemes. The analytical tools outlined in this dissertation can be a useful tool in managing genetic variation of open-pollinated crops and will aid in the conservation of unique genetic diversity. Production stability and global food security are linked to the conservation and exploitation of worldwide genetic resources and this research attempts to add to that body of knowledge. Copyright 2005, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Beyene, YA 2005, Genetic analysis of traditional Ethiopian Highland Maize (Zea Mays l.) using molecular markers and morphological traits : implication for breeding and conservation, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02212006-112610 / > / Thesis (PhD (Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

Les deux sous-espèces annuelles de téosinte qui sont les plus proches parents sauvages du maïs sont d’excellents systèmes pour étudier l’adaptation locale car leur distribution couvre un large éventail de conditions environnementales. Zea mays ssp. parviglumis est distribuée dans un habitat chaud et mésique en dessous de 1800 m d’altitude, tandis que Zea mays ssp. mexicana prospère dans des conditions sèches et fraîches à des altitudes plus élevées. Nous avons combiné des approches d’écologie inverse et de génétique association afin d’identifier les déterminants de l'adaptation locale chez ces téosintes. A partir de données de séquençage haut débit (HTS) de six populations comprenant des populations de basses et hautes altitudes, une étude précédente a identifié un sous-ensemble de 171 polymorphismes nucléotidiques (SNP candidats) présentant des signaux de sélection. Nous avons utilisé ces SNP candidats pour tester l'association entre la variation génotypique et phénotypique de 18 caractères. Notre panel d’association était constitué de 1663 plantes provenant de graines de 11 populations échantillonnées le long de deux gradients d’altitude. Il a été évalué deux années consécutives dans deux jardins communs. Nous avons contrôlé sa structure neutre en utilisant 18 marqueurs microsatellites. La variation phénotypique a révélé l’existence d'un syndrome altitudinal composé de dix caractères. Nous avons ainsi observé une augmentation de la précocité de floraison, une diminution de la production de talles et de la densité en stomates des feuilles ainsi qu’une augmentation de la taille, de la longueur et du poids des grains avec l’élévation croissante du site de collecte des populations. Ce syndrome a évolué malgré des flux de gènes détectables entre populations. Nous avons montré que le pourcentage de SNP candidats associés aux différents caractères dépend de la prise en compte de la structure neutre soit en cinq groupes génétiques (71,7%), soit en onze populations (11,5%), indiquant une stratification complexe. Nous avons testé les corrélations entre les variables environnementales et les fréquences alléliques des SNP candidats sur 28 populations. Nous avons trouvé un enrichissement à la fois pour les SNP présentant des associations phénotypiques et les SNP présentant des corrélations environnementales dans trois larges inversions chromosomiques, confirmant leur rôle dans l'adaptation locale. Pour explorer la contribution de la variation structurale à l'évolution adaptative, nous nous sommes concentrés sur le contenu en éléments transposables (ET) des six populations séquencées (HTS). Ces éléments constituent environ 85% du génome du maïs et contribuent à sa variabilité fonctionnelle. Nous avons effectué la première description populationnelle des ET chez les téosintes pour deux catégories d'insertions, celles présentes et celles absentes du génome de référence du maïs. Nous avons ensuite recherché des polymorphismes liés aux ET présentant des fréquences alléliques contrastées entre populations de basse et de haute altitude. Nous avons identifié un sous-ensemble d'insertions candidates. Enfin, nous avons génotypé, dans un panel d'association, des insertions d’ET connues pour avoir contribué à l'évolution phénotypique du maïs. Contrairement à ce qui a été observé chez le maïs, certaines de ces insertions n'ont montré aucun effet phénotypique chez les téosintes, ce qui suggère que leur effet dépend du fond génétique. Notre étude apporte de nouvelles connaissances sur l’adaptation altitudinale chez les plantes. Elle ouvre la discussion sur les défis soulevés par l'utilisation (1) d'outils de génomique des populations pour identifier la variation adaptative, (2) de populations naturelles en génétique d’association, et (1) de ressources génétiques sauvages pour l'amélioration des espèces cultivées. / Annual teosintes, the closest wild relatives of maize, are ideal systems to study local adaptation because their distribution spans a wide range of environmental conditions. Zea mays ssp. parviglumis is distributed in warm and mesic conditions below 1800 m, while Zea mays ssp. mexicana thrives in dry and cool conditions at higher altitudes. We combined reverse ecology and association mapping to mine the determinants of local adaptation in annual teosintes. Based on high throughput sequencing (HTS) data from six populations encompassing lowland and highland populations growing along two elevation gradients, a previous study has identified candidate regions displaying signals of selection. Within those regions a subset of 171 candidate single nucleotide polymorphisms (SNPs) was selected to test their association to phenotypic variation at 18 traits. Our association panel encompassed 1663 plants from seeds collected from eleven populations sampled along the elevation gradients. We benefit from phenotypic characterization of all the plants in two common gardens located at mid-altitude for two years. In addition, we controlled for neutral structure of the association panel using 18 microsatellite markers. Phenotypic variation revealed the components of an altitudinal “syndrome” constituted of ten traits evolving under spatially-varying selection. Plants flowered earlier, produced less tillers, displayed lower stomata density and carried larger, longer and heavier grains with increasing elevation of population collection site. This syndrome evolved in spite of detectable gene flow among populations. The percentage of candidate SNPs associated with traits largely depended on whether we corrected for five genetic groups (71.7%) or eleven populations (11.5%), thereby indicating a complex stratification in our association panel. We analyzed correlations between environmental variables and allele frequencies of candidate SNPs on a larger set of 28 populations. We found enrichment for SNPs displaying phenotypic associations and environmental correlations in three Mb-scale chromosomal inversions, confirming the role of these inversions in local adaptation. To further explore the contribution of structural variation to adaptive evolution, we focused on transposable element (TE) content of the HTS populations. TEs constitute ~85% of the maize genome and contribute to its functional variability via gene inactivation and modulation of gene expression. We performed the first population-level description of TEs in teosintes for two categories of insertions, those present and those absent from the maize reference genome. We next searched for TE polymorphisms with contrasted allele frequencies between lowland and highland populations. We pinpointed a subset of adaptive candidate insertions. Finally, we genotyped in our association panel TE insertions known to have contributed to maize phenotypic evolution. In contrast to what was found in maize, some of these insertions displayed no measurable phenotypic effects in teosintes, suggesting that their effect depends on the genetic background. Altogether our study brings new insights into plant altitudinal adaptation. It opens discussions on the challenges raised by the use (1) of population genomic tools to discover adaptive variation, (2) of natural populations in association mapping, and (1) of wild genetic resources in crop breeding.

<p dir="ltr">Uncovering the mechanisms of senescence in maize will give us a deeper understanding needed to drive future yield increases. Previous work on senescence response to sink disruption has identified a set of genes and biochemical mechanisms. Still, little is understood about the impact of this phenotype on yield and other commercially relevant traits. Uncovering the genetic basis of senescence in maize and testing the effect of these alleles on yield will provide a mechanistic framework for considering this trait to drive future yield increases.</p><p dir="ltr">Ear removal experiments demonstrated that senescence timing is insensitive to the presence or absence of an ear outside a critical window from 10 to 45 days after pollination. Nitrogen fertilization did not impact the SSRS response measured in the upper canopy. In further characterizing the SSRS phenotype, we have provided a spatial and temporal map of the B73 senescence response to sink disruption from the top of the plant to the ear leaf and discovered that this phenomenon is dose dependent and proportional to the size of the sink across two genotypes and years. This relationship was successfully used to predict kernel numbers and grain weight from spectral leaf properties as early as 4 weeks after pollination using remote sensing under agronomic conditions.</p><p dir="ltr">A population of 343 exPVP inbred lines was evaluated for source-sink regulated senescence and hybrid testcrosses were made for a subset of 200 inbred lines to testers for measurement of yield and ear photometry phenotypes. Source-sink regulated senescence of inbred parents was correlated with the yield of intra-family hybrids but was not generally correlated with the yield of hybrids made from crosses between two heterotic groups. The presence of multiple significant SNP association at the Bonferroni-corrected threshold at loci that are associated both with kernel traits and SSRS suggests shared genetic regulation of two traits that is likely driving the observed trait correlations of SSRS with kernel size and yield.</p><p dir="ltr">The maize nested association mapping (NAM) parents reveal a previously unknown breadth of SSRS phenotypes in the global diversity of maize germplasm. Mapping genes for SSRS in the NAM populations supports previously reported loci with large, dominant effects as well as evidence for previously unreported modifiers that are capable of suppressing the dominant alleles and producing a quantitative distribution in SSRS phenotypes. There are distinct alleles within sub-populations worth further study such as sweetcorn populations with non-senescence responses to sink disruption. A multi-factor analysis for QTL mapping, GWAS, and mutant variant sequencing identified highly significant loci on chromosomes 1 from 30.4Mbp to 35.8Mbp, chromosome 2 from 183.2Mbp to 190.8Mbp, chromosome 4 from 38.2Mbp to 134.8Mbp (crosses a centromere), chromosome 5 from 140.8Mbp to 233.9Mbp, chromosome 8 from 112.5Mbp to 123.8Mbp, and chromosome 8 from 155.7Mbp to 163.9Mbp. Candidate genes co-located with Bonferroni SNP in these regions may contribute to SSRS phenotypes through regulation of autophagy, accumulation of flavonoids, and sequestration of sugar in cell walls as an alternative sink. It is possible that co-regulation of these genes could cause all of them to be involved in the stress response of B73 to sugar accumulation. To find the causal variants for these traits, fine mapping and comparisons of near-isogenic lines will be required to narrow the list of candidate genes. Uncovering the alleles responsible for SSRS in global maize diversity could provide the building blocks for a physiological approach to increasing yields through optimizing the senescence responses to elevated sugar levels during grain-fill.</p>

The inoculation with Azospirillum brasilense is one of the main strategies to supplement the inorganic inputs of nitrogen (N) and to increase the root development in maize. However, the beneficial inoculation effects are not always reached, which, in part, is due to genotypic variation in the plant host, resulting in different degrees of outcome. In this context, we aimed to study the genetic control and genomic prediction of maize traits related to the responsiveness to A. brasilense inoculation. For this, 118 maize hybrids were conducted under N stress and N stress plus A. brasilense treatments in controlled conditions over 2016 and 2017 seasons. We evaluated root and shoot traits and performed diallel analyses, association mapping, and genomic prediction methods considering 59,215 Single-Nucleotide Polymorphism (SNP) markers. Our results revealed a quantitative inheritance of the partnership-related maize traits, with both additive and non-additive genetic effects involved in the genetic control. Furthermore, several candidate genes were identified for the maize-A. brasilense association, especially with heterozygous (dis)advantage effects. In general, the prediction accuracies were higher mostly for the inoculated treatment compared to the non-inoculated. Finally, our findings enable a deeper understanding towards the genetic basis of the maize responsiveness to A. brasilense and may support plant breeders to establish selection strategies aiming the development of superior genotypes for this association. / A inoculação com Azospirillum brasilense é uma das principais estratégias para suplementar os insumos inorgânicos de nitrogênio (N) e aumentar o desenvolvimento radicular do milho. No entanto, os efeitos benéficos da inoculação nem sempre são alcançados, o que, em parte, é devido à variação genotípica da planta hospedeira, que ocasiona diferentes graus de resultados. Neste contexto, nosso objetivo foi estudar o controle genético e a predição genômica de caracteres de milho relacionados à responsividade para a inoculação com A brasilense. Para isso, 118 híbridos de milho foram conduzidos sob estresse de N e estresse de N mais A brasilense em condições controladas nos anos de 2016 e 2017. Nós avaliamos características de raiz e parte aérea e realizamos análises dialélicas, mapeamento associativo e métodos de predição genômica considerando 59.215 marcadores Single-Nucleotide Polymorphism (SNP). Nossos resultados revelaram uma herança quantitativa das características do milho relacionadas à essa parceria, com efeitos genéticos aditivos e não-aditivos envolvidos no controle genético. Além disso, vários genes candidatos foram encontrados para a associação milho-A brasilense, especialmente com efeitos de (des)vantagens de heterozigotos. Em geral, as acurácias de predição foram mais maiores principalmente para o tratamento inoculado em comparação ao não inoculado. Finalmente, nossos resultados possibilitam uma compreensão mais aprofundada das bases genéticas da responsividade do milho à A. brasilense e podem auxiliar os melhoristas de plantas a estabelecerem estratégias de seleção visando o desenvolvimento de genótipos superiores para essa associação.

Au cours des quarante dernières années, l’optimisation des méthodes sylvicoles et l’introduction de variétés améliorées ont permis d’accroître considérablement la productivité du pin maritime. Pour permettre à la filière bois de disposer d’une matière première de qualité sur ce matériel amélioré, un programme de recherches multidisciplinaire a été développé afin d’étudier le déterminisme génétique de la qualité du bois. Neuf facteurs de transcription potentiellement impliqués dans la xylogenèse et l’adaptation des arbres à leur milieu, ont tout d’abord été séquencés dans la population Aquitaine, et leurs patrons de diversité nucléotidique ont été étudiés. Ces patrons ont été comparés à l’attendu de modèles neutres d’évolution et s’en écartent par un niveau élevé de déséquilibre de liaison et l’excès de mutations en fréquences intermédiaires détectés pour trois de ces gènes (HDZ31, LIM2 et MYB1). Ces résultats suggèrent des changements de taille de population affectant l’ensemble du génome, et l’action de sélection balancée sur l’un d’entre eux (MYB1). Les géniteurs de la population d’amélioration Aquitaine ont ensuite été génotypés pour 384 marqueurs moléculaires et évalués pour la croissance et les propriétés chimiques du bois. Ces données moléculaires et phénotypiques ont permis de mettre en évidence des associations significatives entre la variation pour le diamètre du tronc ou la teneur en cellulose du bois et deux marqueurs situés respectivement dans un facteur de transcription HD-Zip (HDZ31) et dans un gène encodant une fascicline. La cohérence des résultats de génétique évolutive et de génétique d’association ouvre ainsi des perspectives encourageantes pour la compréhension de l’architecture génétique de la formation du bois chez cette espèce. Cependant, le faible nombre d’associations significatives pose de nombreux problèmes théoriques et méthodologiques qui sont discutés en vue d’améliorations pour de nouveaux designs expérimentaux. / During the last four decades, the optimization of silvicultural and tree breeding methods has contributed to improve growth and wood homogeneity of maritime pine. In order to provide the different actors of the forestry wood-chain with high quality raw material, the genetic determinism and chemical components of wood quality are being studied in the frame of a multidisciplinary research program. First, nine transcription factors putatively involved in wood formation have been sequenced in the Aquitaine population, and their nucleotide diversity pattern studied. Since these genes potentially play important roles in the adaptation of trees to their environment, their patterns have been compared to those expected under neutral evolution. Strong departures from neutrality were observed, with high levels of linkage disequilibrium and an excess of intermediate frequency variants for three of them (HDZ31, LIM2 and MYB1), which could be linked to population size changes that affected the whole genome, and to balancing selection effects at one of them (MYB1). Secondly, the genitors of the Aquitaine breeding population were genotyped for 384 markers and evaluated for growth and wood chemical properties. Significant associations were detected for two markers, one in a HD-Zip transcription factor (HDZ31) with growth, and the other in a gene coding for a fasciclin protein with cellulose content. The consistency of evolutionary and molecular genetics opens encouraging perspectives for understanding the genetic architecture of wood formation in this species. However, the low number of associations detected raises several theoretical and methodological issues which are discussed for the perspective of improving future experimental designs.