Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Thesis (MScAgric.)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: In cross pollinated crops, recurrent selection is used to increase the frequency of desirable alleles by breaking up existing linkage blocks and forming new gene combinations. Despite promising results from numerous feasibility studies, recurrent selection is seldom routinely used in wheat. A major obstacle has been the inability to readily achieve random interbreeding of large numbers of selected plants. In China the Taigu genetic male sterility gene, Ms2, has however been used to establish a recurrent selection programme in which field grown male sterile plants were pollinated by selected male fertile plants (Huang et al., 1988). Another dominant gene for male sterility, Ms3, was found after EMS treatment of the seeds of an alloplasmie common wheat with Triticum tauschii cytoplasm (Maan et al., 1984) and is located at 3 map units from the centromere on chromosome arm SAS (Maan et al., 1987). In a study done during 1999 at Welgevallen to determine the frequency of natural intererossing under field conditions, Ms3 showed incomplete penetrance and only about two thirds of the seed set on male sterile plants could be attributed to intercrossing. Ms3 has stable expression in plants grown within the normal range of greenhouse temperatures for wheat, 16 - 2SoC. Under warmer field conditions, 21 - 3SoC, its penetrance is, however, incomplete (Maan et al., 1984). The utility of Ms3 under field conditions is therefore unsatisfactory. An attempt to determine the location and origin of an unknown male sterility gene, found in cross 9SK3 of a routine breeding programme, showed that a single locus was not the cause of the male sterility. Chromosome abnormalities and gene imbalances were probably to blame. The male sterility probably relates to a T.urartu addition chromosome in the pedigree of cross 9SK3. To facilitate the production of large numbers of hybrid progeny, a simple hydroponic system was developed in which male sterile tillers cut at the flowering stage can be pollinated and maintained for about 8 weeks, long enough to produce viable seeds. For pollination, florets on male tillers are cut open and placed in a container with a similar number of pollen shedding male tillers. It was found that cut tillers could be maintained in the hydroponic system as long as certain precautions were met: (a) The tillers must be handled with care so as not to damage the flag leaf which must be maintained for as long as period possible. (b) The tillers have a nutrient requirement and a 20% solution showed the best results of the nutrient solutions tested. (c) The sterilizing effect of Jik at O.OS%gave excellent fungal control en helped to sustain the nutrient solution. (d) Although the treatment of tillers with hormones improved seed quality, it was not justified by the additional inputs required. Different selection strategies were used for male and female plants. At the onset of the recurrent selection programme in 1998, a total of 1881 plants were tested for seedling resistance and 597 plants were selected for use as parents and source material for 1999. In total 158 male sterile and 188 male fertile ears were used in the hydroponic pollination and a 63.47% seed set was obtained, resulting in 3410 seeds, forming the 1999 female component. One hundred and fifty seven F2:96K109plants were selected from a field grown population in 1998. These, together with 44 selections from a pedigree programme, formed the male component for 1999. In total 9564 plants were tested for seedling resistance during 1999. A total of 3230 resistant seedling were selected and planted. Again male fertile plants from the previous season were field planted and selected. The selected plants were subjected to mixograph testing. A total of 448 male sterile and 1020 male fertile ears were used for hydroponic pollination. Approximately 12000 seeds were harvested, the seed set being around 75%. The 157 F2:96K109 field selected plants (1999) and 64 selections from a pedigree programme formed the male component for 2000. Seedling resistance testing during 2000 included a total of 6465 plants and 2832 were selected and planted. The hydroponic system was improved during 2000 with new, larger capacity containers being used which improved cross pollination. In total 878 male sterile tillers and 1016 male fertile tillers were cut and intercrossed. In total 25380 seeds were harvested, the seed set being 81.7%. In an attempt to determine the amount of variation within the 157 F2-families selected during 1999, mixograph testing was performed. The data showed variation among families. Seedling resistance testing for leaf and stem rust was performed on the 1999 and 2000 FIs to determine the variation for resistance within the populations. Both populations showed high level of stem rust resistance but lower levels of leaf rust resistance (± 50%). Ms3 can thus be used in combination with hydroponic tiller culture to facilitate recurrent selection. Integration with an excisting pedigree selection programme is viable and requires little additional input. Some of the these results have already been published (Addendum D). / AFRIKAANSE OPSOMMING: Herhalende seleksie word by kruisbestuiwers aangewend om die frekwensie voordelige allele te verhoog deur die opbreek van bestaande koppelingsblokke en vorming van nuwe geen-kombinasies. Hoewel uitstekende resultate m.b.V.herhalende seleksie reeds by koring verkry is, is die roetine aanwending egter beperk weens die gebrek aan effektiewe kruisbestuiwing van groot getalle plante. In China is "Taigu" genetiese manlike steriliteit, Ms2, egter met sukses aangewend vir die vestiging van 'n herhalende seleksieprogram vir landverboude koring. Die manlik-vrugbare plante word vir die bestuiwing van geselekteerde manlik-steriele plante aangewend (Huang et al., 1988). Nog 'n dominante manlike steriliteitsgeen, Ms3, is ontdek na EMS behandeling van sade afkomstig vanaf 'n alloplasmiese gewone koring met 'n Triticum tauschii sitoplasma (Maan et al., 1984) en is gesetelop chromosoom 5AS, 3 kaarteenhede vanaf die sentromeer (Maan et al., 1987). 'n Ondersoek na die frekwensie natuurlike kruisbestuiwing onder landtoestande (Welgevallen, 1999) het getoon dat onvolledige penetrasie van Ms3 lei tot ongeveer 5% selfbestuiwing en dat slegs twee-derdes van die saadset aan kruisbestuiwing toegeskryf kon word. Ms3 word wel stabiel uitgedruk onder normale glashuistemperature tydens blom nl. 16 - 25°C, maar onder warmer landtoestande, 21 - 35°C, is uitdrukking onstabiel met laer penetrasie van die geen (Maan et al., 1984). Die benutbaarheid van Ms3 onder landtoestande was dus onbevredigend. Die ondersoek na die oorsprong en ligging van 'n onbekende, manlike steriliteitsgeen (95K3) wat ontdek is in 'n roetine teelprogram het daarop gedui dat 'n enkellokus waarskynlik me ter sprake is nie, maar eerder chromosoom-abnormaliteite en geenwanbalanse. Die manlike steriliteit kan verband hou met 'n T urartu addisie chromosoom in die stamboom van hierdie bron. Ten einde kruisbestuiwing van 'n groot aantal plante te bewerkstellig, is 'n eenvoudige bestuiwersisteem ontwikkel gegrond op waterkultuurkweking van afgeknipte manlik-steriele (Ms3ms3), are. Manlik-steriele en manlik-vrugbare are is tydens blom geknip. Die manliksteriele are se blommetjies is oopgeknip en toegelaat om deur die manlik-vrugbare are bestuif te word. Die bestuifde manlik-steriele are (Ms3ms3) is hierna vir ongeveer 8 weke gelaat vir saadvorming. Afgeknipte are kan baie suksesvol in voedingsmedium onderhou word mits sekere eenvoudige voorsorgmaatreëls getref word, naamlik: (a) Die are moet met sorg hanteer word en die vlagblaar moet so lank as moontlik behou word. Are moet weekliks teruggeknip word ten einde verstopping en agteruitgang van vaatweefsel teen te werk. Die oorspronklik- afgeknipte halm is dus belangrik. (b) Die are toon 'n definitiewe voedingsbehoefte en 'n 20% voedingsoplossing was die beste van die oplossings wat getoets is. Die voedingsoplossing moet verkieslik weekliks vervang word wanneer are teruggeknip word. Op die tydstip behoort die houers met 'n steriliseringsmiddel gewas te word vir die verwydering van enige moontlike swamgroei aan die houers se wande. (c) Jik was die beter steriliseringsmiddel en het teen 0.05% toediening goeie swaminhibering bewerkstellig. (d) Hormone is nie in die roetinetoepassing gebruik nie aangesien die voordeel hiervan nie die ekstra insette regverdig nie. Verskillende strategieë is aangewend vir die seleksie van manlike en vroulike plante. Met die aanvang van die herhalende seleksieprogram in 1998 is 'n totaal van 1881 plante getoets vir roesweerstand en 597 geselekteer as bronmateriaal vir 1999. In totaal is 158 manliksteriele en 188 manlik-vrugbare are gebruik in die bestuiwersisteem vir die verkryging van die 1999 vroulike komponent. 'n Totaal van 3410 sade is verkry met 'n 63.47% saadset. Tesame met 157 F2:96KI09 landgeselekteerde plante is 44 seleksies vanuit 'n stamboom seleksieprogram gebruik as manlike komponent in 1999. Gedurende 1999 is 9564 plante getoets vir roesweerstand en 3230 geselekteer en geplant. Weereens het landseleksie plaasgevind. Die 157 seleksies is onderwerp aan miksograaf-toetsing. Vierhonderd agt- en - veertig manlik-steriele en 1020 manlik-vrugbare are is gebruik in die bestuiwersisteem. Ongeveer 12138 sade is geoes, teen 'n 75% saadset. Gedurende 2000 is die sade asook 64 seleksies uit 'n stamboom seleksieprogram aangewend as die manlike komponent. Roestoetsing is weereens in 2000 uitgevoer en 6465 plante is geïnokuleer waaruit 2832 plante geselekteer en geplant is. Die bestuiwersisteem is aangepas vir die hantering van groter aantalle are tydens 2000 en in totaal is 878 manlik-steriele are en 'n 1016 manlik-vrugbare are gebruik vir kruisbestuiwing. Die saadset is verhoog na 81.7% en 25380 sade is verkry. Om die hoeveelheid variasie binne die populasie te bepaal, is miksograaftoetsing op die 1999 F2-populasie uitgevoer. Die data het aangetoon dat groot hoeveelhede genetiese variasie beskikbaar is binne die populasie. Roestoetsing van die 1999- en 2000-bestuiwerpopulasies is ook uitgevoer om 'n indikasie te verkry van die verspreiding van weerstand teen blaar- en starnroes. Die blaamoes het 'n relatief lae vlak van weerstand getoon (± 50%) terwyl die stamroesweerstand baie hoë vlakke gehandhaaf het. Ms3 kan dus gebruik word om in kombinasie met waterkultuurkweking van gesnyde halms, 'n herhalende seleksieprogram van stapel te stuur. Integrasie met 'n bestaande stamboom seleksieprogram is ook moontlik en sal relatief min addisionele insette vereis. 'n Gedeelte van die werk is reeds gepubliseer en word hierbyaangeheg as Aanhangsel D.

Doctor of Philosophy / Department of Agronomy / Tesfaye Tesso / Sweet Sorghum [Sorghum bicolor (L.) Moench] is a type of cultivated sorghum grown primarily for its sugar-rich stalks. Because of its high fermentable sugar content, the crop is widely recognized as an alternative feedstock source for bio-fuel production. The extent to which stalk sugar accumulation occurs may be determined by several factors including the sink size. Grain is the most important sink in sorghum and other grain crops. Three experiments were conducted in this study to determine the extent to which the grain sink can reduce sugar accumulation in the stalks, to test and validate a genetic system that allows development of sterile sweet sorghum hybrids, and to assess the potential of sugar-rich hybrids to overcome stalk rot diseases. The first experiment, based on 22 sweet sorghum genotypes, was undertaken to study the effect of eliminating the grain sink (removing the head prior to anthesis) on stalk juice yield, sugar accumulation, and biomass. The data showed that the grain sink had a significant effect on all traits measured. Elimination of the grain sink significantly increased oBrix % (17.8%), dry biomass (27.8%), juice yield (23.9%), and total sugar yield (43.5%). The second experiment was aimed at validating the role of A3 genetic male sterility system for producing sterile sweet sorghum hybrids. Ten sweet sorghum pollinator lines of variable sugar content were selected among the entries included in the previous experiment. The lines were crossed to four A1 and A3 cytoplasmic male sterile (CMS) lines using a Design II mating scheme. The A3 females did not have effective restorers so that the hybrids were expected to be sterile. The parental lines and corresponding hybrids were evaluated for biomass production, oBrix, juice and sugar yield using a randomized complete block design. All A3 hybrids were sterile and did not produce seed when heads were covered prior to pollination. The effect of grain sink represented by the A1 vs. A3 CMS were highly significant for Brix%, biomass, juice, and sugar yield. Comparison of parents vs. crosses component was highly significant, indicating marked heterosis effect for the traits. Both general (GCA) and specific (SCA) combining ability effects were also significant for all traits, indicating the role of both additive and dominance genetic effects in the inheritance of the characters. Earlier studies have shown positive relationships between stalk sugar concentration and stalk rot disease resistance in sorghum. Thus, the objective of the third experiment was to study the effects of the CMS mediated differential accumulation of stalk sugar on severity of charcoal rot disease caused by Macrophomina phaseolina. The experiment provided an opportunity to test the effect of variable stalk sugar in the same genetic backgrounds. The data indicated that hybrids produced from A3 cytoplasm were more resistant to charcoal rot (7.1cm lesion length) compared to those produced from the A1 hybrids (9.5 cm lesion length). The enhanced resistance of hybrids with higher sugar yield could have significant agronomic advantage in sugar based bio-fuel feedstock production.

No description available.

The vast majority of valuable agronomic traits are encoded polygenetically. Chloroplast genetic engineering offers an alternate approach to multigene engineering by allowing the insertion of entire pathways in a single transformation event, while being an environmentally friendly approach. Stable integration into the chloroplast genome and transcription of the phaA gene coding for β-ketothiolase was confirmed by Southern and northern blots. Coomassie-stained gel and western blots confirmed hyperexpression of β-ketothiolase in leaves and anthers, with high enzyme activity. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines followed an accelerated anther developmental pattern, affecting their development and maturation, resulting in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium and vacuolation, decreased the inner space of the locules, affecting pollen grain and resulted in the irregular shape and collapsed phenotype. Reversibility of the male sterility phenotype was achieved by exposing the plants to continuous illumination, producing viable pollen and copious amounts of seeds. This is the first report of engineered cytoplasmic male sterility and offers a new tool for transgene containment for both nuclear and organelle genomes. Detailed characterization of transcriptional, posttranscriptional and translational processes of heterologous operons expressed via the chloroplast genome is reported here. Northern blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons, revealed that in most cases, only polycistronic mRNA was produced or polycistrons were the most abundant form and that they were not processed into monocistrons. Despite such lack of processing, abundant foreign protein accumulation was detected in these transgenic lines. Interestingly, a stable secondary structure formed from a heterologous bacterial intergenic sequence was recognized and efficiently processed, indicating that the chloroplast posttranscriptional machinery can indeed recognize sequences that are not of chloroplast origin, retaining its prokaryotic ancestral features. Processed and unprocessed heterologous polycistrons were quite stable even in the absence of 3'UTRs and were efficiently translated. Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of any cellular control. Finally, we observed abundant read-through transcription in the presence of chloroplast 3'UTRs. Such read-through transcripts were efficiently processed at introns present within native operons. Addressing questions about polycistrons, as well as the sequences required for their processing and transcript stability are essential for future approaches in metabolic engineering. Finally, we have shown phytoremediation of mercury by engineering the mer operon via the chloroplast genome under the regulation of chloroplast native and heterologous 5'UTRs. These transgenenic plants hyperexpress were able to translate MerA and MerB enzymes to levels detectable by coomassie stained gel. The knowledge acquired from these studies offer guidelines for engineering multigene pathways via the chloroplast genome.

Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Understanding the chromosome evolution and mechanisms of speciation can shed light on developing novel ecological-friendly vector control techniques. Sibling species of the An. gambiae complex provide an excellent model system for these topics. To understand the mechanisms of speciation, we investigated the cellular basis and phenotypes of hybrid male sterility in species crosses of the An. gambiae complex. By performing inter-species crosses of An. coluzzii/An. gambiae and An. merus lab strains, we found an asymmetric pattern of hybrid male sterility existed in sons from reciprocal interspecies crosses. Compared with pure species, hybrid males from crosses of ♀An. merus  ♂An. gambiae/An. coluzzii were normal in the morphology of male reproductive tracts; however, the testes of which that process the reductional meiotic division failed to produce primary spermatocytes and were accompanied with unpaired and insufficiently condensed chromosomes. As a result, primary spermatocytes undergo a mitosis-like anaphase division, producing nonmotile and malfunctional diploid sperm with two tails. However, individuals can mate with females normally and form the mating plug to induce the female monogamy. In contrast, hybrid males from the opposite crosses manifest severely underdeveloped reproductive tracts and a premeiotic arrest of germline stem cells in the testis, accompanied by a strong suppression of premeiotic and meiotic genes. In addition, hybrid males from this cross suffered from a shorter copulation time and failed to form mating plugs to induce female monogamous behaviors, albeit the expression of male accessory gland specific genes were similar between hybrids and pure species. To figure out chromosome evolution in the An. gambiae complex, we studied the molecular organization of heterochromatin and investigated the spatial organizations of autosomal regions of polytene chromosomes in soma and germline cells. We found that molecular composition of pericentrometric autosome and sex chromosome repetitive DNA differs among sibling species of An. gambiae complex with highly similarity between An. coluzzii and An. arabiensis. In addition, heterochromatin blocks of chromosomes have distinct compositions of satellite DNA sequences. Next, in order to address the relationship between inter-chromosomal (Chr-Chr) contacts and chromosome-nuclear envelope (Chr-NE) attachments during the development of the organism, we conducted microscopic analyses of the 3D organization of polytene chromosome in An. gambiae, An. coluzzii, and An. merus. Our quantitative study on chromosome territories in larval salivary gland cells and adult ovarian nurse cells showed that, compared with autosomal arms, the X chromosome has a significantly smaller volume and occupies more compact territories. The number of Chr-Chr contacts and the percentage of Chr-NE attachment were conserved among the species within the same cell type. Our data also demonstrated that there is a significantly and consistently inverse relationship between the frequencies of Chr–NE and Chr–Chr attachments on autosomes of two cell types in all tested species. / Doctor of Philosophy / Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Despite being treatable and preventable, malaria is estimated to cause large numbers of deaths every year. Since 2015, the malaria elimination program has stalled largely due to increased insecticide resistance. Novel transgenic techniques have a huge potential in reducing malaria transmission more effectively. However, there are large concerns about the potential negative effects of releasing genetically modified mosquitoes, such as a possibility of accidental spread to non-target species with incomplete reproductive barriers and unpredicted ecological damage. Understanding the mechanisms of speciation about how reproductive isolation occurred and developed as well as chromosome evolution can not only empower the development of ecologically friendly vector control techniques but also improve our basic knowledge. To study mechanisms of speciation, we mated males and females from different closely related species in the Anopheles gambiae complex to investigate the fecundity of hybrid generations. Our study identified two different types of reproductive abnormalities leading to hybrid male sterility. Hybrid males from female An. merus and male An. gambiae or An. coluzzii have normal appearing testes and male accessary glands but the testes produce abnormal sperms, which cannot move and have two tails. Hybrid males from female An. gambiae or An. coluzzii and An. merus have severely underdeveloped testes and male accessary glands. The sperm producing process stops unusually very early in their tiny underdeveloped testes. We also investigated chromosome evolution in species of An. gambiae complex. We found that chromosomal parts containing repetitive DNA, the sequence in the genome not producing proteins, evolve rapidly in An. coluzzii, An. arabiensis, An. quadriannulatus, and An. merus. In contrast, chromosome territories of gene rich regions in giant polytene chromosomes from larval salivary gland cells and adult ovarian nurse cells of An. gambiae, An. coluzzii, and An. merus, were relatively conserved within the same cell type among different species. However, the chromosomal 3D distribution pattern is different among various cell types in these species.

Abstract Demographic history and selection affect patterns of genetic diversity in nature. Timing of growth, reproduction and dormancy are important traits targeted by natural selection, because they are crucial for survival of plants growing in boreal and temperate climates, as reproduction must occur when conditions are favorable and in outcrossing plants it must be synchronized to assure pollination. In addition to adaptation to local environments, evolutionarily diverged populations may contain genomic incompatibilities that result in sterile hybrids in crosses between populations. In several plant families within population crosses can also lead to male sterile progeny, because of conflict between maternally and bi-parentally inherited genomes. In this thesis I used DNA-sequence data to estimate the demographic history of nine Arabidopsis lyrata populations and present genetic variation in some key flowering time genes, evolution of which natural selection has shaped. By crossing experiments I explored genetics of reproductive fitness in hybrids between divergent populations. I found that local climatic conditions have resulted in directional selection in addition to the demographic effects of bottlenecks during colonization events. Flowering time genes have reduced diversity compared to reference loci, which indicates selective sweeps. Selection on nucleotide variation in flowering time genes was found in Scandinavian and Icelandic populations that can be explained by selective sweeps at flowering genes when these populations colonized northern habitats after the last glacial maximum. Cryptic cytoplasmic male sterility was found in a Norwegian population, for which North Carolinian population did not have fertility restorers. It was confirmed that there is only one fertility restorer locus, the genomic location of which was mapped to a 600 kb interval at the top of chromosome two. / Tiivistelmä Populaatioiden levittäytymishistoria ja luonnonvalinta vaikuttavat geneettiseen monimuotoisuuteen ja sen vaihteluun genomin eri osissa. Kukkimisen ja kasvun päättämisen ajoitus ovat tärkeitä luonnonvalinnan kohteena olevia ominaisuuksia, sillä ne ovat välttämättömiä kasvien selviytymiselle lauhkeissa ja pohjoisissa ilmastoissa. Paikallisiin olosuhteisiin sopeutumisen lisäksi populaatioiden erilaistuminen voi johtaa genomisiin yhteensopimattomuuksiin, joiden vuoksi populaatioiden väliset risteymät ovat lisääntymiskyvyttömiä. Monilla kasvisuvuilla myös populaation sisäiset risteytykset voivat johtaa koirassteriileihin jälkeläisiin johtuen konfliktista vain äidin puolelta ja molemmilta vanhemmilta periytyvien genomien välillä. Tässä väitöskirjassa selvitän DNA-sekvenssimuunteluun perustuen yhdeksän idänpitkäpalkopopulaation demografista eli levittäymis- ja populaatiorakennehistoriaa sekä luonnonvalinnan osuutta kukkimisaikaan vaikuttavien geenien evoluutiossa. Risteytyskokeiden avulla tutkin erilaistuneiden populaatioiden risteymäjälkeläisiä selvittääkseni niiden lisääntymiskelpoisuutta ja siihen vaikuttavia geenejä. Geneettinen muuntelu kukkimisgeeneissä oli vähäisempää kuin vertailugeeneissä, joka on merkki kukkimisgeeneihin kohdistuneesta suuntaavasta valinnasta. Kukkimisgeeneihin kohdistuvaa valintaa löytyi eniten skandinaavisista ja islantilaisesta populaatiosta, mikä on selitettävissä niihin kohdistuneella suuntavalla valinnalla aikana, jolloin kasvit levittäytyivät jääkauden jälkeen pohjoisiin elinympäristöihin. Norjalaisesta populaatiosta löytyi piilevä sytoplasminen koirassteriliteetti, jolle Pohjois-Carolinan populaatiolla ei ollut hedelmällisyyden palauttavia geenejä. Tutkimus vahvisti, että hedelmällisyyden palauttaa yksi geeni, joka sijaitsee 600 000 emäsparin kokoisella alueella kromosomin kaksi alkupäässä.

Un objectif central de la biologie évolutive est la compréhension des mécanismes qui conduisent à la formation de nouvelles espèces. Les stérilités hybrides constituent un type de barrières reproductives pouvant mener à la spéciation. Ce travail dissèque les bases génétiques d’une stérilité mâle observée chez l'hybride entre deux accessions naturelles d'Arabidopsis thaliana, Shahdara et Mr-0, lorsque Shahdara est le parent femelle. Par des approches génétiques et cytologiques, nous montrons que deux phénomènes interviennent dans cette stérilité. D'une part le cytoplasme de Shahdara induit une stérilité mâle cytoplasmique (CMS), en interaction avec plusieurs locus nucléaires. D'autre part, une létalité pollinique est due à plusieurs locus distorteurs de ségrégation (pollen killers). La stérilité de l'hybride résulte d'une liaison génétique entre les déterminants nucléaires de la CMS et les pollen killers. L'un des pollen killers a été localisé dans un intervalle de 70 Kb qui contient également des éléments nécessaires à la restauration de la CMS. Ce locus est complexe et présente de nombreuses variations structurales, notamment au niveau de gènes PPR. Ces résultats suggèrent que deux types de conflits génomiques, les distorteurs de ségrégation et la CMS, pourraient coévoluer dans des populations naturelles et conduire à l’élaboration de barrières reproductives au sein d'une même espèce. / Species differentiation and the underlying genetics of reproductive isolation are central topics in evolutionary biology. Hybrid sterility is one kind of reproductive barrier that can lead to differentiation between species. Here, we analyze the complex genetic basis of the intraspecific hybrid male sterility that occurs in offspring of two distant natural strains of Arabidopsis thaliana, Shahdara and Mr-0, with Shahdara as the female parent. Using genetic approaches as well as cytological observation of pollen viability, we demonstrate that this particular hybrid sterility results from two causes of pollen mortality. First, the Shahdara cytoplasm induces gametophytic cytoplasmic male sterility controlled by several nuclear loci. Second, several segregation distorters leading to allele-specific pollen abortion (pollen killers) operate in the hybrids. The complete sterility of the hybrid with the Shahdara cytoplasm results from the genetic linkage of the two causes of pollen mortality, i.e. CMS nuclear determinants and pollen killers. One pollen killer was localized in a 70 Kb interval which also contains restorer alleles for the CMS. This locus is complex and harbors many structural variations, particularly at PPR genes. Our results suggest that two types of genomic conflicts, CMS and segregation distorters, may coevolve in natural populations and contribute to reproductive isolation, and possibly to speciation.

Les stérilités mâles cytoplasmiques (SMC) résultent d'une incompatibilité nucléo-cytoplasmique. Le cytoplasme (presque toujours la mitochondrie) peut porter un gène de stérilité mâle, et le noyau peut restaurer la fertilité pollinique ou non. Les mécanismes physiologiques conduisant à la mort pollinique restent largement incompris. Plusieurs hypothèses ont été proposées, parmi lesquelles une déficience en ATP. Une SMC gamétophytique a été découverte chez A. thaliana. Une phase ouverte de lecture codant possiblement un peptide de 117 acides aminés, appelée orf117Sha, a été identifiée comme facteur de stérilité candidat.Au cours de ma thèse, j'ai cherché à valider le rôle de l'orf117Sha, et à comprendre comment une anomalie mitochondriale pouvait induire cette SMC. Aucune différence n'a pu être détectée au niveau de l'ARNm de l'orf117Sha entre les lignées stérile et restaurée, mais sa protéine semble accumulée uniquement dans la lignée stérile. La phénocopie par transgénèse de la SMC a suggéré un effet délétère de l'ORF117SHA dans les gamétophytes mâle et femelle.La description cytologique de la SMC montre une mort pollinique progressive à partir du stade binucléé. Auparavant, les mitochondries du pollen gonflent puis éclatent, et le développement s'arrête. L’utilisation de senseurs génétiquement encodés mesurant la concentration en ATP (ATeam) et l'état redox du glutathion (roGFP2-Grx1) a permis la mesure de ces facteurs en microscopie confocale, dans des tissus végétatifs et dans le pollen. La production d'ATP ne semble pas affectée dans la lignée stérile, contredisant l'hypothèse de l'ATP. Le glutathion mitochondrial est suroxydé dans la lignée stérile, à la fois dans les tissus végétatifs étudiés et le pollen, qui serait liée à la SMC car annulée par la restauration génétique de fertilité.Avec cette étude, j'apporte des arguments en faveur de l'orf117Sha dans l'induction de la SMC Sha, et je décris les évènements préalables à l'avortement du grain de pollen. Mes résultats permettent de mieux comprendre les évènements physiologiques conduisant à la mort du pollen. / This work aims at better understand the events leading to pollen abortion in a recently discovered gametophytic cytoplasmic male sterility (CMS) in Arabidopsis thaliana. Although CMS have been widely used in hybrid seed production in many crops, the physiological mechanisms leading to pollen death by the mitochondrial sterilizing genes in the permissive (maintainer) nuclear backgrounds are poorly understood. Association genetics previously identified orf117Sha as a candidate mitochondrial CMS-associated gene.In a first part, I analyzed the expression of the orf117Sha gene in sterile plants and in fertile plants carrying nuclear genes restoring male fertility. I observed unusual features of its mRNA, but detected no difference at this level between sterile and restored plants. Oppositely, the ORF117SHA protein seems to be accumulated specifically in the sterile line, supporting its role in CMS. A phenocopy attempt by transgenesis suggested a possible link between a female and male gametophytic lethality and the ORF117SHA, even though few individuals could be analyzed.In a second part, I observed pollen development in sterile plants and fertile controls using different cytological approaches. My results show a progressive pollen death starting from the binucleate stage in the sterile. Prior to abortion, pollen mitochondria swell before rupture, and the development stops. I used confocal microscopy combined with genetically encoded sensors to explore specific physiological features in pollen and vegetative tissues of sterile plants. With ATeam, which allows the assessment of ATP content in the cytosol, I could challenge the generally accepted hypothesis of an ATP deficiency leading to pollen abortion in CMS. Indeed, the ATP production does not seem to be affected in the sterile line. With a mitochondria-addressed roGFP2-Grx1, I was able to assess the redox state of the glutathione pool in vegetative tissues and in the male gametophyte. I observed an overoxydation of the glutathione pool in mitochondria of the sterile line, in vegetative tissue investigated and in the pollen grain. This overoxydation seems to be linked to the CMS as it is annihilated by the presence of restorer genes.My results pave the way for further exploration of the links between the sterility protein, mitochondrial morphology changes, mitochondrial overoxydation, and pollen development arrest and death in the A. thaliana CMS.

<p>In this thesis, I examined variation in sex expression and mating patterns in the sexually polymorphic, wind-pollinated herb <i>Plantago maritima</i>. With a combination of field studies, greenhouse experiments, and genetic analyses, I (a) examined factors influencing sex ratio variation in gynodioecious plants (in which hermaphrodites and females coexist), (b) discovered variation in breeding system, (c) investigated density-dependence of seed production, and (d) documented genetic variation within and among populations close to the northern range margin in Europe. </p><p>In a survey of 104 <i>P. maritima</i> populations, I documented considerable variation in sex ratio (range 0-70% females, median 6.3% females). As predicted, females were more frequently missing from small than from large populations, and the variance in sex ratio increased with decreasing population size. Among twelve populations sampled for seed production, the frequency of females was positively related to relative fecundity of females and negatively related to population size. The results suggest that the local sex ratio is influenced both by the relative fecundity of females and hermaphrodites, and by stochastic processes in small populations.</p><p>A comparative field study showed that plant fecundity decreased with increasing distance to nearest pollen donor both within and among populations in an archipelago in southern Sweden, where self-incompatibility was confirmed in controlled crosses. In contrast, plant fecundity was overall higher and was not density-dependent in the Skeppsvik archipelago in northern Sweden, where controlled crosses showed that plants are self-compatible. The results were consistent with the prediction that evolution of self-fertility should reduce density-dependence of pollination success.</p><p>I quantified the genetic structure within and among populations from eastern Sweden and western Finland based on variation at four polymorphic microsatellite loci. The genetic diversity was low in northern Sweden, which may be the result of a history of small population sizes and periods of frequent self-fertilization.</p>