DNA markers tightly-linked to nuclear fertility restorer genes for cytoplasmic male sterility (CMS) are valuable tools for breeders and researchers working with these genes. Two different targeting approaches were used to identify markers linked to the Rfp1 restorer gene for the pol CMS of canola (Brassica napus L.): nearly isogenic line (NIL) comparison and bulked segregant analysis. These methods were equally efficient in identifying markers linked to Rfp1; combining them allowed a targeting efficiency of 100% to be achieved. The efficiency of bulked segregant analysis was found to be limited by the inadvertent occurrence of shared homozygosity at specific chromosomal regions in the bulks, in contrast with the efficiency of NIL comparison which was limited by the occurrence of residual DNA from the donor cultivar at scattered sites around the genome of the NILs. Eleven DNA markers linked to the Rfp1 gene were identified, one of which perfectly co-segregates with Rfp1. The linkage group on which Rfp1 is localized contains 17 DNA markers. Two restorer genes of the pol CMS, Rfp1 and Rfp2, and a Rfn restorer gene of the nap CMS were found to be at least tightly linked to one another and may all reside at the same locus. A fourth restorer gene, the Rfo restorer for the ogu CMS, was, however, found to be unlinked to the other restorer genes. Different restorer genes for the nap CMS were found in the lines 'Westar-Rf and 'Karat'. A linkage map of the B. napus genome containing 146 markers organized into 23 linkage groups covering a total length of 850.2 cM was constructed from a BC$ sb1$ population. This map contains 63 loci previously localized on the B. napus genome through analysis of an F$ sb2$ population. Comparative analysis indicates that the total length of the BC$ sb1$-derived map is smaller than that of the F$ sb2$-derived map, which suggests that a reduction in recombination frequency is occurring in male gametes. The preferential use of two or three probe-

In order to identify organizational differences between sterile Polima (pol) and fertile Campestris (cam) mitochondrial genomes that could be linked to cytoplasmic male sterility (CMS), the physical map of the pol mitochondrial genome was constructed and compared to the physical map of the cam mitochondrial genome. The only structural differences between the two genomes are confined to a region encompassed by a 4.5 kb segment, present in pol mtDNA but absent in cam mtDNA. This 4.5 kb CMS-associated pol segment contains a chimeric gene called orf224 that is cotranscribed with atpG and comprises the single mtDNA region expressed differently in fertile, sterile and fertility restored plants which makes it a good candidate for specifying the sterility trait. Sequence analysis of the pol 4.5 kb segment has shown that orf224 was the only significant open reading frame (ORF) within the segment that gives rise to abundant transcripts, strengthening the view that the orf224/atp6 gene region is conferring pol male sterility. The pol 4.5 kb segment is also present and similarly organized in the common Brassica napus nap mtDNA but the sequences flanking the two segments are unrelated. Thus, the 4.5 kb segment appears to have transposed during the evolution of the pol and nap mitochondrial genomes and appears to have been lost in the cam mitochondrial genome. Sequence analysis of the nap segment revealed the presence of an ORF related to but divergent from orf224. This open reading frame (orf222) potentially encodes a protein of 222 amino-acids with 79% homology to the predicted product of orf224. orf222 is co-transcribed with the third exon of the trans-spliced gene, nad5, and another ORF of unknown function. Expression of the orf222 gene region is tightly associated with nap CMS since the levels of orf222 transcripts are significantly reduced upon restoration while the expression of 22 other mitochondrial genes do not consistently correlate with nap CMS. Antibodies were rai

Typescript (photocopy) / xxiv, 183 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Physiology, 1982?

La spermatogenèse est le processus par lequel les cellules germinales sont transformées en spermatozoïdes par le déroulement de 3 phases: la phase mitotique et méiotiques et la spermiogénèse. Pendant la spermiogénèse d'importantes structures sont formées afin de générer un spermatozoïde fonctionnel : l’acrosome, la manchette et le flagelle. La manchette est une structure transitoire situé caudalement à l’acrosome, composée par un manteau de microtubules longeant le noyau du spermatide. La manchette est connue pour participer au remodelage du noyau afin de lui conférer une forme falciforme ainsi que pour son rôle dans le développement de l’acrosome et du flagelle. En effet, pendant la spermiogénèse toutes les molécules nécessaires pour la formation du flagelle et de l’acrosome doivent être transportées sur leur site d'assemblage. Les microtubules forment la manchette permettent le mouvement de protéines entre la région pré-acrosomique et la zone d’assemblage du flagelle. Cependant ce transport doit être finement régulé dans l’espace et dans le temps car la localisation aberrante et/ou manquante de certaines protéines peut causer des malformations de l’ acrosome, de la manchette et du flagelle. Un mécanisme qui peut expliquer la façon dont ce processus de transport peut être régulé est la génération de modification post-traductionnelles de la tubuline forment les microtubules car ces modifications peuvent réguler les interactions avec les moteurs moléculaires et les protéines associées aux microtubules. La polyglutamylation correspond à un attachement covalent de chaines de glutamates latérales sur la queue terminale de la tubuline. Cette modification est contrôlée par la coordination des enzymes glutamylase (TTLLs) et déglutamylase (CCPs). De récents études ont souligné l'importance potentielle de certaines de ces enzymes dans la formation et la maintenance du flagelle. Mon projet est centré sur l’étude des fonctions exercées par CCP5 pendant la spermatogenèse chez la souris. CCP5 est le seule enzyme qui a la capacité de couper le glutamate de branchement des chaines latéral et qui peut donc réguler l’équilibre entre présence ou l’absence de glutamate de branchement. L’analyse de la souris CCP5-knockout a permis de souligner le rôle essentiel mené par CCP5 pendant la spermiogénèse. J'ai constaté que les souris CCP5-KO produisent 100 fois moins de sperme, défectueux et immobile, comparé aux contrôles. De plus, des nombreuses cellules haploïdes immatures sont prématurément libérées de l’épithélium germinatif. Une analyse approfondie à révélée que la réduite production de sperme est due à plusieurs défaut ultrastructurelles qui surgissent pendant la spermiogenèse. J’ai observé que l’acrosome n’était pas bien développée et que cela se détachait du noyau chez les spermatides matures condensés. De plus l’organisation des microtubules formant la manchette était aussi affectée par une émanation ectopique, ainsi que par une localisation défectueuse dans le noyau. Ces défauts corrèlent avec la formation des spermatides allongée que n’ont pas la typique forme falciforme. De plus, j’ai constaté la présence de centrioles surnuméraires chez les spermatides allongées CCP5-KO. Ce défaut corrèle avec l’observation de microtubules « doublets » et « singlets » dispersés dans le cytoplasme de la cellule. De plus, les structures accessoires du flagelle se positionnaient, de façon désorganisé, à côté de ces microtubules. On a pu constater que ces microtubules sont très probablement issus de plusieurs axonemes qui s'ouvrent dans leur région. Le processus entier de spermiogenèse semble être défectueux dans la souris CCP5-KO et cela est accompagné par d'importants changements de niveaux de glutamylation chez les spermatides rondes et allongés. Par conséquence la régulation des niveaux de glutamylation faites par CCP5 lors de la spermiogénèse semble être fondamental pour garantir un développement normal des spermatides en spermatozoïdes. / Spermatogenesis is the process by which germ cells are transformed into spermatozoa by three sequential phases: the mitotic- and meiotic- phase followed by spermiogenesis. To allow the final maturation of haploid germ cells into spermatozoa specific structures have to be developed during the spermiogenesis: the acrosome, the manchette and the flagellum. The manchette is a MTs-based structure, located caudally to the acrosome, organizing in a skirt-like fashion. Manchette is known to participate in the shaping of the nucleus conferring it the typical hook-like shape and several studies have underlined its importance in acrosome and flagellum formation. During spermiogenesis all molecules and organelles necessary for both acrosome and flagellum formation have to be transported to their destination sites and manchettal MTs allow the movement of organelles and other proteins between the pro-acrosome region and the spermatid tail. However this MTs-based traffic has to be regulated both in space and time as it has been shown that ectopic or mislocalization of certain proteins can lead to failures in acrosome, manchette and flagellum development. The generation of posttranslationally modified MTs might explain a possible mechanism of traffic regulation since it has been demonstrated that posttranslational modifications (PTMs) can regulate the interaction between MTs and molecular motors and microtubules binding proteins. Polyglutamylation, consist in the addition of glutamate side chains of variable length on α- and β- tubulin carboxy-terminal tails. Glutamylation levels are determined by the combined action of glutamylase (TTLLs) and deglutamylase (CCPs) enzymes. Several reports have recently highlighted the importance of some of these enzymes in flagellum assembly and/or maintenance. During my PhD I investigated about the functional role of CCP5 during mouse spermatogenesis. CCP5 is the only enzyme able to remove the glutamate branching point of the added side chain. Thus, its activity might regulate the equilibrium between presence/absence of glutamate branching points, in turn interfering with polyglutamylation levels. The study of the CCP5-KO mouse reveals that CCP5 has an essential role during mouse spermiogenesis. CCP5-KO male produces 100-fold less sperm cells than controls and released sperm cells are highly defective and immotile. Moreover, haploid immature germ cells are also found in CCP5-KO semen. A deep-analysis reveals that the reduced sperm output is due to several ultrastructural defects emerging during the spermatids differentiation process. The acrosome, although is still formed, it does not appear to develop symmetrically and appears to detach from the nucleus in condensed spermatids. Another structure that is impaired in CCP5-KO spermatids in the manchette. Manchettal MTs, are seen to emanate from ectopic regions of the germ cells without running parallel to the nucleus, and are often observed within the spermatids nuclei. Altogether these defects correlate with an aberrant-shaped spermatid nucleus not showing the typical hook-like shape. Another phenotype observed in CCP5-KO elongating spermatids is the presence of supernumerary basal bodies that correlates with the presence of singlet or doublets microtubules dispersed within the germ cell cytoplasm. Interestingly sperm accessory structures are seen to chaotically organize around the microtubules. Unstable disassembling axonemes are seen together with those MTs, suggesting that CCP5-KO spermatids develop abortive unstable flagella. Interesting all these ultrastructural defects correlate with increased level of glutamylation on round spermatids’ cortical MTs and elongating spermatids’ manchettal MTs. Taken together, this study strongly suggests that CCP5-mediated glutamylation regulation is fundamental for spermatids differentiation into healthy functional spermatozoa.

Kandidátská disertační práce z oblasti asistované reprodukce.

Understanding reproductive isolation and divergence is the focus of speciation research. Recent evidence suggested that some Dendroctonus ponderosae populations produced hybrids with reproductive incompatibilities, a reproductive boundary undetected by phylogeographic analyses using molecular markers. Additionally, the unique bifurcated distribution of D. ponderosae and the proposed isolation-by-distance gene flow pattern around the Great Basin Desert provided a unique opportunity to investigate the evolution of postmating (postyzygotic) isolation while also understanding phenotypic divergence along latitudinal (climatic) gradients. First, I characterized the strength, biological pattern, and geographic pattern of postzygotic isolation in D. ponderosae by crossing increasingly divergent populations in a common garden environment. There was little evidence of hybrid inviability in these crosses, yet geographically distant crosses produced sterile males, consistent with expectations under Haldane's rule. Hybrid male sterility appeared at a threshold among increasingly divergent populations, was bidirectional (reciprocal crosses were affected), and less geographically distant crosses did not show significant gender-specific decreases in fitness. Second, a separate investigation of two critical phenotypic traits (body size and development time) was conducted on intrapopulation F2 generation offspring from a common garden experiment. Genetic differences contributing to phenotypic variance were interpreted within the context of the previously described reproductive incompatibilities, gene flow patterns, and latitudinal gradients. Genetic differences in development time were striking between faster developing and more synchronized northern populations and slower developing, less synchronized southern populations. Differences in development time were not detected between populations at similar latitudes. Body size, although more variable than developmental time, generally conformed to expectations, with northern populations being smaller than southern populations. Average adult size was found to be quite different between many populations and did vary between populations at similar latitudes, yet relative sexual size dimorphism was rather consistent. There was no evidence of correspondence between phenotypic traits (body size and development time) and either reproductive boundaries or gene flow patterns. The results suggest that latitudinally imposed climatic differences are likely driving phenotypic divergence between populations.

No description available.

No description available.

Threshold leak sizes and leak rates were calculated for a number of liquid food products exhibiting a wide range of surface tension and viscosity values. From this data, one can see that mathematically, under typical pressure differentials generated in food packages (less than or equal to ±34.5 kPa), a leak will never start through a 2 μm defect. The calculated leak rates were compared to calculated evaporation rates. The evaporation rate exceeds the leak rate at lower sized microholes (2, and 5 μm diameter) under typical pressure differentials found in food packages. If the liquid, typically aqueous in food products, is evaporating off faster than the leak itself, then there will be solids left behind that could effectively plug the leak. The critical leak size is the size micro-defect that allows microbial penetration into the package. The critical leak size of air-filled defects was found to be 7 μm at all pressures tested. This size is considerably important to food packagers because this is when sterility of the package is lost. Previous leak studies have shown that the critical leak size for liquid-filled defects coincide with the threshold leak size and pressure. If this is in fact true, then air-filled defects should exhibit a larger critical leak size than the liquid-filled defects. In this study, air-filled defects were examined. A bioaerosol exposure chamber was used to test micro-defects, nickel microtubes of known diameters 2, 5, 7, 10, 20, and 50 μm hydraulic diameters, against pressure differentials of 0, -6.9, -13.8, and -34.5 kPa. / Master of Science

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.