Microsatellites are simple repetitive DNA sequences that are used as genetic markers throughout the biological sciences. The high levels of variation observed at microsatellite loci contribute to their utility in studies at the population and individual levels. This variation is a consequence of mutations that change the length of microsatellite repeat tracts. Current understanding suggests that most mutations are caused by polymerase slippage during DNA replication and lead to changes of a single repeat unit in length, but some changes involving multiple repeats can also occur. Despite this simplistic overview, there is evidence for considerable heterogeneity in mutation processes between species, loci and alleles. Such complex patterns suggest that other mechanisms, including those associated with DNA recombination, are also involved in the generation of microsatellite mutations. Understanding which mutational mechanisms are responsible for variation at microsatellite markers is essential to enable accurate data interpretation in genotyping projects, as many commonly used statistics assume specific mutation models. I developed microsatellite markers specific to the X and Y chromosomes and an autosome in the tammar wallaby, Macropus eugenii, and investigated their evolutionary properties using two approaches: indirectly, as inferred from population data, and directly, from observation of mutation events. First, I found that allelic richness increased with repeat length and that two popular mutation models, the stepwise mutation model and the infinite allele model, were poor at predicting the number of alleles per locus, particularly when gene diversity was high. These results suggest that neither model can account for all mutations at tammar wallaby microsatellites and hint at the involvement of more complex mechanisms than replication slippage. I also determined levels of variation at each locus in two tammar wallaby populations. I found that allelic richness was highest for chromosome 2, intermediate for the X chromosome and lowest for the Y chromosome in both populations. Thus, allelic richness varied between chromosomes in the manner predicted by their relative exposure to recombination, although these results may also be explained by the relative effective population sizes of the chromosomes studied. Second, I used small-pool PCR from sperm DNA to observe de novo mutation events at three of the most polymorphic autosomal markers. To determine the reliability of my observations I developed and applied strict criteria for scoring alleles and mutations at microsatellite loci. I observed mutations at all three markers, with rate variation between loci. Single step mutations could not be distinguished because of the limitations of the approach, but 24 multi-step mutations, involving changes of up to 35 repeat units, were recorded. Many of these mutations involved changes that could not be explained by the gain or loss of whole repeat units. These results imply that a large number of mutations at tammar wallaby microsatellites are caused by mechanisms other than replication slippage and are consistent with a role for recombination in the mutation process. Taken as a whole, my results provide evidence for complex mutation processes at tammar wallaby microsatellites. I conclude that careful characterisation of microsatellite mutation properties should be conducted on a case-by-case basis to determine the most appropriate mutation models and analysis tools for each locus. In addition, my work has provided a set of chromosome-specific markers for use in macropod genetic studies, which includes the first marsupial Y chromosome microsatellites. Sex chromosome microsatellites open a new range of possibilities for population studies, as they provide opportunities to investigate gene flow in a male context, to complement data from autosomal and maternally-inherited mitochondrial markers.

Almond (Prunus dulcis) is the most important nut crop in terms of world production. Due to its health benefit and high nutritional value the consumption and world supply of almond is increasing. To remain competitive in the world market, the Australian almond breeding program was established to produce cultivars with better adaptation to Australian conditions. As part of this program an almond mapping population consisting of 93 F₁ progeny derived from a cross between the American cultivar ‘Nonpareil’ (NP) and the European self-compatible cultivar ‘Lauranne’ (LA) was produced to construct the genetic linkage maps. The first almond linkage map developed prior to the commencement of this project failed to produce the eight linkage groups similar to the basic chromosome number of almond (x = 8) and many large gaps were also observed on the linkage groups. Therefore, more markers were needed to saturate the maps. Microsatellite markers are considered one of the best choices for mapping studies. 195 microsatellite markers isolated from Prunus species were obtained from published papers or by personal communication. Polymorphism was revealed by three different methods, and in general, polyacrylamide gel electrophoresis (PAGE) compared to the fluorescent labelled marker detection using an automated DNA sequencer or agarose gel electrophoresis, showed the most efficient and cost effective method of genotyping. A subset of 54 markers which produced reliable and easily interpretable polymorphic bands was selected to screen the whole mapping population. Microsatellites originally isolated from almond species showed the highest rate of amplification and polymorphism followed by peach microsatellites and the least informative markers were isolated from cherry. It seems that the level of transportability and usefulness of microsatellite markers is related to the genetic distance of the closely related species. Almond and peach belong to the same subgenus (Amygdalus) and other Prunus species are classified in Prunophora subgenus. The nut, or kernel, is the commercial part of the almond tree, thus to improve the quality of fruit an understanding of environmental influence, heritability and correlation of traits is required. Pomological and quality characters such as: shell hardness, kernel size, shape, taste, pubescence, colour, and percentage of doubles were measured during three consecutive years (2005-2007) on the total mapping population, but data analysis (ANOVA) was performed only on trees that survived for all three years. Most of the traits showed high broad-sense heritability and kernel shape showed the highest heritability of H² = 0.92 suggesting high genetic control of this trait. Occasionally larger kernels than either parent were found in the progeny indicating potential for improvement of this trait even with smaller kernel size parent that encompass many desirable characters. High correlation was also found between the in-shell and kernel weight (r = 0.74), kernel length / kernel width (r = 0.67), kernel weight to kernel length (r = 0.78) and kernel width (r = 0.80). This correlation estimation pointed out in this study indicates that the improvement of one character may result the progress in another trait. Neither of the parents in the mapping population had bitter or obvious slightly bitter taste but slightly bitter kernels were observed among the progeny. Amygdalin was assumed to be responsible for bitter taste in almond; therefore we measured the amount of amygdalin in sweet and slightly bitter kernel progeny by HPLC. However, the results showed that amygdalin exists in sweet kernels as well. Although the average amount of amygdalin in slightly bitter kernels (20.34 mg kg⁻¹ FW) was higher than sweet kernels (3.67 mg kg⁻¹ FW), some sweet kernels had higher amounts of amygdalin suggesting the impact of other components on slightly bitter kernel. The highest variability within the traits was observed in the percentage of double kernel, which showed the highest standard error. Strong environmental effects, particularly low temperature at pre-blossom time is speculated to produce much higher double kernels. Three genetic linkage maps, one for each parent and an integrated map were constructed by the addition of 54 new microsatellite markers to the previous dataset. All the data was scored and coded according to the coding system necessary by JoinMap3 which was used for map construction. 131 markers including microsatellite, ISSR, RAPD, SCAR and S-allele markers were placed on the integrated map covering 590.7 cM with the average density of 4.5 cM/marker. The minimum number of six microsatellite markers was placed on linkage group 8 and the linkage group 1 which is the longest linkage group has 14 microsatellite markers. Comparative mapping study with other Prunus maps, especially with the highly saturated reference map showed complete synteny and minor changes in the order of four markers on linkage groups compared with Prunus reference map. The conservation of molecular marker order observed in this study supports the idea of looking at Prunus genome as a single genetic system and practical application of this similarity would be in cross-transportability of microsatellite markers from well developed linkage maps to the less studied species in Prunus. Ten microsatellite loci placed on our map have not been reported before and could be used to improve the density of other Prunus maps, especially the reference map. This study contributed to the better understanding of the mode of inheritance and environmental effect on morphological traits and the effect of amygdalin on kernel taste. The most saturated microsatellite based almond linkage map developed in this study can serve as a framework for future almond breeding program in Australia and benefit Prunus improvement programs internationally. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1348850 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Cette thèse étudie l'effet de deux processus cellulaires essentiels, la réplication et la transcription, sur la composition en nucléotides des séquences d'ADN. Ces mécanismes ont un fonctionnement asymétrique par rapport aux deux brins d'ADN, et ils ont comme conséquence une composition asymétrique dans les séquences.<br /> Nous avons étudié la co-orientation entre réplication et transcription chez les procaryotes. Nous proposons une méthode pour l'étude des biais de composition qui découple ces deux sources d'asymétrie. Nous montrons que les biais associés à la réplication sont très variables, même entre espèces proches.<br /><br /> Nous avons ensuite analysé le patron de substitution dans les régions transcrites et autour des origines de réplication du génome humain, et notamment l'effet du contexte 5'-3'. Les biais de voisinage sont similaires pour l'asymétrie associée à la réplication et à la transcription. La variation des taux de substitutions en fonction du patron d'expression des gènes suggère qu'un biais de réparation asymétrique et contexte-dépendant pourrait être en jeu.<br /><br /> Enfin, nous avons proposé une méthode de calcul du patron de substitution dans des séquences à composition biaisée: les microsatellites. Nous avons démontré que les microsatellites transcrits sont sujets au mêmes processus asymétriques que les régions non-répétées.

L'étude de la démographie dans une perspective historique participe à la compréhension des processus évolutifs. Les données de diversité génétique sont potentiellement informatives quant au passé démographique des populations: en effet, ce passé est enregistré avec perte d'information par les marqueurs moléculaires, par l'intermédiaire de leur histoire généalogique et mutationnelle. L'acquisition de données de diversité génétique est de plus en plus rapide et aisée, et concerne potentiellement n'importe quel organisme d'intérêt. D'où un effort dans la dernière décennie pour développer les outils statistiques permettant d'extraire l'information démographique des données de typage génétique.<br />La présente thèse propose une extension de la méthode d'inférence bayésienne développée en 1999 par M. Beaumont. Comme la méthode originale, (i) elle est basée sur le coalescent de Kingman avec variations d'effectif, (ii) elle utilise l'algorithme de Metropolis-Hastings pour échantillonner selon la loi a posteriori des paramètres d'intérêt et (iii) elle permet de traiter des données de typage à un ou plusieurs microsatellites indépendants. La version étendue généralise les modèles démographique et mutationnel supposés dans la méthode initiale: elle permet d'inférer les paramètres d'un modèle de fondation-explosion pour la population échantillonnée et d'un modèle mutationnel à deux phases, pour les marqueurs microsatellites typés. C'est la première fois qu'une méthode probabiliste exacte incorpore pour les microsatellites un modèle mutationnel autorisant des sauts.<br />Le modèle démographique et mutationnel est exploré. L'analyse de jeux de données simulés permet d'illustrer et de comparer la loi a posteriori des paramètres pour des scénarios historiques: par exemple une stabilité démographique, une croissance exponentielle et une fondation-explosion. Une typologie des lois a posteriori est proposée. Des recommandations sur l'effort de typage dans les études empiriques sont données: un unique marqueur microsatellite peut conduire à une loi a posteriori très structurée. Toutefois, les zones de forte densité a posteriori représentent des scénarios de différents types. 50 génomes haploides typés à 5 marqueurs microsatellites suffisent en revanche à détecter avec certitude (99% de la probabilité a posteriori) une histoire de fondation-explosion tranchée. Les conséquences de la violation des hypothèses du modèle démographique sont discutées, ainsi que les interactions entre processus et modèle mutationnel. En particulier, il est établi que le fait de supposer un processus mutationnel conforme au modèle SMM, alors que ce processus est de type TPM, peut générer un faux signal de déséquilibre génétique. La modélisation des sauts mutationnels permet de supprimer ce faux signal.<br />La méthode est succinctement appliquée à l'étude de deux histoires de fondation-explosion: l'introduction du chat Felis catus sur les îles Kerguelen et celle du surmulot Rattus norvegicus sur les îles du large de la Bretagne. Il est d'abord montré que la méthode fréquentiste développée par Cornuet et Luikart (1996) ne permet pas de détecter les fondations récentes et drastiques qu'ont connu ces populations. Cela est vraisemblablement dû à des effets contraires de la fondation et de l'explosion, sur les statistiques utilisées dans cette méthode.<br />La méthode bayésienne ne détecte pas non plus la fondation si l'on force une histoire démographique en marche d'escalier, pour la même raison. La fondation et l'explosion deviennent détectables si le modèle démographique les autorise. Toutefois, les dépendances entre les paramètres du modèle empêchent de les inférer marginalement avec précision. Toute information a priori sur un paramètre contraint fortement les valeurs des autres paramètres. Ce constat confirme le potentiel de populations d'histoire documentée pour l'estimation indirecte des paramètres d'un modèle de mutation des marqueurs.

Les zones hybrides entre taxons proches peuvent offrir un regard privilégié sur les processus évolutifs, en particulier la spéciation. L'évolution de l'isolement reproducteur dans ces zones va dépendre en grande partie de l'intensité des flux de gènes et de la valeur sélective des hybrides. Chez les arbres forestiers, les zones de contact sont souvent très étendues, étant donnés leurs larges distributions, les longues distances de dispersion de pollen et les forts taux d'allofécondation. Nous avons choisi comme modèle d'étude des deux espèces de frênes Fraxinus excelsior L. (frêne commun) et Fraxinus angustifolia Vahl (frêne oxyphylle), autochtones en France. Nous avons d'abord développé des marqueurs moléculaires et physiologiques (liés à la dormance des graines) discriminants, qui se sont révélés être de bon outils pour l'aide au diagnostic de reconnaissance, mais insuffisants pour détecter les hybrides en populations naturelles. Nous avons donc utilisé des marqueurs microsatellites et des données morphologiques pour mettre en évidence plusieurs zones hybrides très différentes à l'échelle de la France. Nous avons montré que la distribution des deux espèces et de leurs hybrides était fortement corrélée aux variations de facteurs environnementaux, suggérant que le climat est déterminant pour limiter l'hybridation naturelle. Les deux espèces observant des dates de floraison très divergentes, nous avons étudié l'effet de la phénologie florale sur la limitation des flux de gènes à échelle locale. Nous avons montré l'existence d'un isolement par le temps. Les flux de gènes ont lieu principalement entre individus fleurissant à la même période, et les flux de pollen sont asymétriques. Les hybrides fleurissant tôt sont les plus nombreux, et ont un succès reproducteur mâle et femelle largement supérieur, produisant plus de fleurs et de graines et subissant sensiblement moins d'attaques de gales florales. Observant par ailleurs des taux d'autofécondation élevés, ils pourraient posséder une valeur sélective supérieure dans ce type d'écotone intermédiaire, pouvant accroître leur potentiel colonisateur.

L'aire de distribution des espèces est conditionnée à la fois par des facteurs biotiques et abiotiques et ses frontières dépendent généralement des limites physiologiques de l'espèce. Ainsi, en bordure d'aire de répartition, les populations se caractérisent par une diversité génétique moindre ainsi qu'une forte différentiation génétique et leur existence dépend d'un équilibre fragile entre événements de colonisation et d'extinction.Depuis les années 1970, l'augmentation et l'accélération des pressions anthropiques exercées sur les écosystèmes bouleversent ces équilibres et des modifications de l'aire de répartition sont observées chez certaines espèces dont le bivalve Macoma balthica, espèce clé des écosystèmes littoraux en Europe, dont la limite sud de répartition s'est décalée vers le nord-est au cours des quarante dernières années.Afin d'évaluer les capacités adaptatives de M. balthica et dans un but de conservation, deux approches complémentaires ont été menées dans des environnements contrastés qui ont permis de mettre en évidence des signes d'adaptation locale.D'une part, l'étude du génome neutre à l'aide des outils méthodologiques et concepts de la génétique des populations a permis d'inférer l'histoire démographique de l'espèce avec une attention particulière portée sur une baie en limite d'aire de répartition (Baie de Marennes Oléron, France) et sur une baie soumise à de fortes pressions physico-chimiques (Baie de Gdansk, Pologne). Nous avons mis en évidence notamment :(i) un polymorphisme significatif dans les populations en limite d'aire en opposition avec les attendus théoriques(ii) des ruptures au flux de gènes dans le golfe de Gascogne soumis au réchauffement des eaux de surface mais aussi le long d'un gradient environnemental dans la baie de Gdansk. D'autre part, l'étude moléculaire de la sélection a été menée par la méthode de pyroséquençage sur le transcriptome d'individus prélevés en milieux contrastés. Cette étude a permis de mettre en évidence des tendances d'expression différentielle de gènes de réponse générale au stress selon le milieu considéré.

<p>The phylogeography of a wide ranging temperate species, the adder, <i>Vipera berus</i>, was investigated using several genetic tools, with special emphasis on the post-glacial colonisation pattern of Fennoscandia. The area was colonised from two directions by adder populations representing different glacial refugia. The two populations meet in three places and the main contact zone is situated in Northern Finland. The two other contact zones are the result of dispersal across the Baltic Sea to the Umeå archepelago and South-Western Finland. Asymmetrically distributed nuclear genetic variation compared to mitochondrial DNA in the northern contact zone suggests a skewed gene flow from the east to the west across the zone. This pattern might reflect differences in dispersal among sexes and lineages, or may be accounted for by a selective advantage for nuclear variation of eastern origin among Fennoscandian adders.</p><p>The phylogeographic pattern for adders across the entire species range was addressed by sequencing part of the mitochondrial genome and scoring microsatellite markers. The adder can be divided into three major genetic groups. One group is confined to the Balkan peninsula harbouring the distribution range of <i>V. b. bosniensis</i>. A second, well differentiated group is restricted to the Southern Alps. These two areas have probably served as refugia for adders during a number of ice ages for the adders. The third group is distributed across the remainder of the species’ range, from extreme Western Europe to Pacific Russia and can be further divided into one ancestral group inhabiting the Carpathians refugial area, and three more recent groups inhabiting areas west, north and east of the Alps. The adder provides an example of a species where the Mediterranean areas are housing endemic populations, rather than the sources for post-glacial continental colonisation. Continent-wide colonisation has instead occurred from up to three cryptic northern refugia. </p>

<p>The white-tailed eagle is a formerly threatened raptor that is commonly used as a flagship and indicator species in conservation work. This thesis uses molecular genetic methods to study sex determination of nestlings, genetic variability, population structure and phylogeography of the white-tailed eagle.</p><p>Fourteen microsatellite markers were developed and tested for the white-tailed eagle.</p><p>A method to sex white-tailed eagle nestlings in the field is presented. The method is based on just one tarsus measure, and is suitable for situations where a single person is handling the nestlings alone in a treetop.</p><p>Most European white-tailed eagle populations underwent extreme declines during the 20th century. The results presented here show that bottlenecked populations have maintained significant levels of genetic diversity. Gene flow between regions is not a main explanation for this, as indicated by both genetic and ringing data. Instead, the long generation time of white-tailed eagles has acted as an intrinsic buffer against rapid loss of genetic diversity. Additionally, local conservation led to protection of more genetic diversity than if conservation had focused on the large remnant population in Norway.</p><p>Mitochondrial DNA of white-tailed eagles is structured in two main clades with a predominantly eastern and western Eurasian distribution. The clades likely correspond to separate Ice Age refugia but do not grant classification as evolutionary significant units given their current extensive overlap across large parts of Eurasia.</p><p>Microsatellite variation was studied in populations across Eurasia. Variability was rather constant across the continent, but clearly lower on Iceland and Greenland. This is best explained by founder effects during their colonisation, but only weak bottlenecks during colonisation of and persistence on the continent. Current population differentiation between Europe and eastern Eurasia is not compatible with a zero gene flow model but requires some amount of gene flow over evolutionary time scales.</p>

<p>Management of wolves is a complex issue, and molecular genetics is an important tool in this work. Molecular genetics can provide important information at the species, population and individual level, which can be essential for the development of management programs aiming at the long term survival of wolf populations.</p><p>In this thesis I developed new genetic markers on the canine Y chromosome to estimate the number of founders of the Scandinavian wolf population. This knowledge is important to reconstruct the history of the population and to design the most appropriate conservation strategies. Next, genetic markers with different pattern of inheritance have been used to identify hybrids between wolves and dogs. This allowed us to determine the direction of hybridization and to evaluate its possible impact on the gene pool of a wolf population. Furthermore, I also developed a method for a more reliable identification of the predator responsible of an attack by using saliva remains left on the prey. Since predation on livestock is perhaps the main reason for the negative opinions about the predator, the correct identification of the responsible for an attack (wolf, dog or hybrid) is essential. </p><p>Finally, this thesis has also been focusing on the domestication of dogs. By using Y chromosome markers (paternally inherited), it has been possible to complement previous studies based on mtDNA sequences (maternally inherited) and autosomal markers (inherited from both parents). In this way I have obtained a more complete picture of the domestication process and of the origin of breeds. This has shown that there has been a bias in the contribution of the two sexes in the origin of dog breeds (fewer males then females contributing to each breed) and that the origin of dogs was not marked by extensive backcrosses with male wolves over the entire species range.</p>

<p>The identification of processes governing genetic structure at different spatial scales remains a major challenge in evolutionary biology and is of considerable applied interest in conservation biology. In <i>Euphrasia stricta</i> five varieties have been identified (<i>brevipila</i>, <i>gotlandica</i>, <i>stricta</i>, <i>suecica</i> and <i>tenuis</i>) based on differences in habitat, phenology and morphology. In this thesis, I examined genetic variation at AFLP and microsatellite marker loci in relation to variation in habitat and morphology within and among varieties of <i>E. stricta</i> on the island Gotland in the Baltic Sea. The results are discussed in relation to evolutionary processes acting within this species complex. </p><p>In a study conducted at the regional scale, the two early-flowering varieties <i>suecica</i> and <i>tenuis</i> each formed a genetically distinct group, while the three late-flowering varieties <i>brevipila</i>, <i>gotlandica</i> and <i>stricta</i> formed a third group. The results suggest that <i>suecica</i> and <i>tenuis</i> have ancient origins since they are genetically different both from the <i>brevipila</i>/<i>gotlandica</i>/<i>stricta</i> group and from each other despite their similar habitat preferences. This pattern was obtained using both marker systems. Discrepancies between AFLP and microsatellites were found in patterns of isolation by distance and in estimates of expected heterozygosity, <i>H</i>_e.</p><p>Focusing on the mixed genetic group <i>brevipila</i>/<i>gotlandica</i>/<i>stricta</i> and the causes behind their clustering together despite differences in morphology and habitat preferences, I performed a study at a smaller geographic scale. Studying a population of <i>E. stricta</i> I found that, although gene flow within the population was strong, it had not prevented the formation of genetic groups associated with micro-habitat properties. </p><p>An important result for conservation of the rare variety <i>suecica</i> is its distinct genetic separation from variety <i>tenuis</i>. If the aim of conservation is to preserve the uniqueness of <i>suecica,</i> the two varieties should be treated as separated entities. </p>