京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13020号 / 論農博第2830号 / 新制||農||1042(附属図書館) / 学位論文||H28||N4966(農学部図書室) / 32948 / (主査)教授 祝前 博明, 教授 今井 裕, 教授 廣岡 博之 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

It is generally accepted that populations which experience severe bottlenecks have a reduction in fitness. One of the most frequently reported fitness costs is increased hatching failure in bottlenecked populations of birds. The mechanism responsible for increased hatching failure is unknown. Research on other animals suggest that reduced population numbers cause unavoidable inbreeding that in turn leads to abnormalities in the gametes. In this thesis I examine some of the possible causes for increased hatching failure in severely bottlenecked populations of introduced birds in New Zealand. I look at three traits identified as a cause for infertility or hatching failure previously and determine whether there is a link with the size of a population s bottleneck. It is possible that reduced numbers of sperm reaching the site of fertilisation is a primary cause of hatching failure. I examined the perivitelline membrane of various species of introduced birds and counted the total number of sperm present to compare to how many would be expected in non-bottlenecked species. Although there was no relationship between the size of the bottleneck and the number of sperm present, all species had lower than expected sperm counts. In many species of mammals, a reduction in the quality of sperm is attributed to inbreeding depression bought about by genetic bottlenecks. I next compared the level of sperm abnormalities, variation in midpiece size sperm, and sperm motility with the size of the bottleneck each species passed through when introduced to New Zealand. There was no significant correlation between either the variation in midpiece size or sperm motility with bottleneck size. However, there was a trend for species that passed through more severe bottlenecks to have a slightly higher level of midpiece size and lower motility. Finally, I examined whether there was a link between abnormalities in the eggshell and the size of the respective bottleneck. There was no significant change in eggshell thickness or any change in the number of pores associated bottleneck size. However, there was a decreased number of round pores in severely bottlenecked species, although the consequences of this are unknown. My findings do not directly link a single cause for increased hatching failure in bottlenecked species of birds, but they do highlight the need for monitoring of reproductive traits in endangered species that have experienced a population bottleneck.

This study reexamines the nuclear microsatellite analysis by Rooney et al. (1999a) of Bering-Chukchi-Beaufort Seas bowhead whales (Balaena mysticetus) to determine if this population underwent a genetic bottleneck as a result of 19th and early 20th Century commercial whaling. This investigation used more accurate laboratory techniques to score alleles, had a larger sample size that was divided into two groups (mainland Alaska and St. Lawrence Island (SLI)), and used a moderately different set of microsatellite loci which are more variable and thus, more informative. The results corroborate the findings of Rooney et al. (1999a) for mainland Alaska showing no evidence of a genetic bottleneck. However, the SLI data analyses provide conflicting conclusions. The Wilcoxon test is significant for a heterozygote excess (p = 0.042) suggesting that a genetic bottleneck has occurred. This is not substantiated by the exact tests of each locus or the table-wide sign test. There is a possibility that a bottleneck has occurred, but due to the small sample size this is not a definitive conclusion and warrants reanalysis with a larger sample size.

This study reexamines the nuclear microsatellite analysis by Rooney et al. (1999a) of Bering-Chukchi-Beaufort Seas bowhead whales (Balaena mysticetus) to determine if this population underwent a genetic bottleneck as a result of 19th and early 20th Century commercial whaling. This investigation used more accurate laboratory techniques to score alleles, had a larger sample size that was divided into two groups (mainland Alaska and St. Lawrence Island (SLI)), and used a moderately different set of microsatellite loci which are more variable and thus, more informative. The results corroborate the findings of Rooney et al. (1999a) for mainland Alaska showing no evidence of a genetic bottleneck. However, the SLI data analyses provide conflicting conclusions. The Wilcoxon test is significant for a heterozygote excess (p = 0.042) suggesting that a genetic bottleneck has occurred. This is not substantiated by the exact tests of each locus or the table-wide sign test. There is a possibility that a bottleneck has occurred, but due to the small sample size this is not a definitive conclusion and warrants reanalysis with a larger sample size.

The European rabbit, Oryctolagus cuniculus, was introduced to Australia in 1859 and quickly became a significant vertebrate pest species in the country across a wide distribution. In arid and semi-arid environments, rabbit populations exist as metapopulations - undergoing frequent extinction recolonisation cycles. Previous studies identified population genetic structuring at the regional level between arid and semi-arid environments, and habitat heterogeneity was suggested as a possible causal factor. For the most part, rabbit behaviour has been overlooked as a factor that could contribute to explaining population genetic structure in arid and semi-arid environments. This study utilised a combination of genetic sampling techniques and a simulated territorial intrusion approach to observing wild rabbit behaviour in arid and semi-arid environments. The genetic component of the study compared population samples from each region using four polymorphic microsatellite loci. The behavioural component examined variation in the level of territoriality exhibited by three study populations in the arid region towards rabbits of known versus unknown origins (resident vs transgressor (simulating dispersal)). A difference was observed in population genetic structure determined from nuclear markers between arid and semi-arid regions, which supports findings of previous research using mitochondrial DNA data in the same area. Additionally, differences in aggressive response to known vs unknown rabbits were identified in parts of the arid region, which together with the effects of habitat heterogeneity and connectivity may explain the observed differences in population genetic structure. Knowledge of behavioural plasticity and its effect on relative dispersal success and population genetic structure may contribute to improved management and control of feral rabbit populations at the regional level within Australia; and may assist with conservation efforts in the species' natural range in Europe.

It is generally accepted that populations which experience severe bottlenecks have a reduction in fitness. One of the most frequently reported fitness costs is increased hatching failure in bottlenecked populations of birds. The mechanism responsible for increased hatching failure is unknown. Research on other animals suggest that reduced population numbers cause unavoidable inbreeding that in turn leads to abnormalities in the gametes. In this thesis I examine some of the possible causes for increased hatching failure in severely bottlenecked populations of introduced birds in New Zealand. I look at three traits identified as a cause for infertility or hatching failure previously and determine whether there is a link with the size of a population s bottleneck. It is possible that reduced numbers of sperm reaching the site of fertilisation is a primary cause of hatching failure. I examined the perivitelline membrane of various species of introduced birds and counted the total number of sperm present to compare to how many would be expected in non-bottlenecked species. Although there was no relationship between the size of the bottleneck and the number of sperm present, all species had lower than expected sperm counts. In many species of mammals, a reduction in the quality of sperm is attributed to inbreeding depression bought about by genetic bottlenecks. I next compared the level of sperm abnormalities, variation in midpiece size sperm, and sperm motility with the size of the bottleneck each species passed through when introduced to New Zealand. There was no significant correlation between either the variation in midpiece size or sperm motility with bottleneck size. However, there was a trend for species that passed through more severe bottlenecks to have a slightly higher level of midpiece size and lower motility. Finally, I examined whether there was a link between abnormalities in the eggshell and the size of the respective bottleneck. There was no significant change in eggshell thickness or any change in the number of pores associated bottleneck size. However, there was a decreased number of round pores in severely bottlenecked species, although the consequences of this are unknown. My findings do not directly link a single cause for increased hatching failure in bottlenecked species of birds, but they do highlight the need for monitoring of reproductive traits in endangered species that have experienced a population bottleneck.

Most staple food crops were domesticated thousands of years ago through independent processes across different regions of the world. Studies of the history of such crops have been essential to our understanding of plant domestication as a process that started with the collection of wild material and continued with subsequent propagation, cultivation, and selection under human care. Domestication often involves a complex genetic structure with contributions from multiple founder populations, interspecific hybridization, chromosomal introgressions, and polyploidization events that occurred hundreds to thousands of years earlier. Such intricate origins complicate the systematic study of the sources of phenotypic variation. The analysis of recently domesticated, non-traditional, non-model species, such as Sinningia speciosa (Gesneriaceae), can expand the knowledge that we have on phenotypic variation under domestication, and help us to comprehend modern patterns of plant domestication and to broaden our understanding of the general trends. S. speciosa is commonly known as the 'florist's gloxinia', and it has been cultivated for 200 years as an ornamental houseplant. In our genomic study of S. speciosa, we examined an extensive diversity panel consisting of 115 individuals that included different species in the genus, wild representatives, and cultivated accessions, as well as 150 individuals from an F2 segregating population. Our analyses revealed that all of the domesticated varieties are derived from a single founder population that originated in or near the city of Rio de Janeiro in Brazil. We identified two loci associated with domesticated traits (flower symmetry and color) and did not detect any major hybridization or polyploidization events that could have contributed to the rapid increase in phenotypic diversity. Our findings, in conjunction with other features such as a small, low-complexity genome, ease of cultivation, and rapid generation time, makes this species an attractive model for the study of genomic variation under domestication. Basic research on non-model organisms with low economic importance is uncommon but necessary to understand the world from a broader perspective. In such cases, reduced representation approaches like Genotyping-by-Sequencing (GBS) are efficient low-cost alternatives to whole genome resequencing. However, most of these technologies are subject to patent protection, licensing processes, and fees that constrain genomic research for small non-profit research organizations. We have designed a protocol to construct reduced representation libraries from genomic DNA. Our approach, called Targeted Amplification of Scattered Sites (TASS), deviates from the traditional digestion-ligation-amplification process that is the subject of intellectual property that protects most current methods. Instead, TASS relies on 1) targeting and duplicating scattered regions in the genome by annealing and expanding long tail primers with short annealing sites, and 2) amplifying these regions using primers that are complementary to the added overhangs. At the moment GBS is more consistent and delivers more variants than TASS. However, we have established a foundation on which further optimization can produce an accessible, easy to implement, high-throughput genotyping approach. / Doctor of Philosophy / Most staple food crops were domesticated thousands of years ago through unrelated processes that were initiated across different regions of the world. Studies of the history of such crops have been essential to our understanding of plant domestication, a process that started with the collection of wild material and continued with subsequent propagation and cultivation under human care. Plant domestication has often involved a complex combination of ancestral lineages that encompass multiple populations, crosses with other species, and large DNA reorganizations that occurred hundreds to thousands of years earlier. Such intricate origins make the systematic study of plant domestication very challenging. The analysis of recently domesticated plants such as the 'florist's gloxinia' (Sinningia speciosa), can help us to better understand some of the changes that have occurred during domestication, as well as to comprehend modern patterns of plant domestication and to broaden our understanding of general trends. Florist's gloxinias are ornamental plants that have been cultivated during the last 200 years. In this study we examined 115 specimens, including wild and cultivated types of florist's gloxinias, as well as closely related species in Sinningia. We also constructed and evaluated an artificial population of 150 individuals from the cross of a wild and a cultivated form. Our analyses revealed that all of the domesticated varieties are descendants from a single wild population that originated in or near the city of Rio de Janeiro in Brazil. We also identified two regions of DNA that are responsible for the changes in flower shape and color, and crosses with other species did not introduce such alterations. Our findings, in conjunction with other features such as its small nuclear DNA content, the ease of cultivation indoors, and a rapid generation time, makes the florists' gloxinia an attractive crop to the study the effects of plant domestication. Research on organisms with low economic importance is uncommon but necessary to understand the world from a broader perspective. In such cases, analyzing the entire genetic information that is stored as DNA may be cost-prohibitive. Instead, approaches that sample small portions of DNA from each individual can be utilized. Most of these technologies are currently patented and subject to licensing processes and fees that limit their implementation by small non-profit research organizations. In this study we designed a protocol to sample small portions of DNA, similarly to existing techniques. However, our approach, called Targeted Amplification of Scattered Sites (TASS), employs a sampling process that deviates from the traditional patented procedure that is used in most current methods. At present, TASS is not as consistent and delivers less information than traditional approaches. However, we have established a foundation on which further optimization can produce an accessible and easy to implement technique.

Mitochondrial DNA (mtDNA) mutations have been widely observed to cause a variety of human diseases, especially late-onset neurodegenerative disorders. The prevalence of mitochondrial diseases caused by mtDNA mutation is approximately 1 in 5,000 of the population. There is no effective way to treat patients carrying pathogenic mtDNA mutation; therefore preventing transmission of mutant mtDNA became an important strategy. However, transmission of human mtDNA mutation is complicated by a large intergenerational random shift in heteroplasmy level causing uncertainty for genetic counseling. The aim of this dissertation is to gain insight into how human mtDNA heteroplasmy is inherited. By working closely with our experimental collaborators, the computational simulation of mouse embryogenesis has been developed in our lab using their measurements of mouse mtDNA copy number. This experimental-computational interplay shows that the variation of offspring heteroplasmy level has been largely generated by random partition of mtDNA molecules during pre- and early postimplantation development. By adapting a set of probability functions developed to describe the segregation of allele frequencies under a pure random drift process, we now can model mtDNA heteroplasmy distribution using parameters estimated from experimental data. The absence of an estimate of sampling error of mtDNA heteroplasmy variance may largely affect the biological interpretation drawn from this high-order statistic, thereby we have developed three different methods to estimate sampling error values for mtDNA heteroplasmy variance. Applying this error estimation to the comparison of mouse to human mtDNA heteroplasmy variance reveals the difference of the mitochondrial genetic bottleneck between these organisms. In humans, the mothers who carry a high proportion of m.3243A>G mutation tend to have fewer daughters than sons. This offspring gender bias has been revealed by applying basic statistical tests on the human clinical pedigrees carrying this mtDNA mutation. This gender bias may partially determine the mtDNA mutation level among female family members. In conclusion, the application of population genetic theory, statistical analysis, and computational simulation help us gain understanding of human mtDNA heteroplasmy inheritance. The results of these studies would be of benefit to both scientific research and clinical application. / Ph. D.

Contexte: Les champignons mycorhiziens à arbuscules (AMF) établissent des relations symbiotiques avec la plupart des plantes grâce à leurs réseaux d’hyphes qui s’associent avec les racines de leurs hôtes. De précédentes études ont révélé des niveaux de variation génétique extrêmes pour des loci spécifiques permettant de supposer que les AMF peuvent contenir des milliers de noyaux génétiquement divergents dans un même cytoplasme. Si aucun processus de reproduction sexuée n’a jusqu’ici été observé chez ces mycorhizes, on constate cependant que des niveaux élevés de variation génétique peuvent être maintenus à la fois par l’échange de noyaux entre hyphes et par des processus fréquents de recombinaison entre noyaux. Les AMF se propagent par l’intermédiaire de spores qui contiennent chacune un échantillon d’une population initiale de noyaux hétérogènes, directement hérités du mycélium parent. À notre connaissance les AMF sont les seuls organismes qui ne passent jamais par un stade mononucléaire, ce qui permet aux noyaux de diverger génétiquement dans un même cytoplasme. Ces aspects singuliers de la biologie des AMF rendent l’estimation de leur diversité génétique problématique. Ceci constitue un défi majeur pour les écologistes sur le terrain mais également pour les biologistes moléculaires dans leur laboratoire. Au-delà même des problématiques de diversité spécifique, l’amplitude du polymorphisme entre noyaux mycorhiziens est mal connue. Le travail proposé dans ce manuscrit de thèse explore donc les différents aspects de l’architecture génomique singulière des AMF. Résultats L’ampleur du polymorphisme intra-isolat a été déjà observée pour la grande sous-unité d’ARN ribosomal de l’isolat Glomus irregulare DAOM-197198 (précédemment identifié comme G. intraradices) et pour le gène de la polymerase1-like (PLS) de Glomus etunicatum isolat NPI. Dans un premier temps, nous avons pu confirmer ces résultats et nous avons également pu constater que ces variations étaient transcrites. Nous avons ensuite pu mettre en évidence la présence d’un goulot d’étranglement génétique au moment de la sporulation pour le locus PLS chez l’espèce G. etunicatum illustrant les importants effets d’échantillonnage qui se produisaient entre chaque génération de spore. Enfin, nous avons estimé la différentiation génétique des AMF en utilisant à la fois les réseaux de gènes appliqués aux données de séquençage haut-débit ainsi que cinq nouveaux marqueurs génomiques en copie unique. Ces analyses révèlent que la différenciation génomique est présente de manière systématique dans deux espèces (G. irregulare et G. diaphanum). Conclusions Les résultats de cette thèse fournissent des preuves supplémentaires en faveur du scénario d’une différenciation génomique entre noyaux au sein du même isolat mycorhizien. Ainsi, au moins trois membres du genre Glomus, G. irregulare, G. diaphanum and G. etunicatum, apparaissent comme des organismes dont l’organisation des génomes ne peut pas être décrit d’après un modèle Mendélien strict, ce qui corrobore l’hypothèse que les noyaux mycorhiziens génétiquement différenciés forment un pangenome. / Background: Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi whose hyphal networks form symbioses with plants. Previous studies have revealed extremely high levels of genetic variation for some loci, which has lead to the proposition that AMF contain thousands of genetically divergent nuclei that share the same cytoplasm, i.e. they are heterokaryotic coenocytes. No reproductive stage has as yet been observed in AMF, yet evidence is accumulating that the observed high levels of diversity could be maintained by the exchange of nuclei between hyphal systems and (meiotic) recombination. AMF spores contain varying fractions of this heterogeneous population of nuclei, which migrate directly from the parent mycelium. To our knowledge, AMF are the only organisms that never pass through a single nucleus stage in their life cycle, which allows nuclei to diverge into genetically distinct nuclei within the same cytoplasm. Thus, estimating genetic diversity in arbuscular mycorrhizal fungi (AMF) is a major challenge, not only for ecologists in the field but also for molecular biologists in the lab. It is unclear what the extent of polymorphism is in AMF genomes. The present thesis investigates different aspects of this peculiar genome organization. Results The second chapter in this thesis confirms the extensive intra-isolate polymorphism that was previously observed for large subunit rDNA (in G. irregulare DAOM-197198) and the polymerase1-like gene, PLS (in G. etunicatum), and shows that this polymorphism is transcribed. In the third chapter I report the presence of a bottleneck of genetic variation at sporulation for the PLS locus, in G. etunicatum. Analyses in the fourth chapter, based on a conservative network-based clustering approach and five novel single copy genomic markers, reveal extensive genome-wide patterns of diversity in two different AMF species (G. irregulare and G. diaphanum). Conclusions The results from this thesis provide additional evidence in favor of genome differentiation between nuclei in the same isolate for AMF. Thus, at least three members of the Glomus genus, G. irregulare, G. diaphanum and G. etunicatum appear to be organisms whose genome organization cannot be described by a single genome sequence: genetically differentiated nuclei in AMF form a pangenome.

Contexte: Les champignons mycorhiziens à arbuscules (AMF) établissent des relations symbiotiques avec la plupart des plantes grâce à leurs réseaux d’hyphes qui s’associent avec les racines de leurs hôtes. De précédentes études ont révélé des niveaux de variation génétique extrêmes pour des loci spécifiques permettant de supposer que les AMF peuvent contenir des milliers de noyaux génétiquement divergents dans un même cytoplasme. Si aucun processus de reproduction sexuée n’a jusqu’ici été observé chez ces mycorhizes, on constate cependant que des niveaux élevés de variation génétique peuvent être maintenus à la fois par l’échange de noyaux entre hyphes et par des processus fréquents de recombinaison entre noyaux. Les AMF se propagent par l’intermédiaire de spores qui contiennent chacune un échantillon d’une population initiale de noyaux hétérogènes, directement hérités du mycélium parent. À notre connaissance les AMF sont les seuls organismes qui ne passent jamais par un stade mononucléaire, ce qui permet aux noyaux de diverger génétiquement dans un même cytoplasme. Ces aspects singuliers de la biologie des AMF rendent l’estimation de leur diversité génétique problématique. Ceci constitue un défi majeur pour les écologistes sur le terrain mais également pour les biologistes moléculaires dans leur laboratoire. Au-delà même des problématiques de diversité spécifique, l’amplitude du polymorphisme entre noyaux mycorhiziens est mal connue. Le travail proposé dans ce manuscrit de thèse explore donc les différents aspects de l’architecture génomique singulière des AMF. Résultats L’ampleur du polymorphisme intra-isolat a été déjà observée pour la grande sous-unité d’ARN ribosomal de l’isolat Glomus irregulare DAOM-197198 (précédemment identifié comme G. intraradices) et pour le gène de la polymerase1-like (PLS) de Glomus etunicatum isolat NPI. Dans un premier temps, nous avons pu confirmer ces résultats et nous avons également pu constater que ces variations étaient transcrites. Nous avons ensuite pu mettre en évidence la présence d’un goulot d’étranglement génétique au moment de la sporulation pour le locus PLS chez l’espèce G. etunicatum illustrant les importants effets d’échantillonnage qui se produisaient entre chaque génération de spore. Enfin, nous avons estimé la différentiation génétique des AMF en utilisant à la fois les réseaux de gènes appliqués aux données de séquençage haut-débit ainsi que cinq nouveaux marqueurs génomiques en copie unique. Ces analyses révèlent que la différenciation génomique est présente de manière systématique dans deux espèces (G. irregulare et G. diaphanum). Conclusions Les résultats de cette thèse fournissent des preuves supplémentaires en faveur du scénario d’une différenciation génomique entre noyaux au sein du même isolat mycorhizien. Ainsi, au moins trois membres du genre Glomus, G. irregulare, G. diaphanum and G. etunicatum, apparaissent comme des organismes dont l’organisation des génomes ne peut pas être décrit d’après un modèle Mendélien strict, ce qui corrobore l’hypothèse que les noyaux mycorhiziens génétiquement différenciés forment un pangenome. / Background: Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi whose hyphal networks form symbioses with plants. Previous studies have revealed extremely high levels of genetic variation for some loci, which has lead to the proposition that AMF contain thousands of genetically divergent nuclei that share the same cytoplasm, i.e. they are heterokaryotic coenocytes. No reproductive stage has as yet been observed in AMF, yet evidence is accumulating that the observed high levels of diversity could be maintained by the exchange of nuclei between hyphal systems and (meiotic) recombination. AMF spores contain varying fractions of this heterogeneous population of nuclei, which migrate directly from the parent mycelium. To our knowledge, AMF are the only organisms that never pass through a single nucleus stage in their life cycle, which allows nuclei to diverge into genetically distinct nuclei within the same cytoplasm. Thus, estimating genetic diversity in arbuscular mycorrhizal fungi (AMF) is a major challenge, not only for ecologists in the field but also for molecular biologists in the lab. It is unclear what the extent of polymorphism is in AMF genomes. The present thesis investigates different aspects of this peculiar genome organization. Results The second chapter in this thesis confirms the extensive intra-isolate polymorphism that was previously observed for large subunit rDNA (in G. irregulare DAOM-197198) and the polymerase1-like gene, PLS (in G. etunicatum), and shows that this polymorphism is transcribed. In the third chapter I report the presence of a bottleneck of genetic variation at sporulation for the PLS locus, in G. etunicatum. Analyses in the fourth chapter, based on a conservative network-based clustering approach and five novel single copy genomic markers, reveal extensive genome-wide patterns of diversity in two different AMF species (G. irregulare and G. diaphanum). Conclusions The results from this thesis provide additional evidence in favor of genome differentiation between nuclei in the same isolate for AMF. Thus, at least three members of the Glomus genus, G. irregulare, G. diaphanum and G. etunicatum appear to be organisms whose genome organization cannot be described by a single genome sequence: genetically differentiated nuclei in AMF form a pangenome.