京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第22791号 / 人博第962号 / 新制||人||228(附属図書館) / 2020||人博||962(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 瀬戸口 浩彰, 教授 市岡 孝朗, 教授 宮下 英明 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

How come the same species of plants can naturally occur under various conditions in different parts of the world? A plant's ability to adapt in response to a changing climate hinges on the presence of genetic variation in traits, such as flowering phenology. In this study, I examine whether flowering start varies genetically within populations and compare this variation to differences between populations. This study quantifies genetic variation in flowering time in two Italian populations of Arabidopsis thaliana whilst using two Swedish populations as a reference. This was done using a randomized block design where plants were grown in a controlled climate. To characterize and measure flowering phenology, time of bolting and flowering were recorded. The experiment included four populations, a total of 150 maternal lines, and 2980 plants. One-way ANOVAs conducted separately by population indicated significant among-line variation in the two Italian populations. Flowering time differed between the Italian and Swedish populations, but not between the two Italian populations. More data would be needed to draw conclusions about the among-line variation in the Swedish populations. The results indicate that the Italian populations have the potential to respond to selection on flowering time, which is a likely consequence of a changing climate.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19051号 / 農博第2129号 / 新制||農||1033(附属図書館) / 学位論文||H27||N4933(農学部図書室) / 32002 / 京都大学大学院農学研究科農学専攻 / (主査)教授 白岩 立彦, 教授 奥本 裕, 准教授 中﨑 鉄也 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Tobacco (<i>Nicotiana tabacum</i> L.) leaf initiation stops when floral induction is completed. Floral induction (an internal biochemical change which signals development of flowers) can occur prematurely, during the pre- and/or post-transplant environment. Plants which flower prematurely have few leaves and low yields. Removal of the influorescence on these plants will break apical dominance and allow production of additional leaves from axillary bud. This practice requires additional labor and increases production costs. Objectives of this study were to 1) determine how application timing, rate, localization, and on-plant duration of ethephon (2-chloroethylphosphonic acid) influences growth and flowering of flue-cured ‘NC 82’ tobacco; 2) examine the relationship between temperatures in the seedling environment and premature flowering and determine how time of plant bed cover removal affects premature flowering; and 3) quantify the ethylene released from buds, leaves, stems, and roots of tobacco seedlings at various days after application of ethephon. Ethephon applied to flue-cured tobacco seedlings before the completion of floral induction significantly reduced premature flowering and increased days to flower, number of leaf nodes per plant, and yield. Multiple applications and increased rates of ethephon did not increase the number of leaf nodes beyond the level obtained from a single application of 960 mg L⁻¹ ethephon solution applied at the rate of 44 mL m⁻² of plant bed. In wash-off studies, maximum benefit was obtained when ethephon remained on the seedlings one to two hours after application. In localization studies, increases in number of leaf nodes per plant were not different when 0.09 and 0.51 mg of ethephon was applied to the bud and largest leaf, respectively. Ethylene released from ethephon-treated greenhouse seedlings remained detectable four weeks after treatment. On-farm test locations with the two highest percentages of premature flowering had the lowest average daily minimum temperatures during the pretransplant period. Premature flowering was significantly increased at two of seven locations by removal of the perforated plastic plant bed covers two weeks compared to one week before transplanting. Floral induction of tobacco seedlings in controlled pretransplant environments was obtained by continuous 15°C temperature and 8 h photoperiods for 3 weeks. / Ph. D.

Plants have spread to almost everywhere in the world. As they disperse, they meet many different environments to which they may be able to adapt. For a plant species to adapt to a new environment, genetic variation is needed. The individuals differ from each other in their genetic composition, which often means differences in phenotypes. Those individuals that manage to reproduce will form the next generation. With different conditions in different environments, it will not be the same phenotypes that reproduce everywhere. In that way, plant species will form into a mosaic of locally adapted populations varying genetically as the species disperses. After the last ice age plants have started to disperse away from the equators. With increasing latitudes come increasing challenges to migrating plants. As plant species disperse northwards along this gradient of varying conditions individuals are selected for cold adaptive traits like flowering time and freezing tolerance, acquired by cold acclimation. In this way, genetic variation from the original populations for these traits becomes sorted out along a latitudinal cline. The aim of this thesis was to understand how selection along a latitudinal gradient has shaped natural variation in cold adaptive traits in plants dispersing northwards, and specifically, to investigate what variation can be observed in phenotypes for these traits and how these traits correlate with genetic variation in genes known to be involved in cold acclimation. In this study significant variation was found in a sample of the model plan Arabidopsis thaliana accessions in cold adaptive traits flowering time and freezing tolerance. A clear latitudinal cline in the cold adaptive traits freezing tolerance for A. thaliana was observed. Analysis of nucleotide polymorphism for the cold responsive ICE1 (inducer of CBF expression 1) transcription factor revealed a haplotype structure with two allelic clades as well as unusually high levels of synonymous polymorphism. Nucleotide polymorphism analysis for the transcription factors CBF1, CBF2 and CBF3 (C-repeat binding factors) that play a key role in regulating the expression of a group of target genes known as the “CBF regulon” showed a distinct geographical haplotype structure. One haplotype was dominant in southern accessions while in the other northern accessions overrepresented. There was a significant effect of CBF haplotype on both freezing tolerance and flowering time even after correcting for latitude. Significant differences in CBF expression levels were found between the different CBF genes as well as between different accessions. Sequence variation at CBF was shown to have a significant effect on expression levels of CBF2. No clear correlations were found between CBF gene expression and freezing tolerance or temperature sensitivity for any of the accessions used in the study. This highlights the complex relationship between sequence variation in candidate genes and gene expression, and the problems associated with unraveling the genetic basis of ecologically important traits.

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&#160;000 emäsparin kokoisella alueella kromosomin kaksi alkupäässä.

Abstract The timing growth and reproduction are critical to the fitness of plants and animals. The timing also has an important role in local adaptation. Locally adapted plants may have different responses to photoperiod and other environmental cues and genes or alleles behind underlying differences may differ between populations. The molecular genetics and physiology of flowering of the plant molecular biology model organism Arabidopsis thaliana is being intensively studied, and this offers a good opportunity to study the genetic basis of flowering time variation in related non-model species. The closely related perennial species Arabidopsis lyrata provides an interesting comparison to A. thaliana because of its different ecology, mating system and life history. The influence of sampling designs on clustering methods was analyzed using simulations and microsatellite data in the selfing A. thaliana. It was found that sample size has a large effect on the resulting number of clusters and sampling too few individuals per locality could lead to a severe underestimation of the real number of subpopulations. Patterns of sequence variation in flowering time genes and association between polymorphisms at FRI (and FLC) and flowering time was studied in A. thaliana and in A. lyrata to find out whether the genes were responsible for flowering time differences between and within natural populations. In A. thaliana there was no significant association between polymorphisms at FLC and FRI and flowering time. In A. lyrata the FRI gene was polymorphic for an indel associated with flowering time variation within two Northern European populations, suggesting that the indel (or a linked polymorphism) was involved in flowering time variation. However, FRI did not explain the flowering time differences between A. lyrata populations, and other loci must be involved. Patterns of diversity and divergence at flowering time related loci were compared against a set of random reference loci to examine the roles of selection and demography. Sequence variation in the studied A. lyrata populations departed from the standard neutral equilibrium model and it has been influenced by recent historical events, most likely bottlenecks. The level of silent and synonymous polymorphisms in flowering time genes was highly reduced and this can be likely explained by selective sweeps at flowering time genes. / Tiivistelmä Kasveilla kukkimisen ajoittaminen suotuisaan ajankohtaan on hyvin tärkeää suvullisen lisääntymisen kannalta. Kukkimisen oikealla ajoituksella on myös tärkeä rooli kasvien sopeutumisessa paikallisiin olosuhteisiin. Kukkimisaikamuunteluun vaikuttavat useimmiten lukuisat geenit sekä ympäristötekijät, jotka voivat vaihdella alueellisesti ja populaatioiden välillä. Vaikka kukkimiseen ja kukkimisaikaan vaikuttavia tekijöitä tunnetaan jo hyvin, luonnonpopulaatioiden muuntelun ja paikallisen sopeutumisen geneettinen tausta on huonommin tunnettu. Väitöstutkimus keskittyy Arabidopsis-populaatioiden paikalliseen sopeutumiseen tarkastelemalla kukkimisajan muuntelua ja siihen vaikuttavia geeneettisiä tekijöitä. Tutkimuksessa käytetyt geneettiset aineistot perustuvat osin neutraaleihin merkkigeeneihin (mikrosatelliittimuunteluun), ja osin sekvenssien nukleotidimuunteluun. Väitöstutkimuksessa on simulointien avulla selvitetty populaatiosta analysoitavien yksilöiden lukumäärän merkitystä populaatiorakenteen selvittämisessä itsesiittoisella lituruoholla (Arabidopsis thaliana). Tulosten mukaan on hyvä analysoida useampia yksilöitä paikallisista populaatioista, sillä liian pienet otoskoot voivat johtaa ryhmien määrän aliarvioimiseen. Koalesenssisimulaatiot osoittavat idänpitkäpalon (Arabidopsis lyrata) populaatioiden poikkeavan tasapainotilasta ja populaatioissa tapahtuneen populaatiokoon muutoksia (ns. pullonkaulailmiö). Tutkimuksessa havaittiin sekvenssimuuntelun olevan alhaisempaa kukkimisaikageeneissä kuin referenssigeeneissä todennäköisesti positiivisen valinnan vaikutuksesta. Tutkimuksessa todettiin, että FRI geenissä tapahtuneet mutaatiot ovat kahdessa tutkitussa lajissa erilaisia luonteeltaan, mutta geenillä on kuitenkin samanlainen rooli kukkimisajan määräämisessä. Assosiaatiokokeissa lituruoholla ei Pohjois-Euroopan populaatioissa löydetty merkitsevää assosiaatiota FRI geenin ja kukkimisajan välillä, kun puolestaa idänpitkäpalolla FRI vaikutti kukkimisaikamuunteluun kahdessa pohjoisessa populaatiossa.

Premise of research. The duration and intensity of flowering overlap among plants are the first determiners of the potential for pollinator-mediated plant-plant interactions. Yet, our ability to describe community-wide patterns of coflowering, and thus understand its impact on the structure of plant-pollinator communities, is limited. Methodology. We present a conceptual framework for how network theory can reveal structural properties that are ecologically relevant in diverse coflowering communities. Coflowering modules, in particular, may suggest that groups of species coflower more strongly (clustering) with each other than with other species (over-dispersion) in the community. Such a finding would indicate that competitive and facilitative interactions do not act alone but instead act simultaneously to mediate the assembly of coflowering communities. We illustrate our conceptual framework in four diverse coflowering communities in the serpentine seeps in northern California. Pivotal results. Our coflowering networks vary in size and degree but not in overall connectance, suggesting that both intrinsic community features (species richness) and ecological constraints (length of flowering season) play a role in mediating coflowering community structure (distribution of frequency and intensity of flowering overlap among plant species). We show, for the first time, that groups of species tend to coflower more strongly with each other than with other species in a community, supporting the idea that competition and facilitation are not mutually exclusive processes mediating coflowering community assembly. Our results show that the degree of modularity is not sensitive to the number of coflowering species within each community, suggesting that ecological factors may be more important in driving this pattern. Conclusions. Coflowering networks have the potential to advance our understanding of the causes and consequences of flowering overlap in diverse plant communities by revealing a more in-depth and novel characterization of coflowering community structure. Such characterization will allow for a better understanding of the importance of coflowering patterns in mediating the structure of plant-pollinator interactions.

The evolutionary relevance of homeotic alterations for the origin of new taxonomic entities is still a controversial objective in plant sciences. In this context, the discovery of a floral homeotic variant of Capsella bursa-pastoris in natural populations offers the unique opportunity to elucidate the evolutionary significance of homeotic mutants in the wild. Since all petals are transformed into additional stamens, the variant was termed Stamenoid petals (Spe). In this thesis, a combination of ecological and molecular characterisation of the variant was performed, to improve the understanding of evolutionary processes in plant populations. Molecular markers were used to analyze genetic differentiation among known provenances and also within a large sympatric population of wild-type and homeotic mutant. The results clearly suggest a repeated evolution of the novel flower morphology. Furthermore, genetic analyses provided substantial evidence, that the two floral variants are well-defined into flower-type dependent sub-samples within one population. The evaluation of phenotypic traits elucidated that the homeotic variant is not hampered in fitness. In greenhouse and field experiments, a significant ecological differentiation in the onset of flowering was detected among variants. Finally, the novel floral phenotype shows a co-dominant inheritance, and a marker-assisted mapping approach exposed a single locus in a genetic map. In conclusion, the comprehensive study of ecological and molecular aspects indicates that the floral homeotic variant may be treated as an established taxonomic entity and proved the predicted role as model for evolutionary objectives. Since morphological alterations like Spe are discussed as a result of macroevolution, the homeotic variant of C. bursa-pastoris provides the opportunity to survey a (macro)evolutionary novelty in association with continuous micro-evolutionary adaptation

L'évolution climatique a été marquée ces dernières décennies par des changements importants, notamment une augmentation de température et une variation de la pluviométrie. Une des conséquences du changement climatique est son impact actuel et futur sur l'agriculture et la sécurité alimentaire. En région sahélienne, la sécurité alimentaire repose essentiellement sur quelques céréales, parmi lesquelles le mil occupe une place fondamentale. Un des traits qui permettent l'adaptation au climat de cette espèce est la date de floraison, dont la variation permet d'accomplir le cycle de vie de la plante dans des saisons pluvieuses plus ou moins courtes. Les bases génétiques de ce caractère adaptatif sont encore peu connues. Nous avons développé une méthodologie de cartographie d'association phénotype-génotype, afin d'identifier des gènes impliqués dans la variation de ce caractère. Cette étude a permis d'identifier deux gènes candidats, PHYC et MADS11, associés à la date de floraison et à la variation morphologique chez le mil. Nous avons validé ces associations par des analyses QTLs. Pour PHYC, nous avons aussi étudié le pattern de déséquilibre de liaison sur une zone d'environ 80 kb autour du gène, et développé une approche de Markov Chain Monte Carlo (MCMC) pour comparer les gènes identifiés dans la région. Cette analyse suggère que les polymorphismes à l'intérieur de PHYC sont les meilleurs candidats expliquant l'effet phénotypique observée dans cette région du génome. La seconde partie de ce projet a été consacrée à l'examen méthodologique de la cartographie d'association pour l'étude des interactions entre le génotype et l'environnement. Les résultats de cette thèse ont été discutés notamment dans la perspective de gestion de l'impact du changement climatique sur le mil, céréale majeure des zones semi-arides / In last decades, climate changes led to temperature increase and rainfall variation across the globe. One of the key consequences of these changes is their impact on agriculture and food security. In sahelian countries, food security relies on a few cereal crops, among which pearl millet plays a crucial role for population food supply. Sahel region is facing the impact of rainfall variability and drought since the 1970s. Flowering time variation is one of the main adaptations that allow pearl millet cultivation in drier and shorter rainy seasons. The genetic bases of this complex trait are still understudied. We developed an association mapping framework for the analysis of genotype-phenotype relationship in pearl millet. We successfully identified two genes associated with flowering time variation in pearl millet (PHYC and MADS11). We confirmed these associations using QTL studies. For PHYC, we also examined the pattern of linkage disequilibri um on a chromosomal region extending to 80 kb around the gene, and we developed a Markov Chain Monte Carlo approach (MCMC) to compare six genes identified in this region. Our results suggest that, among the polymorphisms observed in this region, polymorphisms in PHYC are the best candidate for a direct causative role. The second part of this project addressed methodological examination of association mapping framework to deal with genotype by environment interactions. The results of this work were discussed with regard to the challenge of pearl millet crop adaptation to climate change.