The aim of this study was to gain insight into the nature of resistance genes and mechanisms of resistance present in different ascochyta blight (AB) resistant genotypes of lentil to efficiently select non-allelic AB resistance genes mediating different mechanisms of resistance for gene pyramiding. Recombinant inbred lines (RILs) from all possible crosses among AB resistant Lens culinaris genotypes CDC Robin, 964a-46, ILL 7537 and ILL 1704 were subjected to allelism tests. Efforts were also made to understand the genetics of resistance in the L. ervoides accession L-01-827A. LR-18, a RIL population from the cross CDC Robin × 964a-46 was subjected to quantitative trait loci (QTL) mapping using a comprehensive genetic linkage map previously developed from polymorphic SNPs, SSRs and phenotypic markers. Results of allelism tests suggested that genes conditioning resistance to ascochyta blight in all lentil genotypes were non-allelic. Two complementary recessive resistance genes in L-01-827A were detected. QTL analysis indicated that CDC Robin and 964a-46 were different at two AB resistance QTLs. Histological tests suggested that cell death inhibition in CDC Robin, and reduced colonization of epidermal cells in 964a-46 might be the mechanisms of resistance in these genotypes. Comparing the expression of key genes in the salicylic acid (SA) and jasmonic acid (JA) signaling pathways of CDC Robin and 964a-46 suggested that the SA pathway was strongly triggered in 964a-46. However, the JA pathway was triggered in both, but at a lower expression level in 964a-46 than in CDC Robin. RNA-seq analysis revealed a number of candidate defense genes differentially expressed among genotypes with hypothetical actions in different layers of the plant defense machinery. The expression levels of the six candidate defense genes measured by quantitative real-time PCR analysis was correlated with those of RNA-seq. In conclusion, 964a-46 and CDC Robin mediated resistance to ascochyta blight through different resistant mechanisms, making them ideal candidates for resistance gene pyramiding. Gene pyramiding can be accelerated using closely linked markers to CDC Robin and 964a-46 resistance genes identified through QTL analysis.

Submitted by VITO ANTONIO MASTROCHIRICO FILHO null (vito.oceano@gmail.com) on 2016-04-09T21:47:55Z No. of bitstreams: 1 Dissertação Repositório Vito.pdf: 3114594 bytes, checksum: fe4a528d166d3a59bfac7d3a244a016c (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-04-11T13:38:46Z (GMT) No. of bitstreams: 1 mastrochiricofilho_va_me_jabo.pdf: 3114594 bytes, checksum: fe4a528d166d3a59bfac7d3a244a016c (MD5) / Made available in DSpace on 2016-04-11T13:38:46Z (GMT). No. of bitstreams: 1 mastrochiricofilho_va_me_jabo.pdf: 3114594 bytes, checksum: fe4a528d166d3a59bfac7d3a244a016c (MD5) Previous issue date: 2016-02-29 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Pacu (Piaractus mesopotamicus) é uma espécie de peixe Neotropical amplamente distribuída nas bacias dos rios Paraná e Paraguai, e uma das espécies de peixe neotropicais de maior valor para a aquicultura. Uma melhor compreensão do genoma do pacu é necessária para o manejo genético na conservação dos estoques naturais e cultivados. O principal objetivo foi identificar SNPs (Single Nucleotide Polymorphisms) gene-associados no transcriptoma de fígado do pacu e, em seguida, aplicar em análises de variabilidade genética e de parentesco visando um manejo adequado desta importante espécie não modelo na aquicultura. O sequenciamento do transcriptoma foi realizado por meio da plataforma Roche/454, que resultou na formação de 4.110 contigs não redundantes. Destes, 2.051 genes foram identificados e funcionalmente anotados a fim de revelar genes relacionados às características econômicas interessantes para a aquicultura. Foram encontrados 464 SNPs localizados em 5’UTR (10.0%), 3’UTR (17.2%) e em regiões CDS (71,1%), e classificados como sinônimos (70,6%) e não sinônimos (29,4%). Foram genotipados 32 SNPs por meio da técnica Sequenom MassARRAY, dos quais alguns estavam relacionados com sistema imune. A variabilidade genética foi estimada em populações de indivíduos selvagens (Rio Paraná) e de indivíduos cultivados em sete pisciculturas do estado de São Paulo (FF1, FF2, FF3, FF4, FF5, FF6 e FF7). Não foram observadas diferenças significativas entre heterozigosidade observada (Hobs) e esperada (Hexp) para cada população. Análises de diferenciação genética mostraram baixo nível de estruturação genética entre as populações (Fst = 0.064, AMOVA = 93,59% da variação dentro de populações, P<0,05). Análises de parentesco mostraram que a maioria das estações de piscicultura possuíam pelo menos 40% de indivíduos aparentados, com risco de endogamia e necessidade de realização de um programa de acasalamentos direcionados. Nossos resultados proporcionaram importantes recursos genéticos para o pacu, com aplicabilidade para a aquicultura. / Pacu (Piaractus mesopotamicus) is a Neotropical freshwater fish widely distributed in Parana, Paraguay Basin. Wild populations of pacu are threatened by overfishing and it is one of the fish species of highest commercial value for aquaculture. An understanding of the pacu genome is appropriate to genetic management in the conservation of wild and cultivated stocks. The main objective was identify gene-associated SNPs in liver transcriptome of pacu. We used SNPs (Single Nucleotide Polymorphisms) to perform genetic variability and kinship analysis for suitable management of this important non-model species in aquaculture. Transcriptome sequencing was done with the Roche/454 technology and yielded 4,110 non-redundant contigs. Of these, 2,051 genes were identified and functionally annotated to reveal genes correlated to economical traits in aquaculture. We found 464 SNPs in 5’UTR (10.0%), 3’UTR (17.2%) and CDS (71,1%), classified in synonymous (70,6%) and non-synonymous (29,4%). We genotyped 32 feasible SNPs through Sequenom MassARRAY platform and we obtained some SNPs related to immune system. Genetic diversity was estimated in wild individuals (Parana river) and in seven farm fish populations (FF1, FF2, FF3, FF4, FF5, FF6 and FF7). There were no significant differences between observed heterozygosity (Hobs) and expected (Hexp) for each population; and also between observed heterozygosity, expected heterozygosity and minimum allele frequency (MAF), when the population averages were compared (P <0.05). In addition, genetic differentiation analyzes showed low genetic structure of wild and cultivated populations of pacu (Fst = 0.064; AMOVA = 93.59% of the variation within populations; P<0,05). Kinship analysis showed most hatchery stations had at least 40% of related individuals, at risk of inbreeding and the need to perform a directed mating program. Our results showed unprecedented genomic resources for pacu. / FAPESP: 2014/12412-4

Chez tout organisme, l’évolution et l’acclimatation aux changements du milieu de vie sont orchestrés par de nombreux acteurs moléculaires. Parmi eux, les facteurs de transcription (FTs) jouent un rôle clé en régulant l’expression des gènes. Identifier les FTs impliqués dans la production de composés d’intérêt est donc une étape importante dans un contexte biotechnologique. Le laboratoire dispose d’une souche mutante de la microalgue haptophyte Tisochrysis lutea produisant deux fois plus de lipides de réserve que la souche sauvage en condition de privation azotée. Compte tenu du rôle clé des FTs dans l’établissement du phénotype, cette thèse vise à identifier les FTs impliqués dans la mise en place de ce phénotype mutant.Un pipeline bio-informatique d’identification et classification des FTs présents dans le génome de T. lutea a été élaboré. Le manque de donnée chez les haptophytes constituant un vide dans l’étude de l’histoire évolutive des microalgues, une étude comparative des FTs présents dans le génome d’algues de différentes lignées a été réalisée. Celle-ci révèle que l’étude des FTs aide à comprendre et illustrer l’histoire évolutive des microalgues par la mise en évidence de présences/absences de familles de FTs spécifiques de lignée.Afin de comprendre l’établissement du phénotype de la souche mutante de T. lutea, des données transcriptomiques ont permis la construction de réseaux de co-expression et de régulation des gènes chez les deux souches. Leur analyse croisée a identifié sept FTs candidats potentiellement liés au phénotype mutant. Une approche de p-RT-PCR a confirmé l’implication de deux FTs dans la remobilisation de l’'azote en condition de stress azoté. / In every organism, evolution and acclimation to environmental changes are orchestrated by numerous molecular players. Among them, transcription factors (TFs) play a crucial role by regulating gene expression. Therefore, identify TFs involved in the production of high value products is a significant step in a biotechnological context. The laboratory has at its disposal a mutant strain of the haptophyte microalga Tisochrysis lutea producing twice more storage lipids than the wild type strain when exposed to nitrogen deprivation. Given the key role of TFs in phenotype establishment, this PhD aim at identify the TFs involved in that of the mutant phenotype of T. lutea.A TFs identification and classification pipeline was elaborated and applied to T. lutea’s genome. Since the lack of data in haptophytes constitutes a limit in studies on microalgae evolutionary history, a comparative study of TFs identified in the genome of microalgae belonging to different lineages was carried out. This study reveals that TFs could be used to understand and illustrate microalgae evolutionary history through the highlight of lineage specific presence/absence of TF families.Aiming at understanding T. lutea’s mutant strain phenotype establishment, transcriptomic data were used to build gene co-expression networks and gene regulatory networks for both strains. Their comparative analysis identified seven TFs potentially liked to the mutant phenotype. A q-RT-PCR approach confirmed the involvement of two TFs in nitrogen recycling under nitrogen deprivation.

Orientador: Ney Lemke / Resumo: MicroRNAs (miRNAs) são pequenas moléculas de RNA que regulam pós-transcricionalmente a expressão de genes, modelando o transcriptoma e a produção de proteínas. Em geral, os miRNAs são conservados no genoma de eucariotos, sendo considerados elementos vitais em diversos processos biológicos durante o desenvolvimento, tais como crescimento, diferenciação e morte celular. A grande diversidade de miRNAs identificados está restrita a poucas espécies e apenas uma parte do total de alvos de miRNAs preditos foi caracterizada funcionalmente. Nesse contexto, o uso da tecnologia de sequenciamento de alto rendimento (high throughput sequencing) atrelada à análise de nível transcricional por RT-qPCR possibilitam a identificação do microRNoma. A tilápia do Nilo, Oreochromis niloticus, é considerada um excelente modelo biológico para o estudo de miRNAs em vertebrados devido à sua importância econômica e evolutiva. O presente trabalho teve como objetivos: organizar os dados do sequenciamento dos miRNAs da tilapia do Nilo; disponibilizá-los em forma de uma base de dados para a comunidade científica; integrar as informações dos miRNAs identificados com outros bancos de dados de miRNAs; analisar os dados através de análises de bioinformática para determinação de agrupamentos definidos pelo nível de expressão de cada miRNA em seis tipos de tecido (músculo branco, músculo vermelho, testículo, ovário, fígado, olho, cérebro e coração) com distinção entre os gêneros e nas fases do desenvolvimento (2,... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

abstract: Induced pluripotent stem cells (iPSCs) are an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. We describe the establishment of primary dermal fibroblasts cells lines from 28 autopsy donors. These fibroblasts were used to examine the proliferative effects of establishment protocol, tissue amount, biopsy site, and donor age. As proof-of-principle, iPSCs were generated from fibroblasts from a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. To our knowledge, this is the first study describing autopsy donor-derived somatic cells being used for iPSC generation and subsequent neural differentiation. This unique approach also enables us to compare iPSC-derived cell cultures to endogenous tissues from the same donor. We utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, supported by (i) a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain, (ii) an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue, and (iii) a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. These studies support the utility of autopsy donors' somatic cells for iPSC-based neurological disease models, and provide evidence that in vitro neural differentiation can result in physiologically progression. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2013

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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) analise_transcriptoma_arroz_estresse_frio.pdf: 1983607 bytes, checksum: 89744effdc48619cef6643fb7d1e353c (MD5) Previous issue date: 2014-03-06 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / O estresse por frio pode trazer prejuízos econômicos significativos em diversas fases do ciclo da cultura do arroz. Em arroz (Oryza sativa L.), há grande variabilidade genética para tolerância ao frio sendo a subespécie japonica considerada tolerante e a subespécie indica considerada sensível. Foi realizado sequenciamento de RNA (RNA-Seq) visando identificar genes diferencialmente expressos entre os genótipos BRS Atalanta (indica) (A), Nipponbare (japonica) (N) e bulks formados por RILs (Linhas endogâmicas recombinantes) provenientes do cruzamento entre elas. Bulks (B) com as 15 RILs mais próximas a cada genitor (BA e BN) foram formados com base em dados de genotipagem. As plântulas, tanto da condição tratamento quanto da condição controle, permaneceram por 7d a 28°C. Para análise do transcritoma, as plântulas da condição tratamento (T) foram expostas ao frio numa temperatura de 13°C por 24h e as plantas da condição controle (C) permaneceram mais 24h a 28°C. Após este período foi realizada a coleta das plântulas, extração de RNA total, síntese de cDNA e preparo das bibliotecas para sequenciamento (RNA-Seq). Este foi realizado em sequenciador HiSeq 2000, sendo as reads de 100b single-end. Diversas combinações foram analisadas para obter o número de genes diferencialmente expressos envolvendo as condições BRS Atalanta (controle e tratamento), Nipponbare (controle e tratamento), bulk de RILs mais próximos a cultivar BRS Atalanta (controle e tratamento) e bulk de RILs mais próximos ao genótipo Nipponbare (controle e tratamento). Um número significativo de genes mostra-se diferencialmente expressos em cada condição, sendo de modo geral mais genes estão subexpressos do que superexpressos, exceto para as condições AC x BAC, AT x BAT e NT x BNT. Um dendrograma usando as distâncias de Jensen-Shannon mostra a formação de dois ramos sendo que num deles está a cultivar BRS Atalanta e os bulks formados por sua semelhança genotípica com esta cultivar. No outro ramo estão o genótipo Nipponbare e os bulks formados pela semelhança com o genitor. Genes pertencentes a diversas famílias de fatores de transcrição estão diferencialmente expressos nas diferentes combinações, sendo que para grande parte destas famílias já foi demonstrada sua participação na resposta ao estresse por frio. As conclusões deste trabalho indicam que o número de genes diferencialmente expressos (log2-fold-change ≥ |1.5|) varia grandemente entre as combinações, apresentando como limite superior 1481 genes subexpressos na combinação BNC x BAC e 1017 genes superexpressos em NT x AT, e seis genes subexpressos na combinação NT x BNT e cinco genes superexpressos na combinação NC x BNC. As combinações analisadas mostram a expressão diferencial de um grande número de famílias de fatores de transcrição, muitas delas já descritas como responsivas ao estresse por frio, que apresentam um padrão difuso de expressão entre as subespécies indica e japonica. / Chilling stress can cause significant economic losses in different phases of the cycle of rice. There are a wide of genetic variability for cold tolerance in rice (Oryza sativa L.), japonica subspecies is considered tolerant and subspecies indica is considered sensitive. RNA sequencing (RNA-Seq) was performed to identify differentially expressed genes among the cultivar BRS Atalanta (indica) (A), Nipponbare (japonica) (N) and bulks composed by RILs from crosses between them. Bulks (B) with the 15 closest RILs to each parent (BA and BN) RILs were composed based on SNPs-genotyping data. To analyze the transcriptome, plants of the treatment condition (T), 7d after imbibition were exposed to a chilling temperature of 13°C per 24h and plants of the control condition (C) remained 24h more at 28°C. After this time was performed the collection of seedlings, total RNA extraction, cDNA synthesis and preparation of libraries for sequencing (RNA-Seq) in a HiSeq 2000 sequencer, with 100bp single-end reads. Several combinations were analyzed to obtain the number of differentially expressed genes involving BRS Atalanta conditions (control and treatment), Nipponbare (control and treatment), bulk of RILs closest to BRS Atalanta – BA (control and treatment) and bulks of RILs closest to the cultivar Nipponbare - BN (control and treatment). A significant number of differentially expressed genes are shown in each condition, being generally founded more downregulated than up-regulated genes, except for the combinations AC x AT x NT x BAT and BAC x BNT. A dendrogram using the Jensen-Shannon distance shows the formation of two branches of which one is composed by BRS Atlanta cultivar and the bulks composed by the genotypic similarity with this cultivar. In the other branch is the cultivar Nipponbare and the bulks composed by similarity with the parent. Several differentially expressed genes from transcription factors families are present in different combinations, and for many of these families has been demonstrated its involvement in the response to chilling stress. The conclusions of this study indicate that the number of differentially expressed genes (log2 fold-change ≥ |1.5|) greatly changed between combinations, with an upper limit of 1481 down-regulated genes in the combination BAC x BNC and 1017 up-regulated genes in the combination NT x AT, six genes are up-regulate in the combination NT x BNT and five genes are upregulated in NC x BNC combination. The combinations analyzed showed differential expression of a large number of families of transcription factors, many of which have been described as responsive to cold stress, showing a diffuse pattern of expression between indica and japonica groups.

Les ARN sont des molécules aux propriétés diverses interagissant les uns avec les autres dans le but de médier des fonctions spécifiques. L’évaluation de l’abondance relative des ARN est une étape cruciale à la compréhension de la stoechiométrie nécessaire à ces fonctions. Cependant, plusieurs biais et limitations s’imposent avec l’utilisation de différentes techniques d’évaluation, dont le séquençage d’ARN (RNA-Seq), qui sont problématiques dans l’estimation de l’abondance de différents types d’ARN. De récentes études ont exposé les avantages d’un protocole novateur de RNA-Seq qui utilise une rétrotranscriptase thermostable d’intron de groupe II (TGIRT) d’origine bactérienne afin de réduire ces biais. Ce mémoire fait la comparaison entre différentes techniques de RNA-Seq afin d’élucider si l’utilisation de TGIRT en RNA-Seq offre une représentation plus juste du transcriptome. Les comparaisons avec les valeurs d’abondance de différents types d’ARN décrites dans la littérature ainsi qu’obtenues expérimentalement par notre groupe pointent au fait que TGIRT donne une meilleure estimation de l’abondance relative des ARN, et plus particulièrement des ARN hautement structurés. Cette meilleure estimation de l’ensemble de la composition du transcriptome permet de faire des observations sur les rapports d’abondance entre des ARN codants et non-codants fonctionnellement apparentés. Notamment, des ratios d’expression constants entre les ARN non-codants associés à des RNP et les ARNm codants pour leur facteurs protéiques ont été observés. Ceci suggère la présence d’une régulation transcriptionnelle commune nécessaire à la stoechiométrie de ces complexes. Une forte disparité dans l’expression des snoRNA et de leurs gènes hôtes, dépendant du type de snoRNA et de gène hôte a par ailleurs été constatée, corroborant une régulation distincte de la stabilité de ces transcrits. Dans l’ensemble, nos données suggèrent que la méthode TGIRT-Seq est la plus appropriée dans l’évaluation du transcriptome entier et ouvre donc la voie à des analyses plus holistiques par RNA-Seq en donnant une estimation plus juste de l’abondance relative des transcrits d’ARN. / Abstract : RNA are molecules with a wide range of properties that can interact with one another to mediate specific function. The evaluation of RNA abundance is a crucial step in understanding the stoichiometry needed for these functions. However, many limitations and biases come with the use of different techniques, including RNA sequencing (RNA-Seq), which affects the estimation of the abundance of different RNA types. Recent studies have exposed the advantages of a new RNA-Seq protocol using a thermostable group II intron reverse transcriptase (TGIRT) of bacterial origin to reduce these biases. This thesis makes the comparison between different RNA-Seq techniques to elucidate if the use of TGIRT in RNA-Seq offers a more representative depiction of the transcriptome. The comparisons with the abundance values given in literature and obtained experimentally by our group agree with the fact that TGIRT gives a better estimation of the relative abundance of RNA, especially highly structured RNA. This better estimation of the transcriptomic landscape allows many observations on the abundance relations between coding and non-coding RNA that are functionally related. Namely, constant expression ratios between RNP associated noncoding RNA and the mRNA that codes for their associated proteins have been observed. This suggests the presence of a common transcriptional regulation which is necessary for the stoichiometry of these complexes. A strong disparity in the expression of snoRNA and their host genes depending on snoRNA and host gene types has also been observed and corroborate a distinct regulation of these transcripts’ stability. In summary, our data suggest that the TGIRT-Seq method is the most appropriate to evaluate the transcriptome and thus opens the way to more holistic RNA-Seq analyses by giving a better estimation of RNA transcripts relative abundance.

Background: Transcriptome analytic tools are commonly used across patient cohorts to develop drugs and predict clinical outcomes. However, as precision medicine pursues more accurate and individualized treatment decisions, these methods are not designed to address single-patient transcriptome analyses. We previously developed and validated the N-of-1-pathways framework using two methods, Wilcoxon and Mahalanobis Distance (MD), for personal transcriptome analysis derived from a pair of samples of a single patient. Although, both methods uncover concordantly dysregulated pathways, they are not designed to detect dysregulated pathways with up- and down-regulated genes (bidirectional dysregulation) that are ubiquitous in biological systems. Results: We developed N-of-1-pathways MixEnrich, a mixture model followed by a gene set enrichment test, to uncover bidirectional and concordantly dysregulated pathways one patient at a time. We assess its accuracy in a comprehensive simulation study and in a RNA-Seq data analysis of head and neck squamous cell carcinomas (HNSCCs). In presence of bidirectionally dysregulated genes in the pathway or in presence of high background noise, MixEnrich substantially outperforms previous single-subject transcriptome analysis methods, both in the simulation study and the HNSCCs data analysis (ROC Curves; higher true positive rates; lower false positive rates). Bidirectional and concordant dysregulated pathways uncovered by MixEnrich in each patient largely overlapped with the quasi-gold standard compared to other single-subject and cohort-based transcriptome analyses. Conclusion: The greater performance of MixEnrich presents an advantage over previous methods to meet the promise of providing accurate personal transcriptome analysis to support precision medicine at point of care.

Observations of floral development indicate that floral organ initiation in pentapetalous flowers more commonly results in a medially positioned abaxial petal (MAB) than in a medially positioned adaxial petal (MAD), where the medial plane is defined by the stem and the bract during early floral development. It was proposed that the dominant MAB petal initiation might impose a developmental constraint that leads to the evolution of limited patterns of floral zygomorphy in Asteridae, a family in which the floral zygomorphy develops along the medial plane and results in a central ventral (CV) petal in mature flowers. Here, I investigate whether the pattern of floral organ initiation may limit patterns of floral zygomorphy to evolve in pentapetalous angiosperms. I analyzed floral diagrams representing 405 species in 330 genera of pentapetalous angiosperms to reconstruct the evolution of floral organ initiation and the evolution of developmental processes that give rise to floral zygomorphy on a phylogenetic framework. Results indicate that MAB petal initiation is the most common; it occupies 86.2% of diversity and represents the ancestral state of floral organ initiation in pentapetalous angiosperms. The MAD petal initiation evolved 28 times independently from the ancestral MAB petal initiation. Among the 34 independent originations of floral zygomorphy, 76.5% of these clades represent MAB petal initiation, among which only 47% of the clades result a CV petal in mature flowers. The discrepancy is explained by the existence of developmental processes that result in floral zygomorphy along oblique planes of floral symmetry in addition to along the medial plane. Findings suggest that although the early floral organ initiation plays a constraining role to the evolution of patterns of floral zygomorphy, the constraint diverges along phylogenetically distantly related groups that allow the independent originations of floral zygomorphy through distinct development processes in pentapetalous angiosperms. In additional study, the butterfly-like flowers of Schizanthus are adapted to pollination by bees, hummingbirds, and moths. I investigated the genetic basis of the zygomorphic corolla, for which development is key to the explosive pollen release mechanism found in the species of Schizanthus adapted to bee pollinators. I examined differential gene expression profiles across the zygomorphic corolla of Schizanthus pinnatus, a bee-pollinated species, by analyzing RNA transcriptome sequencing (RNA- seq). Data indicated that CYC2 is not expressed in the zygomorphic corolla of Sc. pinnatus, suggesting CYC2 is not involved in the development of floral zygomorphy in Schizanthus (Solanaceae). The data also indicated that a number of genes are differentially expressed across the corolla.

Philosophiae Doctor - PhD / The growth and development of apple (Malus x domestica Borkh.) fruit occurs over a period of about 150 days after anthesis to full ripeness. During this period morphological and physiological changes occur defining fruit quality. These changes are a result of spatial and temporal patterns of gene expression during fruit development as regulated by environmental, genetic and environmental-by-genetic factors. A number of previous studies partially characterised the transcriptomes of apple leaf, fruit pulp, whole fruit, and peel plus pulp tissues, using cDNA micro arrays and other PCR based technologies. These studies, however, remain limited in throughput and specificity for transcripts of low abundance. Hence, the aim of this project was to apply a high throughput technique to characterise the full mRNA transcriptome of the ‘Golden Delicious’ fruit peels and pulp tissues in order to understand the molecular mechanisms underlying the morphophysiological changes that occur during fruit development.