Mapping of quantitative trait loci for thermosensitive genic male sterility in indica rice

The objective of this work was to select and use microsatellite markers, to map genomic regions associated with the genetic control of thermosensitive genic male sterility (TGMS) in rice. An F2 population, derived from the cross between fertile and TGMS indica lines, was used to construct a microsatellite-based genetic map of rice. The TGMS phenotype showed a continuous variation in the segregant population. A low level of segregation distortion was detected in the F2 (14.65%), whose cause was found to be zygotic selection. There was no evidence suggesting a cause-effect relationship between zygotic selection and the control of TGMS in this cross. A linkage map comprising 1,213.3 cM was constructed based on the segregation data of the F2 population. Ninety-five out of 116 microsatellite polymorphic markers were assembled into 11 linkage groups, with an average of 12.77 cM between two adjacent marker loci. The phenotypic and genotypic data allowed for the identification of three new quantitative trait loci (QTL) for thermosensitive genic male sterility in indica rice. Two of the QTL were mapped on chromosomes that, so far, have not been associated with the genetic control of the TGMS trait (chromosomes 1 and 12). The third QTL was mapped on chromosome 7, where a TGMS locus (tms2) has recently been mapped. Allelic tests will have to be developed, in order to clarify if the two regions are the same or not.

QTL mapping for protein content in soybean cultivated in two tropical environments

The objectives of this study were to detect quantitative trait loci (QTL) for protein content in soybean grown in two distinct tropical environments and to build a genetic map for protein content. One hundred eighteen soybean recombinant inbred lines (RIL), obtained from a cross between cultivars BARC 8 and Garimpo, were used. The RIL were cultivated in two distinct Brazilian tropical environments: Cascavel county, in Paraná, and Viçosa county, in Minas Gerais (24º57'S, 53º27'W and 20º45'S, 42º52'W, respectively). Sixty-six SSR primer pairs and 65 RAPD primers were polymorphic and segregated at a 1:1 proportion. Thirty poorly saturated linkage groups were obtained, with 90 markers and 41 nonlinked markers. For the lines cultivated in Cascavel, three QTL were mapped in C2, E and N linkage groups, which explained 14.37, 10.31 and 7.34% of the phenotypic variation of protein content, respectively. For the lines cultivated in Viçosa, two QTL were mapped in linkage groups G and #1, which explained 9.51 and 7.34% of the phenotypic variation of protein content. Based on the mean of the two environments, two QTL were identified: one in the linkage group E (9.90%) and other in the group L (7.11%). In order for future studies to consistently detect QTL effects of different environments, genotypes with greater stability should be used.

Study of linkage between miniature and singed genes in Drosophila melanogaster

We have developed a practical exercise for undergraduate students whose main aim is to identify, using genetic crosses, a pair of D. melanogaster mutations (miniature and singed). Each student receives a vial with the problem strain containing two unknown mutations. The first step is to observe and describe both mutations. Then, the students carry out genetic crosses between mutant and normal strains: (P) ♀ mutant strain × ♂ normal strain (P) ♀ normal strain × ♂ mutant strain A different offspring is expected in these crosses: in the first one we will obtain normal females and m sn males, whereas in the second all individuals will present normal phenotype. It is possible to deduce that both are sex linked mutations. With this information and to simplify the amount of work, only F1 individuals from the first cross will be used (m+sn+ / m sn × m sn / Y chrom.) to obtain the F2 generation. By counting the number of miniature (recombinant type), singed (recombinant type), miniature-singed (parental type) and normal (parental type) flies it is possible to estimate the recombination frequency between both genes. Knowing the phenotype, their chromosomal location (X chromosome) and the genetic distance between both mutations, it is possible to identify them by finding all this information in a Drosophila melanogaster genetic map. Additionally, a statistical analysis can be carried out to compare the number of expected F2 individuals with those observed in the experiment. As the distance between both genes is 15.1 m.u., then the expected percentages for each phenotype would be: normal (42.45%), miniature-signed (42.45%), miniature (7.55%) and singed (7.55%). Multiplying the frequency of each class by the total number of individuals obtained in the F2 it is possible to estimate the expected number of flies for each class. Finally, a χ2 test can be computed to ascertain whether there are significant differences between expected and observed number of individuals.

Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea

When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s). Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s). Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives). Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador) at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc). This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

Mapping quantitative trait loci for flag leave senescence as a yield determinant in winter wheat under optimal and drought stressed environments

The timing of flag leaf senescence (FLS) is an important determinant of yield under stress and optimal environments. A doubled haploid population derived from crossing the photo period-sensitive variety Beaver,with the photo period-insensitive variety Soissons, varied significantly for this trait, measured as the percent green flag leaf area remaining at 14 days and 35 days after anthesis. This trait also showed a significantly positive correlation with yield under variable environmental regimes. QTL analysis based on a genetic map derived from 48 doubled haploid lines using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, revealed the genetic control of this trait. The coincidence of QTL for senescence on chromosomes 2B and 2D under drought-stressed and optimal environments, respectively, indicate a complex genetic mechanism of this trait involving the re-mobilisation of resources from the source to the sink during senescence.

QTL identification for tolerance to fruit set in tomato by FAFLP markers

This study aimed to detect quantitative trait loci (QTL) by fALP (Fluorescent Amplified Fragment Length Polymorphism) markers associated to the trait tomato fruit set at high temperatures. A biparental cross between line Jab-95 (heat-tolerant) and cultivar Caribe (heat-susceptible) was made. A total of 192 plants of the F2 generation were evaluated, generating 172 polymorphic markers through six primer combinations previously identified by the Bulked Segregant Analysis technique. To construct the genetic map, 106 of the 172 markers that segregated in the expected Mendelian segregation proportion (3:1) were used. The map covered 1191.46 cM of the genome. Six trait-linked QTL were identified in the analysis of simple markers and three others by the interval-mapping methodology. These results could be highly useful in improvement programs, since heat-tolerant plants can be selected rapidly, which improves tomato fruit set.

Identificação de regiões relacionadas ao déficit hídrico em Eucalyptus por meio de marcadores moleculares

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mapeamento genético e identificação de marcadores aflp, microssatélites genômicos e funcionais associados a parâmetros agroindustriais em cana-de-açúcar

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identificação de marcadores microssatélites genômicos e funcionais associados a parâmetros agroindustriais em cana-de-açúcar

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A construção de conceitos relacionados com os movimentos Terra-Lua-Sol por alunos da E.J.A. à luz da teoria histórico-cultural

A construção de conceitos científicos no âmbito escolar ainda precisa ser melhor compreendida. No caso de conceitos relacionados à astronomia, as pesquisas ainda são escassas no Brasil, principalmente quando se trata da Educação de Jovens e Adultos (EJA). O presente estudo objetivou compreender, à luz da Teoria Histórico-Cultural, as formas através das quais estudantes da EJA constroem conceitos relacionados com os movimentos do sistema Terra-Lua-Sol, em suas interações com o professor e os colegas em sala de aula. A pesquisa foi realizada em uma turma do ensino médio da EJA da Escola de Aplicação da Universidade Federal do Pará. A turma era formada por 19 estudantes, com idades variando entre 16 e 37 anos. A coleta de informações foi feita durante um semestre letivo, inicialmente através de questionários. Eles continham perguntas abertas sobre a temática, a fim de investigar as concepções prévias dos estudantes. Posteriormente, as aulas em que o assunto foi ensinado foram gravadas em áudio e vídeo. Nestas aulas os alunos elaboraram individualmente e coletivamente explicações para a sucessão dia-noite na terra. Os grupos foram formados espontaneamente pelos alunos e, em seguida, foram recombinados pelo professor. Os registros foram transcritos e analisados microgeneticamente. As respostas dos estudantes ao questionamento inicial que tratava sobre a sucessão do dia e da noite na Terra foram categorizadas em quatro níveis A, B, C e D desde o mais afastado até o mais próximo do conceito escolar cientificamente aceito. Os resultados obtidos mostraram que 13 estudantes melhoram o perfil conceitual, pois migraram dos níveis A, B ou C para o nível D da categoria de respostas, ou seja, estes estudantes entendiam que a sucessão dianoite era decorrente do movimento de rotação da terra. Os outros seis estudantes, que já se encontravam no nível D, permaneceram nesse nível, porém melhoraram suas explicações em relação as suas respostas iniciais. Foram selecionados três estudantes para a análise microgenética dos percursos da construção de seus conhecimentos. Eles tinham suas respostas escritas iniciais classificadas nos níveis A ou B e durante suas interações com o professor e com os colegas incorporaram elementos do discurso científico, conseguindo elaborar explicações teóricas para o fenômeno observado. Os resultados ilustram diferentes mecanismos de ajuste da ajuda educacional oferecida pelo professor e pelos colegas, que salientam a importância de uma abordagem dialógica e do trabalho com diferentes formatos de grupos em sala de aula.

Contribuições da epistemologia genética para a construção do conhecimento na educação infantil

The kindergarten has as main objective the development of the child from zero to five years in their physical, emotional, cognitive and social domains. This is also the goal of the Centers for Children Living from the Universidade Estadual Paulista (UNESP) that caters to children of teachers, technical and administrative staff and students. In this work we report some of the action of the Technical Commission of the CCIs with reflections on the construction of children's knowledge through observations of the CCIs in different campuses of Unesp, conducted through technical visits. As a theoretical contribution we adopt the Genetic Epistemology of Jean Piaget. Partial results show that, although there are efforts to understand and assist the process of constructing knowledge of children, many challenges remain to be overcome in the realization of an excellent service to children.

Toroidal solitons in 3+1 dimensional integrable theories

We analyze the integrability properties of models defined on the symmetric space SU(2)/U(1) in 3 + 1 dimensions, using a recently proposed approach for integrable theories in any dimension. We point out the key ingredients for a theory to possess an infinite number of local conservation laws, and discuss classes of models with such property, We propose a 3 + 1-dimensional, relativistic invariant field theory possessing a toroidal soliton solution carrying a unit of topological charge given by the Hopf map. Construction of the action is guided by the requirement that the energy of static configuration should be scale invariant. The solution is constructed exactly. The model possesses an infinite number of local conserved currents. The method is also applied to the Skyrme-Faddeev model, and integrable submodels are proposed. (C) 1999 Elsevier B.V. B.V. All rights reserved.

Gametic phase disequilibrium between the syntenic multiallelic HTG4 and HMS3 markers widely used for parentage testing in Thoroughbred horses

Validation of parentage and horse breed registries through DNA typing relies on estimates of random match probabilities with DNA profiles generated from multiple polymorphic loci. Of the twenty-seven microsatellite loci recommended by the International Society for Animal Genetics for parentage testing in Thoroughbred horses, eleven are located on five chromosomes. An important aspect in determining combined exclusion probabilities is the ascertainment of the genetic linkage status of syntenic markers, which may affect reliable use of the product rule in estimating random match probabilities. In principle, linked markers can be in gametic phase disequilibrium (GD). We aimed at determining the extent, by frequency and strength, of GD between the HTG4 and HMS3 multiallelic loci, syntenic on chromosome 9. We typed the qualified offspring (n (1) = 27; n (2) = 14) of two Quarter Bred stallions (registered by the Brazilian Association of Quarter Horse Breeders) and 121 unrelated horses from the same breed. In the 41 informative meioses analyzed, the frequency of recombination between the HTG4 and HMS3 loci was 0.27. Consistent with genetic map distances, this recombination rate does not fit to the theoretical distribution for independently segregated markers. We estimated sign-based D' coefficients as a measure of GD, and showed that the HTG4 and HMS3 loci are in significant, yet partial and weak, disequilibrium, with two allele pairs involved (HTG4*M/HMS3*P, D'(+) = 0.6274; and HTG4*K/HMS3*P, D'(-) = -0.6096). These results warn against the inadequate inclusion of genetically linked markers in the calculation of combined power of discrimination for Thoroughbred parentage validation.

Genomic and transcriptional analysis of allergen genes in apple (Malus x domestica)

Apple consumption is highly recomended for a healthy diet and is the most important fruit produced in temperate climate regions. Unfortunately, it is also one of the fruit that most ofthen provoks allergy in atopic patients and the only treatment available up to date for these apple allergic patients is the avoidance. Apple allergy is due to the presence of four major classes of allergens: Mal d 1 (PR-10/Bet v 1-like proteins), Mal d 2 (Thaumatine-like proteins), Mal d 3 (Lipid transfer protein) and Mal d 4 (profilin). In this work new advances in the characterization of apple allergen gene families have been reached using a multidisciplinary approach. First of all, a genomic approach was used for the characterization of the allergen gene families of Mal d 1 (task of Chapter 1), Mal d 2 and Mal d 4 (task of Chapter 5). In particular, in Chapter 1 the study of two large contiguos blocks of DNA sequences containing the Mal d 1 gene cluster on LG16 allowed to acquire many new findings on number and orientation of genes in the cluster, their physical distances, their regulatory sequences and the presence of other genes or pseudogenes in this genomic region. Three new members were discovered co-localizing with the other Mal d 1 genes of LG16 suggesting that the complexity of the genetic base of allergenicity will increase with new advances. Many retrotranspon elements were also retrieved in this cluster. Due to the developement of molecular markers on the two sequences, the anchoring of the physical and the genetic map of the region has been successfully achieved. Moreover, in Chapter 5 the existence of other loci for the Thaumatine-like protein family in apple (Mal d 2.03 on LG4 and Mal d 2.02 on LG17) respect the one reported up to now was demonstred for the first time. Also one new locus for profilins (Mal d 4.04) was mapped on LG2, close to the Mal d 4.02 locus, suggesting a cluster organization for this gene family, as is well reported for Mal d 1 family. Secondly, a methodological approach was used to set up an highly specific tool to discriminate and quantify the expression of each Mal d 1 allergen gene (task of Chapter 2). In aprticular, a set of 20 Mal d 1 gene specific primer pairs for the quantitative Real time PCR technique was validated and optimized. As a first application, this tool was used on leaves and fruit tissues of the cultivar Florina in order to identify the Mal d 1 allergen genes that are expressed in different tissues. The differential expression retrieved in this study revealed a tissue-specificity for some Mal d 1 genes: 10/20 Mal d 1 genes were expressed in fruits and, indeed, probably more involved in the allergic reactions; while 17/20 Mal d 1 genes were expressed in leaves challenged with the fungus Venturia inaequalis and therefore probably interesting in the study of the plant defense mechanism. In Chapter 3 the specific expression levels of the 10 Mal d 1 isoallergen genes, found to be expressed in fruits, were studied for the first time in skin and flesh of apples of different genotypes. A complex gene expression profile was obtained due to the high gene-, tissue- and genotype-variability. Despite this, Mal d 1.06A and Mal d 1.07 expression patterns resulted particularly associated with the degree of allergenicity of the different cultivars. They were not the most expressed Mal d 1 genes in apple but here it was hypotized a relevant importance in the determination of allergenicity for both qualitative and quantitative aspects of the Mal d 1 gene expression levels. In Chapter 4 a clear modulation for all the 17 PR-10 genes tested in young leaves of Florina after challenging with the fungus V. inaequalis have been reported but with a peculiar expression profile for each gene. Interestingly, all the Mal d 1 genes resulted up-regulated except Mal d 1.10 that was down-regulated after the challenging with the fungus. The differences in direction, timing and magnitude of induction seem to confirm the hypothesis of a subfunctionalization inside the gene family despite an high sequencce and structure similarity. Moreover, a modulation of PR-10 genes was showed both in compatible (Gala-V. inaequalis) and incompatible (Florina-V. inaequalis) interactions contribute to validate the hypothesis of an indirect role for at least some of these proteins in the induced defense responses. Finally, a certain modulation of PR-10 transcripts retrieved also in leaves treated with water confirm their abilty to respond also to abiotic stress. To conclude, the genomic approach used here allowed to create a comprehensive inventory of all the genes of allergen families, especially in the case of extended gene families like Mal d 1. This knowledge can be considered a basal prerequisite for many further studies. On the other hand, the specific transcriptional approach make it possible to evaluate the Mal d 1 genes behavior on different samples and conditions and therefore, to speculate on their involvement on apple allergenicity process. Considering the double nature of Mal d 1 proteins, as apple allergens and as PR-10 proteins, the gene expression analysis upon the attack of the fungus created the base for unravel the Mal d 1 biological functions. In particular, the knowledge acquired in this work about the PR-10 genes putatively more involved in the specific Malus-V. inaequalis interaction will be helpful, in the future, to drive the apple breeding for hypo-allergenicity genotype without compromise the mechanism of response of the plants to stress conditions. For the future, the survey of the differences in allergenicity among cultivars has to be be thorough including other genotypes and allergic patients in the tests. After this, the allelic diversity analysis with the high and low allergenic cultivars on all the allergen genes, in particular on the ones with transcription levels correlated to allergencity, will provide the genetic background of the low ones. This step from genes to alleles will allow the develop of molecular markers for them that might be used to effectively addressed the apple breeding for hypo-allergenicity. Another important step forward for the study of apple allergens will be the use of a specific proteomic approach since apple allergy is a multifactor-determined disease and only an interdisciplinary and integrated approach can be effective for its prevention and treatment.

Genetische Kartierung und QTL-Analyse agronomischer Eigenschaften der Weinrebe unter besonderer Berücksichtigung der Pilzresistenz

153 Nachkommen einer Kreuzung aus der pilzresistenten Rebsorte ‘Regent‘ und ‘Lemberger‘ als klassischer pilzsensitiver Sorte zeigen quantitative Merkmalsvariation bezüglich der Resistenz gegen Plasmopara viticola und Uncinula necator sowie für weitere Eigenschaften, die z.B. das Eintreten der Beerenreife betreffen. Auf dem Weg über die genetische Kartierung mit molekularen Markern und der Lokalisierung von QTL-Effekten konnten Hinweise auf weinbaulich relevante Genomregionen gewonnen werden; dies liefert z.B. die Basis für markergestützte Selektion bei Zuchtvorhaben mit dem Resistenzträger ‘Regent’ (vgl. auch FISCHER et al., 2004). Ein Major-QTL für die Resistenz gegen den Echten Mehltau Uncinula necator sowie zwei Major QTL für die Resistenz gegen den Erreger des Falschen Mehltau, Plasmopara viticola, traten mit hoher Signifikanz auf drei verschiedenen Kopplungsgruppen von ‘Regent‘ auf. Auch Regionen mit Relevanz für das Eintreten der Beerenreife wurden beschrieben. Über die Isolierung, Sequenzierung und anschließende Analyse einzelner Markerfragmente mit Methoden der Bioinformatik ist es gelungen, ein putatives T10P12.4-Ortholog der Weinrebe (ein thioredoxinähnliches Protein) in enger Kopplung zu einem Major-QTL-Maximum für Plasmopara viticola-Resistenz zu identifizieren, das als Kandidat für die Beteiligung an der Pathogenantwort in Frage kommt. Es konnte exemplarisch gezeigt werden, dass die eingesetzten Methoden der Kartierung und QTL-Analyse unter Verwendung PCR-basierter Markertypen wie SSR und AFLP und einer beschleunigten Analyse über computergestützte Kapillargelelektrophorese in vertretbarem Zeitrahmen bis zur Isolation potentieller Schlüsselgene führen können. Die grundsätzliche Eignung der QTL-Analyse als effizientes Werkzeug gezielter Züchtungsplanung für den Weinbau bestätigte sich. Ihre Anwendung im Rahmen der vorliegenden Dissertation hat die Basis für die Nutzung von QTL-Information bei dem Vergleich etablierter und der Entwicklung neuer Sorten gelegt und zum Verständnis von Prozessen beigetragen, die den betrachteten Eigenschaften wie der Pilzresistenz möglicherweise zu Grunde liegen. Ein großer Teil der gewonnenen Daten bringt auch die Untersuchungen anderer Kultivare voran und ist intervarietal übertragbar. Darüber hinaus haben sich Chancen für vergleichende Studien zwischen der Weinrebe einerseits und der Modellpflanze Arabidopsis thaliana sowie weiteren Kulturpflanzen andererseits abgezeichnet. Die Hinweise auf die zentrale Rolle und universelle Natur des Redox-Signalling haben interessante Perspektiven zum Verständnis organismenübergreifender physiologischer Zusammenhänge eröffnet. Dies betrifft z.B. auch die Reaktion auf Verwundung oder die Pathogenantwort.