Philosophiae Doctor - PhD / The domesticated apple (Malus x domestica Borkh), belonging to the Malusgenus of the Rosaceae family, is one of the edible pomaceous fruits. Since it is one of the important commercial fruit crops worldwide, the quality of the fruit is crucial to breeders and farmers as it ultimately determines acceptance of a cultivar for consumption. Fruit quality is also a critical determinant factor that is used to estimate the potential of apples to have a long shelf life / South Africa

>Magister Scientiae - MSc / Recent advances in the field of plant genetics and application of molecular technologies has lead to greater understanding of various crop genomes and their organization.The applications of these techniques include molecular markers which have been used to examine DNA variation within crop species. This allows for the creation of further genetic variation for new and favourable traits.Molecular markers or DNA markers are short fragments of DNA that can be used to locate desirable genetic traits in the genome or show specific genetic differences. The Maloideae subfamily includes fruit species such as pear. Pears (Pyrus communis L.) are large edible fruit that are grown in cool climates, native to coastal regions in Africa, Asia and Europe. The external appearance of this fruit plays a vital role on its rate of sale potential. Thus it is important for the appearances of the pear to meet the expectations of the consumer.External factors affecting the appearance of fruit, such as shape and colour, can have a large influence on the consumer’s first impression and opinion of what the fruit may taste like(Jaeger and MacFie, et al., 2001). The South African pear industry is the fourth largest in the fruit industry after apple, citrus and grape, exporting 3.8% to Europe (Ferrandi, et al., 2005).Increase in production and export of the pear is dependant on the variety of cultivars with desired traits. New cultivars, especially ranges of new cultivars, with harvest dates from early to late in the season, can fill gaps in the marketing strategy of exporters and in the local markets (Human, et al., 2005) Therefore, development of molecular markers allows for their possible use in maker-assisted selection and for the construction of a genetic linkage map thus leading to the location of favourable traits and ultimately the improvement of the quality of the pear.In this study high throughput genomic DNA extractions were performed. The Cetyltrimethyl ammonium bromide (CTAB) method was employed as the results proved to be most promising. Furthermore the screenings of molecular markers were conducted in order to obtain DNA variation. Molecular markers were used to locate specific genetic differences.Multiplexing PCR was conducted using fluorescent primers for further screening and results proved to be useful as many variations could be observed.

Spodoptera frugiperda (J.E. Smith) tem sido uma das pragas mais importantes da cultura do milho no Paraguai e Brasil. Este trabalho foi realizado para subsidiar programas de manejo de resistência no Paraguai, reconhecendo-se a proximidade das fronteiras agrícolas desse país com Brasil e a alta capacidade de dispersão de S. frugiperda. Para tanto, foram realizados estudos de caracterização da suscetibilidade aos inseticidas lufenuron e flubendiamide e de diversidade genética e o fluxo gênico em populações de S. frugiperda coletadas nas principais regiões produtoras de milho do Paraguai e de dois Estados fronteiriços do Brasil (Mato Grosso do Sul e Paraná). O método de bioensaio para a caracterização das linhas-básica de suscetibilidade foi o de tratamento superficial da dieta artificial com o inseticida. As variações na suscetibilidade a lufenuron e flubendiamide entre as populações de S. frugiperda testadas foram baixas (< 4 vezes), comparando-se as populações das regiões Oriental e Ocidental do Paraguai que apresentam caraterísticas climáticas distintas ou entre as populações do Paraguai e do Brasil. Para o monitoramento da suscetibilidade de populações de S. frugiperda no Paraguai foram definidas as doses diagnósticas, baseada na DL99, de 0,16 &mu;g de lufenuron.cm-2 (equivalente a 10 &mu;g de lufenuron.mL-1 de água) e 2,84 &mu;g de flubendiamide.cm-2 (180 &mu;g de flubendiamide.mL-1 de água). Mediante o uso de 12 loci microssatélites, foram verificadas maior diversidade genética dentro das populações (87,70%) quando comparada entre as populações de cada país (9,91%) e entre países (2,39%). Sendo assim, não foi constatada alta estruturação genética entre as populações de S. frugiperda coletadas no Paraguai e Brasil. As populações do Paraguai mostraram-se mais distintas entre si quando comparadas com as populações do Brasil. Nossos resultados apontam baixa estruturação genética e moderado/alto fluxo gênico entre as localidades de coleta de populações de S. frugiperda e entre as populações do Paraguai e Brasil. Os resultados obtidos na presente pesquisa servirão para a implementação de um programa de manejo da resistência de S. frugiperda a inseticidas no Paraguai, com ênfase na necessidade de ações no âmbito regional considerando os estados fronteiriços do Brasil. / Spodoptera frugiperda (J.E. Smith) has been one of the most important corn pests in Paraguay and Brazil. This research was conducted to collect information to insect resistance management programs in Paraguay considering the proximity of agricultural borders of this country with Brazil and the high dispersal capacity of S. frugiperda. We conducted studies to characterize the susceptibility to the insecticides lufenuron e flubendiamide and to evaluate the genetic diversity and gene flow among S. frugiperda populations collected from major corn-producing regions in Paraguay and two frontier States of Brazil (Mato Grosso do Sul and Paraná). The bioassay method to characterize the baseline susceptibility was the diet surface treatment with the insecticide. The variation in the susceptibility to lufenuron and flubendiamide among S. frugiperda populations tested was low (< 4-fold) by comparing populations from Estearn and Western regions of Paraguay with distinct climatic conditions or populations from Paraguay and Brazil. For susceptibility monitoring to lufenuron and flubendiamide in S. frugiperda populations in Paraguay, we defined the diagnostic doses based on LD99 of 0.16 &mu;g of lufenuron.cm-2 (equivalent to 10 &mu;g of lufenuron.mL-1 of water) and 2,84 &mu;g of flubendiamide.cm-2 (180 &mu;g of flubendiamide.mL-1 of water). Using 12 microsatellite loci, higher genetic diversity within populations (87.70%) than among populations within each country (9.91%) and between countries (2.39%). Thus, there was no high genetic structure between S. frugiperda populations collected in Paraguay and Brazil. The populations of S. frugiperda from Paraguay were more distinct among themselves when compared with the populations from Brazil. Our results suggest low genetic structure and moderate/high gene flow among different sampling locations of S. frugiperda populations as well as between populations from Paraguay and Brazil. The results obtained in this research will support the implementation of resistance management programs of S. frugiperda to inseticides in Paraguay with focus on the need of plan of actions at regional level considering the border States of Brazil.

Uma população F2 foi desenvolvida através do cruzamento entre uma linhagem de frangos de corte (TT) e uma de postura (CC). Sete machos TT foram acasalados com sete fêmeas CC para produzir uma população F1 TC. Cada macho F1 foi acasalado com três fêmeas F1 não-aparentadas para produzir aproximadamente 100 descendentes por família de irmãos completos. Neste estudo foram utilizadas as 5 famílias que apresentaram maior número de marcadores informativos nos cromossomos 1 a 5, em estudos realizados anteriormente nesta população. Foram coletados dados fenotípicos de peso ao nascer (PN), peso aos 35 (PV35) e 41 (PV41) dias de idade, ganho de peso (GP35-41), consumo de ração (CR35-41) e eficiência alimentar (EF35-41) dos 35 aos 41 dias de idade. Os indivíduos parentais e F1 foram genotipados com 35 marcadores microssatélites posicionados nos cromossomos 9, 10, 12, 14, 15, 16, 17 e 18. Os marcadores informativos (22) foram genotipados nos indivíduos F2 das famílias escolhidas. O cromossomo 16 não foi mapeado, pois o único marcador disponível não foi informativo. No cromossomo 17 apenas um marcador foi informativo, logo foi utilizada uma análise de marca simples para associação do marcador com as características selecionadas. O mapa de ligação de cada cromossomo foi construído e comparado ao mapa consenso. Os mapas de ligação apresentaram o mesmo ordenamento dos marcadores em relação aos mapas consenso, no entanto foram encontradas pequenas discrepâncias nas distâncias entre os marcadores A análise empregada no estudo de mapeamento de QTL por intervalo utilizou o método de regressão linear e estimação de quadrados-mínimos, utilizando o programa QTL Express, na opção análise de F2. No cromossomo 17, duas características foram associadas ao genótipo do marcador: GP35-41 (P<0,05) e EF35- 41 (P<0,01). Foram encontrados 4 QTL sugestivos no cromossomo 10: para PV35, PV41, GP35-41 e EF35-41. Houve interação do QTL com família para EF35-41. Os QTLs para PV35, PV41 e GP35-41 apresentaram efeito aditivo, apenas EF35-41 apresentou efeito de dominância. / An F2 chicken population was developed by crossing broiler line (TT) with a layer line (CC). Seven males TT were mated with seven females CC to generate an F1 population. Each male F1 was then mated with three unrelated females F1 generating approximately 100 individual per family of dam. On the present study 5 families were used, which showed the greatest number of informative markers in previews studies of chromosomes 1 to 5 of the resource population. Phenotypic dada were collected for body weight at 1 (BW1), 35 (BW35) and 41 (BW41) days of age, weight gain (WG35- 41), feed intake (FI35-41) and feed efficiency (FE35-41) from 35 to 41 days of age. The parental and F1 individuals were genotyped for 35 microsatellite markers distributed on chromosomes 9, 10, 12, 14, 15, 16, 17 and 18. The informative markers were genotyped on the F2 from the selected families. Chromosome 16 was excluded, because the only available marker was not informative. On chromosome 17 only one marker was informative, therefore a single marker analysis was used to associate the marker with the selected characteristics. The linkage map of each chromosome was constructed and compared to the consensus map. The linkage maps showed the same marker ordering compared to the consensus map, however small discrepancy between markers distances were presented. Interval mapping using regression methods was applied to a line-cross and least square analysis, using the QTL Express program, on the F2 analyses option. On chromosome 17, two characteristics were associated to the marker genotype: WG35-41 (P<0.05) and FE35-41 (P<0.01). Suggestive QTLs were detected for BW35, BW41, WG35-41 and FE35-41 on chromosome 10. A QTL x family interaction effect was statistically significant for FE35-41. QTLs for BW35, BW41 and WG35-41 showed addictive effects, only FE35-41 showed dominance effect.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21614号 / 理博第4521号 / 新制||理||1649(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 幸島 司郎, 教授 平田 聡 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Testicular germ cell tumors (TGCTs) are thought to arise during early embryogenesis due to the arrest of germ cell differentiation at primordial germ cells (PGCs) or gonocytes. Oxidative stress (OS) is implicated in cancer development as a factor leading to DNA damage. Reactive oxygen species (ROS) -induced instability occurs as a series of progressive steps. The cell has several defense mechanisms against the deleterious effect of ROS (e.g. antioxidants and DNA repair). When the defense mechanisms are exhausted by increasing OS, DNA damage leads to genomic instability with subsequent mutations that can be transmitted during cell division. On the other hand, male infertility is a representation of testicular dysgenesis syndrome, which carries a risk for TGCTs development. The mechanisms underlying both TGCTs and male infertility are thought to be overlapping to some extent. The central hypothesis of this work is that OS induces germ line genomic instability leading to testicular germ cell tumors. To test this hypothesis, mouse germ cell lines were established and subjected to different doses of OS in the form of H2O2. The mutation frequency was associated with the treatment dose 2 uM at days 3, 6, and 9 (p<0.001, p<0.001, and p<0.0003, respectively). The mBAT27 marker showed a mutation frequency fitting quadratic response surface regression. The mutation frequencies pointed to the possible role of OS leading to accumulation of DNA damage and initiating events that lead to TGCTs development that may occur early in life, possibly during the prenatal period. In addition, different panels of microsatellite markers from across the genome were analyzed to test for differential instability in both somatic cells and germline cells. Blood and semen samples from 18 infertile patients and 7 ethnically matched controls were used. Microsatellite markers were selected; 26 on the Y chromosome, 16 on the X chromosome, and 20 on different autosomes. Microsatellite instability was detected in markers located near genes responsible for testis development, spermatogenesis, cell differentiation, and proteins involved in mismatch repair mechanisms. This supports the hypothesis that testicular germ cell tumors may arise during early embryogenesis through acquiring multiple mutations that accumulate over time.

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