Made available in DSpace on 2016-08-12T19:18:45Z (GMT). No. of bitstreams: 1 leidianeBA_DISSERT.pdf: 951596 bytes, checksum: 769c70c85ea3d6f0353cabbe1093e973 (MD5) Previous issue date: 2014-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Downy mildew, caused by the fungus Pseudoperonospora cubensis, is a major threat to the production of melons in wetlands worldwide. Despite its importance, there are few studies involving the assessment of sources of resistance, as well as the identification of molecular markers that are connected to this feature and that may help breeders in marker-assisted selection (MAS). Given this, the objectives of this work were promote the selection of sources of resistance to downy mildew, from melon accessions, collected from different states in the Northeast region of Brazil and to identify QTLs that co-segregate with the resistance gene of this pathogen, using the F2:3 obtained from parents and Védrantais MR-1, contrasting to the register character. To identify sources of resistance, thirty-six accesses and four commercial cultivars in a randomized block design (RBD) were assessed with three replications and seven plants per plot, which evaluation was made on the field. The evaluation was carried out on 24 days after transplanting the seedlings to the field, when 50% of plants reached the stage of flowering, with the aid of a diagrammatic scale. It was observed that there is variability in melon germplasm for reaction to downy mildew. The accessions A1, A4, A6, A9, A12, A17, A18, A23, A27, A35 and A36 were considered promising for use in breeding programs aiming resistance to P cubensis. All commercial cultivars analyzed were highly susceptible to the pathogen. To identify markers connected the resistance QTLs, MR-1 and Védrantais parents were crossed and subsequently the F2 generation was obtained. It was extracted the DNA for evaluation molecular of the 98 plants obtained with microsatellite primers polymorphic selected from contrasting parents. Of F2 plants were obtained 98 progenies F2:3 that were used for the assessments phenotypic of the severity caused by P. cubensis, it was used the same time assessment described in the previous experiment, the period April-June 2013. The experimental design utilized was lattice simple 10 x 10. The efficiency of lattice was low (100.28%) thus data were analyzed in DBC. Significant genetic differences (P<0.01) between the progenies were identified. The estimated of heritability was considered high (86.75%), indicating successful selection. Were found 23 primers polymorphic, of this sum, thirteen amplified in all the population and were used for analyzes genotypic. Of these, ten markers presented values below level critical specified by FDR test. There was more proportion allele of parent Védrantais for eight loci, while there was more proportion allele the parent MR-1 for two loci. For other loci (three), frequencies allelic did not change. The values coefficients of determination (R2) obtained for the markers were relatively low in population. By regression simple linear, the markers explained the feature in population F2:3 the most were CMBR 139 and CMMS 22-2, being the same identified by regression multiple linear, thus they can also be used in SAM / O míldio, causado pelo fungo Pseudoperonospora cubensis, é uma das principais ameaças à produção do meloeiro em áreas úmidas em todo o mundo. Apesar de sua importância, são poucos os trabalhos envolvendo a avaliação de fontes de resistência, bem como a identificação de marcadores moleculares que estejam ligados a esta característica e que possam auxiliar os melhoristas na seleção assistida por marcadores (SAM). Diante disso, os objetivos deste trabalho foram promover a seleção de fontes de resistência ao míldio, a partir de acessos de meloeiro, coletados em diferentes estados da região Nordeste do Brasil e também identificar QTLs que co-segreguem com genes de resistência a este patógeno, utilizando progênies F2:3 obtidas dos genitores MR-1 e Védrantais, contrastantes para o caráter. Para a identificação de fontes de resistência, foram avaliados trinta e seis acessos e quatro cultivares comerciais em delineamento em blocos casualizados (DBC) com três repetições e sete plantas por parcela, cuja avaliação foi feita em campo. A avaliação se deu aos 24 dias após o transplantio das mudas para o campo, depois que 50% das plantas atingiram o estágio de floração, com o auxílio de uma escala diagramática. Observou-se que existe variabilidade no germoplasma de meloeiro para reação ao míldio. Os acessos A1, A4, A6, A9, A12, A17, A18, A23, A27, A35 e A36 foram considerados promissores para o uso em programas de melhoramento visando resistência a P cubensis. Todas as cultivares comerciais analisadas foram altamente suscetíveis ao patógeno. Para identificação de marcadores ligados a QTLs de resistência, os genitores MR-1 e Védrantais foram cruzados e posteriormente foi obtida a geração F2. Das 98 plantas obtidas foi extraído o DNA para avaliação molecular com primers microssatélites polimórficos selecionados a partir dos genitores contrastantes. Das plantas F2 foram obtidas 98 progênies F2:3 que foram utilizadas para as avaliações fenotípicas quanto a severidade provocada por P. cubensis, seguindo a mesma época de avaliação descrita no experimento anterior no período de abril a junho de 2013. O delineamento utilizado foi o látice simples 10 x 10. A eficiência do látice foi baixa (100,28%), por isso, os dados foram analisados em DBC. Foram identificadas diferenças genéticas significativas (P<0,01) entre as progênies. A estimativa de herdabilidade foi considerada alta (86,75%), indicando sucesso com a seleção. Foram encontrados 23 primers polimórficos. Treze amplificaram em toda a população e foram utilizados para as análises genotípicas. Destes, dez marcadores apresentaram valores abaixo do nível crítico especificado pelo teste FDR. Para oito locos houve maior proporção de alelos do genitor Védrantais, enquanto que para dois locos houve maior proporção de alelos do genitor MR-1. Para os demais locos (três), as frequências alélicas não se alteraram. Os valores dos coeficientes de determinação (R2) obtidos para os marcadores foram relativamente baixos na população. Pela regressão linear simples, os marcadores que mais explicaram a característica na população F2:3 foram CMBR 139 e CMMS 22-2, sendo os mesmos identificados pela regressão linear múltipla, podendo, portanto, serem utilizados na SAM

The tilapias are a group of African and Middle Eastern cichlid fish that are widely cultured in developed and developing countries. With many different species and sub-species, and extensive use of interspecies hybrids, identification of tilapia species is of importance in aquaculture and in wild populations where introductions occur. This research set out to distinguish between tilapia species and sub-species by retrieving species-specific nuclear DNA markers (SNPs) using two approaches: (i) sequencing of the coding regions of the ADA gene; and (ii) next-generation sequencing, both standard RADseq and double-digest RADseq (ddRADseq). The mitochondrial DNA (mtDNA) marker cytochrome c oxidase subunit I (COI) was used to verify tilapia species status. ADA gene sequence analysis was partially successful, generating SNP markers that distinguished some species pairs. Most species could also be discriminated using the COI sequence. Reference based analysis (RBA: using only markers found in the O. niloticus genome sequence) of standard RADseq data identified 1,613 SNPs in 1,002 shared RAD loci among seven species. De novo based analysis (DBA: based on the entire data set) identified 1,358 SNPs in 825 loci and RBA detected 938 SNPs in 571 shared RAD loci from ddRADseq among 10 species. Phylogenetic trees based on shared SNP markers indicated similar patterns to most prior phylogenies based on other characteristics. The standard RADseq detected 677 species-specific SNP markers from the entire data set (seven species), while the ddRADseq retrieved 38 (among ten species). Furthermore, 37 such SNP markers were identified from ddRADseq data from a subset of four economically important species which are often involved in hybridization in aquaculture, and larger numbers of SNP markers distinguished between species pairs in this group. In summary, these SNPs are a valuable resource in further investigating hybridization and introgression in a range of captive and wild stocks of tilapias.

Mefenoxam has been a premier compound for Phytophthora disease control in the nursery industry for 30 years. The primary objectives of this research were to examine whether Phytophthora species have developed resistance to this compound and to investigate fungicide resistance management strategies. Phytophthora nicotianae, a destructive pathogen of numerous herbaceous and some woody ornamental plants, was used as a model system. P. cinnamomi, a major pathogen of a wide range of tree species and shrub plants, was also included for comparison. Twenty-six isolates of P. nicotianae were highly resistant to mefenoxam with a mean EC50 value of 326.5 µg/ml while the remaining 70 were sensitive with an EC50 of <0.01 µg/ml (Label rate: 0.08µg/ml). All resistant isolates were recovered from herbaceous annuals and irrigation water in 3 Virginia nurseries. Resistant isolates were compared with sensitive ones using seedlings of Lupinus "Russell Hybrids" in the absence of mefenoxam for relative competitive ability. Resistant isolates out-competed sensitive ones within 3 to 6 sporulation cycles. Resistant isolates exhibited greater infection rate and higher sporulation ability than sensitive ones. No mefenoxam resistant isolates were identified in P. cinnamomi. All 65 isolates of P. cinnamomi were sensitive to mefenoxam with an EC50 of < 0.04 ï ­g/ml. Attempts to generate mutants with high resistance to mefenoxam through UV mutagenesis and mycelial adaptation were not successful. However, there were significant reductions in sensitivity to mefenoxam; those slightly resistant mutants carried fitness penalties, which may explain why P. cinnamomi remains sensitive to mefenoxam. The effect of propamocarb hydrochloride on different growth stages of Phytophthora nicotianae was evaluated in search for an alternative fungicide. Propamocarb greatly inhibited sporangium production, zoospore motility, germination and infection. However, it has little inhibition of mycelial growth and infections. Propamocarb can be used as an alternative fungicide to mefenoxam where mefenoxam resistance has become problematic. However, it must be used preventively; i.e. before infections occur. The genetic inheritance of mefenoxam resistance in P. nicotianae was studied using F1 progenies of a cross between resistant and sensitive isolates. The F1 progenies segregated for mefenoxam resistance in ratio of 1R:1S, indicating the mefenoxam resistance is controlled by a single dominant gene. One RAPD marker putatively linked to resistant locus in repulsion phase was obtained by bulked segregant analysis and was converted to the SCAR marker. This marker is capable of differentiating mefenoxam resistant populations from sensitive populations included in this study. / Ph. D.

Die vorliegende Studie wurde in einer Gruppe von Dörfern im Ostsudan, Gedaref State, durchgeführt. Bei 100 Patienten mit der Verdachtsdiagnose Kala Azar- oder Post-Kala Azar- Leishmaniose war der Erregernachweis mit der PCR direkt in klinischen Proben, die auf Filterpapier aufgebracht worden waren, ohne vorherige Kultivierung erfolgreich. In dieser PCR wurden die ribosomalen, internal transcribed spacer (ITS1 & ITS2) amplifiziert, weil sie sehr variabel sind, eine klare Speziesidentifizierung gestatten und bei weiterführenden Analysen der PCR-Produkte auch der Nachweis stammspezifischer Unterschiede erwartet werden konnte. Für die Analyse der Diversität von Leishmania donovani-Isolaten aus dem Sudan wurden 4 verschiedene PCR-basierte Methoden eingesetzt: das PCR-Fingerprinting mit unspezifischen Einzelprimern, die RFLP- Analyse des amplifizierten ITS-Locus, ,,single strand conformation polymorphism (SSCP)- Analysen der amplifizierten ITS-Region, des Gens, welches für die Hauptoberflächenprotease (gp63) kodiert, und anonymer DNA- Fragmente sowie Sequenzanalysen der entsprechenden Zielregionen. Das PCR-Fingerprinting und die Restriktionsanalyse der ITS-Region lieferten weitgehend übereinstimmende Fragmentmuster für alle untersuchten L.donovani-Stämme. 12 unterschiedliche Profile wurden bei der SSCP-Analyse des ITS1-Locus für 86 Isolate aus dem Sudan erhalten, während der ITS2-Locus bei diesen Stämmen hochkonserviert war und nur ein Stamm ein unterschiedliches SSCP-Muster aufwies. L. Donovani -Stämme anderer geographischer Herkunft hatten unterschiedliche ITS2-Profile in der SSCP. Für den gp63 - Locus waren 3 polymorphe SSCP-Muster bei 31 untersuchten sudanesischen Isolaten nachweisbar. Für die meisten der anonymen DNA-Fragmente, L510, L413, LK413, L0308 UND L0114, konnten leider nur von 8 kultivierten Stämmen gute PCR-Produkte erhalten werden. Lediglich das Fragment L0110 konnte erfolgreich von 31 auf Filterpapier aufgebrachten Proben direkt amplifiziert werden. Die Suche nach Polymorphismen mit der SSCP ergab keine Unterschiede in diesen anonymen DNA-Regionen, mit Ausnahme des Fragments L0114, das zwei verschiedene Muster aufwies. Die Ergebnisse der SSCP-Analysen und der DNA- Sequenzierung stimmten gut überein, wodurch bestätigt wurde, dass die SSCP genetische Unterschiede auf dem Niveau einzelner Basenaustausche nachweisen kann. Die SSCP- Technik hat Vorteile gegenüber den anderen Methoden, die für die Untersuchung von Sequenzvariationen innerhalb der Spezies L. donovani angewandt wurden. Es konnten keine Korrelationen zwischen der Form der klinischen Manifestation und den Ergebnissen des PCR- Fingerprinting, der ITS-RFLP- und ITS-SSCP- Analysen festgestellt warden. Diese Studie ist von besonderem Nutzen in epidemiologischen Feldstudien, bei denen die Kultivierung der Erreger besonders in Entwicklungsländern extrem schwierig sein kann. / This study was carried out in clusters of villages that represent an endemic focus of visceral leishmaniasis (VL). These villages were located in Gedaref state, eastern Sudan. For diagnostic purposes polymerase chain reaction (PCR) was performed successfully, directly from clinical samples spotted on filter papers with no prior cultivation from 100 patients suspected of having kala-azar or post kala-azar dermal leishmaniasis. Mainly the ribosomal internal transcribed spacer (ITS1 & ITS2) were targeted in PCR because this region is more variable and allows clear species identification and also strain differences could be expected by further analysis of these PCR products. PCR was found to be more sensitive compared to the gold standard microscopic method. Four PCR based approaches were used to analyse diversity within Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single non-specific primers, restriction analysis of the amplified ITS locus (RFLP), single-stranded conformation polymorphism (SSCP) of the ITS region, major surface protease (gp63) gene, anonymous DNA fragments and sequencing of these targeted regions. When PCR fingerprinting and restriction analysis of ITS region were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave 12 different SSCP profiles among the 86 Sudanese isolates, where as the ITS2 locus was highly conserved among the 86 samples with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. The gp63 locus gave 3 polymorphic patterns among 31 Sudanese isolates. Concerning most of the anonymous DNA fragments namely, L510, L413, LK413, L0308 and L0114 unfortunately, we succeeded to get good PCR products only from DNA extracted 8 successful cultures. Only for the fragment L0110 we were able to get good PCR products from 31 samples spotted on filter papers. When these PCR products were investigated for polymorphisms using SSCP no differences were observed with exception of L0114 region, which showed 2 patterns. SSCP analysis correlates well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigating of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics. This study is beneficial particularly in epidemiological studies based on field-work where obtaining cultures can be extremely difficult especially in developing countries.

European hazelnut is a significant crop in the Pacific Northwest, and the US ranks 4th internationally for hazelnut production. Production in the Pacific Northwest is threatened, however, by the disease eastern filbert blight (EFB) caused by the fungus Anisogramma anomala (Peck) E. Müller. To meet the challenges faced by the hazelnut industry in Oregon and Washington, the breeding program at Oregon State University has focused on developing DNA marker technology and producing EFB resistant cultivars. This study focused on developing new microsatellite markers from hazelnut transcriptome sequences and on disease resistance from three accessions ('Culpla,' 'Crvenje,' and OSU 495.072) which showed no disease symptoms following a series of inoculations. DNA markers have been useful in hazelnut breeding for marker-assisted selection, construction of genetic linkage maps, cultivar fingerprinting, and phylogeny studies. Previously developed markers include AFLP, RAPD, ISSR, and microsatellite (SSR) markers developed from enriched libraries and ISSR fragments. This study utilized the transcriptome sequence from 'Jefferson' hazelnut to mine for microsatellites, align with the genomic sequence, design primers, screen for polymorphism, and characterize and map polymorphic markers. A total of 1432 microsatellites were mined from the transcriptome sequence, and the most frequently found motifs were AG (35.8%), AT (13.3%), and AAG (12.7%), and 382 primer pairs were designed. Screening showed that 119 markers were polymorphic, and these were characterized on sets of 50 and 14 accessions. Fifty-three markers that segregated in the mapping population or in three alternate populations were mapped and assigned to linkage groups. A dendrogram showed that accessions clustered mostly according to geographic origin. These results confirm the high level of diversity present in hazelnut, and the markers developed in this study will be useful for further genetics studies in hazelnut. The three EFB resistant parents 'Culpla,' 'Crvenje,' and OSU 495.072 were subjected to two inoculation treatments: greenhouse inoculations and exposure under an inoculation structure. The accessions remained free of disease after both treatments. Progeny segregating for resistance were produced. The progeny were inoculated either in the greenhouse or under the structure, and disease response recorded for each individual. DNA was extracted from seedlings, and sets of 32 seedlings from each resistant parent were screened with previously mapped markers using PCR and capillary electrophoresis. All three resistance sources were correlated with marker A614, allowing the resistance loci to be assigned to linkage group (LG) 6. The progeny were then screened with all known microsatellite markers on LG 6, and linkage maps constructed of the marker loci and resistance loci. Markers KG821, LG628, and LG696 are especially close to the resistance loci and will be useful for marker-assisted selection. Although these resistance loci are located in the same region of LG 6 as the 'Gasaway' resistance gene, they are different from 'Gasaway,' and markers linked to resistance will be useful for introgressing and pyramiding resistance in new cultivars. / Graduation date: 2013