Rozprawy doktorskie na temat „Recombinant inbred lines”

Evaluation of soybean recombinant inbred lines for seed weight yield, agronomic traits, and resistance to sudden death syndrome Sudden death syndrome (SDS) caused by Fusarium virguliforme is a devastating disease in soybean (Glycine max (L.) Merr.) that causes up to 70% of yield losses depending on the developmental stage when the plant become infected. The characterization of resistance is greatly significant for disease management. Therefore, three populations were developed by crossing three resistant lines, `Hamilton', LS90-1920 and LS97-1610 with a susceptible line to SDS, `Spencer'. Ninety-four F5:6 recombinant inbred lines from each population (Hamilton x Spencer, LS90-1920 x Spencer, and LS97-1610 x Spencer) were evaluated for two years (2009 and 2010) at two locations (Carbondale and Valmeyer) in southern Illinois. Population statistics, genotype x environment interaction, and broad-sense heritability were used to reveal any major resistance genes. Genetic correlation coefficients of SDS resistance with important agronomic traits such as lodging, pubescence, growth habit, and plant height were also calculated. The information from this study will be helpful to breeders in developing populations for genetic analyses and enforcing selection practices.

The majority of genetic differences between species and individuals have been hypothesised to impact on the regulation, rather than the structure, of genes. As the details of genetic variation are uncovered by the various genome sequencing projects, understanding the functional effects on gene regulation will be key to uncovering the molecular mechanisms underying the genesis and inheritance of common phenotypes, such as complex human disease and commercially important traits in plants and animals. Unlike coding sequence polymorphisms, genetic variants affecting gene expression will reside in the transcriptional machinery and its regulatory inputs. As these are largely specific to cell- or tissue-types, we would expect that regulatory variants will also affect final mRNA levels in a tissue specific manner. Genetic variation between individuals may therefore be more complex than the sum total of sequence differences between them. Demonstrating this hypothesis is the main focus of this thesis. We use microarrays to measure mRNA levels of approximately 22,000 transcripts in inbred and recombinant inbred strains of mice, and present compelling evidence that the genetic influences on these levels are tissue-specific in at least 85% of cases. We uncover two loci which apparently influence transcript levels of multiple genes in a tissue-specific manner. We also present evidence that failure of microarray data normalisation may cause spurious linkage of expression phenotypes leading to erroneous biological conclusions, and detail a novel, extensible mathematical framework for performing tailored normalisation which can remove such systematic bias. The wider context of these results is then discussed.

Soybeans [Glycine max (L.) Merrill] are a major commercial crop that originated in Eastern Asia, which date back 5,000 years ago in China and are still used worldwide today. Soybeans are considered an oil seed crop that averages twenty percent oil content and consists of thirty-five to forty percent protein. Soybeans are used in most aspects of the modern world as a source of protein for humans and animals alike. It is also used for its oils, which can be found in food, consumables, and plastic. Soybean production came about in the 18th Century in the United States as a hay crop and in some regions as an ornamental plant, but did not start being grown in large-scale production until the early 19th Century. Seed producing companies did not take interest in the plant until 1970, when Congress established the United States Plant Variety Act. This Act allows protection for companies against unauthorized use of proprietary material. Plant breeders focused on improving yield, drought tolerance, and disease resistance. Sudden Death Syndrome (SDS) is a disease of soybeans that affect soybean populations in the Western Hemisphere. SDS is a seedling disease in which a soil-borne fungal infection attacks the roots of a young soybean plant. This infection is more severe in soils highly saturated with water early in the planting year and then followed by cool weather before the soybean plant flowers in late summer. Yield losses commonly do not exceed ten to fifteen percent of a crop, but cases have occurred where yield was reduced over seventy percent due to SDS. Three species that affect the Western Hemisphere; Fusarium virguliforme (FV), formally know as Fusarium solani f. sp. Glycines (FSG); which mainly affects soybean production in the North American continent, Fusarium phaseoli and Fusarium tucumaniae, which affect the South American continent. SDS in the United States can account for yield losses occurring in primarily Arkansas, Iowa, Illinois, Indiana, Kentucky, Missouri, and Tennessee during 1999 to 2002 time period, with Iowa, Illinois, and Indiana having the most severe effects. SDS has rapidly spread throughout the United States and it was estimated to suppress the soybean yield in 2002, with damage that was valued at $157.4 million. There is not a 100 percent proven agronomic practice for controlling SDS, so the identification of host resistant genes are required in order to develop different varieties that will offer the producer the most economically efficient way to manage the disease.

Stem rust and Yellow rust diseases are the two major wheat fungal diseases causing considerable yield losses in Turkey and all around the world. There are studies which are carried out to identify and utilize resistance sources in order to obtain resistant lines of wheat. However, virulent pathotypes are continuously being important threats to wheat production and yield. For that reason, new approaches for rapid identification are needed. The aim of this study was to investigate and to understand the structural and functional differences between the resistant and sensitive durum wheat cultivars to the plant fungal diseases of stem and yellow (stripe) rusts. To aim this, forty durum wheat recombinant inbred lines (RILs), which were previously determined to be resistant or sensitive to stem and yellow rust diseases, were investigated by the noninvasive Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. Also, classification of the resistant and sensitive lines depending on the structural and functional differences has been attempted. The FTIR spectra for stem rust disease showed that, resistant durum wheat lines had a significant increase in the population of unsaturation in acyl chains of lipid molecules, an increase in lipid and in total protein content and also an increase in carboxylic acids and alcohols. For yellow rust disease, resistant lines had a significant increase in hydrogen bonding and they had also a more ordered membrane structure. In Principal Component Analysis for stem rust disease, according to 3700-650 cm-1 region, amide III band (1213-1273 cm-1 region) and C-H stretching region (3020- 2800 cm-1), the resistant and sensitive groups were separated successfully. For yellow rust disease, according to 3700-650 cm-1 region, Amide A and Amide III bands, the resistant and sensitive lines were grouped distinctly. FTIR spectroscopy provides a useful approach to determine the differences in molecular structure of durum wheat RILs regarding resistance of lines to fungal diseases. However, further research is still needed to ensure if the structural and functional differences in biomolecules of the samples could be used as molecular markers for discrimination of rust resistant materials from rust sensitive ones.

Orientadores: Luciana Lasry Benchimol Reis, Matthew Ward Blair
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O feijão comum (Phaseolus vulgaris L.) é uma cultura importante economicamente tanto para o consumo nacional como para a exportação. A seca é um dos principais estresses abióticos em todo o mundo e afeta cerca de 60% da área de cultivo de feijão. O avanço nas tecnologias de marcadores moleculares oferecem poderosos métodos para examinar as relações entre as características, gerando um grande volume de informações potencialmente úteis para assessorar os programas de melhoramento. O presente projeto teve como objetivo o desenvolvimento da Plataforma DArT para feijão comum junto à empresa DArT Pty Ltd, e o mapeamento destes marcadores juntamente com microssatélites e SNPs na população AND 277 x SEA 5 proveniente do CIAT (Colômbia), a fim de localizar os QTLs associados à tolerância à seca. O genitor SEA 5 é uma linhagem avançada do BAT 477, é tolerante à seca e de origem Mesoamericano e o genitor AND 277 é um genótipo resistente à mancha angular e antracnose e de origem Andina. Um total de 4.468 marcadores DArTs, 288 marcadores SNPs e 180 marcadores microssatélites polimórficos foram identificados na população e utilizados na genotipagem para construir um mapa genético saturado. A fenotipagem das 105 linhagens endogâmicas recombinantes (RILs) na geração F8 mais os dois genitores foi realizada avaliando 18 características associadas à tolerância a seca utilizando um delineamento inteiramente casualizado com quatro repetições, aplicando um estresse terminal na fase vegetativa V3/V4. Dois mapas foram construídos, um integrando 80 SSR e 251 SNPs e outro com cinco SSR, 91 SNPs e 4.468 DArTs. A identificação dos QTLs foi realizada através da análise de mapeamento por intervalo composto (CIM) para o mapa SSR - SNPs e mapeamento de precisão (SML) para o mapa SSR-SNPs-DArT. Um total de 12 QTLs foram identificados para o tratamento não irrigado e 29 QTLs para o tratamento irrigado pela análise CIM. Para as análises SML, 23 QTLs foram identificados para o tratamento não irrigado e 11 QTLs para o irrigado. QTLs de maior efeito foram encontrados para clorofila, biomassa fresca do caule e da folha, Massa seco da folia, temperatura da folha, número de vagens, número de sementes, massa de sementes, dias para florescimento, massa seca das vagens e produtividade nos dois tratamentos. Todos os QTLs detectados sob condições de seca apresentaram o alelo do genitor SEA 5. Este estudo é importante para o melhoramento genético não só para entender melhor a herança genética de uma característica tão complexa como a tolerância à seca, bem como para encontrar ferramentas moleculares a serem utilizados para a seleção assistida por marcadores
Abstract: Common bean (Phaseolus vulgaris L.) is the most important food legume for consumption and for exportation. Drought is one of the main abiotic stresses in the world and affects about 60% of bean growing area across the world. The advance in technologies of molecular markers provide a powerful method to examine the relationships between traits, generating large amount of potentially useful information to assist the breeding programs. The objective of this project was the development of DArT platform for common beans with DArT Pty Ltd and the mapping of these markers with microsatellites and SNPs in the population AND 277 x SEA 5 from CIAT (Colombia), in order to locate the QTLs associated with drought tolerance. The SEA 5 parent is a drought tolerant advanced line (Mesoamerican) and the AND 277 is resistant to the angular leaf spot and antracnose (Andean). A total of 4.468 DArT markers, 288 SNP and 180 SSR polymorphic markers were identified in the population and used in genotyping to constructed a saturated genetic map. Phenotyping of 105 recombinant inbred lines (RILs) in F8 generation plus the genitors were performed evaluating 18 traits associated with drought tolerance using a completely randomized design with four replicates, applying terminal stress at vegetative phase V3/V4. Two maps were constructed, one integrating 80 SSR and 251 SNPs and another with five SSR, 91 SNPs and 4,468 DArTs. The identification of QTL analysis was performed by composite interval mapping (CIM) for the SSR - SNPs map and the precision mapping (SML) to map DArT-SSR-SNPs. A total of 12 QTLs were identified for the non-irrigated treatment and 29 QTLs for the irrigated treatment by CIM analysis. For SML analysis, 23 QTLs were identified for the non-irrigated and 11 QTLs for irrigated treatment. QTLs of major effect was found for chlorophyll, fresh biomass of stem and leaf dry weight, leaf temperature, number of pods, number of seeds, seed weight, days to flowering, dry weight of pods and yield in both treatments. All QTLs detected under dry conditions showed the allele of parent SEA 5. This study is important for genetic improvement not only to better understand the genetic inheritance of a trait as complex as drought tolerance, as well as to find molecular tools to be used for marker assisted selection
Doutorado
Genetica Vegetal e Melhoramento
Doutor em Genetica e Biologia Molecular

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The common bean (Phaseolus vulgaris) is grown in Brazil in various locations, soil and climatic conditions. The diseases are among the leading causes of losses in productivity of this legume, and the common bacterial blight (CBB) is the most important bacterioses that affects the culture. The resistance of CBB in common bean is a complex quantitative trait that results from the interaction of several genes. Genetic maps are tools that optimize the search for loci associated with this type of feature, and the most commonly used molecular markers available for this type of study are the SNPs (Single Nucleotide Polymorphism). In this sense, this study aimed to: (i) develop a robust genetic map for common bean using SNP markers and the RIL (Recombinant Inbreed Lines) mapping population derived from Ruda × AND 277; (ii) characterize this RIL population and their parents about the reaction to common bacterial blight in field and greenhouse; and (iii) identifying genomic regions (major genes and/or QTL) that control the bacterial blight in this population. We used 393 individuals of the Ruda × AND 277 RIL population, evaluated for reaction to CBB in two field trials in Ponta Grossa - PR, in the rain growing season of 2012 and 2014 and in an inoculation test at the greenhouse, in Santo Antônio de Goiás - GO. The population was genotyped with 5,398 SNP markers and mapping was performed using the R-OneMap and MapDisto programs. Statistical analyzes were performed in the Genes program, and the Scott-Knott method was used for averages groupingin R platform. The QTL analysis was conducted in QTLCartographer program. Using the chi-square test (1:1), 2,062 markers were selected for mapping. Three genetic maps with high strengt, saturation and resolution were built. Statistical analysis showed that there is genetic variability for the CBB resistance in the population of RILs. The QTL analysis identified 10 QTLs linked to resistance of CBB in the Ruda × AND 277 RIL mapping population, in the chromosomes PV01, PV02, Pv07, Pv09 and PV11, based on results from evaluations carried out in the field and greenhouse. The maps constructed for this population have high strength and resolution and may be used for future work on integrative mapping. The statistical analysis evidenced the quantitative character of resistance to CBB in common bean and showed that the parent Rudá has the CBB resistance alleles. It is expected that the markers linked to these QTLs identified can be used in future studies of marker assisted selection.
O feijoeiro-comum (Phaseolus vulgaris) é cultivado no Brasil em vários locais e diversas condições edafoclimáticas. As doenças estão entre as principais causas de prejuízos na produtividade dessa leguminosa, sendo o crestamento bacteriano comum (CBC) a principal bacteriose que afeta essa cultura. A resistência ao CBC no feijoeiro-comum é uma característica complexa, quantitativa, que resulta da interação de vários genes. Os mapas Genéticos são ferramentas que otimizam a busca de locos associados a esse tipo de característica, e os marcadores moleculares mais utilizados disponíveis para esse tipo de estudo são os SNPs (Single Nucleotide Polymorphism). Neste sentido, o presente trabalho teve como objetivos: (i) construir um mapa genético robusto para o feijoeiro-comum, utilizando marcadores SNP e a população de RILs (Recombinant Inbred Lines, ou linhagens endogâmicas recombinantes) derivada do cruzamento Rudá × AND 277; (ii) caracterizar esta população de RILs e seus genitores quanto à reação ao crestamento bacteriano comum, em campo e em casa de vegetação; e (iii) identificar regiões genômicas (genes de efeito principal e/ou QTLs) que controlam a reação ao crestamento bacteriano comum nesta população. Foram utilizados 393 indivíduos da população de RILs Rudá × AND 277, avaliados quanto à reação ao CBC em dois ensaios de campo em Ponta Grossa – PR, nas águas de 2012 e 2014, e em um ensaio de inoculação em casa de vegetação, em Santo Antônio de Goiás - GO. A população foi genotipada com 5.398 marcadores SNP e o mapeamento das RILs foi realizado utilizando os programas R-OneMap e MapDisto. As análises estatísticas foram realizadas no programa Genes, sendo o agrupamento de médias de Scott-knott realizado na plataforma R. A análise de QTL foi realizada no programa QTLCartographer. Por meio do teste de quiquadrado (1:1) foram selecionados 2.062 marcadores para o mapeamento. Foram construídos três mapas genéticos com elevada robustez, saturação e resolução. As análises estatísticas evidenciaram que há variabilidade genética para a característica de resistência ao CBC na população de RILs. A análise de QTL identificou 10 QTLs ligados à resistência ao CBC na população de RILs Rudá × AND 277 nos cromossomos Pv01, Pv02, Pv07, Pv09 e PV11 com base em dados obtidos a partir de avaliações em campo e casa de vegetação. Os mapas construídos para essa população apresentam elevada robustez e resolução e poderão ser utilizados para futuros trabalhos de mapeamento integrativo. As análises estatísticas evidenciaram o caráter quantitativo da resistência ao CBC em feijoeiro-comum e mostraram que o genitor Rudá possui alelos de resistência ao CBC. Espera-se que os marcadores ligados a esses QTLs identificados possam ser utilizados em futuros trabalhos de seleção assistida por marcadores.

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The common bean (Phaseolus vulgaris) crop plays an important role in the culture and economy of Brazil. It is cultivated in all Brazilian regions and is affected by several diseases like fusarium wilt which is caused by Fusarium oxysporum f. sp. phaseoli (soil-born fungus). This disease brings significant losses in common bean culture and genetic resistance is the primary form of control. One of the core goals of breeding programs is the development of resistant cultivars, therefore the objectives of this work are: i) To select F. oxysporum f. sp. phaseoli resistant F5:7 lines resulted from the crossing between Ouro Branco X CNFP10132, under controlled field and environment conditions ii) To identify SSR markers and QTL-linked SNPs associated with the resistance of common bean to fusarium wilt using 92 recombinat inbred lines(RILs) resulted from the crossing between Ouro Branco x CNFP10132. In the first study, 140 lines, the breeders Ouro Branco and CNFP10132, BRS Esplendor (resistant) and BRS Supremo (susceptible) as controls were evaluated. Field trials were conducted in a center pivot area where natural infestation of the pathogen occurs. The treatments were evaluated in summer and winter crop and the experimental design used was 12x12 triple lattice. The two controlled environment trials were conducted in a completely randomized design. The treatments were inoculated by cutting and immersing the roots in a conidial suspension, which was adjusted to 1x106 conidia/ml for five minutes. The evaluation was performed using a scale of nine grades that represent the severity of the disease: 1 – absence of symptoms and 9 – over 75% of foliage with wilt symptoms. Data were submitted to analysis of variance and Scott-Knott test for both environments. The area under the disease progress curve (AUDPC) and genetic parameters were estimated for controlled environment tests. Significant differences were observed for crops and for controlled environment trials, indicating that environment influences directly the severity of the disease. Highly significant differences were found for lines in all environments evaluated, demonstrating the existence of genetic variability, which allows the selection of resistant lines resistant to fusarium wilt. Treatments were classified in different groups according to the Scott-knott test. When considering the lowest averages in field, controlled environment and AUDPC, the strains Ouro Branco x CNFP 10132.140, Ouro Branco x CNFP 10132.49, Ouro Branco x CNFP 10132.12, Ouro Branco x CNFP 10132.90 and Ouro Branco x CNFP 10132.48 were prominent and are candidates to produce a breeding program. Heritability estimates were high for all environments, mean of 85.48% for field and 95.47% for controlled environment. Therefore, selection for resistance to F. oxysporum f. sp. phaseoli of these lines, will be successful. In the second study it was extracted DNA from 92 lines and from genitors for genotyping with SSRs and SNPs. In order to obtain the localization of these markers, sequences of the primers were aligned to the andean genome of the common bean. The method of single marker (analysis of QTLs based on linear regression) was used to identify QTLs associated with fusarium wilt resistance. These markers were considered significant when brought up p-value <0.05. Ninety-three markers were linked to 104 QTLs associated with fusarium wilt resistance and among these, were considered significant in more than one environment PV 115, PV 251, BARC-PV-0004089, BARC-PV-0004548, BARC-PV-0003450, BARC-PV-0006051, BARC-PV-0003368 , BARC-PV-0005477 and BARC-PV-0004897. However only the BARC-PV-0003450 marker was highly significant in the two environment controled trials (p <0.001) and winter crop (p <0.01) and explained up to 21.5% of the phenotypic variance. Subsequently, the gene annotation was made considering the location of all markers that were significant at p <0.01 comprising 500 kb before and after the localization. 960 coded transcripts were annotated. It was observed in gene annotation that BARC-PV-0003450 marker is located on the chromosome 8, 338.54 kb distant of the gene Phvul.008G014700 which is associated with the putative protein RPP13 related to disease resistance, identified in Arabidopsis thaliana. This protein belongs to the third class of resistance genes that encloses the domain called Leucine-Rich Repeats (LRR). This domain is involved in the recognition of the pathogen by the host during the infection process. Therefore, this marker is suitable for marker- assisted selection aiming the development of cultivars resistant to fusarium wilt.
A cultura do feijoeiro-comum (Phaseolus vulgaris) tem importância cultural e econômica no Brasil. O feijoeiro-comum é cultivado em todas as regiões brasileiras e é acometido por várias doenças, como a murcha-de-fusarium, causada pelo fungo habitante de solo Fusarium oxysporum f. sp. phaseoli. Esta doença causa significativas perdas na cultura e a principal forma de controle é a resistência genética. Desenvolver cultivares resistentes é um dos alvos dos programas de melhoramento, portanto os objetivos deste trabalho foram: i) selecionar linhagens resistentes obtidas de população F5:7 oriunda do cruzamento entre Ouro Branco e CNFP10132 para F. oxysporum f. sp. phaseoli, em condições de campo e de ambiente controlado e ii) identificar marcadores SSR e SNP's ligados a QTLs associados à resistência do feijoeiro-comum à murcha-de-fusarium utilizando 92 linhagens recombinantes endogâmicas (RILs) derivadas do cruzamento Ouro Branco x CNFP10132. No primeiro estudo 140 linhagens, os genitores Ouro Branco e CNFP10132, duas testemunhas BRS Esplendor (resistente) e BRS Supremo (suscetível) foram avaliados. Os ensaios de campo foram conduzidos em área de pivô central onde ocorre infestação natural do patógeno. Os tratamentos foram avaliados em duas safras (safra das águas e de inverno) em delineamento de látice triplo 12x12. Os dois ensaios em ambiente controlado foram conduzidos em delineamento inteiramente causalizado. As plantas foram inoculadas utilizando o método de corte de raiz e imersão destas na suspensão de conídios, que foi ajustada para 1x106 conídeos/mL durante cinco minutos. A avaliação foi feita utilizando uma escala de notas de nove graus que representam a severidade da doença: sendo 1 - ausência de sintomas e 9 - acima 75% da folhagem com sintomas de murcha. Os dados foram submetidos à análise de variância e teste de Scott-Knott para os ambos ambientes. Para os ensaios em ambiente controlado foram estimados área abaixo da curva do progresso da doença (AACPD) e parâmetros genéticos. Foram observadas diferenças significativas para safras e para ensaios de ambiente controlado, indicativo de que o ambiente influencia diretamente na severidade da doença. Foram encontradas diferenças altamente significativas para linhagens em todos os ambientes avaliados, evidenciando a existência de variabilidade genética, o que possibilita seleção de linhagens resistentes à murcha-de-fusarium. Ao considerar as menores médias em campo, ambiente controlado e ACCPD as linhagens Ouro Branco x CNFP 10132.140, Ouro Branco x CNFP 10132.49, Ouro Branco x CNFP 10132.12, Ouro Branco x CNFP 10132.90 e Ouro Branco x CNFP 10132.48 se destacaram e são candidatas para compor o programa de melhoramento. As estimativas de herdabilidade foram altas para todos os ambientes, média de 85,48% para campo e 95,47% para ambiente controlado. Portanto, a seleção para resistência à F. oxysporum f. sp. phaseoli dentre estas linhagens, será bem sucedida. No segundo estudo foi extraído o DNA de 92 linhagens e dos genitores para genotipagem com marcadores SSRs e SNPs. Para obtenção da localização destes marcadores as sequências dos primers foram alinhadas no genoma andino do feijoeiro-comum. O método de mapeamento por marcas simples (análise de QTLs por meio da regressão linear) foi utilizado para identificar QTLs associados à resistência à murcha-de-fusarium. Foram considerados marcadores significativos os que apresentaram p-valor<0,05. Noventa e três marcadores foram identificados ligados a 104 QTLs associados à resistência à murcha-de-fusarium. Dentre estes marcadores destaca-se os que foram significativos em mais de um ambiente PV 115, PV 251, BARC-PV-0004089, BARC-PV-0004548, BARC-PV-0003450, BARC-PV-0006051, BARC-PV-0003368, BARC-PV-0005477 e BARC-PV-0004897. Dentre os marcadores, somente o marcador BARC-PV-0003450 foi altamente significativo nos dois ensaios, em ambiente controlado (p<0,001) e na safra de inverno (p<0,01), e explicou até 21,5% da variância fenotípica. Foi feita a anotação gênica considerando a localização de todos os marcadores que foram significativos à p<0,01 e abrangeu 500 kb anterior e posterior à localização. Foram anotados 960 transcritos codificados. Ainda observou-se que o marcador BARC-PV-0003450 está localizado no cromossomo 8 distante 338,54 kb do gene Phvul.008G014700 o qual está associado à proteína putativa RPP13 relacionada com resistência à doenças, identificada em Arabidopsis thaliana. Esta proteína pertence à terceira classe de genes de resistência que engloba o domínio denominado de Repetições Ricas em Leucina (LRR; Leucine Rich Repeats). Este domínio está envolvido no reconhecimento do patógeno pelo hospedeiro durante o processo de infecção. Portanto há a possibilidade de selecionar linhagens resistentes à murcha-de-fusarium e identificar QTLs que possivelmente estão ligados aos marcadores utilizados

Des changements de méthylation de l'ADN peuvent affecter l'expression des gènes et pour certains être transmis au travers des générations. De telles " épimutations " qui concernent des groupes de cytosines à proximité ou dans les gènes sont donc une source potentielle de variation phénotypique héritable en absence de changements de la séquence de l'ADN. Chez les plantes la méthylation de l'ADN est cependant principalement observée au niveau des séquences répétées. Il reste à déterminer dans quelle mesure les changements de méthylation au niveau de ce type de séquences peuvent être héritées et affecter les phénotypes. Afin de répondre à ces questions, plus de 500 épiRIL (epigenetic Recombinant Inbred Lines) quasi-isogéniques a été générée chez Arabidopsis thaliana. Cette population a été obtenue par le croisement d'un parent sauvage et d'un parent mutant pour le gène DDM1 présentant une très forte réduction du taux de méthylation de l'ADN. Après un rétrocroisement de la F1 avec une plante sauvage, les individus sauvages pour le gène DDM1 ont été sélectionnés et propagées sur 6 générations par autofécondation. Nous avons montré par l'analyse du méthylome de plus de 100 épiRIL que l'hypométhylation induite par ddm1 présente selon les séquences affectées différents degrés de transmission au travers des générations. La réversion de l'hypométhylation concerne des régions associées à une abondance élevée en sRNA de 24 nt. Nous avons utilisé l'hypométhylation stablement transmise dans les épiRIL induite par ddm1 afin de détecter des QTL (Quantitative Trait Loci) affectant le temps de floraison et la longueur de la racine primaire, deux caractères pour lesquels les variations observées dans les épiRIL présentent une héritabilité importante. En dernier lieu, nous avons recherché par différentes approches les variations causales de ces QTL.

碩士
國立臺灣大學
農藝學研究所
103
In this study comparative maps of rice were constructed with a reference physical map (Nippobare) using SSR DNA markers and two recombinant inbred lines (RILs) populations, which were generated by a modified Single Seed Descendent (SSD) method. One population including 121 RILs was generated from an inter-subspecific cross between cultivars Taichung Sen No.10 (TCS10) and Koshihikari (KH). While the other population including 146 RILs was generated from an intra-subspecific cross between cultivar Koshihikari and Tai Nung No.67 (TNG67) with the same method.More misposition and conversion events were found in the linkage groups of the population TCS10 × KH. Besides, the genetic contribution of indica variety TCS10 is larger than that of japonica variety KH in the populations TCS10 × KH, and japonica TNG67 also has larger genetic contribution than KH. In addition, TCS10 × KH has more obvious segregation distortion (SD) were observed in the population TCS10 × KH than in the population KH × TNG67. There were several SD events at the corresponding positions on most chromosomes of the two populations, suggesting that SD events were not taken place in random. The rates of recombination event or double crossing over event are higher in the population KH × TNG67. Also, linkage disequilibrium (LD) decay was slower in the population TCS10 × KH than KH × TNG67. According to LD decay data, the amount of SSR molecular markers were sufficient in both populations; these markers were not sufficiently distributed evenly. To sum up, more projenies and selfing generations in a population developed from a cross between indica and japonica varieties. The results of this study could provide informations for rice breeding.

碩士
國立臺灣大學
農藝學研究所
101
Genetic markers such as DNA have long been used to represent the genotype of an individual (precisely, a lineage) by geneticists and breeders. These markers are developed by some means throughout the genome of the particular organism and being genotyped. Polymorphism of each marker characterizes different individuals. The characterization would be much more specific with the amount of polymorphic genetic markers we recognized. The genotypes of these markers are associated with the phenotypic values in the mapping of quantitative trait loci (QTL). In this study, we derived the multilocus genotypic frequencies for recombinant inbred and advanced intercrossed populations from 2- and 4-way crosses of inbred lines. We provide the mathematical proof for the relationship between the theoretical genotypic frequencies and the recombination scores of individual in the selfed populations derived from biparental cross of inbred lines. It is showed that genotypes with the same recombination score would have the equal probability to show up in any generation beyond the F2. This arisen symmetry also has its similar variants in 2-way random mating as well as 4-way selfing and random mating populations. Multi-level recombination score is proposed to identify the gametes with the same theoretical frequency among the random-mated 4-way cross derivatives. By using these symmetries, we reduced the dimensions of frequencies-transition matrix for each population. The reduction of matrix size lightens the computation effort in the multiplications for obtaining the advanced generation genotypic frequencies. At the end of this study, we provide a simple simulated case studying involving a biparental selfed F6 population and its multiple interval QTL mapping.

Desired base upper half mean length (UHML) of upland cotton (G. hirsutum) in the U.S. has been set a 27.0 mm and is shorter than the standard set by the international community. Upland cotton genotypes from China, South Africa, West Africa, and the U.S. were test crossed to an extra long staple upland (ELSU) and a short staple upland (SSU) and selected genotypes that included both ELSU and MSU phenotypes were crossed in a half-diallel mating scheme to estimate general combing ability (GCA) effects and specific combining ability (SCA) effects. A recombinant inbred line (RIL) population was established to determine the narrow sense heritability (h^2) of AFIS short fiber content by weight (SFCw) and lower half mean length (LHML) and to estimate SFCw using HVI fiber properties. Obsolete cultivars from China are not likely sources for UHML improvement, cultivars from Africa and the U.S. could harbor alleles not being used in current elite short staple cultivars or modern ELSU cultivars. Two ELSU lines used in this study derived through interspecific hybridization with G. barbadense could contain alleles for UHML improvement in modern ELSU cultivars developed without any apparent G. barbadense introgression. A third line D&PL 45-867, might contain alleles for UHML improvement in long staple upland cotton genotypes. Narrow sense heritability estimates indicated a much higher heritability of LHML than AFIS SFCw. Correlation between AFIS SFCw and LHML did not agree with previous studies when using an ELSU X MSU cross. Further study is needed to understand this complex relationship.

Recent initiatives for biofuel production have increased research and development of sweet sorghum. Currently, the initial major limitation to integrating sweet sorghum into existing production systems is the lack of sweet sorghum hybrids adapted to industrial production systems. Hybrid development is now underway, and the application of genetic markers can be used to define the genetic basis of sugar yield and its components, as well as reduce the time required to deliver new sweet sorghum hybrids to market. The purpose of this research was to further characterize the genetic components that influence sweet sorghum productivity, agronomics, and composition. Specifically, a grain x sweet sorghum recombinant inbred line (RIL) population developed for quantitative trait locus (QTL) analysis related to sugar production was evaluated for 24 phenotypic traits including brix, percent moisture, and biomass yield across four environments. The 185 F4 RILs were derived from the parents 'BTx3197' and 'Rio', which are pithy stalk grain and juicy stalk sweet sorghums respectively. Following screening, two genetic maps were constructed with 372 and 381 single nucleotide polymorphisms (SNPs) evaluated using an Illumina GoldenGate assay. Analysis of the data in QTL Cartographer revealed a major and previously reported QTL for soluble solids on chromosome 3, but in contrast to previous studies, this QTL co-localized with other QTLs that have a negative influence on biomass and seed production. Therefore, selection for this QTL may not be advantageous. Because only a few QTLs for percent moisture were found, the results indicated that the pithy stalk phenotype does not have a major effect on percent moisture as measured in this study. Thus, breeding for high or low moisture content will be more challenging than previously expected. The absence of dominance effects indicated that brix must be high in both parents to produce high brix in the hybrid.

Previously seed coat color in flax has been used as a phenotypic marker for specialty quality traits and currently there is an increasing demand to use seed coat color in flax to market flax for human and animal nutrition uses. Seed coat color was studied to 1) understand the inheritance of seed coat color conditioned by the b1 locus, to 2) understand the relationship of other important flax traits with seed coat color as well as to 3) identify markers that are linked to seed coat color for future marker assisted selection of seed coat color. Spearman’s rank correlation and an allelism test was used to show the inheritance of the alleles at the b1 locus. Bulked segregant analysis (BSA) was used to identify putatively linked markers with the b1 locus, these were then screened on the CDC Bethune x M96006 recombinant inbred line population. Furthermore, the CDC Bethune x M96006 and CDC Bethune x USDA-ARS Crystal recombinant inbred line populations were used to identify any important flax traits that had a significant relationship with seed coat color. It was shown that seed coat color conditioned by the b1 locus was stably inherited and that b1vg and b1 are allelic to one another. The results of the BSA showed that there were 17 candidates for linkage but when these markers were screened on the population only the Lu456 from linkage group (LG) six was identified to have linkage (χ²=3.90; P<0.05) with the b1 locus. Additionally, it was shown that the b1 seed coat color allele of the b1 locus had a pleiotropic effect on flower color and flower shape and that seed coat color was associated with linolenic fatty acid content. None of the traits examined were found to be associated with the b1vg allele of this locus. These results show that the b1 locus is likely present on linkage group six, more marker coverage on linkage group six of markers that are polymorphic between the two seed coat color parents would increase the accuracy of detection. Lastly, this study showed that plant breeders should consider using the b1vg allele that conditions the variegated seed coat color to mark unique lines with important combinations of traits because it sorted independently for seed quality traits. Whereas, the yellow seed coat color conditioned by the b1 allele was found to be associated with higher linolenic fatty acid content and the semi-lethality of this allele would make it not suitable for use in parental lines.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.
Dissertation

Chickpea (Cicer arietinum L.) was recently introduced to the Canadian prairies, a region which has a short growing season in which crop maturation often occurs under cool and wet conditions. To improve the yield of chickpea, crop duration must closely match the available growing season. The objectives of this study were to: i) examine the days to flowering of diverse chickpea accessions grown in either long or short-days; ii) examine the days to flowering of selected chickpea accessions grown in a range of thermal regimes combined with either long or short days and to examine the interaction between photoperiod and day and night temperatures on crop duration; iii) determine the timing and duration of the photoperiod-sensitive phase in selected chickpea accessions, and vi) determine the genetic basis of the association between flowering time and reaction to ascochyta blight in chickpea. A wide variation was observed in chickpea accessions for their response to flowering under long (16/8 hours day /night) and short days (10/14 hours day/night). Earlier flowering was observed under long photoperiod regimes compared with the short photoperiod regimes. Variability was detected among chickpea accessions for their flowering responses when different temperatures were combined with different photoperiods. Earlier flowering was observed under long days (16/8 hours day/night) coupled with high to moderate temperature regimes (24/16 ºC and 20/12 ºC, day and night respectively) compared to short-days (10/14 hours day and night) and moderate to low temperature regimes (20/12 ºC and 16/8 ºC day and night, respectively). Those chickpea accessions such as ICC 6821 and ICCV 96029 which originated from the lower latitudes of Ethiopia and India, respectively, flowered earlier compared to accessions such as CDC Corinne and CDC Frontier which originated from the higher latitudes and cooler temperate environments of western Canada. Photoperiod sensitivity phases were detected in chickpea accessions adapted to the cold environments of western Canada, whereas no photoperiod sensitivity phase was identified in the extra-early flowering cultivar ICCV 96029. The duration of the photoperiod sensitive phase in the chickpea accessions was longer under short days compared to long days. Field and growth chamber evaluation of a chickpea RIL population (CP-RIL-1) revealed the presence of variability among the lines and the two parents for their days to flowering and level of resistance to ascochyta blight. Broad sense heritability across different site-years for days to flower 0.45 to 0.78, plant height 0.48 to 0.78, ascochyta blight resistance 0.14 to 0.68, days to maturity 0.26, photoperiod sensitivity 0.83 and nodes number of first flowering 0.37 to 0.75 were estimated. Days to flower and photoperiod sensitivity were significantly r = -0.21 to -0.58 (P ≤ 0.05 to 0.001) and -0.28 to -0.41 (P ≤ 0.01 to 0.001), respectively and negatively correlated with ascochyta blight resistance in the CP-RIL-1 population. A genetic linkage map consisting of eight linkage groups was developed using 349 SNP markers. Seven QTLs were identified for days to flowering under growth chamber and field conditions on chromosomes 3, 5, 6 and 8 each and 3 QTLs on chromosome 4. The total phenotypic variation explained by QTLs for days to flowering ranged from 7 to 44%. Two QTLs for days to maturity were identified on chromosomes 3 and 8. Three QTLs, one each on chromosomes 3, 4 and 5 were identified for photoperiod sensitivity. The total phenotypic variation explained by each QTL for photoperiod sensitivity ranged from 7 to 41%. A total of three QTL for node of first flowering, one on chromosomes 3 and 8 each, and two on chromosome 4 were identified. The two QTL on chromosome 4 explained total phenotypic variations of 11 and 32%, respectively. Ten QTLs distributed across all chromosomes, except chromosomes 2 and 5, were identified for ascochyta blight resistance. The phenotypic variability explained by each QTL for ascochyta blight resistance ranged from 7 to 17%. The molecular markers associated with these QTLs have potential for use in chickpea breeding.