Association mapping of Stagonospora nodorum blotch resistance in modern European winter wheat varieties

Association mapping in populations relevant for wheat breeding has a large potential for validating and fine-mapping QTLs identified in F2- or DH (double haploid)-derived populations. In this study, associations between markers in the region of QSng.sfr-3BS, a major QTL for resistance to Stagonospora nodorum glume blotch (SNG), and SNG resistance were investigated by linkage and association analyses. After increasing marker density in 240 F5:7 recombinant inbred lines (RILs), QSng.sfr-3BS explained 43% of the genetic variance and peaked 0.6cM proximal from the marker SUN2-3B. Association between SNG resistance and markers mapped in the region of QSng.sfr-3BS was investigated in a population of 44 modern European winter wheat varieties. Two genetically distinct subpopulations were identified within these lines. In agreement with linkage analyses, association mapping by a least squares general linear model (GLM) at marker loci in the region of QSng.sfr-3BS revealed the highest association with SNG resistance for SUN2-3B (p<0.05). Association mapping can provide an effective mean of relating genotypes to complex quantitative phenotypes in hexaploid wheat. Linkage disequilibrium (r 2) in chromosome 3B extended less than 0.5cM in 44 varieties, while it extended about 30cM in 240 RILs, based on 91 SSR and STS marker-pair comparisons. This indicated that the association mapping population had a marker resolution potential at least 390-fold higher compared to the RIL populatio

Identification of a QTL on chromosome 7AS for sodium exclusion in bread wheat

Tese de doutoramento em Engenharia Física (Instrumentação), apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraO número de ciclotrões com capacidade para acelerar protões até cerca de 20 MeV tem vindo a aumentar em todo o mundo. Apesar de o objectivo principal das instalaçõoes que contêm estas máquinas ser a produção de radionuclídeos para tomografia por emissão de positrões (PET, do inglês positron emission tomography), algumas dessas instalações estão equipadas com várias linhas de feixe que podem ser adaptadas para investigação científica. Por exemplo, radiobiologia, radiofisiolgia e outros estudos de dosimetria podem ser realizados utilizando uma destas linhas de feixe devidamente adaptada para o efeito. Neste trabalho, uma das linhas de feixe do ciclotrão PET da Universidade de Coimbra foi complementada e instrumentada por forma a possibilitar a irradiação de um arranjo experimental com um feixe de protões de elevada qualidade. Esta nova linha de feixe foi construída de raíz e sem causar qualquer interferência com as demais linhas do ciclotrão, dedicadas à produção de radionuclídeos. São apresentados resultados tanto experimentais como de simulação, estes últimos obtidos através dos pacotes de simulação SRIM/TRIM e Geant4, tendo como objectivo a medição do pico de Bragg depositado pelo feixe de protões do ciclotrão PET, com uma energia nominal de 18 MeV. Utilizando um bloco de plástico cintilador e uma câmara fotográfica com ligação à internet da marca D-link foi possível medir tanto o alcance como a largura do feixe de protões com uma resolução espacial inferior a 0,1 mm. Os alcances do feixe medidos após este passar pelo meio de um tubo de alumínio com vácuo com 40 cm de comprimento e um segundo tubo com 2,4 m de comprimento permitiram confirmar que a energia efectiva do feixe é de 18 MeV. Mediu-se também o pico de Bragg utilizando um alvo constituído por várias folhas de alumínio intercaladas com lâminas de polietileno. O sinal de corrente foi recolhido das várias folhas de alumínio através de amplificadores de transimpedância fabricados no âmbito desta tese. Verificou-se que o pico de Bragg assim medido é consistente com simulações realizadas utilizando o pacote SRIM/TRIM. Após a instalação da linha de feixe no perímetro exterior do ciclotrão, esta foi caracterizada, calibrada e validada. Para tal, o sinal induzido pela passagem do feixe por uma folha de alumínio com 20 μm de espessura é lido através do amplificador de transimpedância mencionado. Este sinal amplificado providencia informação de dose em tempo real através de um programa desenvolvido em C/C++. Para além da dose, as principais variáveis de monitorização que este programa providencia incluem a corrente do feixe, a carga integrada em conjunto com a taxa de dose. Deste modo a dose e a corrente integrada (carga total) entregue até um dado instante na montagem experimental pode ser controlada por meio de um obturador controlado por computador. Feixes de protões com correntes tão baixas como 10 pA podem deste modo ser aferidas. A folha de alumínio foi escolhida por ser resistente à radiação, possuir baixa densidade e baixa probabilidade de radioactividade induzida pelo feixe e, finalmente, por representar um custo negligenciável. Junta-se a estas vantagens o facto de o método potenciar o cálculo da dose entregue a um alvo durante uma irradiação, com uma perda mínima da energia do feixe de protões, e com dispersão igualmente mínima. Resultados experimentais e simulações com o Geant4 são apresentados que revelam a aplicação, pela primeira vez, de um feixe de 18 MeV proveniente de um ciclotrão para irradiação de uma região seleccionada de um alvo. Fazendo uso do sistema de dosimetria apresentado no parágrafo anterior foi possível irradiar de modo homogéneo uma região circular com 18 mm de diâmetro. Torna-se assim possível irradiar culturas celulares localizadas em placas multi-poços com um diâmetro por poço de 16 mm, como é usual em experiências de radiobiologia. Verificou-se que o controlo do campo magnético aplicado dentro do ciclotrão é crucial para se obter uma irradiação uniforme em todo o campo do alvo. Para tal, efetua-se antes de cada irradiação e com o obturador fechado, um varrimento à corrente que gera o campo magnético dentro do ciclotrão, medindo-se um perfil quase gaussiano e tomando-se o seu valor central para se obter uma irradiação homogénea. As taxas de dose no alvo (entre 500 mGy/s e 5 mGy/s) são obtidas através de um disco em rotação posicionado na trajectória do feixe. O disco, com 150 mm de raio e uma fenda de 0,5 mm na sua extermidade, permite reduzir a taxa de dose por um factor de 5×10−4. Finalmente, vários filmes do tipo Gafchromic EBT2 foram expostos a diferente valores de dose por forma a validar toda a instalação para irradiação de um alvo com feixes de protões. Para tal validação fez-se uso do sistema de dosimetria em filme 2D do Serviço de Radioterapia do Centro Hospitalar Universitário de Coimbra. A dose absoluta nos filmes irradiados com protões foi verificada neste sistema e apresentou uma precisão melhor que 2%.The number of cyclotrons capable of accelerating protons to about 20MeV is increasing throughout the world. Originally aiming at the production of positron emission tomography (PET) radionuclides, some of these facilities are equipped with several beamlines suitable for scientific research. Radiobiology, radiophysiology, and other dosimetric studies can be performed using these beamlines. In this work, a PET cyclotron was fitted with a long beam transport line to irradiate with a good quality proton beam experimental setups. The beamline was configured as a natural extension of one of the cyclotron beam ports, while keeping available the other beam ports for PET radionuclides production. Experimental results are reported, together with SRIM/TRIM and Geant4 simulations, which aim at measuring the Bragg peak of the 18 -MeV proton beam from the PET cyclotron. By using a piece of plastic scintillator and a D-link Ethernet-based camera, the proton beam range and width were measured with a spatial resolution of 0.1mm. The ranges of the proton beam in the plastic scintillator were used to assess its energy after trespassing one or two Havar R windows and either a 40-cm-long or a 2.4-m-long aluminum pipe. The initial energy of the proton beam from the PET cyclotron was found to be 18 MeV. Additionally, the Bragg peak of the protons from the PET cyclotron was assessed using a stacked target consisting of several aluminum foils interleaved with polyethylene sheets, readout by in-house made transimpedance electronics. The measured Bragg peak is consistent with simulations performed using the SRIM/TRIM simulation toolkit. An out-of-yoke irradiation setup using the accelerated proton beam coming from the PET cyclotron was developed, characterized, calibrated, and validated. A 20-μm-thick aluminum transmission foil is readout by in-house made transimpedance electronics, providing online dose information via a C/C++ program. xvii Overview The main monitoring variables include beam current, integrated charge together with dose rate. The beam monitor is able to readout and deliver these variables in real-time. Hence the dose and integrated current (total charge) delivered upto a given instant to an experimental setup may be controlled via a computercontrolled shutter that was installed in the beam path. Proton beam currents down to 10 pA can be assessed using the thin aluminum foil. The aluminum was chosen for this task because it is radiation hard, it has low density and low radiation activity, and finally because it is easily available at negligible cost. In addition, this method allows for calculating the dose delivered to a target during an irradiation with high efficiency, and with minimal proton energy loss and scattering. Experimental results and Geant4 simulations are reported, which aim at using for the first time the 18 -MeV proton beam from a PET cyclotron to irradiate a selected region of a target using the developed dosimetry system. By using this system, a homogeneous beam spot on target with a diameter of 18mm can be controlled. This allows controlled irradiation of cell cultures located in typical biological multi-well dishes with diameters of 16mm each. It was found that the control of the magnetic field applied inside the cyclotron plays a major role for achieving said homogeneity. For that, scanning the magnet current and measuring the corresponding dose rate reveals a quasi-Gaussian shaped curve that must be known before any irradiation procedure (the final shutter is closed during such measurements). The optimum magnet current is taken from the center of the Gaussian-shaped curve, hence producing a homogenous dose on target. The measured dose rate on target ranges from 500mGy/s down to 5mGy/s. This is achieved with a 150mm radius rotating disk with a slit of 0.5mm width, that decreases target dose rates by a factor of 5 × 10−4. Several Gafchromic R EBT2 films were exposed to different values of dose for validating the developed irradiation setup using the 2D film dosimetry system of the Department of Radiotherapy of Coimbra University Hospital Center. The absolute dose in the irradiated films were assessed with a precision better than 2%. It is planned, in the near future, to irradiate small animals, cell cultures, or other materials or samples

QTL analysis of resistance to Fusarium head blight in Swiss winter wheat ( Triticum aestivum L.)

Fusarium head blight (FHB) of wheat is a widespread and destructive disease which occurs in humid and semi-humid areas. FHB epidemics can cause serious yield and quality losses under favorable climatic conditions, but the major concern is the contamination of grains with mycotoxins. Resistance to FHB is quantitatively inherited and greatly influenced by the environment. Its evaluation is costly and time-consuming. The genetic basis of FHB resistance has mainly been studied in spring wheat. The objective of this study was to map quantitative trait loci (QTLs) for resistance to FHB in a population of 240 recombinant inbred lines (RILs) derived from a cross between the two Swiss winter wheat cultivars Arina (resistant) and Forno (susceptible). The RILs were genotyped with microsatellite and RFLP markers. The resulting genetic map comprises 380 loci and spans 3,086cM. The 240 RILs were evaluated for resistance to FHB in six field trials over 3 years. Composite interval mapping (CIM) analyses carried out on FHB AUDPC (i.e. mean values across six environments) revealed eight QTLs which altogether explained 47% of the phenotypic variance. The three main QTLs were mapped on the long arms of chromosomes 6D (R 2=22%), 5B (R 2=14%) and 4A (R 2=10%). The QTL detected on 5B originated from the susceptible parent Forno. Other QTLs with smaller effects on FHB resistance were detected on chromosomes 2AL, 3AL, 3BL, 3DS and 5A

Dissection of quantitative and durable leaf rust resistance in Swiss winter wheat reveals a major resistance QTL in the Lr34 chromosomal region

The Swiss winter bread wheat cv. ‘Forno' has a highly effective, durable and quantitative leaf rust (Puccinia triticina Eriks.) resistance which is associated with leaf tip necrosis (LTN). We studied 240 single seed descent lines of an ‘Arina×Forno' F5:7 population to identify and map quantitative trait loci (QTLs) for leaf rust resistance and LTN. Percentage of infected leaf area (%) and the response to infection (RI) were evaluated in seven field trials and were transformed to the area under the disease progress curves (AUDPC). Using composite interval mapping and LOD>4.4, we identified eight chromosomal regions specifically associated with resistance. The largest and most consistent leaf rust resistance locus was identified on the short arm of chromosome 7D (32.6% of variance explained for AUDPC_% and 42.6% for AUDPC_RI) together with the major QTL for LTN (R 2=55.6%) in the same chromosomal region as Lr34 (Xgwm295). A second major leaf rust resistance QTL (R 2=28% and 31.5%, respectively) was located on chromosome arm 1BS close to Xgwm604 and was not associated with LTN. Additional minor QTLs for LTN (2DL, 3DL, 4BS and 5AL) and leaf rust resistance were identified. These latter QTLs might correspond to the leaf rust resistance genes Lr2 or Lr22 (2DS) and Lr14a (7BL

Detection of QTLs for Stagonospora glume blotch resistance in Swiss winter wheat

Stagonospora nodorum is the causal agent of the Stagonospora glume blotch disease in hexaploid wheat. The Swiss winter bread wheat cv. 'Arina' has a highly effective, durable and quantitative glume blotch resistance. We studied 240 single seed descent (SSD)-derived lines of an 'Arina × Forno' F5:7 population to identify and map quantitative trait loci (QTLs) for glume blotch resistance under natural infestation. Using composite interval mapping (CIM) and LOD>4.5, we detected two chromosomal regions on chromosome arms 3BS and 4BL which were specifically associated with glume blotch resistance. These identified QTLs were designated QSng.sfr-3BS and QSng.sfr-4BL, respectively. QSng.sfr-3BS peaked at the locus Xgwm389 in the telomeric region of the short arm of chromosome 3B and explained 31.2% of the observed phenotypic variance for the resistance within the population. The responsible QSng.sfr-3BS allele originated from the resistant parent 'Arina'. The QTL QSng.sfr-4BL (19.1%) mapped to chromosome arm 4BL ('Forno' allele) very close to two known genes, TaMlo and a catalase (Cat). Both QTL alleles combined could enhance the resistance level by about 50%. Additionally, they showed significant epistatic effects (4.4%). We found PCR-based microsatellite markers closely linked to QSng.sfr-3BS (gwm389) and QSng.sfr-4BL (gwm251) which make marker-assisted selection (MAS) for Stagonospora glume blotch resistance feasible. We also found one resistance QTL, QSng.sfr-5BL, on the long arm of chromosome 5B which overlapped with QTLs for plant height as well as heading tim

An integrative genetic linkage map of winter wheat ( Triticum aestivum L.)

We constructed a genetic linkage map based on a cross between two Swiss winter wheat (Triticum aestivum L.) varieties, Arina and Forno. Two-hundred and forty F5 single-seed descent (SSD)-derived lines were analysed with 112 restriction fragment length polymorphism (RFLP) anonymous probes, 18 wheat cDNA clones coding for putative stress or defence-related proteins and 179 simple-sequence repeat (SSR) primer-pairs. The 309 markers revealed 396 segregating loci. Linkage analysis defined 27 linkage groups that could all be assigned to chromosomes or chromosome arms. The resulting genetic map comprises 380 loci and spans 3,086cM with 1,131cM for the A genome, 920cM for the B genome and 1,036cM for the D genome. Seventeen percent of the loci showed a significant (P < 0.05) deviation from a 1:1 ratio, most of them in favour of the Arina alleles. This map enabled the mapping of QTLs for resistance against several fungal diseases such as Stagonospora glume blotch, leaf rust and Fusarium head blight. It will also be very useful for wheat genetic mapping, as it combines RFLP and SSR markers that were previously located on separate map