Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection.

Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Submitted by Valquíria Barbieri (kikibarbi@hotmail.com) on 2018-03-20T20:44:06Z No. of bitstreams: 1 DISS_2014_Solange Ossuna Fernandes.pdf: 5309319 bytes, checksum: 27262f2dd082e3af96047c24419b6983 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-04-10T19:02:31Z (GMT) No. of bitstreams: 1 DISS_2014_Solange Ossuna Fernandes.pdf: 5309319 bytes, checksum: 27262f2dd082e3af96047c24419b6983 (MD5) / Made available in DSpace on 2018-04-10T19:02:31Z (GMT). No. of bitstreams: 1 DISS_2014_Solange Ossuna Fernandes.pdf: 5309319 bytes, checksum: 27262f2dd082e3af96047c24419b6983 (MD5) Previous issue date: 2014-07-30 / CAPES / A couve-flor é considerada uma olerícola de grande importância econômica, especialmente para pequenos produtores, por ser uma cultura rentável em pequenas áreas. No Brasil, as principais regiões produtoras são Sudeste e Sul. O tamanho e a qualidade de sua cabeça são os aspectos mais importantes à sua comercialização, sendo influenciados pelos nutrientes absorvidos durante o ciclo da cultura, destacando-se, entre eles, o nitrogênio e o boro. O nitrogênio (N) é o nutriente que mais influencia o crescimento das plantas, pois está presente na composição das mais importantes biomoléculas, tais como ATP, NADH, NADPH, clorofila, proteínas e inúmeras enzimas. Em couve-flor, a deficiência deste nutriente influencia de forma significativa as variáveis vegetativas da planta, diminuindo o número de folhas, a altura da planta, o diâmetro da haste, a área foliar e a matéria seca. A fertilização nitrogenada acelera o desenvolvimento da planta, aumentando a produtividade e o diâmetro da cabeça de couve-flor, já que o N é responsável pelo desenvolvimento vegetativo rápido e vigoroso da planta, o que está associado ao bom desenvolvimento da inflorescência. O boro (B) é essencial para o crescimento celular, principalmente nas partes jovens da planta, como gemas, meristemas apicais e estruturas reprodutivas, sendo essencial também para a estrutura e função da parede celular, pois estabiliza a rede péctica e regula o tamanho do poro da parede celular. A adubação com B para brássicas no Brasil é recomendada com frequência, visto que os resultados existentes indicam resposta positiva para utilização deste micronutriente, que há muito já teve sua essencialidade comprovada, apesar de sua função primária não ter sido totalmente esclarecida. As doses de fertilizantes atualmente indicadas subestimam a real necessidade da cultura nas diferentes regiões produtoras do país, o que dificulta a obtenção de altas produtividades e padrões comerciais. Com isso, o objetivo deste estudo foi avaliar a influência de N e B no cultivo de couve-flor ‘Verona CMS’. / Cauliflower is considered a greenery of great economic importance, especially to smallholders, to be a culture profitable in small areas. In Brazil, the main producing regions are South and Southeast. Curd size and quality are the most important aspects of your marketing, being influenced by nutrients absorbed during the crop cycle, standing out, among then, nitrogen and boron. Nitrogen (N) is the nutrient that most affects plant growth, because it is present in the composition of the most important biomolecules, such as ATP, NADH, NADPH, chlorophyll, proteins and numerous enzymes. In cauliflower, N deficiency affects the vegetative plant variables, decreasing number of leaves, plant height, stem diameter, leaf area, and dry matter. Nitrogen fertilization accelerates the development of the plant, increasing yield and cauliflower curd diameter, whereas N is responsible for fast and vigorous vegetative developing, that is associated with good development of the blossom. Boron (B) is essential for cellular growth, mainly in young plant parts like buds, apical meristems, and reproductive structures, being essential for cell wall structure and function, because stabilizes pectic network and regulates de pore size of cell wall. Boron fertilization for Brassicacea in Brazil is often recommended, whereas an existents result indicates positive response for this micronutrient utilization, which has long since had its essentiality proven, although their primary function is not clarified. Fertilizers levels currently recommended underestimate the real need of culture in different places cultivation, making it difficult to obtain high productivity and trade patterns. Therefore, the aim of this study was evaluate N and B influence in the cultivation of cauliflower ‘Verona CMS’.

Made available in DSpace on 2015-03-26T13:36:42Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1139190 bytes, checksum: 7d7600e51fc96aab96c671593415f23b (MD5) Previous issue date: 2011-07-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Arsenic is the more dangerous element to human health. Phytoremediation is an important technology to reduce the antropogenic effect in the environmental contamination, but it is necessary a detailed knowledge of tolerance mechanism, which is the goal of this work. We choose Brassica juncea, tolerant plant to several heavy metals, such as cadmium, and a plant of rapid growth. The arsenate was added to nutritive solution at final concentrations of 10, 50, 200 and 500 μM, and data was collected after 6, 10, 13, 14, 15 and 16 days of exposition. Higher the arsenic in the treatment, higher the level found in leaves, stalk and in roots reach the highest level (5457 mg Kg-1). This amount was 26 and 34 times higher than that found in stalk and leaves, respectively. No alteration in phosphorus level was found at any treatment . This absence of phosphate uptake inhibition seems to have a contribution to the tolerance found in this genotype. In contrast, arsenic concentrations equal or higher than 50 μM were associated with increase in sulphur content in roots in parallel with an increase in total phenol content in these organs, while it reduced sulfur levels in leaves. The two highest doses of arsenate led to the appearance of toxicity symptoms in leaves, characterized by marginal chlorosis and purple coloring in the abaxial faces of old leaves. Despite the highest levels of arsenic in roots, no change in root length and dry weight was detected. In shoot however, we observed reduction in leaf area, leng th and fresh weight in plants treated with 200 and 500 mM arsenate. Additionally, only the highest dose has produced a reduction in net photosynthesis (A) and stomatal conductance (gS) by the 13th day. On the 14th day it was possible to observe the reduction of these parameters in plants treated with 200 mM arsenate. Longer periods in the presence of hight doses allowed us to observe that, besides the reduction in stomatal opening, arsenic could also produce biochemical limitation to photosynthesis, which was associated to reduction in electron transport rate (ETR) and in the quantic efficiency of photosystem II [Y(II)], concomitantly with increase in the level of energy loss as heat [Y(NPQ)]. The second higher dose (200 μM) has not altered the A and other fluorescence parameters, by the 13th day and only negligible effects in leaf area and dry mass in parallel to no effect in roots was observed. Altogether these data support that the genotype of Brassica juncea under study is tolerant to arsenic. Despite of accumulation of high levels of arsenic in roots, no oxidative damage was observed, and no effects in root growth was noted. The fact that high levels of arsenic has occurred in roots, with minor effects in growth indicate the great potential of this plant to be used in phytoremediation as a phytostabilizer. / O arsênio é considerado o elemento mais perigoso para a saúde humana. Fitorremediação é uma importante tecnologia para amenizar o efeito antropogênico na contaminação ambiental, mas para o seu sucesso é necessário a compreensão detalhada dos mecanismos de tolerância de metais pesados, objetivo deste trabalho. A espécie escolhida foi a Brassica juncea, tolerante a vários metais, como cádmio e zinco, e que possui um crescimento rápido. O arsênio foi fornecido a solução nutritiva na forma de arsenato de sódio nas concentrações de 10, 50, 200 e 500 μM, sendo coletados dados aos 6, 10, 13, 14, 15 e 16 dias após a exposição. Quanto maiores os níveis de arsênio presentes no tratamento, maiores os níveis encontrados na planta, havendo um acúmulo de até 5457 mg Kg-1 de As nas raízes expostas a 500 μM de arsenato. Esta quantidade foi 26 e 34 vezes superior aquela observada em caules e folhas, respectivamente. Em nenhum dos tratamentos foram observadas alterações na concentração de fósforo. Esta ausência de inibição de acúmulo de fósforo pode significar a presença de um mecanismo de tolerância à toxidez do arsênio neste genótipo. Em contraste, doses de arsênio iguais ou superiores a 50 μM aumentaram os níveis enxofre nas raízes, em paralelo com um aumento no teor de fenóis, ao passo que reduziu os níveis de enxofre na folha. As duas maiores doses de arsenato levaram ao aparecimento de sintomas de toxidez nas folhas, caracterizados por clorose marginal e coloração arroxeada nas faces abaxiais de folhas velhas. Apesar dos altos níveis de arsênio em raízes, não houve redução no comprimento e na massa seca deste órgão. Na parte aérea por outro lado, foi observada a redução na área foliar, comprimento e massa fresca em plantas tratadas com 200 e 500 μM de arsenato. Adicionalmente observou-se que somente a maior dose reduziu a fotossíntese líquida (A) e condutância estomática (gS) até o 13º dia. A partir do 14º dia já foi possível observar a queda destes parâmetros em plantas tratadas com 200 μM de arsenato. Maiores períodos na presença altas doses de arsenato permitiram observar que alem da redução da abertura estomática, há uma limitação bioquímica contribuindo para a redução da fotossíntese. A diminuição em A esteve associada a limitações na etapa fotoquímica onde ocorreu uma redução na taxa linear de transporte de elétrons (ETR) e na eficiência quântica do fotossistema II [Y(II)], concomitante com o aumento da perda da energia luminosa na forma de calor [Y(NPQ)]. A segunda maior dose (200 μM) não provocou redução em A e nos parâmetros fotoquímicos até o 13º dia, com reduções negligíveis na área e massa foliar, com nenhum efeito no crescimento das raízes. Em conjunto, estes resultados permitem evidenciar que o genótipo de Brassica juncea em estudo possui tolerância aos níveis de arsênio. Apesar de acumular níveis altíssimos de arsênio nas raízes, estas plantas não apresentaram dano oxidativo, nem redução na acumulação de massa seca nas raízes. O fato de que altos níveis de As seja acumulado nas raízes, com efeitos reduzidos no seu crescimento, indica o grande potencial desta espécie para o seu uso na fitorremediação como uma espécie fitoestabilizadora.

As formigas da tribo Attini são obrigatoriamente dependentes da simbiose que mantêm com fungos cultivados no interior de suas colônias.N as Attini mais derivadas (gêneros Atta e Acromyrmex)esse fungo é cultivado com substratos vegetais frescos cortados pelas formigas, constituindo a única fonte de nutrientes durante os estágios imaturos (larvas e pupas) e fonte parcial de nutrientes durante a fase adulta. N esta fase, as formigas obtêm grande parte de seus nutrientes através da ingestão de seivas foliares durante o corte e o processamento do materiala ser incorporado ao fungo.Assim,dado o duplo destino que os produtos vegetais têm nessa relação simbiótica entre fungos e formigas, os metabólitos secundários contidos nas plantas cortadas pelas formigas são também capazes de atuar nessas duas instâncias.D entre os metabólitos secundários, os glucosinolatos são conhecidos por intermediarem muitas das interações das plantas que os contêm e por possuírem um mecanismo de ação envolvendo a hidrólise pela enzima mirosinase. O presente estudo avaliou os efeitos da sinigrina sobre o crescimento radialdo fungo mutualista de Atta sexdens e sobre a mortalidade das operárias isoladas das colônias.Para isso os fungos foram cultivados in vitro em meio de cultura M EA-LP contendo sinigrina e incubados a 25° C em BO D .A mortalidade dos insetos foi avaliada de duas maneiras:com o oferecimento sinigrina em dietas sólidas ou em solução aquosa.O s resultados mostraram que a sinigrina intacta não apresentou efeitos sobre qualquer um dos dois. Porém, apresentou indícios de que os produtos de hidrólise podem vir a inibir o crescimento do fungo. Também foram analisadas as composições de glucosinolatos em plântulas de 6 variedades de B. oleraceae utilizando-se Cromatagrafia Líquida de Alta Eficiência com detecção em U V (CLAE) e em espectrômetro de massas (CLAE/M S).Dez glucosinolatos foram encontrados,dentre os quais glucoiberina, sinigrina e progoitrina foram os mais abundantes. O s resultados também apontaram que, apesar de se tratarem da mesma espécie, o conteúdo de glucosinolatos é bastante distinto entre as variedades. Entretanto, as amostras da mesma variedade mostraram um padrão relativamente bem estável, apresentando sempre os mesmos glucosinolatos em proporções relativas semelhantes. / vide dissertação

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study was to evaluate the agronomic and ecophysiological responses of canola cultivars grown under water excess in the soil, determining which crop stages are more sensitive, the length time of water excess tolerated and the tolerant cultivars. Four experiments were installed, two field and two in the greenhouse. Field experiments were conducted in two crop years (2014 and 2015), in Universidade Federal de Santa Maria, Santa Maria, RS, in a randomized block design, with split plot design, in a factorial 2 x 3, with factors drainage (with drain and no drain) and canola cultivars (Hyola 433, Hyola 411 and Hyola 61), with four replications. Were quantified plant height, number of leaves, leaf area, thermal time length of the development subperiods, leaf conductance, photosynthetic ratio and grain yield components. The experiments in greenhouse were conducted in Santa Maria and São Vicente do Sul, in 2015, in a factorial completely randomized design 4 x 5 in Santa Maria and with an additional treatment 5 x 4 + 1 in São Vicente do Sul, whose factors were phenological stages and time length with soil water excess (0, 24, 48, 96 and 192 h), with 3 replications. Were quantified the index of emergency velocity, emergency rate and yield components. The water excess in the soil causes morphological, phenological, physiological and productive changes in the canola crop. In the sowing-emergence subperiod, the soil water excess for a long time reduces the plant population. After the crop emergence, the rosette formation and onset of anthesis are the most critical stages to the canola crop. The plant population and the number of siliques per plant are the variables that most influence the productivity of canola grain in areas with water surplus. The use of drains is effective for the establishment and development of the canola in areas or periods with water excess. The length time that the canola crop supports water excess without reducing grain yield depends on the phenological stage, however, 24 h of water excess can reduce emergency velocity index, number of siliquas per plant, dry mass of shoots and grain yield. Productivity is most affected when the water excess occurs in the rosette formation and in onset of anthesis. There is a decreasing relationship between the increase in water surplus period and grain yield of the canola crop. The Hyola 411 and Hyola 433 cultivars may be promising for cultivation in areas with water excess. The Hyola 61 cultivar has productive potential, however, seems to be more sensitive to water excess in the soil. / Objetivou-se com esse trabalho avaliar as respostas agronômicas e ecofisiológicas de cultivares de canola submetidas ao excesso hídrico no solo, determinando-se quais os estádios de maior sensibilidade da cultura, o período de tempo com excesso hídrico tolerado e cultivares mais tolerantes. Quatro experimentos foram instalados, dois a campo e dois em casa de vegetação. Os experimentos de campo foram realizados em dois anos agrícolas, 2014 e 2015, na Universidade Federal de Santa Maria, Santa Maria, RS, em delineamento em blocos casualizados, com parcelas subdivididas, em esquema fatorial 2 x 3, sendo os fatores, dreno (com dreno e sem dreno) e cultivares de canola (Hyola 433, Hyola 411 e Hyola 61), com quatro repetições. Determinou-se altura de plantas, número de folhas, área foliar, duração dos subperíodos de desenvolvimento, condutância foliar, taxa fotossintética e componentes de rendimento de grãos. O experimento de casa de vegetação foi conduzido no ano de 2015, em delineamento inteiramente casualizado em esquema fatorial 4 x 5 em Santa Maria, RS, e fatorial com tratamento adicional 5 x 4 + 1 em São Vicente do Sul, RS, sendo os fatores estádios fenológicos e 0, 24, 48, 96 e 192 h de excesso hídrico contínuo, com 3 repetições. Determinou-se o índice de velocidade de emergência, porcentagem de emergência e componentes de rendimento. O excesso hídrico no solo causa alterações morfológicas, fenológicas, ecofisiológicas e produtivas na cultura da canola. No subperíodo semeadura-emergência, o excesso hídrico no solo por períodos prolongados resulta na redução da população de plantas. Após a emergência das plantas, os estádios de formação de roseta e início da antese são os mais críticos para a cultura da canola. A população de plantas e o número de síliquas por planta são as variáveis que mais influenciam a produtividade de grãos da canola em locais com excesso hídrico. O uso de drenos é eficiente para permitir o estabelecimento e desenvolvimento da canola em condições de excesso hídrico. O tempo em que a canola suporta o excesso hídrico sem comprometer a produtividade de grãos depende do estádio fenológico, porém, 24 h de excesso hídrico são capazes de reduzir a velocidade de emergência, o número de síliquas por planta, a massa seca da parte aérea e a produtividade de grãos. A produtividade de grãos é mais prejudicada quando o excesso hídrico ocorre na formação de roseta e no início da antese. Existe uma relação decrescente entre o aumento no período de excesso hídrico e a produtividade de grãos na cultura da canola. As cultivares Hyola 411 e Hyola 433 podem ser promissoras para cultivo em locais com excesso hídrico. A cultivar Hyola 61 tem potencial produtivo, porém, parece ser mais sensível ao excesso hídrico no solo.

Leptosphaeria maculans est l'agent de la nécrose du collet des crucifères, principale maladie fongique du colza (Brassica napus). Lorsque des variétés de colza possédant la résistance Rlm7 ont été commercialisées au début des années 2000, la totalité des souches européennes étaient avirulentes vis-à-vis de ce gène de résistance. A l'inverse, AvrLm3 était considéré comme absent de ces populations. Un précédent projet de thèse réalisé dans l'équipe a montré la rapide capacité d'adaptation de L. maculans à la pression de sélection exercée par Rlm7 (Daverdin et al. 2012) et le phénotypage des populations isolées lors de cette étude avait montré que 98% des souches virulentes vis-à-vis de Rlm7 étaient avirulentes vis-à-vis de Rlm3. L'hypothèse d'un masquage du phénotype avirulent dû à la présence d'AvrLm4-7 a alors été validée par la complémentation d'une souche avirulente vis-à-vis de Rlm3 par AvrLm4-7. Sur ces bases, l'objectif de mon projet de thèse était d'identifier AvrLm3 afin d'analyser l'antagonisme entre les phénotypes AvrLm3 et AvrLm4-7, ainsi que les mécanismes menant au contournement de Rlm3 et Rlm7.Le gène AvrLm3 avait été identifié comme génétiquement lié à AvrLm4-7. Toutefois il n'avait pas été possible d'identifier un gène candidat. Par la combinaison d'approches de génétique et de génomique (clonage positionnel, RNA-seq, séquençage de novo d'une souche avirulente et de clones BAC), j'ai pu identifier AvrLm3, dont la séquence était absente du génome de référence de L. maculans. AvrLm3 est très fortement exprimé lors des phases précoces de l'infection et code pour une petite protéine sécrétée et ne présentant pas d'homologies avec d'autres protéines fongiques. Ce gène a donc les caractéristiques classiques des gènes codant pour des effecteurs, mais présente la particularité d'être le premier gène d'avirulence de L. maculans localisé en région télomérique. Bien que le mécanisme par lequel la présence du gène AvrLm4-7 supprime la reconnaissance d'AvrLm3 dans l'interaction entre B. napus et L. maculans n'ait pas été élucidé au terme de ma thèse, plusieurs hypothèses ont pu être invalidées, notamment l'absence de régulation de l'expression d'AvrLm3 par AvrLm4-7. De plus, une approche double hybride a mis en évidence une absence d'interaction directe entre les deux protéines d'avirulence. L'isolement de souches de L. maculans issues de parcelles expérimentales en 2012 et 2013 a mis en évidence le contournement actuel de la résistance Rlm7, avec environ 10 % de souches virulentes. Le phénotypage de ces collections a confirmé la présence majoritaire d'un allèle avirulent d'AvrLm3 chez celles-ci, moins de 0,5 % des souches isolées étant virulentes à la fois vis-à-vis de Rlm3 et de Rlm7. L'analyse de 592 souches d'origine mondiale a montré qu'AvrLm3 est toujours présent chez L. maculans, sous diverses formes alléliques, suggérant un rôle majeur d'AvrLm3 dans la fitness fongique. La résurgence du phénotype avirulent vis-à-vis de Rlm3 suite au contournement de Rlm7 fait envisager l'opportunité d'exploiter l'interaction entre les deux gènes d'avirulence pour une gestion plus durable des gènes de résistance de B. napus, par l'alternance de cultivars possédant Rlm3 et Rlm7 ou le pyramidage de ces deux gènes dans des variétés de colza. De plus, le rôle démontré d'AvrLm3 et d'AvrLm4-7 dans l'agressivité lors de l'infection suppose un fort coût de fitness lié à la virulence. Toutefois, des mécanismes originaux de compensation permettant au champignon d'échapper à la reconnaissance par Rlm3 et Rlm7 tout en conservant a priori fonctionnelle la fonction effectrice d'AvrLm3 et AvrLm4-7 ont été identifiés. Les résultats obtenus lors de ce travail de thèse ont permis la meilleure caractérisation d'une interaction gène-pour-gène inhabituelle. La diversité des mécanismes moléculaires permettant à L. maculans de contourner la résistance Rlm3 illustre la complexité de la course aux armements entre les plantes et les agents pathogènes. / Leptosphaeria maculans is a Dothideomycete responsible for stem canker on oilseed rape (Brassica napus). Genetic control, encompassing mostly the use of major resistance genes, is the most effective method to control this pathogen. When the first cultivars harboring Rlm7 have been deployed in the early 2000's, all of the European isolates were avirulent towards this resistance gene and AvrLm3 was considered to be absent from the populations. In 2012, Daverdin et al. showed that L. maculans has the ability to rapidly overcome the Rlm7 resistance whenever a strong selection pressure is applied. The phenotyping of isolates sampled by Daverdin et al. also showed that more than 98 % of the isolates virulent towards Rlm7 had become avirulent towards Rlm3. This result led to the hypothesis that the presence of AvrLm4-7 can suppress AvrLm3 recognition by Rlm3 and this was validated by the complementation of an isolate avirulent towards Rlm3 with a functional allele of AvrLm4-7. AvrLm3 is genetically linked to AvrLm4-7, at a distance of 20 cM. However, no candidate gene was identified before the beginning of my PhD. The objective of my PhD project was to identify AvrLm3, in order to better understand the antagonistic relationship between AvrLm3 and AvrLm4-7 phenotypes. The combination of genetic and genomic approaches (genetic mapping, RNA-seq, de novo sequencing of an isolate avirulent towards Rlm3 and BAC clone sequencing) allowed me to identify AvrLm3, whose sequence was absent from the reference genome assembly. AvrLm3 has common characteristics with others genes encoding fungal effector: it codes for a small, cysteine-rich protein, is highly expressed at early infection stages and shows no homology with others fungal genes. Although the mechanism allowing the suppression of AvrLm3 recognition due to the presence of AvrLm4-7 has not been elucidated, several hypotheses were invalidated: the presence of AvrLm4-7 has no impact on AvrLm3 expression and the two avirulence proteins do not interact physically.The sampling of field isolates in 2012 and 2013 showed the currently ongoing breakdown of Rlm7, with ca. 10% of virulent isolates, and the resurgence of the AvrLm3 phenotype in these populations, only 0.5 % of isolates being virulent towards both Rlm3 and Rlm7. A large collection of isolates from worldwide origin was genotyped for AvrLm3. It revealed that all isolates possess AvrLm3, with a high level of allelic diversity. The resurgence of the avirulent phenotype towards Rlm3 following the breakdown the Rlm7, along with the important role of AvrLm3 and AvrLm4-7 in fungal fitness, suggest that the antagonistic relationship between these two avirulence phenotypes could be an opportunity to propose original strategies to increase the durability of Rlm3 and Rlm7, by alternating both resistance genes at the landscape level or using pyramiding strategies. However, we identified novel isoforms of AvrLm4-7 allowing the fungus to escape Rlm7 recognition, while maintaining the suppression of Rlm3 recognition effective. This work allowed to better characterize an unusual gene-for-gene relationship. Indeed, only one other example of antagonism between avirulence phenotypes has been identified to date, in the phytopathogenic fungus Fusarium oxysporum (Houterman et al., 2008). The diversity of mechanisms allowing L. maculans to overcome the Rlm3 resistance illustrates perfectly the complexity of the arms race between plants and pathogens.

La recombinaison méiotique produite par les Crossing Overs (COs) est un facteur limitant pour l’efficacité de la sélection variétale. Une possibilité pour produire des plantes hyper-recombinantes serait d’exploiter la variabilité intraspécifique pour les fréquences de recombinaison. L’identification des polymorphismes causaux, liés à la séquence ou l’expression, représente un travail de longue haleine. Une approche alternative serait de produire des mutants pour des régulateurs négatifs des fréquences de recombinaison. Chez le colza, jeune allotétraploïde (AACC, 2n=38), il est possible de jouer sur ces 2 approches. Dans un premier temps j’ai cherché à vérifier dans quelle mesure pouvait varier le transcriptome méiotique entre 2 variétés ayant servi à cartographier un QTL pour le contrôle de la recombinaison entre chromosome homoéologues (hérités des génomes parentaux). Ce transcriptome méiotique s’est révélé de façon inattendue très variable ; les principales sources de cette variation étant notamment la nature du génome (A ou C) ainsi que l’effet variété. J’ai montré que les HEs (le remplacement d’une région chromosomique par la duplication de la région homoéologue) contribuent de façon importante aux différences d’expression observées à la fois entre variétés ou au sein d’un même génotype. Dans un second temps, j’ai vérifié que FANCM décrit chez Arabidopis thaliana comme un régulateur négatif pour les fréquences de recombinaison avait bien la même fonction chez les Brassica. Chez Brassica rapa j’ai vérifié qu’un mutant fancm complémente comme attendu un mutant déficient pour la voie majoritaire de formation des COs. Chez Brassica napus j’ai observé une faible augmentation à la fois des fréquences de recombinaison entre chromosomes homologues et homoéologues. Ce travail souligne l’importance de la caractérisation des HEs chez les allopolyploïdes. Au-delà de leurs impacts sur le contenu et l’expression génique, les HEs ont très certainement des conséquences phénotypiques. Cette étude présente aussi un exemple de biologie translationnelle pour un trait important en amélioration des plantes. / Meiotic recombination driven by Crossing-Over (CO) is a limiting factor for the efficiency of plant breeding. One way to produce hyper-recombinant plants is to use the existing interspecific variability for recombination frequencies. Identification of the causal polymorphisms, either link to gene sequence or expression, represents a long-term endeavour. Another possibility is to mutate anti-meiotic CO genes. In rapeseed, a young allotetraploid species (AACC, 2n=38), both of these approaches are possible. First I wanted to check how much varies the meiotic transcriptome between 2 varieties that differ in term of recombination between homoeologous chromosomes (inherited from parental genomes). Unexpectedly, the meiotic transcriptome turned out to be very variable, the main source of this variation being notably the origin of the genome (A or C) and the variety. I also showed that homoeologous exchanges (HEs; the replacement of one chromosomal region with a duplicate of the homeologous region) contributed to this variation and led to large changes in expression both between and within varieties. Then I assessed whether FANCM, an anti-CO protein identified in Arabidopis thaliana had the same function in the Brassica genus. In Brassica rapa, a fancm mutant complements as expected a meiosis mutant defective in the main formation pathway for the formation of meiotic COs. In Brassica napus, I observed a slight increase in both homologous and homoeologous recombination frequencies. This work emphasizes the importance of characterizing HEs in allopolyploids species. Beyond their impact on gene content and expression, HEs most have likely phenotypic consequences. This study also presents an example of translational biology for an important trait in crop breeding.

Tesis por compendio / [ES] El incremento en la concentración de sales en suelos y agua es una de las problemáticas que más amenazan a la humanidad por su efecto directo sobre la productividad de las cosechas. En líneas generales la pregunta a la que queremos contribuir es ¿cómo enfrentarnos a una situación de salinización en un contexto de cambio climático y crecimiento demográfico? Más concretamente estudiamos la problemática en el sureste español desde un enfoque en que los resultados puedan ser explotados en el corto y medio plazo por el sector empresarial. La Región de Murcia es una de las principales zonas productoras del sureste español donde además existen graves problemas de salinización. En la presente tesis doctoral se propone utilizar aguas salinas para riego e investigar las implicaciones de ello a nivel de absorción de agua y nutrientes, estudiando también los cambios fisiológicos en la planta para obtener aplicaciones que permitan el crecimiento económico. En este sentido se abordan dos estrategias: por un lado, el estudio de plantas halofitas, y por otro, la bioestimulación en plantas glicofitas. La mayoría de los cultivos productivos son plantas glicofitas, ven afectadas sus producciones cuando hay elevadas concentraciones de sal en el medio de cultivo. Por el contrario, las plantas halofitas han desarrollado diferentes mecanismos para resistir los efectos negativos de la salinidad. Es por ello que la explotación directa de halofitas se ha identificado como una salida económica para las zonas afectadas. Otra forma de mitigación de los efectos del cambio climático es el desarrollo de la bioestimulación para incrementar la resistencia a estreses o para incrementar la concentración de compuestos bioactivos en los alimentos. Para los estudios realizados, se seleccionaron 6 especies vegetales que fuesen representativas de los diferentes comportamientos frente a la salinidad, que fuesen propias de la localización seleccionada (Región de Murcia) y de interés para la empresa promotora del proyecto de investigación (Sakata Seed Ibérica). Las especies estudiadas fueron: Brassica oleracea L., Brassica rapa L., Cakile maritima, Salicornia fruticosa y Atriplex halimus. El primer objetivo se centró en el análisis de los efectos de la salinidad sobre las especies objeto de estudio. Más concretamente intentando identificar rasgos que pudiesen servir para discriminar entre especies y/o cultivares las mejores respuestas a la salinidad. Por un lado, las interrelaciones entre distintos rasgos fisiológicos en respuesta a la salinidad se analizaron estadísticamente mediante un Discriminant Canonical Analysis (DCA), en el que se observó una relación entre el crecimiento y la suculencia de las hojas como medida de adaptación a la salinidad. Patrones como la acumulación de iones, el contenido en proteínas y ácidos grasos insaturados en respuesta a la salinidad pueden emplearse para discriminar entre distintas respuestas al estrés salino. Por otro lado, se estudió el efecto de la salinidad sobre la concentración y el perfil de glucosinolatos de distintos cultivares de brócoli (Brassica oleracea L.) en distintos periodos productivos. También se observó el efecto estimulador de los estreses como la temperatura y la salinidad sobre el perfil de glucosinolatos y la posibilidad de inducir complejos de glucosinolatos con fenólicos que no habían sido descritos previamente en Brassicales. El segundo objetivo se centró en la búsqueda de formas de explotación económica de las distintas respuestas fisiológicas. Por un lado, se desarrolló una metodología que permitió incrementar el contenido en glucosinolatos de las plantas de brócoli, sin que se viese en detrimento ni la producción ni los parámetros organolépticos del órgano comercial, lo cual fue posible mediante la aplicación de metil jasmonato y un polieter polisiloxano de forma foliar. Por otro lado, en las especies halofitas se analizó su aptitud par / [CA] L'augment de la concentració de sals en sòls i aigua és un dels problemes que més amenacen la humanitat pel seu efecte directe sobre la productivitat dels cultius. En termes generals, la qüestió a la qual volem contribuir és: com afrontar una situació de salinització en un context de canvi climàtic i creixement de la població? Més concretament, hem estudiat els problemes del sud-est d'Espanya des d'un enfocament en què els resultats poden ser explotats a curt i mitjà termini pel sector empresarial. La Regió de Múrcia és una de les principals zones productores del sud-est d'Espanya on també hi ha greus problemes de salinització. En aquesta tesi s'utilitzen aigües salines per al reg i s'investiguen les implicacions que això comporta a nivell d'absorció d'aigua i nutrients, estudiant també els canvis fisiològics en la planta per obtenir aplicacions que permetin el creixement econòmic. En aquest sentit s'aborden dues estratègies: d'una banda, l'estudi de les plantes d'halòfites i, de l'altra, la bioestimulació en plantes glicòfítes. La majoria dels cultius productius són plantes glicòfites, la seva produccions es veu afectada quan hi ha altes concentracions de sal en el medi de creiximent. Per contra, les plantes halófites han desenvolupat diferents mecanismes per resistir els efectes negatius de la salinitat. És per això que l'explotació directa d'halòfits s'ha identificat com una sortida econòmica per a les zones afectades. Una altra forma de mitigació dels efectes del canvi climàtic és el desenvolupament de bioestimulació per augmentar la resistència a estrès o augmentar la concentració de compostos bioactius en els aliments. Per als estudis realitzats en aquesta tesi doctoral es van seleccionar 6 espècies vegetals representatives dels diferents comportaments contra la salinitat, típics de la ubicació seleccionada (Regió de Múrcia) i d'interès per a l'empresa promotora del projecte de recerca (Sakata Seed Ibérica). Les espècies estudiades van ser: Brassica oleracea L., Brassica rapa L., Cakile maritima, Salicornia fruticosa i Atriplex halimus. El primer objectiu es va centrar en l'anàlisi dels efectes de la salinitat sobre les espècies objecte d'estudi. Més específicament tractant d'identificar trets que podrien servir per discriminar entre espècies i/o cultivars amb les millors respostes a la salinitat. D'una banda, les interrelacions entre diferents trets fisiològics en resposta a la salinitat van ser analitzades estadísticament per una Anàlisi Canònica Discriminant (DCA), en la qual s'observava una relació entre el creixement i la suculència de les fulles com a mesura d'adaptació a la salinitat. Patrons com l'acumulació d'ions, el contingut de proteïnes i àcids grassos insaturats en resposta a la salinitat es poden utilitzar per discriminar entre diferents respostes a l'estrès salí. D'altra banda, s'ha estudiat l'efecte de la salinitat en la concentració i perfil de glucosinolats de diferents cultius de bròquil (Brassica oleracea L.) en diferents períodes de producció. També vam observar l'efecte estimulant d'estrès com la temperatura i la salinitat en el perfil dels glucosinolats i la possibilitat d'induir complexos glucosinolatats amb fenòlics que no s'havien descrit prèviament en brassicales. El segon objectiu es va centrar en la recerca de formes d'explotació econòmica de les diferents respostes fisiològiques. D'una banda, es va desenvolupar una metodologia que permetia augmentar el contingut de glucosinolat de plantes de bròquil, sense perjudici ni de la producció ni dels paràmetres organolèptics de l'òrgancomercial, que va ser possible mitjançant l'aplicació de jasmonat de metil i un polieter de polisiloxà de forma foliar. D'altra banda, per a les espècies d'halòfits, es va realitzar una anàlisi per determinar la seva idoneïtat per a possibles usos tant culinaris com industrials. / [EN] The increase in the concentration of salts in soils and water is one of the problems that most threatens humanity due to its direct effect on crop productivity. In general terms, the question we want to contribute to is how to deal with salinization in the context of climate change and population growth? More specifically, we study the problems in south-eastern Spain from an approach in which the results can be exploited in the short and medium-term by the business sector. The region of Murcia is one of the main producing areas in south-eastern Spain where there are also serious salinization problems for various reasons. In this doctoral thesis, it is proposed to use saline water for irrigation and to investigate the implications that this entails at the level of water and nutrient absorption, also studying the physiological changes in the plant to obtain applications that allow economic growth. In this sense, two strategies are approached: on the one hand, the study of halophytic plants, and on the other hand, biostimulation in glycophyte plants. Most of the productive crops are glycophyte plants, their productions are affected when there are high concentrations of salt in the culture medium. On the contrary, halophyte plants have developed different mechanisms to resist the negative effects of salinity. That is why the direct exploitation of halophytes have been identified as an economic outlet for the salt-affected areas. Another way of mitigating the effects of climate change is the development of biostimulation to increase resistance to stresses or to increase the concentration of bioactive compounds in food. For the studies carried out in this doctoral thesis, 6 plant species were selected that were representative of the different behaviours towards salinity, that were specific to the selected location (The region of Murcia), and of interest to the company promoting the research project (Sakata Seed Ibérica). The species studied were: Brassica oleracea L., Brassica rapa L., Cakile maritima, Salicornia fruticosa, and Atriplex halimus. The first objective of this thesis focused on the analysis of the effects of salinity on the species understudy. More specifically trying to identify traits that could serve to discriminate between species and/or cultivars that best respond to salinity. Moreover, the interrelationships between different physiological traits in response to the salinity were statistically analysed using a Discriminant Canonical Analysis (DCA), in which a relationship between growth and succulence of the leaves were observed as a measure of adaptation to salinity. Patterns such as ion accumulation, protein content, and unsaturated fatty acids in response to salinity can be used to discriminate between different responses to salt stress. On the other hand, the effect of salinity on the concentration and profile of glucosinolates of different cultivars of broccoli (Brassica oleracea L.) indifferent productive periods were also studied. The stimulating effect of stresses such as temperature and salinity on the glucosinolate profile and the possibility of inducing glucosinolates with phenolics complexes that had not been previously described in Brassicales were also observed. The second objective focused on the search for forms of economic exploitation of the different physiological responses. On the one hand, a methodology was developed that allowed increasing the glucosinolate content of broccoli plants, without affecting the production or any organoleptic parameter of the commercial organ. This was possible through the application of methyl jasmonate and a polyether polysiloxane in foliar form. On the other hand, for the halophyte species, an analysis was carried out to determine their aptitude for possible culinary and industrial uses. / This work was funded by the Spanish Ministerio de Ciencia, Innovación y Universidades (AGL2016-80247-C2-1-R and RTC-2015-3536-2). / Agudelo Sánchez, A. (2021). Absorción de agua y nutrientes y respuesta fisiológica de plantas halofitas y glicofitas bajo condiciones de estrés salino [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/177647 / TESIS / Compendio