Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-27T12:03:27Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_SilviaPereira..pdf: 1700069 bytes, checksum: de95e5af421e528465694aadb5cdee58 (MD5) / Made available in DSpace on 2017-07-27T12:03:27Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_SilviaPereira..pdf: 1700069 bytes, checksum: de95e5af421e528465694aadb5cdee58 (MD5) Previous issue date: 2016-02-23 / CAPES / Adaptações e mecanismos de tolerância à seca são fatores fundamentais para a sobrevivência das espécies vegetais de clima árido ou semiárido. É devido a adaptações como área foliar, profundidade de raízes, manutenção do status hídrico, espessura da cutícula e densidade estomática que tais espécies obtêm sucesso neste ambiente. Contudo, diante das mudanças climáticas há a necessidade de avaliação da eficiência desses mecanismos diante de uma condição ainda mais limitante. Dessa forma, nosso objetivo foi analisar os principais mecanismos fisiológicos de tolerância a seca de Cynophalla flexuosa (L.) J.Presl., uma espécie lenhosa e sempre verde de uma floresta tropical sazonalmente seca, avaliando de que forma a cera epicuticular (CE) das folhas dessa espécie são eficientes diante das condições do seminário em diferentes épocas do ano. O trabalho foi realizado em três períodos (julho de 2014, dezembro de 2014 e fevereiro de 2015), ao longo de um dia em uma área de Caatinga, utilizando folhas intactas e com parte da CE removida mecanicamente. Foi calculado o balanço hídrico do solo e mensurado o potencial hídrico foliar, trocas gasosas, fluorescência da clorofila a, bioquímica e anatomia foliar. Foi observado que C. flexuosa reduz seu potencial hídrico foliar nos horários mais quentes, sendo uma espécie anisohídrica, capaz de manter sua condutância estomática mesmo diante de um balanço hídrico negativo do solo em todos os períodos de avaliação. A concentração da CE foi maior no mês de fevereiro e seu principal componente foram as cadeias de n-alcanos, compostos muito eficientes em manter a impermeabilidade da cutícula e proteger as folhas. O desempenho fotossintético não mostrou alterações ao longo do dia em folhas que tiveram a CE removida, além da manutenção do metabolismo bioquímico, com poucas variações nos diferentes períodos avaliados. Diante disso, C. flexuosa possui eficientes mecanismos de adaptação à seca, podendo suportar ambientes ainda mais limitantes, como o previsto para as próximas décadas devido às mudanças climáticas. / Adaptations and drought tolerance mechanisms are key factors for the survival of plant species in arid or semi-arid climate. It is due to adaptations such as leaf area, depth of roots, maintenance of water status, cuticle thickness and stomatal density these species succeed in this environment. However, given the climate change there is a need of efficiency evaluation of these mechanisms before a further limiting condition. Therefore, our objective was to analyze the main physiological mechanisms of drought tolerance of Cynophalla flexuosa (L.) J.Presl., a woody and evergreen specie in a seasonally dry tropical forest, assessing how the leaves epicuticular wax (EW) of this species are efficient before the semiarid conditions in different seasons. The study was conducted in three periods (July 2014, December 2014 and February 2015), throughout the one day in an area of Caatinga, using intact leaves and the EW removed mechanically. We calculated the the hydric balance of the soil and measured leaf water potential, gas exchange, chlorophyll fluorescence, biochemistry and leaf anatomy. It was observed that C. flexuosa reduces its leaf water potential at the hottest times, one anisohydric species, able to maintain its stomatal conductance in the face of a negative soil water balance in all evaluation periods, which reduced its xylem potential. The concentration of EW was higher in the month of February and its main component was n-alkane chains, very efficient compounds in maintaining a waterproof cuticle and protect the leaves. The photosynthetic performance showed no change throughout the day in leaves that had the EW removed, as well as maintenance the biochemical metabolism, with few variations in different periods. Therefore, C. flexuosa has efficient mechanisms of adaptation to drought and can withstand even more limiting environments, as predicted in the coming decades due to climate change.

Sweetpotato (Ipomoea batatas (L.) Lam.) is an important food crop in East Africa including Kenya. The crop incurs high yield losses in production due to biotic (insect pests and diseases) and abiotic (drought, and heat) constraints. Among abiotic constraints, drought is the most important. Prolonged periods of drought in arid and semi-arid areas of Kenya have led to reduced quantity and quality of sweetpotato storage roots and in severe cases caused total crop failure. The objectives of this study were to: 1) determine sweetpotato production system constraints and farmers’ coping strategies; 2) evaluate sweetpotato clones for yield performance and drought tolerance; 3) analyse genotype x environment interaction and stability for storage root yield of selected clones, 4) determine mechanisms of drought tolerance in sweetpotato, and 5) determine combining ability and heterosis for yield and drought tolerance traits under managed drought stress conditions. To determine the production constraints and farmers’ coping strategies, a survey was conducted in central, eastern and western Kenya. Out of 345 farmers interviewed, 60% were women, and 40% men. Farm sizes ranged from 0.4-0.8 ha, with 90% of sweetpotato cultivated on 0.2 ha or less. The main sweetpotato varieties were Vitaa, Kabonde and Bungoma and the majority of farmer’s used their own conserved planting material which was conserved by leaving them in the field after harvest. About 35% of the farmers identified weevils as the major pest, and sweetpotato virus disease (SPVD) as the major disease, while 28% of the farmers identified drought as a major constraint. The farmers used clean seed, high yielding varieties, high planting density, and manure application as the main strategies to cope with sweetpotato production constraints. Eighty four sweetpotato clones were evaluated under managed drought stress environments at KARI-Kiboko and KARI-Thika. Drought reduced the fresh weight of storage roots (FSR) (72.5%), fresh biomass weight (FB) (74.0%), marketable fresh storage root (MFSR) (80.7%), number of storage roots (NSR) (24.5%), days to permanent wilting point (DPWP) (0.3%), but seemed to increase percent root dry matter (% RDM) (-4.7%), harvest index (HI) (-2.6%), and chlorophyll content (CC) (-2.7%). Across the environments, genotypes 194555.7 (1.06), 421066 (1.05), Chingovu (0.94), 420014 (0.91), Excel (0.9), 199062.1 (0.87) and Unawazambane06-01 (0.81) gave higher FSR yields (kg plant-1) than the local checks. Genotypes Nyarmalo and Polista were among the lowest yielding in the irrigated and non-irrigated conditions. Clones W119, 441725, and Xiadla-xa-kau were the highest yielding under the drought stress conditions. The performance of 24 improved clones was evaluated in replicated trials at KARI-Thika and KARI-Kiboko using 24 sweetpotato clones grown under managed drought stress conditions for two seasons. AMMI, GGE biplots and regression analyses were conducted to determine stability of the clones. Mean FSR was significantly different (P < 0.001) in the two research sites with the environment contributing to 92.7% of the total variation, genotype 1.8%, and interactions 0.4%. AMMI and GGE biplots, and regression indicated the most stable clones to be 441725, Unawazambane06-01 and 189150.1, while Xiadla-xa-kau was the least stable. To gather more information on drought tolerance mechanisms expressed in the sweetpotato genotypes, an experiment was conducted in the greenhouse using clones expressing different levels of drought tolerance in the field. The results showed more under-developed roots (pencil roots) in the drought stressed regimes than in the irrigated regimes and in the drought susceptible genotypes. Drought tolerant genotypes produced more FSR and NSR. The number of vine branches (NVB), vine tip pubescence (VTP) and mature leaf pubescence (MLP) increased with reduction of drought stress however, drought tolerant clones had reduced NVB. Drought stressed clones had shorter basal vine length (BVL), reduced CC, and reduced leaf growth (LG). Drought stress reduced growth of vines in terms of internodes length, internodes diameter, vine length, petioles length, and leaf CC. Overall, for the first time, the study demonstrated that drought stress in the first three months after planting, leads to the proliferation of non-edible pencil roots, which do not become edible storage roots even when drought stress is removed. Therefore, water is critical during this period for improved sweetpotato storage root yield, probably as a drought tolerant mechanism. Combining ability for yield and drought tolerance of 15 F1 sweetpotato families generated through a half diallel mating of six parents was evaluated at KARI-Kiboko in 2012. Significant (P≤0.05) general combining ability (GCA) and specific combining ability (SCA) effects were recorded for root yield in both drought stress and no stress conditions, indicating that both additive and dominance gene effects were important in the inheritance of resistance to drought stress. Progenies from families G2, G5, G7, G8, G10, G12 and G15 had good SCA for fresh storage root yield, total biomass, number of days to permanent wilting point (DPWP), harvest index, and drought stress index (DSI). Progeny 8 from family G4, 5 and 8 from G15, had the highest mid and best parent heterotic effect (117-270%) for fresh storage root yield in both drought stress and no stress conditions. Progeny 6, and 7 from family G10, had the highest mid and best parent heterotic effect (165-234%) for fresh total biomass yield under no drought stress conditions. In conclusion, the progenies from families G2, G5, G7, G8, G10, G12 and G15 that had high yield and biomass specific combining ability under drought and no drought stress indicated that they could be having drought tolerant genes, and therefore could be incorporated into advanced drought screening trials with the aim of releasing the best performing drought tolerant varieties. Secondly, the findings in this study lay a foundation for sweetpotato breeding programmes on drought tolerance. Thirdly, for the first time, this study uniquely combines yield performance, combining ability estimates, days to permanent wilting point and heterosis under contrasting moisture regimes to unmask the gene action of drought tolerance in sweetpotato, a milestone in science. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Cowpea (Vigna unguiculata (L.) Walp) is an important protein-rich grain legume of major economic importance. It is widely grown by small-scale farmers in the arid and semi-arid regions of the world where it is cultivated for its leaves, fresh immature pods and dry grains. However, it is also an underutilized grain legume. In sub-Saharan Africa where most of the cowpea is produced, drought stress is one of the major factors limiting its productivity. Despite the inherent capacity to survive drought stress, several cowpea varieties are affected by mid and late season drought. Therefore, varieties with a higher tolerance to drought stress are required to obtain higher and more stable yields. The objectives of this study were: (i) to determine morphological responses of four dual-purpose cowpea landraces to water deficits during vegetative and reproductive stages (ii) to determine physiological responses of four dual-purpose cowpea landraces to water deficits and recovery during the reproductive stage (iii) to determine yield performance of cowpea landraces after recovery from water stress and how this relates to (ii) above. Four cowpea landraces namely; Lebudu, Lehlodi, Sejwaleng and Morathathane collectedfrom Kgohloane and Ga-Mphela villages, Limpopo Province, South Africa were used in the study. Pot experiments were conducted under glasshouse conditions at the Controlled Environment Facility (CEF), University of KwaZulu-Natal. The first pot experiment evaluated the morphological responses of four cowpea landraces to water stress and recovery. The study was conducted as a single factor experiment laid out in randomized complete block design (RCBD). The treatments (four cowpea landraces) were each planted in 40 pots giving a total of 160 experimental units (drained polyethylene pots with a 5 litre capacity). Each plant in each pot was treated as a replicate. Plants were well-watered until the formation of six fully expanded trifoliates, then irrigation was withheld for 28 days to simulate drought stress during the vegetative growth. The imposition of drought stress was terminated by re-watering all plants after 28 days. The cowpea plants were re-watered sufficiently and allowed to grow until the four landraces reached 50% flowering stage. Watering was withheld again at 50% flowering for a two-week period for all the four landraces to simulate drought stress during the reproductive growth. The second experiment was conducted to investigate physiological responses of the four cowpea landraces to water stress during the reproductive stage. The experiment was laid out as a 4 x 2 factorial treatment structure in randomized complete design (CRD) with the following three factors: cowpea landraces – 4 levels (Lebudu, Lehlodi, Sejwaleng and Morathathane), water regimes – 2 levels (stressed and well-watered) treatment combinations each replicated 20 times (20 pots each containing one plant) giving a total of 160 experimental units (drained polyethylene pots with a 5 litre capacity). Data on morphological responses were collected and included: number of green vs. senesced leaves, visual assessment of leaf greenness, stem, branch greenness and survival percentage. Physiological responses to water stress were determined during the reproductive stage and included: leaf water potential, relative water content, stomatal conductance, proline content, chlorophyll content, carotenoid content, chlorophyll a content, phenolics (free and membrane-bound), total antioxidant capacity and chlorophyll fluorescence parameters (Fv/Fm). Genstat 14th edition (VSN International, UK) was used to perform analyses of variance (ANOVA) and differences between means were determined by the Least Significant Differences (LSD) at the 5% probability level. Landraces showed different morphological responses during both vegetative and reproductive growth stages. Lebudu, Lehlodi and Sejwaleng displayed a strong ability to maintain stem greenness longer as compared to Morathathane during vegetative growth. Lebudu delayed leaf senescence more than other landraces; no differences in survival were observed. All landraces survived for 28 days without water and resumed growth after re-watering. During the reproductive stage, Lebudu displayed a strong ability to maintain leaf, branches and stem greenness longer and showed relatively higher tolerance to drought stress compared to other three landraces. Water stress caused a decline in leaf water potential, relative water content, carotenoid content, chlorophyll content, stomatal conductance and increased proline content, phenolics, chlorophyll a content, total antioxidant capacity and while chlorophyll fluorescence parameter, Fv/Fm, was not affected. All landraces maintained higher relative water content above a critical threshold with Sejwaleng maintaining a significantly higher RWC (69%) than Lehlodi, Lebudu and Morathathane. Morathathane developed a more negative leaf water potential at maximum stress than Lebudu, Lehlodi and Sejwaleng. Stomatal closure was observed in all cowpea landraces during water stress, but re-opened after re-watering. Chlorophyll content was considerably reduced in Morathathane as compared to Lebudu, Lehlodi and Sejwaleng. No significant differences were observed between the cowpea landraces with respect to carotenoid content at maximum stress. Chlorophyll a content increased significantly for Morathathane as compared to Lebudu, Lehlodi and Sejwaleng. High accumulation of proline was observed for Lebudu, Lehlodi and Morathathane as compared to Sejwaleng, which showed a very slow accumulation of proline. Lebudu, Lehlodi and Sejwaleng showed an increase in phenolic compounds while a decline was observed for Morathathane. Total antioxidant capacity (TAOC) was high in all cowpea landraces during water stress. Also, all chlorophyll fluorescence parameters showed that cowpea landraces had efficient photo-protection mechanisms during drought stress. After re-watering, relative water content, leaf water potential, stomatal conductance, chlorophyll content, carotenoids, chlorophyll a, proline content and TAOC recovered and reached the same level as that of well-watered plants. All four landraces were re-watered after the imposition of stress and above ground biomass, pod mass and number and seed yield determined. Although there was a reduction in the total above-ground biomass, pod mass and number in all four landraces under water stress compared to the well–watered treatment; this was not statistically significant (P > 0.05). Furthermore, no significant differences (P > 0.05) were observed between the four landraces with respect to seed yield under stressed and well-watered conditions. This study established that cowpea landraces vary with respect to the various morphological and physiological adaptive mechanisms in response to water deficits. Such adaptive mechanisms probably ensure their survival under severe water stress conditions until the next rainfall and therefore allowing them to produce reasonably relatively higher leaf and seed yield. Detailed knowledge of these mechanisms in the landraces could be useful in the genetic enhancement and breeding for drought tolerance in the existing cowpea germplasm. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

A caracterização e o entendimento das variações genômicas e morfológicas, bem como a estrutura genética de variedades locais armazenadas em bancos de germoplasma é importante para sua efetiva utilização em programas de melhoramento visando tolerância a estresses. Neste trabalho um conjunto de 192 variedades oriundas de diferentes regiões geoclimáticas do Japão foram testadas quanto à suas características morfológicas e produtivas, utilizando ensaios de campo e metodogias de fenotipagem de alto desempenho. A fenotipagem por meio da metodologia de camadas de herbicida (Aminotriazol+Diuron+2,4D, 100mg/plant) alocado a 30 centímetros de profundidade foi possível detectar variação entre as variedades para comprimento de raiz e velocidade de emissão de raiz sendo possível a distinção de variedades com sistema radicular profundo e sistema radicular superficial baseando-se na sua pontuação no ensaio de herbicida, destacando-se 20 genótipos como possíveis doadores de genes para comprimento, densidade e velocidade de emissão de raízes. Ensaios a campo foram conduzidos em 4 localidades expondo as variedades as mais distintas condições climáticas, buscando analisar a diversidade fenotípica para caracteres agromorfológicos. Os dados fenotípicos obtidos pelos marcadores morfológicos geraram um total de 15 grupos de acessos quando utilizados os 13 caracteres avaliados. A média do índice de sensibilidade a seca foi de 0,99 havendo materiais tolerantes com índices próximos a 0,6 e materiais sensíveis com índice próximos a 1,12. Os 384 marcadores SNP detectaram um total de 73728 alelos indicando alta porcentagem de A (40,8%) e G (34,6%) comparado com C (15,6%) e T (3,6%). Quanto aos heterozigotos, a maior porcentagem foi observada de A/G (0,54%) e a menor porcentagem de A/T (0,04%), sendo a maior parte dos heterozigotos observados nos cromossomos 3 e 8 comparado com outros cromossomos. As análises caracterizaram os acessos japoneses como 98,4% pertencentes à subespécie Japônica. Para associação entre marcadores e fenótipos, foi utilizada a abordagem de modelo linear misto (MLM), o qual incorpora informações de estrutura populacional e parentesco. Os resultados obtidos deverão ser investigados futuramente a fim de confirmar as associações em diferentes populações. O aumento do estresse hídrico teve efeito significativo no desempenho dos acessos, e a interação genótipo e estresse hídrico foi significativa para rendimento final e tamanho de panícula. Entre os acessos estudados foi observada variação genética para as características relacionadas com a tolerância a estresse hídrico e encontraram-se acessos com reduções no rendimento devido ao déficit hídrico comparáveis com o das testemunhas, embora com tamanho de panícula menor, inclusive em condições ótimas. Dos 384 marcadores utilizados, 10 foram responsáveis por associações significativas com o índice de sensibilidade a seca, com base nos diferentes métodos de correção para múltiplos testes. Estas associações foram selecionadas para verificar o efeito alélico sobre o genótipo observado, gerando informações preliminares para a aplicação futura de seleção assistida por marcadores (SAM). O tamanho dos blocos de ligação foram estimados em ~100 kb (r < 0,05) e ~75kb (r < 0,1). / Germplasm characterization and the knowledge of its diversity and population structure are important to effective utilization of genetic resources in breeding programs specially drought breeding program. In this work 192 landraces from all over Japan were evaluate for their morphological and productive characteristics, using field trials and high throughput screening methods. The screening using herbicide barrier approach at 30 cm depth was able to detect genetic diversity between the landraces for root length and clearly distinguish between deep root landraces and shallow root landraces. With this approach was possible to select 20 landraces with deep root system as possible donor of drought tolerance genes. Aiming characterize the landraces to agromorphological characteristics field trials were carried in 4 different locations exposing the landraces for a diversity environment effect. Using 13 traits phenotypically data generate a total of 15 groups during cluster analysis. The average result for drought sensitive index was 0.99, however this results presents a huge variability having landraces with scores about 0.6 to landraces with score up to 1.12. Drought effect was huge and statistically significant affecting directly yield and panicle size. The landraces presented genetic variability for drought tolerance and some landraces presenting yield and panicle size reductions due to drought comparable with drought-tolerant controls were detected. A total of 73728 alleles were detected by the SNP markers, indicated a high percentage of A (40,8%) and G (34,6%) alleles compared to C(15,6%) and T (3.6%). Heterozygocity of A/G was highest (0,54%) and lowest in A/T (0.04%). Of 3 chromosomes of rice, chromosome 8 produced highest percentage of heterozygocity compared to other chromosomes. Accessions were classified as 98.4% belonging to japônica subspecies. Association between markers and phenotypes was performed using a mixed linear approach (MLM), which incorporates information regarding population structure and kinship. Among the 384 markers used, 10 were responsible for significant associations with drought sensibility index, based on different criteria to correct for multiple tests.These associations were selected to determine the allelic effects over the traits, in order to generate preliminary data for marker assisted selections (MAS). Estimated size of haplotype blocks were ~100 kb (r <0.05) and ~75 kb (r <0.1). Future studies should confirm marker trait associations here found using different populations.

<p>Arbuscular mycorrhizal fungi (AMF) are best known for their potential to help plants acquire nutrients, especially phosphorous. These microbes improve soil health by promoting soil aggregation and carbon sequestration, and further benefit plants by helping them withstand biotic and abiotic stress. Currently, there are 200 recognized species of AMF within the phylum Glomeromycota. Recent studies indicate that individual AMF species differ in the benefits they provide, with some even acting as parasites. Moreover, AMF community composition can be altered by soil and crop management practices, but the effect of these changes on the benefits conferred by AMF are still not well understood. Consequently, the goal of this study was to determine how two widely used crop management systems can alter the composition of AMF species, and affect the potential for these communities to promote the productivity and drought tolerance. To accomplish this goal, we collected AMF inoculum from a long-term crop systems trial comparing organic and conventional management for use in greenhouse trials where we subjected plants to drought. We collected AMF inoculum during mid-summer when differences between the two management systems were likely cause larger effects on AMF communities, and again in autumn after harvest to see if differences in AMF communities would persist. We determined AMF species composition using next generation sequencing. Results of this study confirm that soil-building practices commonly used in organic farming systems can improve soil health and increase the productivity of food-grade soybeans. They also demonstrate that AMF communities in Indiana croplands are highly diverse, and some of these taxa can improve soybean growth and help plants tolerate water stress. Although the overall diversity of AMF communities did not differ between the organic and conventional management systems in mid-summer, individual AMF taxa did differ between the systems, which were likely responsible for the greater tolerance to water stress observed when plants were amended with inoculum from the organic system. AMF communities present during autumn were significantly different between the two crop management systems, but did not result in differences in drought tolerance of soybeans, indicating that the loss of key AMF taxa in the organic system from the first relative to the second experiment was likely responsible. Finally, plants grown using inoculum from both crop management systems in autumn had greater tolerance to water stress than plants that received a AMF commercial inoculum. This provides further evidence that individual AMF species vary in the benefits they provide, and that the presence of a diverse consortium of AMF species is needed to optimize plant health and productivity in agricultural systems. Agricultural producers should consider incorporating soil-building practices that are commonly used in organic farming systems such as planting winter cover crops, to improve the health of their soil and enhance the productivity of their crops. <b></b></p> <br>

Common bean is a major dietary component in several countries, but its productivity is negatively affected by abiotic stresses. Dissecting candidate genes involved in abiotic stress tolerance is a paramount step toward the improvement of common bean performance under such constraints. Thereby, this thesis presents a systematic analysis of the DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) gene subfamily, which encompasses genes that regulate several processes during stress responses, but with limited information for common bean. First, a series of in silico analyses with sequences retrieved from the P. vulgaris genome on Phytozome supported the categorization of 54 putative PvDREB genes distributed within six phylogenetic subgroups (A-1 to A-6), along the 11 chromosomes. Second, we cloned four novel PvDREB genes and determined their inducibility-factors, including the dehydration-, salinity- and cold-inducible genes PvDREB1F and PvDREB5A, and the dehydration- and cold-inducible genes PvDREB2A and PvDREB6B. Afterwards, nucleotide polymorphisms were searched through Sanger sequencing along those genes, revealing a high number of single nucleotide polymorphisms within PvDREB6B by the comparison of Mesoamerican and Andean genotypes. The nomenclature of PvDREB6B is discussed in details. Furthermore, we used the BARCBean6K_3 SNP platform to identify and genotype the closest SNP to each one of the 54 PvDREB genes. We selected PvDREB6B for a broader study encompassing a collection of wild common bean accessions of Mesoamerican origin. The population structure of the wild beans was accessed using sequence polymorphisms of PvDREB6B. The genetic clusters were partially associated with variation in latitude, altitude, precipitation and temperature throughout the areas such beans are distributed. With an emphasis on drought stress, an adapted tube-screening method in greenhouse conditions enabled the phenotyping of several drought-related traits in the wild collection. Interestingly, our data revealed a correlation between root depth, plant height and biomass and the environmental data of the location of the accessions. Correlation was also observed between the population structure determined through PvDREB6B and the environmental data. An association study combining data from the SNP array and DREB polymorphisms enabled the detection of SNP associated with drought-related traits through a compressed mixed linear model (CMLM) analysis. This thesis highlighted important features of DREB genes in common bean, revealing candidates for further strategies aimed at improvement of abiotic stress tolerance, with emphasis on drought tolerance / O feijoeiro é um componente essencial na dieta em diversos países, no entanto, sua produção é afetada negativamente por estresses abióticos. O estudo de genes candidatos envolvidos na adaptação aos estresses é uma etapa fundamental para o melhoramento da performance do feijoeiro sob tais estresses. Desse modo, esta tese apresenta uma análise sistemática da subfamília de genes DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB), que reúne genes envolvidos em diversos processos em resposta a estresses, mas pouco estudados no feijoeiro. Primeiramente, uma série de análises in silico com sequências de feijoeiro obtidas da plataforma Phytozome possibilitaram a categorização de 54 genes PvDREB putativos, distribuídos em seis subgrupos (A-1 até A-6) nos 11 cromossomos da espécie. Posteriormente, quatro novos genes PvDREB foram clonados e seus padrões de inducibilidade foram determinados. PvDREB1F e PvDREB5A foram induzidos por desidratação, baixa temperatura e salinidade, enquanto PvDREB2A e PvDREB6B foram predominantemente induzidos por desidratação e baixa temperatura. Polimorfismos de nucleotídeos foram buscados através de sequenciamento por método derivado de Sanger, revelando elevado número de SNP no gene PvDREB6B. A nomenclatura desse gene foi discutida detalhadamente ao longo da tese. A plataforma de marcadores SNP BARCBean6K_3 foi acessada para identificar o SNP mais próximo de cada um dos 54 PvDREB. O gene PvDREB6B foi selecionado para um estudo mais amplo, envolvendo uma coleção de acessos selvagens de origem Mesoamericana. A estrutura populacional destes genótipos foi analisada a partir de polimorfismos na sequência de PvDREB6B. Os grupos genéticos apresentaram associação parcial com variação da latitude, altitude, precipitação e temperatura das áreas em que os acessos naturalmente ocorrem. Com ênfase no estudo do déficit hídrico, uma plataforma de fenotipagem destes acessos em casa de vegetação, utilizando um sistema de tubos plásticos, foi elaborada para a análise de diversos parâmetros relacionados ao estresse por déficit hídrico. Os dados revelaram correlação entre profundidade de raízes, altura das plantas e a biomassa e as variáveis ambientais de cada local. A correlação também foi detectada entre a estrutura populacional estudada por PvDREB6B e os dados ambientais. Finalmente, um estudo de associação genética foi realizado entre os SNP da plataforma e ligados a DREB e os parâmetros fenotípicos, permitindo a identificação de marcadores SNP associados a caracteres específicos, usando um modelo linear misto (CMLM). Esta tese apresentou importantes aspectos sobre os genes DREB em feijoeiro, revelando candidatos para seu uso em estratégias de melhoramento para tolerância a estresses abióticos, com ênfase em déficit hídrico

Des épisodes climatiques de sècheresse et/ou de hautes températures peuvent engendrer de fortes pertes de rendement pour les cultures de céréales au champ. Un QTL associé au rendement et à ses composantes a été détecté dans quatre populations de blé (Triticum aestivum L.) sur le bras long du chromosome 3B « qYDH.3BL ». Deux populations d’haploïdes doublés (RAC875/Kukri et Excalibur/Kukri) et deux populations de lignées recombinantes (RAC875/Kukri et Gladius/Drysdale) ont été utilisées pour cartographier finement le QTL, au même titre que l’identification de gènes candidats. Ces quatre populations ont été testées sous des conditions environnementales variées, incluant des périodes de sécheresse et/ou hautes températures en Australie et au Mexique. Des modèles statistiques mixtes et linéaires décomposant les variations génétiques et non-génétiques ont été utilisés pour la détection de QTL en considérant dans un premier temps chaque environnement unique, puis en considérant les environnements multiples dans une analyse commune. Les allèles de RAC875, Drysdale et Excalibur à ce locus ont montré une hausse du rendement de 5 à 12.5 % comparées à celles de Gladius ou Kukri. Un total de trente-sept marqueurs moléculaires a été cartographié dans la région du QTL. Les marqueurs moléculaires ont été sélectionnés (i) par comparaison avec une carte génétique publiée du chromosome 3B, ou (ii) en désignant de nouveaux marqueurs moléculaires sur les séquences de BAC-end, de contig ou de gènes provenant du projet de séquençage du chromosome 3B (3BSEQ, http://urgi.versailles.inra.fr/, cv. Chinese Spring). Ceci a permis la construction d’une carte génétique consensus du locus qYDH.3BL . A ce jour, aucun QTL associé au rendement ou ses composantes en condition de sécheresse et/ou de hautes températures n’a encore été cloné positionellement chez le blé tendre. Les marqueurs moléculaires de la région d’intérêt ont été utilisés pour cartographier physiquement des contigs, soit par PCR, soit par comparaison de séquences in silico. La région du QTL inclus un total de huit contigs physiques comprenant 85 gènes annotés. L’utilisation de base de données de transcris biologiques publiques ou internes ont été utilisées pour détecter la présence de ces gènes, réduisant la liste à soixante-cinq gènes. Sur les contigs ayant une confiance élevée, aucun des vingt gènes n’a été exprimé différentiellement entre RAC875 et Kukri. Cependant, un gène présentant du polymorphisme dans sa séquence ainsi qu’une délétion/insertion d’un segment portant 12 gènes ont été découvert permettant ainsi de continuer à affiner la liste de gènes candidats. Les trois lignées parentales (RAC875, Drysdale et Excalibur) qui ont l’allèle liée au haut rendement ont le même haplotype pour ce gène, et la même délétion/insertion en opposition au deux autres lignées parentales Gladius et Kukri. Ainsi, dans ce travail de thèse nous avons pu confirmer la présence d’un QTL répondant aux stresses environnementaux sur le chromosome 3BL dans différentes populations et différents environnements, identifier des gènes candidats sous le QTL, et proposer une liste restreinte pour de futures analyses sur la base de données d’expression et de polymorphismes entre les parents des populations de cartographie. / Drought and heat can occur during the growth cycle of crops and severely reduce yield. A QTL associated with yield and yield-related component was found in four wheat populations (Triticum aestivum L.) on the long arm of chromosome 3B “qYDH.3BL”. The four populations were grown under various climatic conditions including drought, heat and combinations of both in a number of different areas (Australia and Mexico). Linear mixed models that partition and account for genetic and non-genetic or extraneous variation were used to detect loci in single-environment and/or multi-environment QTL analysis using ASReml-R. The alleles carried by RAC875, Excalibur or Drysdale improved grain yield by between 5% and 12.5%. Two doubled haploid populations (RAC875/Kukri and Excalibur/Kukri) and two recombinant inbred line populations (RAC875/Kukri and Gladius/Drysdale) were used to fine map qYDH.3BL and identify candidate gene(s). A total of thirty-seven molecular markers were mapped on one or both genetic maps of chromosome 3B enabling development of a consensus genetic map of the qYDH.3BL region. The markers were selected based on comparisons with a published “neighbour map” of chromosome 3B or designed using either BAC-end, contig or gene sequences from the chromosome 3B sequencing project; 3BSEQ http://urgi.versailles.inra.fr/ (cv. Chinese Spring). A positional cloning approach was used to identify candidate genes for qYDH.3BL. Molecular markers from the targeted region were assigned to physical contigs by screening the chromosome 3B BAC library experimentally using PCR or in silico by sequence comparison. A total of eight physical contigs containing 85 genes, were anchored to the qYDH.3BL region. Public and in-house resources of wheat transcript sequences were used to restrict the gene list to 65 expressed genes. Based on comparison of the 65 gene sequences to gene probes in a drought transcriptomic database, three genes were found to be differentially expressed between RAC875 and Kukri under drought conditions. Short genomic sequence reads (10× coverage) from each of the five parental lines (RAC875, Kukri, Excalibur, Gladius and Drysdale) were mapped against the 65 genes for polymorphism discovery. One gene exhibited sequence polymorphism between the drought tolerant parents (RAC875, Excalibur and Drysdale) and the drought-sensitive parents (Gladius and Kukri). In addition, presence/absence polymorphisms were consistently detected throughout a region containing 12 genes, indicating that the drought tolerant parents may have a deletion (or alien introgression) in this region. Thus, in this work, we confirmed the genetic effect of qYDH.3BL in multiple environments and multiple populations, saturated the target region with new molecular markers and defined a preliminary list of genes located in the qYDH.3BL region and selected candidate genes for further investigations.

Abstract not available

Cowpea (Vigna unguiculata. L) is an important African crop. However, it is also an underutilized grain legume. Consequently, there is not enough research data on cowpea seed physiology. Whereas there is evidence of cowpea being a drought tolerant crop, there is no evidence to associate plant drought tolerance with seed quality in response to water stress. This study sought to understand the effect of production site and water stress on cowpea seed quality development with respect to germination capacity and vigour. Patterns of raffinose family of oligosaccharides (RFO) during seed development to mature dry stage were used to physiologically relate seed performance to water stress. The effect of water stress and exogenous ABA on the accumulation of stress LEA proteins (dehydrins) in relation to seed quality development and germination was investigated. RFOs are known for their roles in desiccation sensitivity but no studies have shown their significance in cowpeas. Seeds of six cowpea cultivars were produced at two distinct growth sites characterised by irrigated and dry land conditions. The seeds were assessed during six developmental stages, for water content, dry matter accumulation, and performance. Harvested seeds were then planted in a pot experiment under controlled conditions to examine the effect of water stress on seed quality development and data collected during three developmental stages. Harvested seeds from the pot experiment were subsequently analyzed for changes in RFO accumulation during development using gas chromatography. The seeds were also used to investigate the effect of water stress and ABA on the accumulation of stress LEA proteins (dehydrins) in relation to seed quality development in cowpea. In addition, this study evaluated the use of image analysis as a method that can be used to objectively determine seed coat colour variation in cowpea. Statistical variation in individual seed’s solute leakage for cowpea cultivars differing in seed coat colour and produced under different environmental conditions was explored and correlations were done between seed conductivity test with other aspects of seed performance during germination. Furthermore the results of the conductivity test were compared with accelerated aging test, in relation to seed performance. The study provided evidence that cowpea seed lots produced under different environmental, and possibly management conditions may not differ with respect to seed quality as determined by germination capacity and vigour. However, significant differences between sites with respect to seed maturation patterns determined by water content and dry matter accumulation were observed. Adverse maternal environmental effects on the subsequent performance of seeds in a drought tolerant crop may not necessarily lead to poor performance. Cultivar differences in response to simulated drought conditions at the whole plant and tissue level can be considerable and highly variable; however, these differences may not have adverse effects on the germination and vigour of the seeds. Drought avoidance mechanisms at the whole plant level in cowpea are quite efficient in allowing the species to adapt to simulated drought conditions. These mechanisms may allow the cowpea cultivars to maintain metabolism and restore conditions for their continued growth under water stress; and produce few seeds of high germination capacity and vigour. Stachyose was found to be the predominant member of the raffinose family of oligosaccharides in cowpea. It is suggested that stachyose accumulation could be used as an indicator of stress tolerance in cowpea. However, the relationship between RFO concentration and the acquisition of desiccation remained as a matter of speculation in the present study and is still generally inconclusive. There was no evidence to suggest the acquisition of maximum desiccation tolerance is associated with maximum seed vigour. It is suggested in cowpea, which is drought tolerant, that maximum vigour does not necessarily imply the acquisition of maximum desiccation tolerance; rather there is a minimum level of desiccation tolerance that is required for the development of optimal seed vigour. The use of an in vivo approach in the study of LEA function in cowpea enabled the accurate comparison of two different groups of LEA proteins in developing cowpea seeds under conditions of water stress and in relation to germination and vigour. Both group 1 LEA and group 2 LEA (dehydrin) were shown to increase in concentration in response to water stress. In addition group 1 LEA protein was observed to be relatively abundant in cowpea seeds. A maternal influence on LEA protein gene expression under conditions of water stress, which may induce dehydrin accumulation vii during the earlier stages of seed development, was implied by the observation that dehydrin-like proteins were induced after two weeks of development in cowpea plants subjected to stress during the vegetative phase. In addition, the exogenous application of ABA delayed radicle protrusion; this was associated with a delay in the disappearance of LEA proteins and is suggestive of a relationship between LEA protein accumulation and the acquisition of desiccation tolerance. The study has demonstrated that image analysis can objectively discriminate seed coat colour variation in cowpea. Dark coloured seeds in general performed better than light coloured seeds; however seed coat colour was not always associated with better performance. A newly developed Aging Stress Differential Index (ASDI) has been used in this study to demonstrate a link between seed coat colour and sensitivity to water stress. The ASDI correlated well with the observations relating stress tolerance to stachyose accumulation. The skewed distribution patterns in individual electrical conductivity and the presence of extreme values may have implications with respect to the suitability of using standard statistical analyses which compare mean values to evaluate such data. In addition variation in individual electrical conductivity may also be influenced by cultivar differences and the chemical composition of the seed coat. Therefore associations between seed coat colour and electrical conductivity as a measure of performance should be treated with caution. The AA test does reflect changes in seed vigour, however ranked electrical conductivity values after AA did not consistently reflect differences in seed performance between cultivars and sites, and they did not correlate well with other aspects of performance. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

There are few maize varieties that are drought tolerant in semi-arid eastern Kenya and farmer perceptions of drought tolerant maize cultivars have not been studied in this region. Farmers in this region use maize landraces that have not been studied for their potential future hybridization. The main objectives of this study were therefore to: (i) study farmer perceptions of drought and preference for maize varieties, (ii) improve drought tolerance in maize populations in the semi-arid eastern Kenya using S1 family recurrent selection, and (iii) classify maize landraces according to their heterotic patterns. A participatory rural appraisal (PRA) was conducted in Machakos and Makueni districts in semi-arid eastern Kenya. A total of 175 farmers were involved in focus group discussions. An open ended questionnaire and a checklist were used to guide the farmers during the discussion sessions. Scoring and ranking techniques were used to assess farmers’ preferences of maize varieties and constraints to maize production. The farmers grew maize as their major crop followed by beans. Nearly 60% of the farmers grew local maize landraces, whose seed they recycled from season to season; 40% grew improved varieties, but mainly composites rather than hybrids. The key farmers’ criteria for choosing a maize variety in order of importance were drought tolerance, early maturity, high yield, and disease resistance. The major constraints to maize production were drought, lack of technical know-how, pests, poor soils, and inadequate seed supply. Maize traits preferred by farmers in a drought tolerant variety included high yield, recovery after a dry spell and the stay green characteristic. Two maize landrace populations MKS and KTU from semi-arid eastern Kenya and three CIMMYT populations V032, ZM423, and ZM523 were subjected to two cycles of S1 progeny recurrent selection for drought tolerance in yield and traits indicative of drought tolerance were measured during flowering and grain filling from February 2005 to September 2007. Evaluation to determine selection gains was done in one trial replicated five times. It was laid out as a 4x4 lattice design and drought was imposed at reproductive stage by withholding irrigation one week before flowering and resumed during grain filling. The trial was repeated under well-watered conditions which served as a control experiment. After two cycles of selection under drought stress conditions, KTU population had a realized gain in yield of 0.2 t ha-1, MKS population 1.2 t ha-1 and ZM423 0.4 t ha-1, whereas in V032 and ZM523, grain yield reduced by 1.1 t ha-1 and 0.6 t ha-1, respectively. Under well watered conditions, the realized gains in grain yield were positive in all the populations except V032, where there was a reduction of 0.1 t ha-1. Selection increased the genetic variability and heritability estimates for yield in S1 lines of MKS and ZM423 populations, but decreased in KTU, V032 and ZM523 populations. The research to identify heterotic patterns was undertaken using ten maize landraces from the semi-arid eastern Kenya, six maize landraces from coastal Kenya, and three maize populations from CIMMYT. These populations were planted at Kiboko Research Farm during the short rains of October-December 2005 and crossed to two population testers, Embu 11 and Embu 12. The evaluation of the test crosses was done during the long rains of March-June 2006. Percentage heterosis for yield ranged from -17.7% to 397.4%, -79.4 to 22.2% for anthesis-silking interval, -23.9% to 29.2% for ear height, -0.1 to 1.1 for ear diameter, -7.1 to 21.2% for ear length and -5.9% to 30.3% for plant height. iii General combining ability (GCA) effects were significant (p=0.05) for all the traits, while specific combining ability (SCA) effects were not significant (p>0.05), implying that variation among these crosses was mainly due to additive rather than nonadditive gene effects. Since SCA was not significant (p>0.05) for yield, maize populations were classified based on percentage heterosis for yield alone. The maize populations therefore, were grouped into three different heterotic groups P, Q and R. Twelve landrace populations and two CIMMYT populations showed heterosis with Embu 11 and no heterosis with Embu 12 were put in one group P. Two landrace populations that showed no heterosis with either tester were put in group Q. Two landrace populations and one CIMMYT population showed heterosis with both testers were put in group R. None of the populations showed heterosis only with Embu 12 and no heterosis with Embu 11. The main constraint to maize production was drought and the farmers preferred their landraces whose seed they recycled season to season. After two cycles of recurrent selection, the landrace populations showed improved progress in yield. Thus, further selection will be beneficial in the populations where genetic variability increased. Therefore, these populations can further be improved per se and released as varieties and/or incorporated into the existing maize germplasm to broaden their genetic base, given that their heterotic patterns have been identified. Considering that farmers recycle seed, breeding should be towards the development of open-pollinated varieties which are drought tolerant. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.