Dissertations / Theses on the topic 'Cotton Molecular genetics'

Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the interconversion of carbon dioxide and carbonate and has been found to play a wide range of roles in animals, plants and bacteria. Cotton genomic and cDNA libraries were screened for the plastidial isoform of carbonic anhydrase. The nucleotide sequences of two 1.2 Kb partial cDNA clones were determined. These clones exhibit high homology to carbonic anhydrases from other dicot plants and possess all the expected peptide motifs. For example, serine and threonine rich chloroplastic targeting peptide and conserved zinc binding residues are both present. These clones were utilized to isolate two carbonic anhydrase genes that were shown to encode different isoforms by PCR and RFLP analysis.

A cotton PATE cDNA clone has a 1.7-kb insert with an coding region for 410 amino acids, lacking codons for the three N-terminal amino acids. The predicted amino acid sequence of the PATE preprotein has a characteristic stromal-targeting domain and a 63% identity to the Arabidopsis FatB1 thioesterase sequence. A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-ACP thioesterase (FatB1) gene. The gene spans 3.6 kb with six exons and five introns. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein is identified as a FatB thioesterase from its deduced amino acid sequence similarity to those of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential promoter/enhancer elements including basic helix-loop-helix elements (E box). Alkaline blot hybridization of cotton genomic DNA suggests the presence at least two FatB1 thioesterase genes in cotton. Four plasmid constructs for both constitutive and seed-specific anti-sense RNA suppression and gene-transgene co- suppression of PATE gene expression were successfully generated. Two overlapping cotton genomic clones were found to encompass a Δ-12 fatty acid desaturase (FAD2-3) gene. The continuous FAD2-3 coding region is 1,155 bp and would encode a protein of 384 amino acids. The FAD2-3 gene has one large intron of 2,967 bp entirely within its 5'-untranslated region. Several potential promoter/enhancer elements, including several light responsive motifs occur in the 5'-flanking region. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.

Algodão geneticamente modificado que expressa a toxina Cry1Ac de Bacillus thuringiensis Berliner tem sido plantado no Brasil desde 2006. Entre as pragas-alvo da tecnologia, Alabama argillacea (Hüeb.) é uma espécie monófoga e apresenta alto potencial de risco de evolução da resistência. Para a implantação de um programa de manejo da resistência de A. argillacea à toxina Cry1Ac no Brasil, os principais objetivos do trabalho foram: a) estabelecer a linhas-básicas de suscetibilidade à toxina Cry1Ac em populações de A. argillacea e definir concentrações diagnósticas para o monitoramento da resistência e b) isolar e caracterizar locos microssatélites para avaliar a variabilidade e estruturação genética de populações de A. argillacea no Brasil. As linhas-básicas de suscetibilidade foram estimadas por meio de bioensaio de imersão de discos de folhas em soluções contendo a toxina Cry1Ac para populações de A. argillacea coletadas nos estados da Bahia, Goiás, Mato Grosso e Mato Grosso do Sul, durante as safras agrícolas de 2008 e 2009. Foram isolados e caracterizados dez locos microssatélites. Para avaliar a variabilidade genética foram estimadas as heterozigosidades observadas e esperadas. Para o estudo da estruturação genética foram estimadas as estatísticas F e feita a análise de agrupamento (distância de Nei) e análise Bayesiana. Baseado na estimativa da CL50 foram encontradas variações naturais de até seis vezes na suscetibilidade à toxina Cry1Ac entre as populações testadas. A partir da análise conjunta dos dados de concentração-mortalidade das populações testadas, foram definidas as concentrações diagnósticas de 10 e 32 µg de Cry1Ac/ml de água para futuros programas de monitoramento da resistência. O número médio de alelos por loco foi de 7,1 (variando de dois a 23 alelos). As heterozigosidades observada e esperada médias foram de 0,532 e 0,329. O índice de fixação intrapopulacional médio (f FIS) foi de 0,268, com variação entre os locos de -0,008 a 0,736. O índice de fixação da espécie (FIS) estimado através da análise de variância foi de 0,244 (IC 95% de 0,093 a 0,418). O valor de FST estimado foi de 0,036 (IC 95% de 0,007 a 0,080). Esse valor de FST não diferiu significativamente de zero, indicando a ausência de estruturação genética. Contudo foi detectado certo grau de endogamia intrapopulacional. A estruturação espacial da variabilidade genética não foi detectada, pois as populações avaliadas apresentaram uma coesão que é mantida pela alta taxa de migração (6,7 migrantes por geração). Entretanto, foi identificada indícios de estruturação genética determinada pelo tempo, uma vez que tanto o agrupamento baseado em distâncias genéticas quanto à análise Bayesiana identificaram grupos que são formados por populações coletadas em safras agrícolas diferentes. As causas ligadas a essa mudança na variabilidade genética não puderam ser identificadas, entretanto pode se inferir que possivelmente causas naturais ou práticas de manejo estejam determinando eventos de gargalo genético. Devido ao intenso fluxo gênico entre populações de A. argillacea no Brasil, estratégias de manejo da resistência devem ser implantadas no âmbito nacional.
Genetically modified cotton expressing Cry1Ac toxin of Bacillus thuringiensis Berliner has been planted in Brazil since 2006. Among target pests of this technology, Alabama argillacea (Hüeb.) is a monophagous species and offers a high potential risk of resistance evolution. In order to implement a resistance management program of A. argillacea to Cry1Ac toxin in Brazil, the objectives of this research were: a) to establish baseline susceptibility to Cry1Ac toxin in A. argillacea populations and define diagnostic concentrations for resistance monitoring and b) to isolate and characterize microsatellite loci to evaluate the variability and genetic structure of A. argillacea populations in Brazil. The baseline susceptibility data were estimated with leaf-disc bioassays by dipping into different concentration of Cry1Ac solution. Populations of A. argillacea were collected in Bahia, Goiás, Mato Grosso and Mato Grosso do Sul States, during 2008 and 2009 cotton-growing seasons. Ten microsatellite loci were isolated and characterized. The genetic variability was evaluated estimating observed and expected heterozygosities. For the studied of genetic structure, the F statistics was estimated, and Cluster analysis (Nei´s distance) and Bayesian analysis were performed. Based on estimation of LC50, natural variation up to 6-fold was detected in the susceptibility to Cry1Ac among tested populations. Based on analysis of concentration-mortality data by combining all populations, diagnostic concentrations of 10 and 32 µg of Cry1Ac/ml of water were defined for monitoring resistance. The mean number of alleles per loci was 7.1 (varying from 2 to 23 alleles). The observed and expected heterozigosities was 0,523 e 0, 395. The mean intrapopulation fixation index (f FIS) 0,268, varying from -0.008 to 0.736 between loci. The species fixation index (FIS) estimated by analysis of variance was 0.244(95% CI of 0.093 to 0.418). The estimated value of FST was 0.036 (95% CI of 0.007 to 0.080). The FST value was not significantly different from zero, indicating absence of genetic structure However, some degree of intrapopulational inbreeding was detected. Spatial structure of genetic variability was not detected because tested populations showed cohesion kept by high migration rate (6.7 migrants per generation). However, evidence of genetic structure across time was detected by Cluster analysis of genetic distance as well as by Bayesian analysis with group formation by population collection seasons. Factors affecting changes in genetic variability were not identified; however, natural factors or management practices may be determining some genetic bottleneck events. Due to intense gene flow among A. argillacea populations in Brazil, resistance management strategies must be implemented in a national basis.

Com a crescente expansão de cultivos agrícolas no Brasil, tem sido bastante comum o sistema de produção de algodão e milho em uma mesma região. Como uma possível conseqüência deste sistema de cultivo, os problemas com Spodoptera frugiperda (J. E. Smith) têm aumentado nestas duas culturas nos últimos anos. Assim, para o estabelecimento de um manejo mais efetivo desta praga, foi levantada a hipótese de que as mesmas populações de S. frugiperda atacam as culturas do algodão e milho em uma determinada região. Para testar esta hipótese, foram realizados os seguintes estudos: 1) a avaliação a suscetibilidade ao piretróide deltametrina em populações de S. frugiperda, e 2) a estimativa da similaridade e da estrutura genética em populações de S. frugiperda com o uso de marcadores moleculares RAPD e AFLP. A avaliação da suscetibilidade a deltametrina das populações de S. frugiperda foi realizada mediante bioensaio de aplicação tópica. Além disso, foi avaliada a resposta à seleção com deltametrina em uma população de S. frugiperda em condições de laboratório. As populações de S. frugiperda coletadas na cultura do algodão foram significativamente menos suscetíveis a deltametrina do que as populações coletadas no milho. Estes resultados poderiam ser entendidos como uma conseqüência da pré-seleção de indivíduos resistentes a deltametrina A população de S. frugiperda selecionada em laboratório apresentou uma razão de resistência a deltametrina de ≈14 vezes. Pela técnica de RAPD, os dendrogramas (Simple Matching e Jaccard) classificaram as populações da praga em grupos proximamente relacionados com a região geográfica de coleta dos insetos. Não foi identificado nenhum ramo capaz de separar e associar as populações de S. frugiperda a nenhuma das duas plantas hospedeiras avaliadas. Estes resultados sugeriram que as populações da praga em algodão e milho em uma determinada região do Brasil apresentam um nível significativo de fluxo gênico. Os resultados da técnica AFLP foram organizados em um dendrograma UPGMA (índice de Jaccard), o qual também não classificou as populações da praga em grupos relacionados às plantas nas quais os insetos foram coletados. Não foi detectada correlação significativa entre a dissimilaridade genética e distância geográfica nas populações testadas. Foi detectada variação molecular entre as populações da praga atribuída à origem geográfica dos insetos. A análise molecular de variância indicou que 7% da variação molecular total poderiam ser atribuídos à divisão das populações de S. frugiperda em grupos de insetos coletados no Brasil e na Argentina. Além disso, foi detectado 0% de variação genética, e um valor de fluxo gênico Nm=1,32 entre os grupos de populações da praga coletadas nas culturas do algodão e do milho. Assim, as infestações de S. frugiperda que ocorrem em campos de milho e algodão podem ser consideradas como subunidades potencialmente intercruzantes de uma mesma população. Portanto, conclui-se que há necessidade de um planejamento bastante estratégico nos plantios de algodão e de milho, no delineamento de programas de manejo de S. frugiperda no Brasil.
Due to the expansion of the cultivated areas in Brazil, it has been very common to find the cultivation of cotton and maize in the same region. As a potential consequence to this cultivation system, the problems with Spodoptera frugiperda (J.E. Smith) have increased for the last years at both crops. In order to provide elements to the establishment of a more effective management of this pest it was hypothesized that the same populations of S. frugiperda attack the cotton and maize crops in Brazil. To test this hypothesis the following studies were conducted: 1) the evaluation the susceptibility to the pyrethroid deltamethrin in S. frugiperda populations and 2) the assessment of the molecular variability and genetic structure in S. frugiperda populations by using RAPD and AFLP molecular markers. The evaluation of the susceptibility to deltamethrin in S. frugiperda populations was conducted by using topical application bioassays. Furthermore, the response of this pest to the selection pressure with the insecticide was evaluated under laboratory conditions. The insect populations collected in the cotton crop were significantly less susceptible to deltamethrin than the ones collected in the maize crop. These results might be consequence of a pre-selection of deltamethrin-resistant individuals. The deltamethrin-resistant population selected under laboratory conditions had the resistance ratio of ≈14 fold. The dendrograms obtained by using RAPD markers (Simple Matching and Jaccard) classified the populations into clusters related to the geographical origin of the samples. Any branch of the dendrograms underpinned a molecular association of S. frugiperda with neither of the two host plants. These results suggested the existence of considerable gene flow between cotton and maize populations of S. frugiperda collected at the same region in Brazil. The AFLP results were organized in a UPGMA dendrogram (Jaccard index) which also did not classify the populations of S. frugiperda into clusters related to the host plant in which the insects were collected. It was not found a significant correlation between genetic dissimilarity and geographical distances. It was detected genetic variation attributable to the geographical origin of the populations. The analysis of molecular variance highlighted 7% of the variation due to the division of the S. frugiperda populations into Brazilian and Argentine groups. Also, no molecular variation (0%) and a gene flow rate equal to Nm=1.32 were estimated between fall armyworm group of populations collected at maize and cotton fields in Brazil. The same populations of S. frugiperda infest cotton and maize crops in Brazil and could be considered as interbreeding subunits of the same population. Therefore, there is a need of strategic action plans to the planting of cotton and maize crops for designing of management programs of S. frugiperda in Brazil.

Cotton (Gossypium spp.) is the worlds leading textile fiber crop, and an important source of oil and protein. Insufficient candidate gene derived-markers suitable for genetic mapping and limited information on genes that control economically important traits are the major impediments to the genetic improvement of Upland cotton (G. hirsutum L.). The objectives of this study were to develop a SNP marker discovery strategy in tetraploid cotton species, SNP characterization and marker development from fiber initiation and elongation related genes, chromosomal assignment of these genes by SNP marker-based deletion analysis or linkage mapping, and genetic and molecular analysis of mutants affecting cotton fiber development. Phylogenetic grouping and comparision to At- and Dt-genome putative ancestral diploid species of allotetraploid cotton facilitated differentiation between genome specific polymorphisms (GSPs) and marker-suitable locus-specific polymorphisms (LSPs). By employing this strategry, a total of 222 and 108 SNPs were identified and the average frequency of SNP was 2.35% and 1.30% in six EXPANSIN A genes and six MYB genes, respectively. Both gene families showed independent and incongruent evolution in the two subgenomes and a faster evolution rate in Dt-genome than that in At-genome. SNPs were concordantly mapped to different chromsomes, which confirmed their value as candidate gene marker and indicated the reliability of SNP discovery stragey. QTL mapping by two F₂ populations developed from fiber mutants detected major QTL which explain 62.8-87.1% of the phenotypic variation for lint percentage or lint index in the vicinity of BNL3482-138 on chromosome 26. Single marker regression analyses indicated STV79-108, which was located to the long arm of chromosome 12 (the known location of N₁ and perhaps n₂ loci), also had significant association (R² % value 15.4-30.6) with lint percentage, lint index, embryo protein percentage and micronaire. Additional QTL and significant markers associated with other seed and fiber traits were detected on different chromosomes. Inheritance analysis indicated that both genetic models N₁N₁n₂n₂ and n₂n₂li₃lisub>3 could lead to the fiberless phenotype. The observation of fuzzless-short lint phenotype indicated fiber initiation and elongation were controlled by different mechanisms. The penetrance of Li₂ gene expression was observed in this study.

Aphis gossypii Glover (cotton-melon aphid) is a major pest of agriculture worldwide. Cucumis melo L. (melon) possesses monogenic resistance to this aphid, and is a good model for the study of aphid resistance mechanisms in plants. This dissertation presents analyses of the effects of the resistance gene on A. gossypii, and of the gene's effects on biochemical and molecular-genetic properties of melon plants. Nearly isogenic lines (NILs) of melon, either resistant or susceptible to A. gossypii, were compared for their influence on aphid life history traits and feeding behavior. The resistance trait delayed development, increased mortality, and markedly decreased reproduction of aphids confined to leaves of resistant plants. Aphids on resistant plants salivated into phloem sieve elements significantly longer, and were less likely to begin sap ingestion after salivation, suggesting that the resistance factor acts within phloem sieve elements. Biochemical properties of callose synthase were compared between NILs to test the hypothesis that callose deposition plays a role in the resistance mechanism. No differences were detected between resistant and susceptible melon genotypes with respect to callose synthase subunit abundance or in vitro enzyme activity. Sixty-four F₃ families from a melon mapping population were tested for aphid resistance to place the resistance locus on a genetic map of the melon genome. Four molecular markers were found to be linked to the aphid resistance phenotype. The name Agr ( Aphis gossypii resistance) is proposed for this locus. The closest flanking markers were positioned at 4.3 and 7.0 cM from Agr. Evidence suggests Agr might be a member of the nucleotide binding site-leucine-rich repeat (NBS-LRR) family of plant resistance genes, which are known to cluster in plant genomes. Melon genomic DNA sequences homologous to this gene family were isolated to test the hypothesis that Agr is an NBS-LRR homolog. Two of these sequences were tested for genetic linkage to Agr in a population of F₂ plants segregating for the resistance trait. DNA gel blot analysis determined that one sequence, NBS-2, is approximately 2.7 cM distant from Agr, which suggests Agr resides in a cluster of NBS-LRR homologs and could be a member of this gene family.

Coniothyrium zuluense is the causal agent of a serious Eucalyptus stem canker disease in South Africa (Wingfield et al., 1997). Eucalypts are the most important hardwood plantations in the world, and in South Africa these hardwoods occupy approximately 1.5 million hectares of plantation area, an area that is soon to be increased by an additional 600 000 hectares. As exotics, Eucalyptus plantations are constantly exposed to infection by fungal pathogens such as C. zuluense, which by secreting cell-¬wall degrading enzymes contribute to the degradation of plant cell walls and subsequent reduction and in the quality of timber produced. This ultimately affects the South African paper, pulp and timber industries. Selection of resistant clones through traditional breeding methods is the most common method currently employed in overcoming the problem of fungal infection. The genetic manipulation of Eucalyptus trees for enhanced resistance to fungal diseases is an alternative to the time-consuming and tedious approach of conventional breeding. The identification of several antifungal proteins, particularly polygalacturonase-inhibiting proteins (PGIPs) from various plant species including Eucalyptus, lead to the hypothesis that over-expression of these proteins could potentially reduce pathogen attack. However, prior to the expression of PGIPs in plants, isolation and molecular characterization of these genes are required. The aims of this study were therefore (l) to clone and characterize the complete Eucalyptus grandis pgip gene, (2) to transform Nicotiana tabacum (tobacco) plants with the E. grandis pgip gene and (3) to test for inhibition of C. zuluense PGs by PGIPs extracted from transgenic tobacco plants. This forms the first step towards the generation of E. grandis clones that are more disease tolerant. A review of the role of fungal endopolygalacturonases and polygalacturonase¬inhibitors in plant-pathogen interactions are presented in chapter I. Strategies employed to isolate and characterize pgip genes from a range of plant species are highlighted and the importance ofPGIPs in disease resistance is discussed. In chapter 2, the molecular cloning and characterization of the E. grandis pgip gene is discussed. The work presented in this chapter is a follow up on work previously conducted by Chimwamurombe (2001). Previously, a partial Eucalyptus pgip gene sequence was obtained with the use of degenerate oligonucleotide primers. In this study, the complete Eucalyptus pgip gene was obtained through the employment of genome walking strategies. Transformation of Nicotiana tabacum cv LA Burley plants with the Eucalyptus pgip gene and the molecular characterization of transgenic tobacco plants is discussed in chapter 3. The transformation and expression of foreign genes in tobacco plants is a well-established protocol, making tobacco the most appropriate candidate plant for assessing the functionality of the plant transformation construct. The production of endopolygalacturonases from virulent C. zuluense isolates and the subsequent PGIP assays conducted to determine levels of PG inhibition are included in this chapter. This thesis consists of three independent chapters representing studies on the molecular characterization of an E. grandis pgip gene and focusing on the potential for inhibition of PGs produced by C. zuluense by Eucalyptus PGIP extracted from transgenic tobacco plants. Repetition of certain aspects in the individual chapters has been unavoidable and the thesis is presented following a uniform style.
Dissertation (MSc)--University of Pretoria, 2003.
Genetics
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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Cotton (G. hirsutum L.) has the most important natural textile fiber in the world. The Gossypium remainders in Pernambuco state are in risk of disappearing due to ecological and agricultural process. The study of species in situ, about they genetic and morphological characteristics gives support in order to find and implement preservation options. The aims of this work was characterize the genetic variability of G. hirsutum e G. barbadense accesses of Sertão, Agreste and Zona da Mata of Pernambuco state by morphological and molecular marks. On the in situ study were evaluated 82 accesses in the 29 counties during expedition in 2005 and 2006 by interview and observation about morphological and phenological characters, and condition of conservation. The identified plants uses were: medical, ornamental and for confection of lamp wick. The genetic erosion risk observed were: change and loss of cultural human habit, animal pastry and environmental depredate. In the molecular study was used fifteen pair of microssatelites primers in order to evaluate the allelic frequency, genetic diversity and genetic distance by Nei statistical. The average of heterozygosis expected (He) that correspond the genetic diversity was 0.321, it was biggest for upland cotton (0.481). For the three studied biomes on the Sertão and the Agreste where predominate of the upland cottonthe He had highest value, 0.548 e 0.444, respectively. The average of inbreeding index was 0.806, highest for G. barbadense (0.968). Cluster shoed an interspecific hybrid between G. barbadense and G. hirsutum (PE 0553). On the morphological characterization ex situ were evaluated 38 morphological characters and nine fiber characters of 72 Gossypium accesses PE0553 (G. barbadense), PE0516 (upland cotton) and PE0524 (moco cotton) were the most morphological divergent and showedmore variability for studied characters. The majority of accesses of three types of cotton shoed good index for fiber being what herbaceous cotton were better about fiber uniformity, reflectance, resistance and percentage of fiber. The information obtained on morphological and molecular cauterization Gossypium genus on Pernambuco state showed high genetic diversity that should be conserving in order gives genes to breeding programs.
O algodoeiro (G. hirsutum L.) possui a mais importante fibra têxtil natural no mundo. Os remanescentes de Gossypium no Estado de Pernambuco correm sérios riscos de desaparecerem em decorrência de fatores ecológicos e agrícolas. O estudo das espécies in situ, em relação suas características genéticas e morfológicas, contribui potencialmente à implantação de medidas de manejo e preservação de espécies naturalizadas. O presente trabalho teve por objetivo caracterizar a variabilidade genética de acessos de G.hirsutum e G. barbadense do Agreste, Sertão e Zona da Mata do Estado de Pernambuco, por meio de marcadores morfológicos e moleculares. No estudo in situ, avaliou-se os 82 acessos durante expedições em 29 municípios nos anos 2005 e 2006, investigando-se por entrevistas e observações, sobre caracteres morfológicos, fenológicos e de conservação. Os usos das plantas identificados foram: medicinal, asséptico, ornamental e confecção de pavios de lamparina. Os riscos de erosão genética constatados foram: mudança e abandono de hábitos culturais, pastejo e depredação do ambiente. Na análise molecular, utilizou-se quinze pares de primers microssatélites para avaliar freqüências alélicas, diversidade genética e distância entreos genótipos pelas estatísticas de Nei. A heterozigosidade média esperada (He), que correspondem a diversidade genética, foi 0,321, sendo mais alta para o algodoeiro mocó (0,481). Considerando-se os três biomas estudados, no Sertão e no Agreste, locais em que predominou algodoeiro mocó, tiveram os maior valore de He, 0,548 e 0,444, respectivamente. O coeficiente de endogamia média foi de 0,806, tendo sido mais elevado em G. barbadense (0,968). O agrupamento revelou um híbrido interespecífico entre G. barbadense e G. hirsutum (PE 0553). Na caracterização morfológica ex situ, avaliou-se 38 caracteres morfológicos e nove caracteres de fibra, de 72 acessos de Gossypium. Dos três tipos de algodoeiros caracterizados, constatouse maior diversidade morfológica nos acessos de algodoeiro mocó. Os acessos PE0553 (G. barbadense), PE0516 (algodoeiro herbáceo) e PE0524 (algodoeiro mocó) foram os mais divergentes morfologicamente, apresentando maior variabilidade nos descritores avaliados. A maioria dos acessos dos três tipos de algodoeiros revelaram índices satisfatórios de características intrínsecas de fibra, sendo que os algodoeiros herbáceos foram superiores em relação ao índice de uniformidade de fibra, reflectância,resistência e percentagem de fibra. As informações obtidas na caracterização molecular e morfológica mostraram elevada diversidade genética nos acessos de Gossypium, no Estado de Pernambuco, que deve ser conservada para servir de fonte de genes para os programas de melhoramento genético.

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The germination of cotton seeds and the seedlings emergency are generally delayed and reduced by the salinity. Although the cotton is considered a tolerant culture, it can suffer substantial reductions in regarding its growth and production when exposed to salinity condition. The aims of this study went evaluate the effect of the saline stress in the germination phase to four cotton genotypes (BRS Rubi, BRS Safira, BRS 201 and CNPA 187 8H), using different osmotic potentials generated with increment of sodium chloride (NaCl). The saline stress was simulated using NaCl aqueous solutions in the potentials: 0.0 (Control); -0.2; -0.4; -0.6; -0.8 and -1.0 MPa. The treatments were monitored by means of tests for analysis of seeds, germination, first counting, speed germination index, length of shoot, radicle length, dry weigth of embrionic axis and shoot/radicle ratio. The tests for germination, first counting and index of germination speed were accomplished using 50 seeds for repetition and for the study of length of shoot, radicle length, dry weigth of embrionic axis and shoot/radicle ratio were used 20 seeds by repetition. For both tests four repetitions were accomplished by genotype for each one of the potentials. The seeds of each repetition were involved in papers Germitest humidified with NaCl solution corresponding to the potential. The repetitions of both tests were maintained in a germinator with saturated humidity. The analysis were initiate four days after the induction of the saline stress. The evaluations of the first three variables analyzed were accomplished daily; the seeds were remove and counted when its germinated. For the length tests just the repetitions corresponding to the potential of NaCl 0,0 MPa were analysis 4 days after the beginning of the induction of the saline stress. The analysis of the repetitions of the potentials -0,2 and -0,4 and of the potentials -0,6, -0,8 and -1,0 MPa they were accomplished with 12 and 20 days, respectively. For accomplishment of the analisis of this test the shoot of the 20 plantules of each repetition was separate from the radicle and both parts were measured. The statistical analyses were performed using the GENMOD and GLM procedures of the SAS. For the variable germination, the cultivates CNPA 187 8H and BRS Safira stood out for the potential -0.8 MPa, with averages of 89% and 81%, respectively. The test of speed germination index to cultivate BRS Safira presented the largest averages for the two higher saline potentials. It was observed that the increase of the saline potential reduces the germination percentage and speed germination index. For each day of evaluation it was verified that the increase of the saline potential causes a reduction of the length both of the shoot and of the radicle. The radicle tends to grow more than the shoot until the potential -0,4 MPa
A germina??o de sementes de algod?o e a emerg?ncia de pl?ntulas s?o geralmente retardadas e reduzidas pela salinidade. Embora o algod?o seja considerado uma cultura tolerante, pode sofrer redu??es substanciais no seu crescimento e na produ??o quando exposta ? condi??o de salinidade. O objetivo deste estudo foi avaliar o efeito do estresse salino na fase de germina??o em quatro gen?tipos de algod?o (BRS Rubi, BRS Safira, BRS 201 e CNPA 187 8H), empregando-se diferentes potenciais osm?ticos, gerados com acr?scimo de cloreto de s?dio (NaCl). O estresse salino foi simulado, utilizando-se solu??es aquosas de NaCl nos potenciais 0,0; -0,2; -0,4; -0,6; -0,8 e -1,0 MPa. Os tratamentos foram monitorados por meio de testes para an?lise de sementes: germina??o, primeira contagem, ?ndice de velocidade de germina??o (IVG), comprimento de parte a?rea, comprimento de rad?cula, peso seco de eixo embrion?rio e rela??o rad?cula/parte a?rea. Os testes para germina??o, primeira contagem e IVG foram realizados utilizando-se 50 sementes por repeti??o; para o estudo de comprimento de parte a?rea, comprimento de rad?cula, peso seco de eixo embrion?rio e rela??o rad?cula/parte a?rea, foram utilizadas 20 sementes por repeti??o. Para ambos os testes, foram realizadas quatro repeti??es por gen?tipo para cada um dos potenciais. As sementes de cada repeti??o foram envolvidas em pap?is Germitest umedecidos com a solu??o de NaCl correspondente ao potencial. As repeti??es de ambos os testes foram conduzidas em germinador e a umidade mantida ao ponto de satura??o. As leituras das tr?s primeiras vari?veis analisadas foram iniciadas quatro dias ap?s a indu??o do estresse salino. As avalia??es foram realizadas diariamente; as sementes foram retiradas e contabilizadas ? medida que ocorria a germina??o. Para os testes de comprimento, apenas as repeti??es correspondentes ao potencial de NaCl 0,0 MPa foram lidas, quatro dias ap?s o in?cio da indu??o do estresse. As leituras das repeti??es dos potenciais -0,2 e -0,4 e dos potenciais -0,6, -0,8 e -1,0 MPa foram realizadas, respectivamente, aos 12? e 20? dias. Para a realiza??o das leituras deste teste, a parte a?rea das 20 plantas de cada repeti??o foi separada da rad?cula e ambas mensuradas. As an?lises estat?sticas foram efetuadas, utilizando-se os procedimentos GENMOD e GLM do SAS. Para a vari?vel germina??o, as cultivares CNPA 187 8H e BRS Safira destacaram-se para o potencial -0,8 MPa, com m?dias de 89% e 81%, respectivamente. Foi observado que o aumento do potencial salino reduziu a porcentagem do IVG. Para cada dia de avalia??o, verificou-se que o aumento do potencial salino provoca uma redu??o do comprimento da parte a?rea e da rad?cula. A rad?cula tende a crescer mais que a parte a?rea at? o potencial -0,4 MPa

Heliothis virescens (Fabricius) é uma das pragas-alvo do algodão geneticamente modificado que expressa a proteína Cry1Ac de Bacillus thuringiensis Berliner (Bt). Estudos sobre a suscetibilidade de H. virescens à proteína Cry1Ac e sobre a estrutura genética e padrões de fluxo gênico nas escalas locais e regionais desse inseto são fundamentais para a implementação de programas de Manejo da Resistência de Insetos (MRI) no Brasil. Dessa forma, os principais objetivos do trabalho foram: (a) avaliar a suscetibilidade à proteína Cry1Ac em populações de H. virescens coletadas nas principais regiões produtoras de algodão no Brasil (Bahia, Goiás, Mato Grosso e Mato Grosso do Sul) durante as safras agrícolas 2007/08 e 2008/09; e (b) avaliar a variabilidade genética e fluxo gênico de populações de H. virescens provenientes das culturas de algodão (safras 2007/08, 2008/09 e 2009/10) e de soja (safra 2009/10) utilizando sequências de DNA mitocondrial (DNAmit). As linhas-básica de suscetibilidade à proteína Cry1Ac foram estabelecidas mediante o uso de lagartas neonatas, por meio de bioensaios de incorporação das diferentes concentrações da proteína em dieta artificial. Para avaliar a variabilidade genética e o fluxo gênico entre populações de H. virescens utilizou-se sequências de DNAmit das subunidades I e II da citocromo oxidase COI e COII e a subunidade 6 da desidrogenase dinucleotídica da adenina nicotinamida nad6. As CLs50 estimadas variaram de 0,18 a 0,66 µg de Cry1Ac/mL de dieta para as populações coletadas na safra 2007/08 (variação de 3,7 vezes). Da mesma forma, as concentrações efetivas médias para a inibição do desenvolvimento larval (CE50) variaram de 0,0053 a 0,0161 µg de Cry1Ac/mL dieta (variação de 3,0 vezes). A partir da análise conjunta dos dados de concentração-mortalidade de todas as populações avaliadas, foram definidas e validadas as concentrações diagnósticas de 3,1 e 5,6 µg de Cry1Ac/mL de dieta para programas de monitoramento da resistência de H. virescens à proteína Cry1Ac no Brasil. Baseadas em análises de agrupamento (Neighbor-Joining e Análise de Componentes Principais) e Bayesiana (Structure) foram verificadas uma baixa estruturação entre as populações de H. virescens de diferentes regiões, bem como para aquelas coletadas em plantas hospedeiras diferentes. As análises de AMOVA também indicaram baixa estruturação genética entre as populações de H. virescens estudadas independente da cultura (Fst= 0,019) ou escala geográfica (Fst= 0,012), sugerindo um nível significativo de fluxo gênico. Em média, a distância genética entre as amostras foi de 0,1%. Uma rede de haplótipos obtida com os dados combinados resultou em 35 haplótipos, com quatro haplótipos únicos presentes somente nas amostras coletadas em soja. A principal característica dessa rede é a forma de estrela na distribuição dos haplótipos, bem como a ocorrência de muitos alelos em baixa frequência. Esse tipo de rede é característico de populações que passaram por uma recente expansão populacional e, de fato, a história demográfica de H. virescens, baseada no teste de distribuição da diferença genética par-a-par entre haplótipos (distribuição de Mismatch) e nos resultados negativos nos testes de neutralidade seletiva indicam também um episódio de expansão populacional recente. As informações obtidas no presente trabalho serão fundamentais para o acompanhamento da efetividade das estratégias de manejo da resistência de H. virescens à proteína Cry1Ac no Brasil.
The tobacco budworm, Heliothis virescens (Fabricius), is one of target pests of genetically modified cotton expressing Cry1Ac insecticidal protein derived from Bacillus thuringiensis Berliner. Studies on susceptibility of H. virescens to Cry1Ac and the genetic structure and gene flow patterns at local and regional levels are crucial for establishing an Insect Resistance Management (IRM) program for Bt cotton in Brazil. Thus, the objectives of this study were (a) to evaluate the susceptibility of field-collected populations of H. virescens to Cry1Ac from major cotton-growing regions in Brazil (Bahia, Goiás, Mato Grosso and Mato Grosso do Sul) in the cropping seasons of 2007/08 and 2008/09; and (b) to evaluate the genetic variability and gene flow among H. virescens populations from cotton (2007/08, 2008/09 and 2009/10 cropping seasons) and soybean (2009/10 cropping season) with mitochondrial DNA markers. Baseline susceptibility data to Cry1Ac protein were estimated with neonate larvae thereby using diet incorporation bioassays. Genetic variation and gene flow among H. virescens populations were evaluated by using mitDNA sequences of cytochrome oxidase subunities I and II COI e COII and the subunity 6 of dinucleotide dehydrogenase of adenine nicotinamide nad6. The estimated LC50 values varied from 0.18 to 0.66 µg of Cry1Ac/mL of diet among the 2007/08 populations (3.7 fold variation). Similarly, the EC50 values based on growth inhibition ranged from 0.0053 to 0.0161 µg of Cry1Ac/mL of diet for the 2007/08 populations (3.0 fold variation). A joint analysis of the mortality data across all tested populations was used to develop candidate diagnostic concentrations for future monitoring programs. The proposed diagnostic concentrations of 3.1 and 5.6 µg of Cry1Ac/mL of diet were validated against field-collected populations from 2008/09 and will form the basis for future resistance monitoring programs with H. virescens. Based on cluster analysis (Neighbor-Joining and Principal Coordinate Analysis) and Bayesian analysis (Structure), a low structure was detected among H. virescens populations either by regions or host plants. AMOVA analysis also indicated low genetic structure among H. virescens populations across crops (Fst= 0.019) or geographic scale (Fst= 0.012), suggesting a significant gene flow. The mean genetic distance among samples was 0.1%. The haplotype network obtained with joint data resulted in 35 haplotypes, with four unique haplotypes present only in samples collected from soybean crop. The major characteristics of the haplotype network were the star-like pattern and the occurrence of many alleles at low frequencies. This type of network is typical for populations that passed through a recent population expansion and, in fact, the demographic history of H. virescens, based on distribution test of pair-wise genetic difference among haplotypes (Mismatch distribution) and negative results from tests of selective neutrality also indicate an episode of a recent population expansion.

Estudos de genética de populações de pragas agrícolas têm destacado a importância de se conhecer a estruturação genética e os padrões de fluxo gênico entre populações para o refinamento de estratégias de Manejo Integrado de Pragas (MIP). A lagarta-da-maçã do algodoeiro, Heliothis virescens (F.), é um inseto praga amplamente distribuído e importante economicamente por causar danos consideráveis à cultura do algodão no Brasil. O controle dessa praga tem sido feito principalmente pelo uso de inseticidas e de plantas geneticamente modificadas (GM) que expressam proteína(s) de Bacillus thuringiensis Berliner e o potencial de evolução da resistência é alto. O conhecimento de quanto as populações de H. virescens são capazes de trocar informação genética entre si é de fundamental importância para a implantação de estratégias de manejo dessa praga. No entanto, pouco se sabe sobre a estrutura genética e os padrões de fluxo gênico em H. virescens em escalas locais e regionais no Brasil. Assim, o objetivo desse trabalho foi avaliar a variabilidade genética em populações de H. virescens utilizando marcadores microssatélites. Foram amostrados indivíduos de H. virescens oriundos de populações coletadas nas safras de 2007/08, 2008/09 e 2009/10 nas principais regiões produtoras de algodão e soja no Brasil. Foram estudados nove locos polimórficos em 12 populações, em um total de 205 indivíduos. O número médio de alelos por loco foi de 14,11. Os valores de heterozigosidade média esperada (HE) e observada (HO) foram de 0,303 e 0,438, respectivamente. O coeficinete de endocruzamento da espécie f foi de 0,294 (IC 95% de 0,178 a 0,406). As estimativas de estruturação genética foram = 0,132 (IC 95% de 0,072 a 0,218) e RST = 0,252. Esses valores indicam uma estruturação genética moderada entre as populações. Estimativas do número de migrantes indicaram um pequeno fluxo gênico, principalmente no sentido Centro- Oeste Nordeste, embora a maioria dos indivíduos dentro das populações seja residente; adicionalmente, foi verificado que o estabelecimento das populações do algodão ocorre a partir de indivíduos migrantes da soja ou descendentes desses indivíduos. Análises de Componentes Principais e de atribuição usando inferência Bayesiana revelaram a formação de dois grupos, porém não foi possível identificar um padrão de agrupamento (por região, safra ou hospedeiro). Desta forma os resultados do presente trabalho sugerem uma estruturação genética incipiente para as populações de H. virescens no Brasil. Desse modo, é importante levar esses resultados em consideração para que o MIP em geral, e especificamente para que as abordagens para retardar a evolução da resistência sejam implementadas de forma efetiva para o manejo de H. virescens no Brasil.
Agricultural pests population genetics studies have emphasized the importance of genetic structure and patterns of gene flow knowledge for Integrated Pest Management (IPM) strategies. The tobacco budworm, Heliothis virescens (F.), is a widespread and economically important insect pest renowned for causing considerable damage in cotton fields in Brazil. This pest has been controlled by the use of insecticides and genetic modified plants (GM) expressing proteins from Bacillus thuringiensis Berliner, and it has already been shown to present a high potential to develop resistance to these control technologies. To a successful application of these strategies it is needed to know the capacity of the pest populations to exchange genetic information among them. However, for H. virescens a scarce amount of information about genetic structure and patterns of gene flow is available at local and regional scales in Brazil. In this way, the main objective of this study was to evaluate the genetic variability of H. virescens based on microsatellite markers. Specimens were sampled during 2007/08, 2008/09 and 2009/10 from the main cotton and soybean producers regions in Brazil. From this, nine polymorphic loci from 12 populations were studied in 205 specimens. The average number of alleles was 14.11. Expected (HE) and observed (HO) heterozygosity were 0.303 and 0.438, respectively. Inbreeding coefficient f was 0.294 (IC 95% 0.178 - 0.406). Genetic structure indices were: = 0.132 (IC 95% 0.072 0.218) and RST = 0.252. These values point to a moderate genetic structure among H. virescens populations. Migrants estimative indicate a low gene flow, mainly in the Center-Western Northern direction, although most individuals are residents within populations; additionally it was suggested that immigrants to cotton populations come from soybean fields. Genetic relationships inferred by Principal Component Analysis and Bayesian assignment tests identified two groups, although no group pattern was recognized, even by geographic region, year of sampling or host plant. These results suggest an incipient genetic structuring for H. virescens populations within Brazil. Thus, such results should be considered for IPM strategies aiming in an efficient control of H. virescens in Brazil.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library.
Cotton fibre growth and development are regulated by the expression of several thousand genes in the fibre cell. These genes are often expressed in both cotton fibres and other plant tissues, although a proportion are 'fibre-specific' (expressed predominantly or exclusively in the fibre). Many fibre-specific genes have important functions in fibre development, and their expression is generally regulated at the level of transcription. However, the mechanisms that restrict gene expression to the cotton fibre have not been well characterised. An understanding of these mechanisms is central to a molecular model of fibre development, and may be important in the generation of improved cotton varieties with fibre-specific trans gene expression. The aim of this project was to identify the promoter sequences and transcription factors involved in regulating the fibre-specific expression of FSltp6, a gene encoding a cotton lipid transfer protein (LTP). The FSltp6 gene is abundantly and specifically transcribed in elongating cotton fibres. In this project, the fibre-specificity of the FSltp6 promoter was analysed using constructs containing the FSltp6 promoter sequence driving expression of the reporter gene -glucuronidase (GUS). Cotton fibres and other cotton tissues were transiently transformed with an FSltp6::GUS construct and analysed for GUS expression. The FSltp6 promoter restricted GUS expression primarily to the cotton fibre. Successive 5' deletions of the FSltp6 promoter were then used to isolate the regions necessary for fibre-specific expression. An 84 bp fibre-specificity region (FSR) was found to be essential for GUS expression exclusively in the cotton fibre, while a 49 bp region was necessary for expression in any of the tissues tested. The fibre-specificity of the FSltp6 promoter was also analysed by the stable transformation of tobacco with FSltp6::GUS. The transgenic tobacco plants demonstrated strong GUS expression in the leaf trichomes. This result provided further support for the fibre-specificity of the FSltp6 promoter and (in line with previous studies) suggested the utility of tobacco trichomes as a model for cotton fibre development. A yeast one-hybrid assay was used to identify transcription factors that may regulate fibre-specific expression by interacting with the FSR. This experiment identified three novel classes of cotton protein with potential roles in fibre specificity: HMGA-like proteins, Mutator transposase-like proteins and an AT -hook protein. The full-length cDNA of the AT -hook protein was isolated and analysed for its potential function as a transcription factor and regulator of fibre-specificity. This project has identified a promoter region and several novel transcription factors with a potential role in the regulation of fibre-specific gene expression. These results provide further insight into the molecular regulation of gene transcription in cotton fibres. Application of these results in the generation of transgenic cotton may produce varieties with enhanced gene expression in the cotton fibre.
http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1188821
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2005

Cotton fibre growth and development are regulated by the expression of several thousand genes in the fibre cell. These genes are often expressed in both cotton fibres and other plant tissues, although a proportion are 'fibre-specific' (expressed predominantly or exclusively in the fibre). Many fibre-specific genes have important functions in fibre development, and their expression is generally regulated at the level of transcription. However, the mechanisms that restrict gene expression to the cotton fibre have not been well characterised. An understanding of these mechanisms is central to a molecular model of fibre development, and may be important in the generation of improved cotton varieties with fibre-specific trans gene expression. The aim of this project was to identify the promoter sequences and transcription factors involved in regulating the fibre-specific expression of FSltp6, a gene encoding a cotton lipid transfer protein (LTP). The FSltp6 gene is abundantly and specifically transcribed in elongating cotton fibres. In this project, the fibre-specificity of the FSltp6 promoter was analysed using constructs containing the FSltp6 promoter sequence driving expression of the reporter gene 􀁚-glucuronidase (GUS). Cotton fibres and other cotton tissues were transiently transformed with an FSltp6::GUS construct and analysed for GUS expression. The FSltp6 promoter restricted GUS expression primarily to the cotton fibre. Successive 5' deletions of the FSltp6 promoter were then used to isolate the regions necessary for fibre-specific expression. An 84 bp fibre-specificity region (FSR) was found to be essential for GUS expression exclusively in the cotton fibre, while a 49 bp region was necessary for expression in any of the tissues tested. The fibre-specificity of the FSltp6 promoter was also analysed by the stable transformation of tobacco with FSltp6::GUS. The transgenic tobacco plants demonstrated strong GUS expression in the leaf trichomes. This result provided further support for the fibre-specificity of the FSltp6 promoter and (in line with previous studies) suggested the utility of tobacco trichomes as a model for cotton fibre development. A yeast one-hybrid assay was used to identify transcription factors that may regulate fibre-specific expression by interacting with the FSR. This experiment identified three novel classes of cotton protein with potential roles in fibre specificity: HMGA-like proteins, Mutator transposase-like proteins and an AT -hook protein. The full-length cDNA of the AT -hook protein was isolated and analysed for its potential function as a transcription factor and regulator of fibre-specificity. This project has identified a promoter region and several novel transcription factors with a potential role in the regulation of fibre-specific gene expression. These results provide further insight into the molecular regulation of gene transcription in cotton fibres. Application of these results in the generation of transgenic cotton may produce varieties with enhanced gene expression in the cotton fibre.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2005

Coniothyrium zuluense is the causal agent of a serious Eucalyptus stem canker disease in South Africa (Wingfield et al., 1997). Eucalypts are the most important hardwood plantations in the world, and in South Africa these hardwoods occupy approximately 1.5 million hectares of plantation area, an area that is soon to be increased by an additional 600 000 hectares. As exotics, Eucalyptus plantations are constantly exposed to infection by fungal pathogens such as C. zuluense, which by secreting cell-¬wall degrading enzymes contribute to the degradation of plant cell walls and subsequent reduction and in the quality of timber produced. This ultimately affects the South African paper, pulp and timber industries. Selection of resistant clones through traditional breeding methods is the most common method currently employed in overcoming the problem of fungal infection. The genetic manipulation of Eucalyptus trees for enhanced resistance to fungal diseases is an alternative to the time-consuming and tedious approach of conventional breeding. The identification of several antifungal proteins, particularly polygalacturonase-inhibiting proteins (PGIPs) from various plant species including Eucalyptus, lead to the hypothesis that over-expression of these proteins could potentially reduce pathogen attack. However, prior to the expression of PGIPs in plants, isolation and molecular characterization of these genes are required. The aims of this study were therefore (l) to clone and characterize the complete Eucalyptus grandis pgip gene, (2) to transform Nicotiana tabacum (tobacco) plants with the E. grandis pgip gene and (3) to test for inhibition of C. zuluense PGs by PGIPs extracted from transgenic tobacco plants. This forms the first step towards the generation of E. grandis clones that are more disease tolerant. A review of the role of fungal endopolygalacturonases and polygalacturonase¬inhibitors in plant-pathogen interactions are presented in chapter I. Strategies employed to isolate and characterize pgip genes from a range of plant species are highlighted and the importance ofPGIPs in disease resistance is discussed. In chapter 2, the molecular cloning and characterization of the E. grandis pgip gene is discussed. The work presented in this chapter is a follow up on work previously conducted by Chimwamurombe (2001). Previously, a partial Eucalyptus pgip gene sequence was obtained with the use of degenerate oligonucleotide primers. In this study, the complete Eucalyptus pgip gene was obtained through the employment of genome walking strategies. Transformation of Nicotiana tabacum cv LA Burley plants with the Eucalyptus pgip gene and the molecular characterization of transgenic tobacco plants is discussed in chapter 3. The transformation and expression of foreign genes in tobacco plants is a well-established protocol, making tobacco the most appropriate candidate plant for assessing the functionality of the plant transformation construct. The production of endopolygalacturonases from virulent C. zuluense isolates and the subsequent PGIP assays conducted to determine levels of PG inhibition are included in this chapter. This thesis consists of three independent chapters representing studies on the molecular characterization of an E. grandis pgip gene and focusing on the potential for inhibition of PGs produced by C. zuluense by Eucalyptus PGIP extracted from transgenic tobacco plants. Repetition of certain aspects in the individual chapters has been unavoidable and the thesis is presented following a uniform style.
Dissertation (MSc)--University of Pretoria, 2006.
Genetics
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