Physiological characteristics of the response to water stress of two maize cultivars, the one drought resistant (PAN473) and the other drought sensitive (SR52), were compared. Mature plants were grown in one of two treatments, control and water stress. The drought resistant cultivar had a higher growth rate and a greater amount of roots in the lower soil levels than the drought sensitive cultivar in the water stress treatment. There was no difference between the cultivars in physiological characteristics in the control treatment, but in the water-stress treatment the drought resistant cultivar had a higher transpiration rate during the onset of water stress, and higher relative water content and levels of abscisic acid and proline throughout the period of water stress. A comparison between the cultivars in their gene expression response was done to determine if a correlation could be made with the difference in physiological response. A differential screening of water stress cDNA libraries identified nine different cDNA species which gave a signal with the water stress probe but not the control probe. Three of these cDNAs were represented by more than one cDNA clone. The cDNAs occurred in both libraries, therefore there was no difference between the cultivars in the presence or absence of the water stress responsive genes. The three genes represented by these cDNAs were named rws7, rws16 and rws5. All three genes showed increased transcription in response to water stress in whole plants, and to desiccation and osmotic stress of detached leaves. No increase in transcription was found in response to exogenously applied abscisic acid or proline. No difference between the cultivars was observed in the pattern of transcription response. Two of the three cDNA species that are represented more than once were sequenced. Rws7 had an open reading frame. A BLAST search found no homologous amino acid sequences, but the characteristics of the polypeptide suggest that it is a dehydrin. Rws 16 had a partial coding sequence. A BLAST search found two homologous amino acid sequences, and both were chloride channel proteins. The remaining seven cDNA species were sequenced at their 5' ends, and no complete homologous nucleotide sequences were found. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1996.

Orientador: Paulo Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T21:19:28Z (GMT). No. of bitstreams: 1 Ferreira_NataliaCristinaVerza_D.pdf: 9495810 bytes, checksum: 18cf9ae055a2b9f39de109abc6de469c (MD5) Previous issue date: 2006 / Resumo: O seqüenciamento de ESTs (etiquetas de seqüências expressas) e a sua organização em bancos de dados constituem poderosas ferramentas para identificar genes de interesse expressos em determinados tecidos e/ou tipos celulares. Neste trabalho criou-se um banco de seqüências expressas chamado MAIZESTdb, que contém ESTs de diversos tecidos de milho, porém enriquecido com seqüências provenientes do endosperma de milho em desenvolvimento. O MAIZESTdb contém 227.431 ESTs vindos de mais de 30 órgãos e tecidos de milho diferentes, 30.531 seqüenciados em nosso laboratório a partir de bibliotecas construídas com RNA mensageiro de endosperma. Estas seqüências representam uma grande contribuição na identificação de novos genes expressos no endosperma. A análise deste banco de ESTs possibilitou a identificação de 4.032 transcritos preferencialmente expressos no endosperma, e a sua anotação revelou uma ampla variedade de prováveis genes novos envolvidos no desenvolvimento e no metabolismo do endosperma. O banco MAIZESTdb foi utilizado neste trabalho para a identificação de fatores de transcrição (TFs) expressos no endosperma de milho, e, especialmente, na identificação de fatores preferencialmente expressos no endosperma, que podem desempenhar papéis regulatórios importantes durante a formação da semente. Foram identificados 1.233 TFs expressos em milho, 414 dos quais expressos no endosperma em desenvolvimento. Foram identificados ainda, através de análises in silico, 113 TFs preferencialmente expressos no endosperma, conjunto este que representa 9.2% dos TFs expressos identificados em milho, e que possivelmente contém reguladores importantes dos processos de especificação celular e desenvolvimento do endosperma de milho. Esta é a maior coleção de fatores de transcrição já descrita para este tecido, e representa uma fonte de dados importante para identificação de reguladores dos principais processos relacionados ao desenvolvimento do endosperma, como metabolismo de nitrogênio e carboidratos e controle da massa da semente. Uma das famílias mais representadas entre os TFs preferencialmente expressos no endosperma foi a família NAC de fatores de transcrição. Esta família apresentou 12 membros preferencialmente expressos no endosperma de milho. Um novo membro da família NAC, chamado de EPN-1 (Endosperm Specific NAM 1), teve seu perfil de expressão caracterizado. Sua expressão pode ser detectada desde os 5 DAPs, embora o pico de expressão ocorra entre 20 e 25 DAP, e ele apresenta expressão preferencial no endosperma. O promotor do gene EPN-1 foi clonado, seqüenciado e analisado quanto aos seus possíveis elementos CIS regulatórios; foram encontrados elementos conservados relacionados à endosperma-especificidade, elementos relacionados à regulação por ácido abscísico e giberelinas, e elementos conservados presentes nos promotores de a-amilases, indicando uma possível relação deste gene com o processo de transição entre a maturação e a germinação da semente. Ensaios de expressão transitória com o promotor do gene EPN-1 revelaram que sua expressão está dirigida à camada de aleurona do endosperma de milho, o que constitui mais uma evidência de sua possível função na regulação de genes relacionados aos processos de maturação e germinação da semente / Abstract: The sequencing of ESTs (expressed sequence tags) and its organization in databases constitute powerful tools to identify genes of interest in certain tissues and/or cell types. In this work we have created MAIZESTdb, a database of ESTs expressed in diverse maize tissues. The importance of this database, however, is that it is enriched with sequences from developing maize endosperm. The MAIZESTdb contains 227,431 ESTs coming from more than 30 different maize tissues and organs, 30,531 of which sequenced from endosperm cDNA libraries constructed in our laboratory. These sequences represent a great contribution for the identification of novel genes expressed in endosperm. The analysis of this ESTs database led to the identification of 4,032 transcripts preferentially expressed in the endosperm, and its annotation revealed a great variety of new genes involved in endosperm metabolism and development. The MAIZESTdb was then used to identify transcription factors (TFs) expressed in maize endosperm, and, mainly, in the identification of TFs preferentially expressed in the endosperm. We identified 1,233 TFs expressed in diverse maize tissues, 414 of which expressed in developing endosperm. We also identified, through in silico comparison of transcript abundance and library source, 113 TFs with preferential expression in endosperm, representing 9,2% of the TFs identified in this work. This dataset probably contains important regulators of cellular specification of the endosperm development. This is the biggest TFs collection reported for this tissue, and represents an important source of data for identification of regulators for main processes related to the endosperm development such as nitrogen and carbohydrate metabolism and control of seed mass. One of the most represented families among the TFs preferentially expressed in endosperm was the NAC family of transcription factors. This family presented 12 members with preferential expression in the endosperm. A new member of the NAC family, called EPN-1 (Endosperm Specific NAM 1), was characterized. Its expression / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Orientadores: Marcelo Menossi Teixeira, Augustina Gentile / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T18:33:40Z (GMT). No. of bitstreams: 1 Feltrim_Daniela_M.pdf: 2255232 bytes, checksum: c1f34d09fda3a2de4f7c62d0c7b9ec71 (MD5) Previous issue date: 2013 / Resumo: O presente trabalho possui como objetivo caracterizar fisiológica e funcionalmente um gene de milho que têm expressão relacionada com a tolerância ao cultivo em solo ácido contendo níveis tóxicos de alumínio (Al). Este gene, denominado Zmwrky69, foi identificado na linhagem de milho Cat100-6, tolerante ao Al (Mattiello et al., 2010). A análise da sequência da proteína ZmWRKY69 indica que ela pertence ao grupo III dessa família de fatores de transcrição, apresentando o motivo WRKYGKQ na região amino terminal e o motivo dedo de zinco Cx7Cx23HxC na região carboxi terminal. A proteína ZmWRKY69 está localizada no núcleo, conforme inferido por ensaio de localização subcelular em epitélio de cebola empregando uma fusão da proteína de milho com a proteína verde fluorescente (GFP). Ensaios de duplo híbrido em levedura indicam que a proteína ZmWRKY69 interage com um receptor de giberelina, uma proteína envolvida com a regulação por auxinas e uma proteína com função desconhecida. Plantas transgênicas de tabaco superexpressando Zmwrky69 apresentaram menor inibição do crescimento radicular na presença de Al, comparativamente às plantas selvagens. Esses resultados indicam que o gene Zmwrky69 codifica um fator de transcrição que apresenta um papel importante na tolerância ao Al em milho / Abstract: The objective of the present work is to characterize a gene differentially expressed in the maize inbred line Cat100-6 (aluminum tolerant) grown in acidic soil containing toxic levels of aluminum. This gene called Zmwrky69 was identified in the aluminum tolerant Cat100-6 inbred line (Mattiello et al., 2010). The sequence analysis of the ZmWRKY69 protein indicated that it belongs to the group III of this transcription factor family classification. The protein has the motif WRKYGKQ in the amino terminal region and a zinc finger motif Cx7Cx23HxC in the carboxi terminal region. This protein is localized in the nucleus as inferred by subcellular localization in onion epidermis using a maize protein fused to the Green Fluorescent Protein (GFP). Yeast two-hybrid essays indicate that the protein ZmWRKY69 interacts with a gibberellin receptor, a protein involved in auxin regulation and a protein with an unknown function. Transgenic tobacco plants overexpressing Zmwrky69 presented a lower degree of root growth inhibition in the presence of Al compared to wild type plants. These results indicate that Zmwrky69 gene encodes a transcription factor that presents an important role in Al tolerance in maize / Mestrado / Genetica Vegetal e Melhoramento / Mestra em Genética e Biologia Molecular

The importance of maize in sub-Saharan Africa and the potential of quality protein maize (QPM) to alleviate the nutritional gap caused by lack of access to adequate protein rich foods were highlighted. Frustrations from complex inheritance systems of the QPM trait leading to calls for more information on the inheritance and stability of the QPM trait, fear of total loss of the QPM trait due to the recessive nature of the opaque-2 gene to the wild type gene in normal endosperm maize when QPM and normal endosperm maize coexist, lack of information on the nutritional value of contaminated QPM grain, and poor linkages with the smallholder farmers were all cited as drawbacks in the promotion and adoption of QPM. Therefore the objectives of the study were: 1) To solicit the participation of smallholder farmers in the development and setting up of QPM breeding goals, objectives and dissemination strategies; 2) To estimate general combining ability (GCA), specific combining ability (SCA) and reciprocal cross effects on anthesis days, quality traits and grain yield among the publicly available elite QPM inbred lines; 3) To compare experimental QPM hybrids with selected check cultivars, and normal endosperm maize hybrids for grain yield performance and kernel endosperm modification scores; 4) To evaluate QPM hybrids for grain yield and kernel endosperm modification scores in selected sub-Saharan Africa target environments. 5) To determine the level of normal endosperm maize pollen contamination that can occur in quality protein maize without loss of nutritional superiority; 6) To estimate the average levels and the patterns of foreign maize pollen contamination in QPM crops coexisting with normal endosperm maize varieties. The contribution of smallholder farmers in setting breeding goals and dissemination strategies for QPM was solicited. One major finding was that the kernel endosperm qualities of landrace “Hickory King” need to be incorporated into new QPM varieties so as to encourage adoption. Farmers preferred getting information on QPM varieties through their local Agricultural Research and Extension (AREX) officers. A diallel study of 36 F1 QPM hybrids and their reciprocals was conducted across seven environments for agronomic traits and three environments for nutritional value traits. There were significant differences for all traits analysed using Griffing Method 3 model 1. General combing ability effects were significant and important in the control of anthesis days, kernel endosperm modification, protein content, tryptophan content, and Quality Index (QI). Specific combining ability effects were highly significant and important in the control of grain yield. There were significant SCA effects for anthesis days and QI but the proportions were lesser than the corresponding GCA effects in both traits. Kernel endosperm modification had significant GCA effects and nonsignificant SCA effects. Reciprocal-cross differences were significant for anthesis days, tryptophan content and QI. Nonmaternal effects were significant for tryptophan content whilst both maternal and nonmaternal effects were significant for QI and anthesis days. Nonmaternal effects were relatively more important than maternal effects in all the cases where there were significant reciprocal-cross differences. The cross with the highest SCA effects for grain yield was CZL03016/CML144. The most desirable cross with the lowest anthesis days was CZL03016/CML144 whilst the most desirable inbred line with the lowest anthesis GCA effects was CZL03016. The inbred line with the most desirable GCA effects for protein content, tryptophan content and QI was CML264Q. Inbred line CML264Q crossed to CZL03016 had significant SCA effects for QI. The most desirable GCA effects for kernel endosperm modification were associated with inbred line CZL03016 followed by CZL01006. Maternal effects for both tryptophan content and QI were associated with inbred line CML264Q. Genotype by environment interaction effects across all the seven environments were significant for grain yield and kernel endosperm modification. Check hybrids performed better than experimental hybrids for grain yield but were not different for kernel endosperm modification. The normal endosperm maize hybrids were significantly better for both grain yield and kernel endosperm modification. However, in all the comparisons the best check or normal endosperm maize hybrid was not significantly better than the best experimental or QPM hybrid, respectively. The most desirable score for kernel endosperm modification was from the cross of CZL01006 to CZL03016 though not significantly different from the check hybrid with the best score. AMMI1 was the best model for kernel endosperm modification scores and AMMI2 was suitable for grain yield. Both environments and hybrids were diverse. Grain yield of most hybrids was not stable with specific adaptation to environments. The most stable hybrid with no specific adaptation was CML176/CML181f with a mean yield of 6.51t ha-1. The putative nutritional superiority of normal endosperm maize pollen contaminated QPM as measured by the QI depended on the environmental conditions. The moisture stressed environment (CIMMYT Harare) had a lower QI value (0.858) and a lower tolerance to pollen contamination of 15.3% whereas the grain produced under near to optimum growing conditions (ART farm) had a higher QI value (0.915) and a higher tolerance to pollen contamination of 31.9% before total loss of nutritional superiority. Thus contaminated QPM grain had nutritional superiority up to a certain point before total loss of nutritional superiority. Geostatistical analysis was used to determine the levels and patterns of pollen contamination that occur when QPM and normal endosperm maize crops coexist under conditions minimising both temporal and geographical isolation to the lowest possible levels for the two independent crops. Higher pollen contamination levels were restricted mostly to the sections of the QPM crop proximal to the rows of normal endosperm maize crop, with the central parts of the QPM crops experiencing relatively low levels of contamination. For the four experiments (QCS200711, QCS200712, QCS200721 and QCS200722) in which the thresholds to nutritional superiority were determined, 87.9%, 94.8%, 62.2% and 65.6% of the crop areas passed for superior QPM grain, respectively. Estimates for average contamination levels of homogenous mixtures of grain from each of the nine experiments were below 20% contamination. The contamination levels were far less than previously thought. “Hickory King” kernel quality attributes were important in breeding QPM varieties for the smallholder farmers. Parents of the 72 hybrids were diverse for the agronomic characters studied and three of the experimental hybrids were found to be adapted and comparable to the check varieties. Quality protein maize tolerance to foreign pollen contamination without loss in nutritional superiority depended on growing conditions. The coexistence of QPM and normal endosperm maize without total loss of QPM nutritional superiority was feasible. / Thesis (PhD.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Maize grain contains high levels of phytic acid which chelates iron, zinc and other micronutrients as it passes through the digestive systems of monogastric consumers reducing their bioavailability. Breeding for low phytic acid (LPA) content to improve micronutrient bioavailability is hampered by a tedious and destructive colorimetric method on the grain, low yields compared to the wild-types and reduced seed germination and vigour of LPA mutants. Breeding for LPA therefore should also incorporate breeding for improved germination and vigour in the mutants. Molecular markers to speed up the selection process and studies on gene action and combining ability for germination, vigour and yield parameters of the LPA mutants in combinations with other different maize germplasm will speed up breeding for this trait. The objectives of this study were: to develop a molecular marker linked to the lpa1-1 gene and apply this marker for foreground selection in a backcross breeding programme and to use amplified fragment length polymorphism (AFLP) markers for background selection to recover the recurrent parent genome to speed up the backcrossing process; to study gene action and combining ability for seed germination, vigour and yield from diallel crosses involving LPA mutants, QPM and normal endosperm maize inbred lines by replicated laboratory seed tests ( standard germination test and accelerated aging test) and field evaluations in South Africa and Zimbabwe. A co-dominant single nucleotide polymorphism (SNP) marker which detects the transition base change of C/T nucleotides was developed from the gene sequence to identify the lpa1-1 trait. The 150 bp lpa1-1 SNP marker was validated by forward and reverse DNA sequencing of the parental amplification products which confirmed the C to T base change resulting in the LPA phenotype. The lpa1-1 SNP marker was used for foreground selection in 250 BC(2)F(1) progenies of CM 32 (LPA) x P 16 as the recurrent parent. This SNP marker was used to genotype the lines into homozygous dominant (wild type) and homozygous recessive (LPA) genotypes by their melting profiles and heterozygous genotypes by the normalised difference plots using high resolution melt (HRM) analysis. Seventeen heterozygous and 11 homozygous recessive lines were identified for background selection by fingerprinting with AFLP markers to determine the amount of recurrent parent (P 16) genome present. There were six EcoRI/MseI primer combinations tested with 277 data points scored (84% polymorphism rate). The amount of recurrent parent (P 16) genome recovered ranged from 62% to 92% with 13 lines showing greater than 83% of the recurrent parent genome. The effects of diallel crosses generated between four LPA, three QPM and three normal endosperm maize lines were determined for seed germination and vigour using the standard germination and accelerated aging seed tests under laboratory conditions in accordance with the procedures of the International Seed Testing Association. The specific combining ability (SCA) effects and general combining ability (GCA) effects were significant for the seed germination and vigour traits, indicating that genes with non-additive and additive effects were important in controlling these traits. However, the SCA effects were greater than GCA effects suggesting that genes with non-additive effects were predominant. The LPA parents showed reduced vigour compared to the normal and QPM inbred lines under both conditions, with LPA lines CM 31 and CM 32 showing stress tolerance. There were some combinations involving LPA lines, such as LPA x normal, LPA x QPM and LPA x LPA that retained high vigour and high germination rates under accelerated aging conditions, suggesting that they could be stress-tolerant.. A 10 x 10 diallel involving four LPA, three QPM and three Nm inbred lines was evaluated in replicated trials across six environments. Results show that both additive and non-additive gene effects were significant for resistance to northern corn leaf blight (NCLB), grey leaf spot (GLS) and Phaeosphaeria leaf spot (PLS) diseases. The additive gene effects were predominant for the yield and associated secondary traits such as days to mid-pollen shed (DMP), days to mid-silking (DMS), ear per plant (EPP) and grain moisture content (GMC) and grain yield. The LPA lines were early flowering and had quick grain dry down rate but all showed undesirable negative and significant GCA effects for yield. The yield of the LPA x LPA, LPA x Nm and LPA x QPM group of crosses was lower than the check hybrids by about 32 to 67% showing the need for yield improvement of the LPA combinations. An eight x eight diallel involving two LPA and six normal endosperm lines was evaluated over two seasons in five locations with two replications for grain yield components and foliar diseases. There was significant additive and non-additive gene action for both seed germination and vigour traits with predominance of non-additive gene effects. Both additive and non-additive gene effects were significant for yield and associated traits such as anthesis dates and number of ears per plant. However, the additive gene action was predominant for yield and associated traits. Generally the LPA lines and their combinations showed lower germination and vigour. The LPA line, CM 32 showed stress tolerance under accelerated aging conditions. There were three LPA x Nm crosses that showed improvements to the means of seed germination and vigour and yield traits. Results indicated that there was not any significant correlation between yield and seed germination and between yield and vigour. Yield was, however, significantly and positively correlated with anthesis dates and GMC, indicating that higher yield was associated with longer growing cycles. This study was able to successfully develop and apply the lpa1-1 SNP marker for foreground selection and AFLP markers for background selection in a backcross breeding programme. Problems of low seed germination, seedling vigour and grain yield of LPA lines and their combinations were confirmed. However results also indicated some potential of combining the LPA and QPM traits in a single cultivar. In general, procedures such as reciprocal recurrent selection, that emphasise both GCA and SCA effects would be recommended to improve seed germination, seedling vigour and yield in developing varieties with LPA trait. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Maize (Zea mays L.) is an important agronomic crop with the maize industry forming an important component of the South African economy. Considerable effort has been directed towards the genetic improvement of maize through both conventional breeding and biotechnology. Genotype identification by DNA fingerprinting is becoming an important activity in plant breeding. A widely used molecular based and relatively inexpensive method for DNA fingerprinting is the randomly amplified polymorphic DNA (RAPD) technique. The RAPD technique was tested in this study for its potential use in maize breeding programmes. Initial results using the technique showed a low degree of reproducibility, therefore both the DNA isolation and RAPD protocols were extensively optimised. DNA quality and quantity, and choice of Taq polymerase buffer were three of the variables found to be influential in ensuring reproducibility. The ability of the RAPD technique to characterise seven maize genotypes was evaluated. Sixty random oligonucleotide primers were screened. Forty two primers scored a total of 233 fragments (an average of 5.5 per primer), but not all primers gave reproducible profiles. Eighteen primers scored a total of 110 loci for the presence (1) and absence (0) of DNA fragments. RAPD markers were able to distinguish between all seven genotypes with five primers producing specific fragments for four genotypes. Genetic similarity matrices were calculated using two software programmes i.e. Genstat 5™ release 4.1 (1993) and PAUP (Phylogenetic Analysis Using Parsimony) 4.0 beta version (Swafford, 1998). Cluster analysis was used to generate dendrograms to visualise the genetic relationships of the seven maize genotypes (only minor differences were observed between the Genstat or PAUP method of analysis). Genetic diversity ranged from 0.62 to 0.96. The estimation of genetic relationship was in accordance with the presumed pedigree of the genotypes showing that the RAPD technique demonstrates potential for genome analysis of maize. The applicability of the technique for marker assisted selection was also evaluated. Near-isogenic lines (NILs) for leaf blight (Helminthosporium spp.) were screened for polymorphisms using a total of 120 primers. Ten primers identified polymorphisms between the NILs. Four primers produced five polymorphic fragments present in the resistant inbred K0315Y and absent in the susceptible inbred D0940Y. A small F2 population of 14 individuals was produced by selfing the F1 of a cross between K0315Y and D0940Y. To speed up the generation time, the F1 and F2 plants were cultured by embryo rescue from 18d old harvested seed. One fragment of 627 base pairs produced by primer OPB-01 (5' GTTTCGCTCC 3') showed a 3: 1 segregation in the small F2 population and was considered putatively linked to the HtN gene for leaf blight resistance. This study shows that the RAPD technique does have application in maize breeding programmes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.