The isolation, characterization, and application of WRKY genes as useful molecular markers in tropical trees

The improvement of tropical tree crops using conventional breeding methods faces challenges due to the length of time involved. Thus, like most crops, there is an effort to utilize molecular genetic markers in breeding programs to select for desirable agronomic traits. Known as marker assisted breeding or marker assisted selection, genetic markers associated with a phenotype of interest are used to screen and select material reducing the time necessary to evaluate candidates. As the focus of this research was improving disease resistance in tropical trees, the usefulness of the WRKY gene superfamily was investigated as candidates for generating useful molecular genetic markers. WRKY genes encode plant-specific transcriptional factors associated with regulating plants' responses to both biotic and abiotic stress. ^ One pair of degenerate primers amplified 48 WRKY gene fragments from three taxonomically distinct, economically important, tropical tree crop species: 18 from Theobroma cacao L., 21 from Cocos nucifera L. and 9 from Persea americana Mill. Several loci from each species were polymorphic because of single nucleotide substitutions present within a putative non-coding region of the loci. Capillary array electrophoresis-single strand conformational polymorphism (CAE-SSCP) mapped four WRKY loci onto a genetic linkage map of a T. cacao F2 population segregating for resistance to witches' broom disease. Additionally, PCR primers specific for four T. cacao loci successfully amplified WRKY loci from 15 members of the Byttneriae tribe. A method was devised to allow the reliable discrimination of alleles by CAE-SSCP using only the mobility assigned to the sample peaks. Once this method was validated, the diversity of three WRKY loci was evaluated in a germplasm collection of T. cacao . One locus displayed high diversity in the collection, with at least 18 alleles detected from mobility differences of the product peaks. The number of WRKY loci available within the genome, ease of isolation by degenerate PCR, codominant segregation demonstrated in the F2 population, and usefulness for screening germplasm collections and closely related wild species demonstrates that the WRKY superfamily of genes are excellent candidates for developing a number of genetic molecular markers for breeding purposes in tropical trees. ^

A molecular basis for polyketide biosynthesis in marine dinoflagellates with emphasis on Karenia brevis

Polyketides derived from dinoflagellates are among the most complex and unique structures identified to date. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). No studies of the biosynthesis of dinoflagellate derived polyketides at the genomic level have been reported to date. Nine strains representing seven different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKS) by PCR and RT-PCR. Seven of the nine strains yielded products that were homologous with known and putative type I polyketide synthases. In each case, the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S rRNA gene. However, residual phylogenetic signals, resistance to methylation sensitive restriction enzymes and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes. ^ A more detailed analysis of Karenia brevis, a toxic marine dinoflagellate endemic to the Gulf of Mexico, also supports the hypothesis that dinoflagellates have polyketide synthase genes. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. PKS encoding genes amplified from K. brevis culture were found to be similar to PKS genes from the closely related protist, Cryptosporidium parvum. This suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. This dissertation reports the localization of these PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate. ^

Conservation implications of the reproductive biology of the endangered vine Ipomoea microdactyla Griseb. (Convolvulaceae)

A plant's reproductive biology exerts a significant influence on both population persistence within changing environments and successful establishment of new populations. However, the interaction between extrinsic (i.e. ecological) and intrinsic (i.e. genetic) factors also is an important driver of demographic performance for plant populations. It is light of this that I performed a multidisciplinary investigation of the breeding system, seed and seedling establishment dynamics, and population genetic structure of the endangered Caribbean vine Ipomoea microdactyla Griseb. (Convolvulaceae). The results from the breeding system study show individuals from Florida, USA and Andros Island, Bahamas to be self-incompatible. Plants from the two regions are cross-compatible but there is evidence for outbreeding depression in their progeny. Significant regional differences were found in floral traits and progeny traits that suggests incipient speciation for the Florida populations. The results from the seed and seedling establishment dynamics experiment demonstrate that the restoration of small populations in Florida via seed and seedling augmentation is a successful strategy. The demographic performance of the outplanted individuals was driven significantly by ecological factors (e.g. herbivory) rather than by genetic factors which emphasizes that the ecological context is very important for successful restoration attempts. The results from the population genetic study using an analysis of molecular variation (AMOVA) reveal significant differences in genetic variation among individuals from Florida, Andros, and Cuba. A Bayesian analysis of population genetic structuring coincided with the previous AMOVA results among the three regions. The Mantel test indicated significant 'isolation by distance' for these regional populations implying restricted gene flow over relatively short distances. Overall, the Florida populations had the lowest measures of genetic diversity which is most likely due to the effects of both colonization founder events and habitat fragmentation. The results of my study highlight the value of performing multidisciplinary studies in relation to species conservation as knowledge of both extrinsic and intrinsic factors can best guide decisions for species preservation.

Investigation of molecular mechanisms of LKB1 function in Dictyostelium development and stress responses and the function of superoxide dismutase C (SodC) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium . During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC− cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC− cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC− cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC − cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.