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. ^

Phylogenetics, conservation, and historical biogeography of the West Indian members of the Adelieae (Euphorbiaceae)

The Caribbean Island Biodiversity Hotspot is the largest insular system of the New World and a priority for biodiversity conservation worldwide. The tribe Adeliae (Euphorbiaceae) has over 35 species endemic to this hotspot, representing one of the most extraordinary cases of speciation in the West Indies, involving taxa from Cuba, Hispaniola, Jamaica, and the Bahamas. These species form a monophyletic group and traditionally have been accommodated in two endemic genera: Lasiocroton and Leucocroton. A study based on: (1) scanning electron microscopy of pollen and trichomes, (2) macromorphology, and (3) molecular data, was conducted to reveal generic relationships within this group. Phylogenies were based on parsimony and Bayesian analyses of nucleotide sequences of the ITS regions of the nuclear ribosomal DNA and the non-coding chloroplast DNA spacers psbM-trnD and ycf6-pcbM. One species, Lasiocroton trelawniensis, was transferred from the tribe into the genus Bernardia. Of the remaining species, three major monophyletic assemblages were revealed, one was restricted to limestone ares of Hispaniola and was sister to a clade with two monophyletic genera, Lasiocroton and Leucocroton. Morphological, biogeographical, and ecological data provided additional support for each of these three monophyletic assemblages. The Hispaniolan taxa were accommodated in a new genus with four species: Garciadelia. Leucocroton includes the nickel hyperaccumulating species from serpentine soils of Cuba, while the rest of the species were placed in Lasiocroton, a genus restricted to limestone areas. The geographic history of the islands as well as the phylogenetic placement of the Leucocroton-alliance, allows the research to include the historical biogeography of the alliance across the islands of the Caribbean based on a dispersal-vicariance analysis. The alliance arose on Eastern Cuba and Hispaniola, with Lasiocroton and Leucocroton diverging on Eastern Cuba according to soil type. Within Leucocroton, the analysis shows two migrations across the serpentine soils of Cuba. Additional morphological, ecological, and phylogenetic analyses support four new species in Cuba (Lasiocroton gutierrezii) and Hispaniola ( Garciadelia abbottii, G. castilloae, and G. mejiae). ^

Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155

Cocaine and other drugs of abuse increase HIV-induced immunopathogenesis; and neurobiological mechanisms of cocaine addiction implicate a key role for microRNAs (miRNAs), single-stranded non-coding RNAs that regulate gene expression and defend against viruses. In fact, HIV defends against miRNAs by actively suppressing the expression of polycistronic miRNA cluster miRNA-17/92, which encodes miRNAs including miR-20a. IFN-g production by natural killer cells is regulated by miR-155 and this miRNA is also critical to dendritic cell (DC) maturation. However, the impact of cocaine on miR-155 expression and subsequent HIV replication is unknown. We examined the impact of cocaine on two miRNAs, miR-20a and miR-155, which are integral to HIV replication, and immune activation. Using miRNA isolation and analysis, RNA interference, quantitative real time PCR, and reporter assays we explored the effects of cocaine on miR-155 and miR-20 in the context of HIV infection. Here we demonstrate using monocyte-derived dendritic cells (MDCCs) that cocaine significantly inhibited miR-155 and miR-20a expression in a dose dependent manner. Cocaine and HIV synergized to lower miR-155 and miR-20a in MDDCs by 90%. Cocaine treatment elevated LTR-mediated transcription and PU.1 levels in MDCCs. But in context of HIV infection, PU.1 was reduced in MDDCs regardless of cocaine presence. Cocaine increased DC-SIGN and and decreased CD83 expression in MDDC, respectively. Overall, we show that cocaine inhibited miR-155 and prevented maturation of MDDCs; potentially, resulting in increased susceptibility to HIV-1. Our findings could lead to the development of novel miRNA-based therapeutic strategies targeting HIV infected cocaine abusers.