Inference of genetic diversity in popcorn S3 progenies.

Molecular markers are a useful tool for identification of complementary heterotic groups in breeding programs aimed at the production of superior hybrids, particularly for crops such as popcorn in which heterotic groups are not well-defined. The objective of the present study was to analyze the genetic diversity of 47 genotypes of tropical popcorn to identify possible heterotic groups for the development of superior hybrids. Four genotypes of high genetic value were studied: hybrid IAC 125, strain P2, and varieties UENF 14 and BRS Angela. In addition, 43 endogamous S3 progenies obtained from variety UENF 14 were used. Twenty-five polymorphic SSR-EST markers were analyzed. A genetic distance matrix was obtained and the following molecular diversity parameters were estimated: number of alleles, number of effective alleles, polymorphism information content (PIC), observed and expected heterozygosities, Shannon diversity index, and coefficient of inbreeding. We found a moderate PIC and high diversity index, indicating that the studied population presents both good discriminatory ability and high informativeness for the utilized markers. The dendrogram built based on the dissimilarity matrix indicated six distinct groups. Our findings demonstrate the genetic diversity among the evaluated genotypes and provide evidence for heterotic groups in popcorn. Furthermore, the functional genetic diversity indicates that there are informative genetic markers for popcorn.

Isolation of Pantoea ananatis from sugarcane and characterization of its potential for plant growth promotion.

Each year, approximately 170 million metric tons of chemical fertilizer are consumed by global agriculture. Furthermore, some chemical fertilizers contain toxic by-products and their long-term use may contaminate groundwater, lakes, and rivers. The use of plant growth-promoting bacteria may be a cost-effective strategy for partially replacing conventional chemical fertilizers, and may become an integrated plant nutrient solution for sustainable crop production. The main direct bacteria-activated mechanisms of plant growth promotion are based on improvement of nutrient acquisition, siderophore biosynthesis, nitrogen fixation, and hormonal stimulation. The aim of this study was to isolate and identify bacteria with growth-promoting activities from sugarcane. We extracted the bacterial isolate SCB4789F-1 from sugarcane leaves and characterized it with regard to its profile of growth-promoting activities, including its ability to colonize Arabidopsis thaliana. Based on its biochemical characteristics and 16S rDNA sequence analysis, this isolate was identified as Pantoea ananatis. The bacteria were efficient at phosphate and zinc solubilization, and production of siderophores and indole-3-acetic acid in vitro. The isolate was characterized by Gram staining, resistance to antibiotics, and use of carbon sources. This is the first report on zinc solubilization in vitro by this bacterium, and on plant growth promotion following its inoculation into A. thaliana. The beneficial effects to plants of this bacterium justify future analysis of inoculation of economically relevant crops.

Genetic characterization of papaya plants (Carica papaya L.) derived from the first backcross generation.

The limited number of papaya varieties available reflects the narrow genetic base of this species. The use of backcrossing as a breeding strategy can promote increases in variability, besides allowing targeted improvements. Procedures that combine the use of molecular markers and backcrossing permit a reduction of the time required for introgression of genes of interest and appropriate recovery of the recurrent genome. We used microsatellite markers to characterize the effect of first-generation backcrosses of three papaya progeny, by monitoring the level of homozygosity and the parental genomic ratio. The homozygosity level in the population ranged from 74 to 94%, with a mean of 85% for the three progenies (52-08, 52-29 and 52-34). The high level of inbreeding found among these genotypes increases the expectation of finding more than 95% fixed loci in the next generation of self-fertilization of superior genotypes. The mean proportion of the recurrent parent genome found in first-generation backcross progeny was 50.1%; 52-34 had a larger genomic region in common with the recurrent genitor and the lowest level of homozygosity. The progeny 52-08 was genetically closest to the donor genitor, and it also had the highest level of homozygosity. We found that linking conventional procedures and molecular markers contributed to an increase in the efficiency of the breeding program.

Methodological improvements on extraction of nuclear proteins and its preliminary analysis during the maize (Zea mays L.) endosperm development.

A procedure to obtain endosperm protein extracts was standardized. After confirming the enrichment with nuclear proteins by immunodetection, the protein profiles of extracts from different seed development stages were compared by SDS-PAGE that showed the existence of several differentially expressed proteins.

Identification of a DNA-binding factor that recognizes an alpha-coixin promoter and interacts with a Coix Opaque-2 like protein.

Transient expression and electrophoretic mobility shift assay were used to investigate the cis elements and the DNA-binding proteins involved in the regulation of expression of a 22 kDa zein-like alpha-coixin gene. A set of unidirectional deletions was generated in a 962 bp fragment of the alpha-coixin promoter that had been previously fused to the reporter gene GUS. The constructs were assayed by transient expression in immature maize endosperm. There was no significant decrease in GUS activity as deletions progressed from -1084 to -238. However, deletion from -238 to -158, which partially deleted the O2c box, resulted in a dramatic decrease in GUS activity emphasizing the importance of the O2 box in the quantitative expression of the gene. The -238 promoter fragment interacted with Coix endosperm nuclear proteins to form 5 DNA-protein complexes, C1-C5, as detected by EMSA. The same retarded complexes were observed when the -158 promoter fragment was used in the binding reactions. Reactions with nuclear extracts isolated from Coix endosperms harvested from 6 to 35 days after pollination revealed that the 5 DNA-protein complexes that interact with the alpha-coixin promoter are differentially assembled during seed development. Deletion analysis carried out on the -238/ATG promoter fragment showed that a 35 bp region from -86 to -51 is essential for the formation of the complexes observed. When nuclear extracts were incubated with an antiserum raised against the maize Opaque-2 protein, the formation of 4 complexes, C1, C3, C4 and C5, was prevented indicating that an Opaque-2 like protein participates in the formation of those complexes. Complex C2 was not affected by the addition of the O2 antibody, suggesting the existence of a novel nuclear factor, CBF1, that binds to the promoter and makes protein-protein associations with other proteins present in Coix endosperm nuclei.

Sequence analysis of 22 kDa-like alpha-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements.

Several genomic and cDNA clones encoding the 22 kDa-like alpha-coixin, the alpha-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like alpha-coixin genes designated alpha-3A, alpha-3B and alpha-3C were found in the 15 kb alpha-3 genomic clone. The alpha-3A and alpha-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the alpha-3B gene, suggesting that the three alpha-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication. Comparison of the deduced amino acid sequences of alpha-coixin clones with the published sequences of 22 kDa alpha-zein and 22 kDa-like alpha-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15-20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like alpha-prolamins and the 19 kDa alpha-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins. Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5' and 3' flanking regions of alpha-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. -300 prolamin box are present at conserved positions in alpha-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in alpha-3B that occupies approximately the same positions as those identified for the 22 kDa alpha-zein and alpha-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes. The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like alpha-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.