Expression analyses of candidate resistance genes in the Rpp4 Asian Soybean Rust resistance locus.

Asian Soybean Rust (ASR), caused Phakopsora pachyrhizi, is considered the most severe soybean disease around the world. Infection of susceptible genotypes leads to early defoliation, incomplete seed development, and yield losses as high as 80%. Five ASR resistance genes have been identified in soybean: Rpp1, Rpp2, Rpp3, Rpp4 and Rpp5. Of particular interest is Rpp4, which has remained stable and confers resistance against P. pachyrhizi isolates from around the world. Rpp4 was mapped to soybean linkage group G (chromosome 18), 1.9 cM from simple sequence repeat (SSR) marker Satt288. Sequencing of this region in the susceptible genotype Williams 82 identified a cluster of three CC-NBS-LRR resistance genes. Virus Induced Gene Silencing was used to demonstrate that orthologous genes were responsible for resistance. We have now sequenced a >460 kb region of the Rpp4 locus in the resistant mapping parent PI459025B. Eight CC-NBS-LRR resistance genes have been identified in this region. In order to obtain more information about Rpp4 function, we are using real time quantitative PCR (qRT-PCR) to analyze the expression of all eight genes in different plant tissues, in different stages of development and after inoculation with P. pachyrhizi. We have developed a single pair of primers from the NBD domain that monitors the expression of all eight genes. Direct sequencing of the RT-PCR product differentiates between the eight genes. Detailed sequence analyses of the Rpp4 locus suggest that intra- and intergenic duplications and recombination have played an important role in creating genetic diversity. Alternative splicing of intragenic duplications may create additional sequence diversity at an RNA level. We are developing primers that will allow us to monitor alternative splicing events. Sequencing of the RT-PCR products will determine if alternative splicing plays a role in generating additional sequence diversity at the Rpp4 locus.Edição de Poster da 11. Annual National Outreach Scholarship Conference, Raleigh

Agrobacterium-mediated genetic transformation of a tropical elite maize line.

ABSTRACT: The efficiency of maize transformation mediated by Agrobacterium tumefaciens is influenced by various factors. The aim of this study was to test the effect of different concentrations of N6 salts (50% ? half strength, and 100% - full strength) in the infection and cocultivation media on genetic transformation efficiency of the L3 tropical elite maize line. Immature embryos were transformed via A. tumefaciens harboring the binary vector pTF102 containing the genes uidA and BAR under control of the CaMV35S promoter. The efficiency of the transgenic events produced was 3% for full strength and 1.1% for half strength N6 salts. Although under a lower concentration of salts, GUS expression was stronger; under this concentration, regeneration was less efficient. Thus, the results showed that the presence of 100% N6 salts in the infection and cocultivation media favored genetic transformation of the L3 maize inbred line mediated by A. tumefaciens

Assessment of Seed Viability by Laser Speckle Techniques

This work presents a new technique as a potential methodology to analyse seeds. The technology is known as dynamic speckle, or biospeckle, an optical phenomenon produced when active materials, such as biological tissue, are illuminated by laser light. In the present work, the biological activity of seed tissues has been inferred from quantitative and qualitative measurements of their speckle activity. The aim is to show that the biospeckle technique has a potential as a methodology to assess seed viability. One aspect that needs to be investigated is how the water content in the seeds affects bio-speckle activity. An experiment has been performed to determine the effect of humidity in the results. Seed activity for different levels of humidity was determined using quantitative and qualitative methods. Also, in others experiments, viable and non-viable seeds with different specific humidity levels could be classified using the same technique.Facultad de Ingeniería (FI