Objective:
Identify molecular markers specific for sucrose synthesis in Saccharum spontaneum and utilize the technology to hasten the introgression of these genes into commercial germplasm.

Approach:
In December 2002, a core collection of 52 S. spontaneum clones, currently vegetatively maintained at the USDA, ARS, Sugarcane Research Unit (SRU), were evaluated for juice quality characteristics. In order to account for genotype by environment interaction, we will collect another set of data on the same clones in 2006. Forward Polymerase Chain Reaction (PCR) primers will be designed from 20 expressed sequence tags (EST) for genes coding for enzymes involved in sugar metabolism, using the software Primer 3 (Whitehead Institute for Biological Resarch, http:www.genome.wi.mit.edu/). PCR primers for one of these genes have already been developed. To date we have developed 31 Target Region Amplified Polymorphism (TRAP) primers for different agronomic traits. These primers were utilized in PCR reactions with the same arbitrary primers that were designed to tackle polymorphic regions within the genes. Markers produced by these primers were given a sequential number not related to the EST from which they were developed. Plant DNA has been extracted, using the CTAB method. PCR reactions will be run in MJ PTC-100 thermocycler. Arbitrary primers will be synthesized with an infrared modification (either IRDye700 or IRDye800) for visualization of PCR amplification in the DNA analyzer (Li_Cor 43000). Association between markers and phenotype will be measured using the analysis of variance model, which is a regression model containing only qualitative independent variables. Regression analysis will be performed using the SAS PROC REG procedure (SAS Institute), using the genotype information generated by each marker as the indicator variable. This method is equivalent to the linear regression of sugar content on the genotype (presence/absence of the marker) and is a special case of the Method of Maximum Likelihood. Using this procedure we already identified putative association of one marker with sucrose, showing the potential applications of the methodology being proposed.