HIGH-THROUGHPUT AFLP GENOTYPING OF INTERSPECIFIC BACKCROSS FAMILIES OF Eucalyptus USING LI-COR IR2 AUTOMATED DNA SEQUENCERS

Forest Biotechnology Group, Department of Forestry, NC State University, Raleigh, NC 27695 USA

The high multiplex ratio of amplified fragment length polymorphism (AFLP) markers makes the AFLP marker system ideal for high-throughput genotyping of a wide variety of organisms. We are using LI-COR automated sequencers and dye-labeled PCR primers for AFLP genotyping of interspecific (E. grandis x E. globulus) backcross populations of Eucalyptus trees. Two populations, each consisting of approximately 350 individuals, are being genotyped using 24 AFLP primer combinations. Approximately 650 polymorphic markers will be scored in each of the backcross families, requiring 455,000 marker determinations. To facilitate timely completion of the mapping experiment, we have developed a modified AFLP protocol that allows high-throughput AFLP genotyping on one-dye (model 4000) and two-dye (model 4200) LI-COR automated sequencers. This approach allows approximately 6,000 marker data point determinations on a single one-dye gel and approximately 12,000 marker determinations on a two-dye gel. The marker data will be used to construct AFLP genetic maps for E. grandis, E. globulus and a hybrid of these two species using a "three-way pseudo testcross" approach. This approach will yield comparative AFLP maps of the three parental trees that will serve as frameworks for mapping candidate genes and quantitative trait loci (QTLs) for wood properties in these trees.