High-Throughput Multiplexed AFLP^® Analysis of Interspecific Hybrids of Eucalyptus Tree Species

Alexander A Myburg, David M. O'Malley, Ronald R. Sederoff and Ross Whetten
Forest Biotechnology Group, Department of Forestry
North Carolina State University, Raleigh, NC 27695

Abstract

The high multiplex ratio of amplified fragment length polymorphism (AFLP) markers makes the AFLP marker system (1) 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 families of Eucalyptus trees. Two populations, each consisting of approximately 350 individuals, are being genotyped for approximately 650 polymorphic markers, requiring 455,000 marker genotype determinations. To facilitate timely completion of the mapping experiment, we have developed a modified AFLP protocol that allows high-throughput AFLP genotyping on LI-COR IR² automated sequencers (2). We have succeeded in doubling the multiplex ratio of AFLP analysis by using a three-primer amplification system (different IRD700 and IRD800-labeled EcoRI adapter primers against a single Msel adapter primer). The labeled AFLP fragments are resolved on 96-well gels on a two-dye (model 4200) LI-COR automated sequencer. This approach allows approximately 12,000 marker determinations on a two-dye gel from only 192 PCR amplification reactions (60,000 marker genotype determinations per week). Throughput is facilitated by obtaining high quality DNA samples using QIAGEN DNeasy 96 Plant kits and scoring digital AFLP gel images using AFLP-Quantar scoring software (Keygene Products). The marker data is being used to construct comparative AFLP genetic maps for E. grandis, E. globulus and a hybrid of these two species.

References:

1. VOS P, HOGERS R, BLEEKER M, REIJANS M, VAN DE LEE T, HORNES M, FRIJTERS A, POT J, PELEMAN F, KUIPER M, ZABEAU M. 1995. AFLP a new technique for DNA fingerprinting. Nucl Acid Res 23:4407-4414
2. MYBURG AA, REMINGTON DLR, 2000. High-throughput AFLP analysis using IR-dye labeled primers and automated sequencers. In prepararion.

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