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

Conclusions

High-throughput AFLP analysis was achieved by:

A. High-throughput sample grinding approach (BIO101 Fastprep Instrument)

B. 96-well DNA extraction procedure (DNeasy™ 96 Plant kit - QIAGEN)

C. Multiplexed AFLP analysis on two-dye (model 4200) LI-COR IR² automated DNA sequencers

D. Ability to load LI-COR gels twice (collect four images per 2-dye gel = 384 AFLP lanes per gel)

E. Fast semi-automated scoring of LI-COR TIFF images using AFLP-Quantar™ (Keygene Products)

A throughput of 40,000 - 60,000 marker genotype determinations per week per two-dye LI-COR sequencer can be achieved, depending on the type of mapping population and level of genetic diversity between the parents.

Acknowledgements

• This work is supported by the NCSU Forest Biotechnology Research Consortium.

• The backcross families are maintained by Pablo Santini of Shell Uruguay Renewable Energy and Compania Forestal Oriental - Paysandu, Uruguay.

• Derek Harkins (NC State Forest Biotechnology Group) was instrumental in developing the new high-throughput DNA extraction procedure for Eucalyptus leaves.

• Christina marques and David Remington optimized the AFLP protocol for Eucalyptus and for detection on LI-COR automated sequencers.

• Pablo Santini (Shell Uruguay Renewable Energy) kindly collected and shipped the leaf samples for DNA extraction for Paysandu, Uruguay to NCSU, North Carolina.

• Alexander Myburg is suppported by the Fulbright Scholarship program (United States Information Agency) and by the National Research Foundation of South Africa.