This proposal seeks cost-sharing support for the purchase of an ABI PRISM 377 Automated DNA Sequencer and necessary peripherals. The Lewis B. and Dorothy Cullman Program for Molecular Systematics Studies, which is a joint initiative of The New York Botanical Garden and the American Museum of Natural History, was formed through a charitable donation from the Lewis B. and Dorothy Cullman Foundation. The New York Botanical Garden's Cullman Program occupies laboratories at both locations, which includes entitlement to user-fee based operation of the Museum's ABI 377 sequencer. The Cullman Program resides primarily in newly renovated laboratory space in the Garden's Harding Laboratory building. A matching challenge grant of $116,000 from the Cullman Foundation has been used toward the total costs of equipping the new laboratories for molecular systematics research. This proposal requests the balance of necessary funds from the NSF. This proposal seeks matching funds from NSF to complete the planned equipping of the Garden laboratories with automated DNA sequencing and DNA fingerprinting capacities. DNA sequencing is not currently possible at the Garden, although all other relevant molecular techniques (e.g., DNA and RNA extraction and purification, Southern and northern blot analyses, PCR amplification of DNA, in situ hybridization and in situ PCR against DNA or cDNA, visualization of fluorochrome labeled in situ probes, and molecular cloning) are supported with the newly purchased equipment. The sequencing facility at the American Museum is crucial for present research initiatives, but it is no long term substitute for in-house DNA sequence and DNA fingerprint determination at the Garden. Aspects such as cost-effectiveness, throughput, flexibility, and enhanced research and educational development argue strongly for the Garden's completion of its own automated DNA analysis facility, as has been planned. The general goal of the Cullman Program is to enr ich and invigorate the Botanical Science Division of The New York Botanical Garden with the modern tools of molecular systematics research. Molecular systematics not only entails DNA sequence production for use in phylogenetic analysis; it also encompasses bold new attempts to link systematics reasoning with the burgeoning field of plant molecular development. The Cullman Program currently supports standard molecular systematics projects as well as systematically oriented projects focusing on flower and root development. Molecular systematic work in Lecythidaceae, Arecaceae, Anacardiaceae, Neckeraceae, Gentianaceae, and Cactaceae is proceeding in collaboration with Cullman Program Adjunct Curators, other staff, and graduate students. These projects are primarily oriented toward classificatory, character evolutionary, and biogeographic issues. Other projects focusing on population level issues will profit from the ABI 377's capacity to perform rapid genotype analysis (e.g., using the amplified fragment length polymorphism AFLP approach). Likewise, the evolutionary-oriented developmental research of the Cullman Program requires active DNA sequencing capacity. A Garden sequencing facility will act much like a local workstation for plant molecular systematics activity, especially given the Garden's academic collaborations with Columbia University (The Center for Environmental Conservation and Research--CERC), Cornell University, Lehman College--CUNY, New York University, and Yale University. Immediate commitments for use of an automated sequencer are substantial (16 investigators), and installation of this instrumentation at The New York Botanical Garden should permit a considerable molecular systematics research capacity in the years to come. The ABI PRISM 377 Automated DNA Sequencer (manufactured by Perkin Elmer's Applied Biosystems Division) is a sophisticated instrument representing the forefront of second-generation sequencer technology. The instrument' s use of simultaneous, scanning-laser detection of four different fluorescent dyes permits samples to be resolved in single gel lanes (as opposed to the four gel lanes occupied by other vendors' equipment, which have a single-dye approach). Throughput time has been increased by a factor four from earlier models through refinements to the scanning procedure. Combined with appropriate software (some software and hardware are included within the base purchase price), gel-plate and loading comb combinations, apparatus for PCR-based DNA labeling, and a dedicated water purification system, the ABI 377 will complete the equipping of The New York Botanical Garden's molecular laboratories.
