Plant & Animal Genome IX Conference

Poster: Large Insert Libraries, Gene Isolation, Etc.
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DEVELOPMENT OF PHYSICAL MAPS INTEGRATED WITH GENETIC MARKERS AND EST: PRELUDE TO GENOME SEQUENCING

¹ Department of Plant, Soil and General Agriculture, Room 176. Southern Illinois University at Carbondale, Carbondale IL 62901-4415, USA

² Crop Biotechnology Center, Institute of Plant Genomics and Biotechnology, 2123 TAMUS, Texas A&M University, College Station, Texas 77843-2123.

The development of robust techniques for physical mapping of entire complex genomes provides an new strategy for gene isolation from eukaryotes. Using a proprietary PAGE fingerprinting method and enzyme kit we are developing large insert clones libraries (BAC) and physical maps of several plant (egs. soybean, rice, Arabidopsis, Lotus, Pea, Moss) and fungal (Ustilago, Fusarium) genomes. We will integrate the physical maps with genetic maps and portions of EST libraries. The soybean physical map currently consists of 90,786 fingerprinted BACs covering 11.1 x soybean haploid genomes generated by PAGE after digestion with two restriction endonucleases. The soybean BACs are generated with EcoRI, HindII or BamHI from cultivar æForrestÆ.The developing physical map was integrated with 77 genetic markers on 14 linkage groups (see www.siu.edu/~pbgc), 313 microsatellites are in the later stages of integration. We are integrating unique ESTs in groups of 512 by multiplexed hybridization methods. The physical map can thereby be confirmed. A robust physical map can be used to increase the efficiency of whole genome sequencing by providing a minimum tile path for DNA sequencing, identifying gaps in a genomic clone libraries and identifying regions with high gene content for selective sequencing. The physical map has been used to develop new genetic markers (micro-satellites InDels and SNPs) in regions of the genome targeted for a lack of conventional genetic markers. We will describe automated, multiplexed methods based on fluorescent tags for physical map generation, minimum tile sequencing and the identification of ESTs with BAC clones and contigs.