Plant & Animal Genome VI Conference Plant & Animal Genome VI Conference
W50,P21
Clemson Univ. Genome Center, Box 340359 Poole Bldg., Clemson, SC 29634
We have constructed a sugarcane BAC library suitable for physical mapping and positional cloning of agronomically important genes. Cultivated sugarcane is a complex aneuploid polyploid with a nuclear DNA content of about 3,000 megabases. The cultivar R570 was chosen for BAC library construction because it contains many valuable economic traits and is the parental clone utilized for the development of a molecular genetic map (Grivet et al., 1996; Genetics 142:987-1000). The library was constructed using megabase DNA isolated from nuclei preparations that were partially digested with HindIII and subjected to various double size selection schemes. The cloning vector was pBeloBAC11. At the present time, the BAC library contains 67,200 clones arrayed in 175 384-well microtiter plates. Plates 1-81 contain clones with an average insert size of 110 kb (range = 36-186 kb) while plates 82-175 have an average insert size of 140 kb (range = 62-212 kb). The library at it's current size represents about three genome equivalents. Our goal is to achieve a final coverage of 3 to 4 genome equivalents. An initial set of high density filters for hybridization were gridded out using the Genetix Q-bot in a 4x4 array for library plates 1-80. Probing with three different barley chloroplast genes has indicated that the library has an exceptionally low chloroplast DNA content of less than 1% (0.29 %). Data from additional library construction/characterization efforts will be discussed. We thank the International Consortium for Sugarcane Bitotechnology for funding this work.