CRITICAL FACTORS IN CONSTRUCTING AN EcoRI BAC LIBRARY FOR SOYBEAN

¹ Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108 USA

² Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108 USA

We previously constructed a soybean bacterial artificial chromosome library in the EcoRI site of pECSBAC4 vector. The library, which consists of 30720 clones, is being used for map-based cloning, comparative genomics, characterizing disease resistance, and genome sequencing. Currently we are expanding the library to facilitate progress toward these goals and used this opportunity to study some of the possible limiting factors in BAC library construction: electroporation conditions, cloning enzyme, and solvent for phenyl-methyl-sulfonyl-fluoride (PMSF) treatment of plant nuclear DNA. Electroporations of ligation reactions were tested in a Bio-Rad Gene Pulser at 25 µFD, 100 OHM, and different voltage gradients. Average insert sizes were 90 kb @ 14 kv/cm, 120 kb @ 15 kv/cm, 145 kb @ 16 kv/cm, 127 kb @ 17 kv/cm, and 107 kb @ 18 kv/cm. Genome coverage was the highest at 16 and 17 kv/cm, but 4.8-fold lower at 14 kv/cm. Cloning enzyme also affected transformation efficiency. In parallel experiments, EcoRI- and HindIII-cut vector and size-selected soybean DNA were ligated and transformed. On two occasions the number of white colonies was 2- to 3-fold higher with HindIII than EcoRI using comparable size-selected plant DNA. Treatment of nuclear soybean DNA with PMSF stocks (0.1 mM) in ethanol or isopropanol also had a considerable impact on ligation reactions. This treatment was performed before partial digestion, pulsed field gel electrophoresis, size selection, electroelution and ligation . In parallel experiments PMSF-ethanol treated DNA led to successful ligation results, whereas PMSF-isopropanol partially or completely inhibited ligation reactions. PMSF-isopropanol treated DNA not only failed to self-ligate, but also inhibited self-ligation of the vector. It is recommended that in any BAC cloning effort, electroporation conditions be critically tested, the most suitable cloning enzyme selected, and PMSF-isopropanol treatment of DNA avoided.