Development of a new efficient method for cloning of microsatellite regions using magnetic beads and its application to the research on reproductive strategies of insects.

We searched a new efficient method for cloning of the microsatellite regions in the genome of the gypsy moth，Lymantria dispar。The genomic DNA fragments which were obtained digestion with restriction enzyme were ligated into plasmai vector and transformed into Escherichia coli JM109competent cells.The cells were infected with helper phage M13KO7and single-stranded DNA(SsDNA)was prepared by standard procedures.Dynabeads M-280 Streptavidin(DYNAL)，bonded with biotin-(GT)I D 225个D 2，were mixed with the ssDNA to select the DNA segments carrying(AC)n sequences.The efficient annealing temperature was from 70℃to 75℃.Subsequently，double-strand DNA was synthesized from those selected ssDNA and transformed into E.coli JM109。We found 24 clones containing(GT/AC)n sequences and 11 of them were unique.Those microsatellite sequences enabled us to perform the paternity test in the gypsy moth。Each female was allowed to mate with two males sequentially.DNA fragments were obtained from each female，two males and pre-larvae in eggs that the female laid.The proportion of progeny sired by the last mated male，P2value，was calculated。The P I D 22 ii D 2 value was variable between 0.22 and 1.00 and was negatively correlated with the interval between first and second mating of the female.These results suggest that mate guarding behavior of gypsy moth males is efficient to ensure their paternity if population density is high，because females seem to remate soon after termination of copulation under such conditions。

We searched a new efficient method for cloning of the microsatellite regions in the genome of the gypsy moth, Lymantria dispar. The genomic DNA fragments which were obtained digestion with restriction enzyme were ligated into plasmai vector and transformed into Escherichia coli JM109 competent cells. The cells were infected with helper phage M13KO7 and single-stranded DNA (ssDNA) was prepared by standard procedures. Dynabeads M-280 Streptavidin (DYNAL), bonded with biotin-(GT)ィイD225ィエD2, were mixed with the ssDNA to select the DNA segments carrying (AC)n sequences. The efficient annealing temperature was from 70℃ to 75℃. Subsequently, double-strand DNA was synthesized from those selected ssDNA and transformed into E. coli JM109. We found 24 clones containing (GT/AC)n sequences and 11 of them were unique.Those microsatellite sequences enabled us to perform the paternity test in the gypsy moth. Each female was allowed to mate with two males sequentially. DNA fragments were obtained from each female, two males and pre-larvae in eggs that the female laid. The proportion of progeny sired by the last mated male, P2 value, was calculated. The PィイD22ィエD2 value was variable between 0.22 and 1.00 and was negatively correlated with the interval between first and second mating of the female. These results suggest that mate guarding behavior of gypsy moth males is efficient to ensure their paternity if population density is high, because females seem to remate soon after termination of copulation under such conditions.