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.

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 09839018
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 自然史科学
• Research Institution: Naruto University of Education
• Principal Investigator: SHIMIZU Koji Naruto University of Education, Professor, 学校教育学部, 教授 (40090427)
• Co-Investigator(Kenkyū-buntansha): KUDO Chiharu (小汐 千春) Naruto University of Education, Research Associate, 学校教育学部, 助手 (60263878) ; KUDO Shin-ichi Naruto University of Education, Associate Professor, 学校教育学部, 助教授 (90284330) ; TAKENAKA Osamu Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Professor, 霊長類研究所分子生理研究部門, 教授 (00093261)
• Project Period (FY): 1997 – 1999
• Keywords: microsatellite DNA / magnetic beads / cloning / reproductive strategy / paternity test / gypsy moth / post copulatory mate guarding behavior / PィイD22ィエD2 value

Research Abstract

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.