Creation of a microsatellite-enriched genomic library from Mayetiola destructor (Hessian Fly)
Brandi Schemerhorn, email@example.com, USDA-ARS, Crop Protection and Pest Control Unit, 170 South University Street, WSLR 201, West Lafayette, IN
Mayetiola destructor is a major insect pest of wheat crop production. It is estimated that the monetary loss caused by this pest in the United States has been $100 million a year. Little information is currently available on the population dynamics of M. destructor in the United States. Microsatellite markers are highly polymorphic, and therefore are good for following migration and breeding history in this species. On the basis of existing markers and a search for microsatellites in the existing Hessian fly entries in the gbest EST database, 11 dinucleotide and trinucleotide motifs were investigated. Hessian fly genomic DNA was digested with Sau3A1 and size-selected for fragments 400 to 1000 bp in length. These fragments were ligated to a biotinylated adaptor and captured by hybridization with specific microsatellite oligonucleotides bound to paramagnetic beads. Enrichment succeeded with nine of the eleven target sequences: (GT)12, (GTG)8, (CAG)8, (GAA)8, (AAC)8, (TTA)8, (GA)12, (TA)12, (AC)12 and failed with (GT)15 and (AAT)8. In total over the nine libraries, 52,224 clones were recovered and confirmed by hybridization with specific biotinylated target sequence with negative controls. From 67.8-95.6% of the clones were positive upon hybridization. Clones were additionally verified by PCR with adaptor and microsatellite-specific primers. Colonies were chosen for sequencing on the basis of PCR screening results. Microsatellite markers will be tested for reliable amplification and for polymorphism among multiple Hessian fly populations.
Species 1: Diptera Cecidomyiidae Mayetioladestructor (Hessian fly) Keywords: microsatellite