Submitted to: Academia Journal of Agricultural Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/3/2018
Publication Date: 8/1/2018
Citation: Crane, Y.M., Crane, C.F., Sanmiguel, P., Schemerhorn, B.J. 2018. A multiplexing strategy for mesoscale targeted sequencing of populations. Academia Journal of Agricultural Research. 8(8):221-228. https://doi.org/10.15413/ajar.2018.0168 .
Interpretive Summary: Hessian fly is known to be one of the most devastating insect pests in wheat worldwide. Hessian fly has many different biotypes that are characterized by their virulence/avirulence to wheat varieties. To identify possible genes that allow the fly to infest different types of wheat, we sequenced large numbers of salivary proteins that are secreted by the fly into the wheat as it feeds. We took a concept, known as barcoding, to identify specific gene variations within these proteins. We were able to detect several gene variations that we will further examine to see if they are responsible for the Hessian fly's devastating impact to wheat. This information will be important for wheat breeders as well as basic scientists working to improve resistance to Hessian fly.
Technical Abstract: Primer pairs were chosen from Hessian fly (Mayetiola destructor (Say)) genomic sequence for orthologs of 52 dipteran salivary proteins that possessed a signal peptide, indicating secretion. Illumina sequence was obtained for 19968 barcoded amplicons representing 48 individuals for each of eight biotypes over all 52 primer pairs. With eight row-specific and 12 column-specific barcodes, judicious spacing of primers, and two PCR steps, it was possible to reduce 208 96-well plates to 16 plates for the second PCR and 4 plates for sequencing, a 98% reduction in sequencing cost. The experiment identified 997 polymorphic sites over all 52 genes.