AphidMASH (Aphid Multi-Allele Scanning Haplotags) v1

This protocol describes a multiplex-PCR amplicon sequencing approach designed to facilitate the genotyping ofSitobion avenae, the identification of the L1014F mutation, the screening of regions harbouring additional mutations known to infer insecticide resistance in other species, and the detection of yellow dwarf virus in aphids. The design of aphid targets was facilitated by re-sequencing data from 12 S. avenae clonal lineages (BioProject: PRJNA1039626 ) and the design of virus targets using sequence data from a HTS Survey (1). This methodology is based on the original GT-seq protocol described by Campbell (2) and adapted from methods described in https://dx.doi.org/10.17504/protocols.io.e6nvw53zdvmk/v2 and (3). 1. S. Byrne, M. Schughart and V. Ballandras et al. The first survey using high-throughput sequencing of cereal and barley yellow dwarf viruses in Irish spring and winter barley crops.Irish Journal of Agricultural and Food Research.2024. Vol. 63(1):1-16. DOI: 10.15212/ijafr-2023-0110 2. Campbell, N.R., Harmon, S.A. and Narum, S.R. (2015), Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing. Mol Ecol Resour, 15: 855-867. https://doi.org/10.1111/1755-0998.12357 3. Leyva-Pérez, M.d.l.O.; Vexler, L.; Byrne, S.; Clot, C.R.; Meade, F.; Griffin, D.; Ruttink, T.; Kang, J.; Milbourne, D. PotatoMASH—A Low Cost, Genome-Scanning Marker System for Use in Potato Genomics and Genetics Applications.Agronomy2022,12, 2461. https://doi.org/10.3390/agronomy12102461