Résumé
Allele frequency fingerprinting of heterogeneous plant populations of outbreeding species can be used for variety identification, association mapping, genomic selection or characterization of genetic resources. In the FACCE-JPI GrassLandscape project, we empirically validated a pool-GBS method for Genome-Wide Allele Frequency Fingerprinting (GWAFF). As pool-GBS cannot be targeted to predefined loci such as candidate genes, we integrated it with targeted resequencing using a highly multiplexed amplicon sequencing strategy to measure allele frequencies (pool-HiPlex). A pool-HiPlex assay was designed that amplifies 185 amplicons in 41 L. perenne genes in just two parallel PCRreactions. We validated pool-GBS and pool-HiPlex using pools of 48 individuals, chosen to represent a wide range of genetic diversity in L. perenne, and addressed completeness, reproducibility and accuracy of allele frequencies with >1000 HiPlex SNPs and >150.000 GBS SNPs. We consistently found high correlations between allele frequencies obtained by genotyping individual plants and pool genotyping on leaf tissue pools and DNA extract pools. We also analyzed the error introduced at various steps of the protocol such as weighing, DNA-quantification, pooling, ligation and/or PCRamplification. Applying a minor allele frequency threshold of 5% or 3% effectively removed nonreproducible SNPs in pool-GBS and pool-HiPlex, respectively. Allele frequency spectra could be obtained for single SNPs as well as for haplotypes spanning neighboring SNPs using read-backed phasing. Application of this methodology to a set of 470 natural populations of L. perenne sampled across Europe and the fertile Crescent revealed a geographical pattern of genetic differentiation in this species.