Plant genetic diversity (PGD) is related to many beneficial effects to the ecosystem functioning of grasslands. The monitoring of PGD in temperate grasslands is complicated by the multiplicity of species present and by a shortage of methods for large-scale assessment. Among them, amplicon sequencing stands out as a robust and cost-effective method.
Grasslands are widespread and economically relevant ecosystems at the basis of sustainable roughage production. Plant genetic diversity (PGD; i.e., within-species diversity) is related to many beneficial effects to the ecosystem functioning of grasslands. The monitoring of PGD in temperate grasslands is complicated by the multiplicity of species present and by a shortage of methods for large-scale assessment. However, the continuous advancement of high-throughput DNA sequencing approaches have improved the prospects of broad, multispecies PGD monitoring. Among them, amplicon sequencing stands out as a robust and cost-effective method. Here we report a set of twelve multispecies primer pairs that can be used for high-throughput PGD assessment in multiple grassland plant species. The loci targeted by the amplicons were selected and tested in two phases: a "discovery phase" based on a sequence capture assay (611 target nuclear loci assessed in 16 grassland plant species), which resulted in the selection of eleven loci; and a "validation phase", in which the selected loci were targeted and sequenced using twelve multispecies primers in test populations of Dactylis glomerata L., Lolium perenne L., Festuca pratensis Huds., Trifolium pratense L. and T. repens L. The resulting multispecies amplicons had overall nucleotide diversities per species ranging from 5.19 x 10-3 to 1.29 x 10-2, which is in the range of flowering-related genes but slightly lower than pathogen resistance genes. We conclude that the methodology, the DNA sequence resources, and the amplicon-specific primer pairs reported in this study provide the basis for large-scale, multispecies PGD monitoring in grassland plants.