Genetic diversity unveiled: cost-effective methods for grassland species

Permanent grasslands are predominantly composed of allogamous plant species that exhibit high levels of plant genetic diversity (PGD) within their populations. Grasslands with high PGD are more resilient to environmental stress and constitute valuable reservoirs of genetic resources for plant breeding. Therefore, monitoring PGD is the basis for detecting changes in PGD and correspondingly intervening. However, PGD monitoring is often neglected in biodiversity reports due to difficulties in taking representative samples and in using standardised and affordable indicators of PGD. Here we successfully applied two common approaches, multispecies amplicon sequencing (MSAS) and genotyping-by-sequencing (GBS), to assess PGD of agronomically relevant grassland species. Using MSAS, we were able to taxonomically distinguish five species (Dactylis glomerata L., Festuca pratensis Huds., Lolium perenne L., Trifolium pratense L., Trifolium repens L.) from multispecies samples and differentiate accessions within species, with fixation index (FST) values ranging from 0.014 for T. repens to 0.089 for L. perenne. Based on an extended L. perenne sample set containing mixtures of two cultivars at different ratios, mixtures containing both cultivars at 50% separated from the corresponding cultivars according to this ratio using MSAS and GBS. Furthermore, GBS enabled separation of samples containing two cultivars at a 75:25-ratio from the corresponding cultivars and the 50:50-ratio samples. These results indicate complementing applications of the two approaches in PGD monitoring. While we anticipate that MSAS with its cost-effectiveness could be applied to large-scale PGD monitoring, GBS with its lower detection limit could be applied to studies where cultivar composition shifts are of interest.