A GBAS marker system for assessing genetic diversity and population differentiation in Norway Spruce

Effective management of Picea abies (Norway spruce), a widely planted conifer species in Europe, requires high-resolution, scalable genotyping tools capable of capturing genetic variation across complex genomes. The primary aim of this study was to establish a cost-effective marker toolkit for Picea abies suitable for population genetics, provenance studies, and long-term monitoring. We employed a Genotyping by Amplicon Sequencing (GBAS) approach to investigate sequence-level variation in Norway spruce, using 124 highly multiplexed markers. The markers included 43 novel microsatellite markers from anonymous genomic regions, 33 elongated established microsatellite markers and 48 EPIC (Exon-Primed Intron-Crossing) markers targeting introns. As a proof of concept, the markers were applied to 85 individuals from five populations in Austria. Genotyping was performed using the Genotyping by Amplicon Sequencing (GBAS) pipeline, enabling the extraction of both traditional length-based genotypes and whole amplicon information. Diversity metrics and clustering analyses revealed spatial genetic structure among populations, likely reflecting neutral demographic processes such as elevation-related isolation.