DOI: 10.5061/dryad.8pk0p2npw
title: VCF files of common grassland plants from wild collected seeds of 19 common European grassland species with up to 4 consecutive generations grown in monoculture for seed production for restoration

description: A growing number of restoration projects require large amounts of seeds.
 As harvesting natural populations cannot cover the demand, wild plants are
 often propagated in large-scale monocultures. There are concerns that this
 cultivation process may cause genetic drift and unintended selection,
 which would alter the genetic properties of the cultivated populations and
 reduce their genetic diversity. Such changes could reduce the pre-existing
 adaptation of restored populations, and limit their adaptability to
 environmental change. We used single nucleotide polymorphism (SNP) markers
 and a pool-sequencing approach to test for genetic differentiation and
 changes in gene diversity during cultivation in 19 wild grassland species,
 comparing the source populations and up to four consecutive cultivation
 generations. We then linked the magnitudes of genetic changes to the
 species’ breeding systems and seed dormancy, to understand the roles of
 these traits in genetic change. The propagation changed the genetic
 composition of the cultivated generations only moderately. The genetic
 differentiation we observed as a consequence of cultivation was much lower
 than the natural genetic differentiation between different source regions.
 The propagated generations harbored even higher gene diversity than
 wild-collected seeds. Genetic change was stronger in self-compatible than
 in self-incompatible species, probably as a result of increased
 outcrossing in the monocultures. Synthesis and applications: Our study
 indicates that large-scale seed production maintains the genetic integrity
 of natural populations. Increased genetic diversity may be indicative of
 increased adaptive potential of propagated seeds, which would make them
 especially suitable for ecological restoration. Yet, it remains to be
 tested whether these patterns observed on the level of molecular markers
 will be mirrored also in plant phenotypes. Further, we used seeds produced
 in Germany and Austria, where the seed production is regulated and
 certified. Whether other seed production systems perform equally well
 remains to be tested.
description: We obtained seeds of grassland plants from two producers of seeds
 for ecological restoration, one in Austria and one in Germany, which we
 call Producer 1 and Producer 2 in the remainder of this paper. The seed
 propagation process starts with the collection of seeds in the wild.
 Producer 1 bases its propagation on a mixture of seeds from multiple wild
 populations, whereas Producer 2 on seeds coming from only one population.
 The wild-collected seeds (F0) thus consist of a mixture of five
 populations in Producer 1, or a single population in Producer 2. The F0
 seeds are often first germinated in a greenhouse to produce plugs which
 are then planted into an agriculture field. The seeds of this first
 cultivated generation (F1) are then harvested and used to establish the
 next cultivation (F2, Figure 1). The F2 seeds are mostly sold, but some
 are kept to establish the F3 generation. When a certain farm propagates
 multiple cultivation lines of the same species, there must be at minimum
 500 m (more for grasses) between the production fields to minimize gene
 flow between them (Prasse et al. 2010). This process is repeated until F5,
 then new seeds must be collected from the wild, to avoid that plants
 (presumably) adapt to their propagation environments or lose genetic
 diversity (Figure 1) (Espeland et al. 2017). The seeds are usually
 mechanically harvested, using agricultural machinery.
 For our study, we obtained wild-collected and cultivated seeds of
 19 different plant species. Because the seed producers carefully stored
 the seeds from both the wild collection and almost every consecutive
 generation in cultivation, we were able to test for possible genetic
 changes during this cultivation process from generation to generation up
 to the fourth cultivated generation. Four of the species were provided by
 both producers, and one from two regions from the same producer (Table 1).
 In total we obtained 24 independent cultivation lines (19 species, 5 of
 them from two regions) which resulted in 83 accessions. To obtain material
 for the genetic analysis, we sowed seeds on seeding substrate, and sampled
 leaves from 18 random individuals per species and generation when the
 plants had become large enough. Molecular
 Analysis Because of the large sample size, we used a
 population pool approach (Futschik & Schlötterer 2010), where we
 pooled 18 individuals per generation and cultivation line into one sample.
 We used a reduced-representation sequencing approach for SNP (single
 nucleotide polymorphism) detection and genotyping and followed the ddRAD
 protocol (Peterson et al. 2012) with slight deviations (see Supplementary
 Information). We used process_radtags from the Stacks
 2.0 pipeline (Catchen et al. 2013; Rochette et al. 2019) to demultiplex
 reads. We then used the dDocent 2.6.0 pipeline (Puritz et al. 2014;
 O’Leary et al. 2018) for contig assembly, SNP detection and assessment of
 allelic read counts for each cultivation line. SNP filtering of the
 resulting VCF file with vcftools removed indels, kept only biallelic loci
 with minimum Phred-scores of 30 and kept only one SNP per contig. To allow
 a comparison of genetic diversity between generations that is not biased
 by different sequencing depths, we further filtered the data using R. We
 used only markers with a minor allele frequency of at least 0.05, and
 genotypes that had a minimum read depth of 36. We corrected for unequal
 sequencing depth of the same locus across pools of the same cultivation
 line by rarefaction, i.e. drawing the minimum number of reads from each
 pool to assess allelic read counts. Pools with less than 500 genotyped
 SNPs were removed (Tab. 1). See Supplementary information, Table S2, and
 supplemental R code for details. The final data sets consisted of 657 -
 9721 (average 5137) biallelic SNP loci per pool across the 19 species and
 24 cultivation lines, with between 0% and 14.6% missing data.
description: Generation 3 and 5 from anthoxanthum odoratum and generation 2
 from salvia pratensis need to be discarded as they are most likely not
 part of the cultivation lines and are therefore not consecutive
 generations.