DOI: 10.5061/dryad.gb5mkkwqw
title: Raw data: multispecies amplicon sequencing (Loera, Studer, and Kölliker, 2021, Molecular Ecology Resources)

description: Grasslands cover close to two fifths of Earth's land. They provide
 many ecosystem services related to the maintenance of soil integrity, and
 the regulation of water, carbon and nitrogen flows. Grasslands constitute
 the basis for sustainable roughage production for ruminant feeding.
 In Switzerland, grasslands cover more than 70% of the total
 agricultural land, which highlights their importance in the domestic food
 production chains. Plant genetic diversity (PGD), a component of
 biodiversity, influences ecosystem functioning in grasslands. High levels
 of grassland PGD are related to resistance against invasive
 plants and yield stabilization during environmental stress (e.g., drought
 or frost). The PGD of grasses and legumes —the two most
 economically relevant plant families found in grasslands, which naturally
 grow in a wide climate spectrum— harbors valuable genetic resources for
 forage breeding. Nevertheless, most PGD studies of natural or semi-natural
 grasslands (i.e., grasslands that are not sown) focus on a single or a few
 related species. Traditional PGD monitoring methods (e.g., simple sequence
 repeats, or SSRs) are ill-suited for large-scale, multispecies
 assessments.  This limits our ability to study the ecological
 effects of grassland PGD, its spatiotemporal patterns, and its
 significance for grassland management. Looking to provide cost-effective
 tools for multispecies PGD monitoring in grasslands, we performed
 a sequence capture assay targeting 611 single-copy nuclear loci, followed
 by multispecies amplicon sequencing (i.e., amplicon sequencing using
 primer pairs that can be used in multiple species) on eleven selected
 loci. Our results indicate that multispecies amplicon sequencing
 is a cost-effective tool for genetic diversity assessment in grassland
 plant species. Furthermore, the sequence capture data provides
 the means to extend the number of multispecies amplicons for further
 research.
description: We surveyed the genetic diversity of 611
 single-copy nuclear loci that are shared across grass and legume species.
 We followed a sequence capture approach to perform targeted sequencing of
 such loci in 5 genotypes of 16 grass and legume species of economic
 relevance in temperate grasslands: <i>Alopecurus pratensis
 </i>L., <i>Arrhenaterum elatius
 </i>L., <i>Cynosurus cristatus </i>L., <i>Dactylis
 glomerata </i>L., <i>Festuca pratensis
 </i>Huds., <i>Festuca rubra
 </i>L., <i>Lolium perenne </i>L., <i>Lolium
 multiflorum </i>Lam., <i>Lotus corniculatus
 </i>L., <i>Medicago sativa </i>L., <i>Onobrychis
 viciifolia </i>Scop., <i>Phleum pratense
 </i>L., <i>Poa pratensis
 </i>L., <i>Trifolium pratense </i>L., <i>Trifolium
 repens </i>L., and <i>Trisetum flavescens
 </i>L. We then ranked the
 target loci according to their nucleotide diversity (π) and
 normalized <em>k</em>-mer richness (NKR) within each species.
 We finally selected eleven loci for amplicon sequencing. Those loci showed
 a combination of moderate to high genetic diversity estimates (i.e., π and
 NKR) and suitability for multispecies primer design. The selected loci
 were amplified and sequenced in test populations of <em>D.
 glomerata</em>, <em>F. pratensis</em>, <em>L.
 perenne</em>, <em>T. pratense</em>, and <em>T.
 repens</em>. The test populations consisted of 16 genotypes per
 species (three cultivars per species) and included single- and
 pooled-plant samples.
description: File Description 
 01 FORAGE-611 baits.zip
 Contains a FASTA file with the probe sequences for the sequence
 capture assay. 02 Discovery
 phase raw reads.zip Contains the FASTQ files with the
 raw reads of the sequence capture assay. 
 03 Sequence capture loci assemblies.zip
 Contains the FASTA files of the assemblies produced from the
 data of the sequence capture assay. The assemblies are grouped by
 locus. 04 Validation phase raw
 reads.zip Contains the FASTQ files with the raw reads
 of the multispecies amplicon sequencing assay. 
 05 Haplotyping scripts.zip
 Contains the scripts used to determine SNP-based haplotypes with
 data from the multispecies amplicon sequencing assay.
 README.md Contains a
 detailed description of all the previous files, including sample
 information (i.e., species and cultivars), sample names, locus names, loci
 orthologues in reference genomes, and the naming pattern of the loci
 assemblies.