A multispecies amplicon sequencing approach for genetic diversity assessments in grassland plant species.

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 on 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 assessments. However, the continuous advancement of high-throughput DNA sequencing approaches has 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 12 multispecies primer pairs that can be used for high-throughput PGD assessments in multiple grassland plant species. The target loci were selected and tested in two phases: a "discovery phase" based on a sequence capture assay (611 nuclear loci assessed in 16 grassland plant species), which resulted in the selection of 11 loci; and a "validation phase", in which the selected loci were targeted and sequenced using multispecies primers in test populations of Dactylis glomerata L., Lolium perenne L., Festuca pratensis Huds., Trifolium pratense L. and T. repens L. The multispecies amplicons had nucleotide diversities per species from 5.19 × 10-3 to 1.29 × 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 primer pairs reported in this study provide the basis for large-scale, multispecies PGD monitoring in grassland plants.

DNA barcode trnH-psbA is a promising candidate for efficient identification of forage legumes and grasses.

OBJECTIVE: Grasslands are widespread ecosystems that fulfil many functions. Plant species richness (PSR) is known to have beneficial effects on such functions and monitoring PSR is crucial for tracking the effects of land use and agricultural management on these ecosystems. Unfortunately, traditional morphology-based methods are labor-intensive and cannot be adapted for high-throughput assessments. DNA barcoding could aid increasing the throughput of PSR assessments in grasslands. In this proof-of-concept work, we aimed at determining which of three plant DNA barcodes (rbcLa, matK and trnH-psbA) best discriminates 16 key grass and legume species common in temperate sub-alpine grasslands. RESULTS: Barcode trnH-psbA had a 100% correct assignment rate (CAR) in the five analyzed legumes, followed by rbcLa (93.3%) and matK (55.6%). Barcode trnH-psbA had a 100% CAR in the grasses Cynosurus cristatus, Dactylis glomerata and Trisetum flavescens. However, the closely related Festuca, Lolium and Poa species were not always correctly identified, which led to an overall CAR in grasses of 66.7%, 50.0% and 46.4% for trnH-psbA, matK and rbcLa, respectively. Barcode trnH-psbA is thus the most promising candidate for PSR assessments in permanent grasslands and could greatly support plant biodiversity monitoring on a larger scale.