An improved chromosome-level genome assembly of perennial ryegrass (Lolium perenne L.).

This work is an update and extension of the previously published article "Ultralong Oxford Nanopore Reads Enable the Development of a Reference-Grade Perennial Ryegrass Genome Assembly" by Frei et al. The published genome assembly of the doubled haploid perennial ryegrass (Lolium perenne L.) genotype Kyuss (Kyuss v1.0) marked a milestone for forage grass research and breeding. However, order and orientation errors may exist in the pseudo-chromosomes of Kyuss, since barley (Hordeum vulgare L.), which diverged 30 million years ago from perennial ryegrass, was used as the reference to scaffold Kyuss. To correct for structural errors possibly present in the published Kyuss assembly, we de novo assembled the genome again and generated 50-fold coverage high-throughput chromosome conformation capture (Hi-C) data to assist pseudo-chromosome construction. The resulting new chromosome-level assembly Kyuss v2.0 showed improved quality with high contiguity (contig N50 = 120 Mb), high completeness (total BUSCO score = 99%), high base-level accuracy (QV = 50), and correct pseudo-chromosome structure (validated by Hi-C contact map). This new assembly will serve as a better reference genome for Lolium spp. and greatly benefit the forage and turf grass research community.

A Community-Curated DokuWiki Resource on Diagnostics, Diversity, Pathogenicity, and Genetic Control of Xanthomonads.

Xanthomonads, including Xanthomonas and Xylella species, constitute a large and significant group of economically and ecologically important plant pathogens. Up-to-date knowledge of these pathogens and their hosts is essential for the development of suitable control measures. Traditional review articles or book chapters have inherent limitations, including static content and rapid obsolescence. To address these challenges, we have developed a Web-based knowledge platform dedicated to xanthomonads, inspired by the concept of living systematic reviews. This platform offers a dynamic resource that encompasses bacterial virulence factors, plant resistance genes, and tools for diagnostics and genetic diversity studies. Our goal is to facilitate access for newcomers to the field, provide continuing education opportunities for students, assist plant protection services with diagnostics, provide valuable information to breeders on sources of resistance and breeding targets, and offer comprehensive expert knowledge to other stakeholders interested in plant-pathogenic xanthomonads. This resource is available for queries and updates at https://euroxanth.ipn.pt. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

High genomic plasticity and unique features of Xanthomonas translucens pv. graminis revealed through comparative analysis of complete genome sequences.

BACKGROUND: Xanthomonas translucens pv. graminis (Xtg) is a major bacterial pathogen of economically important forage grasses, causing severe yield losses. So far, genomic resources for this pathovar consisted mostly of draft genome sequences, and only one complete genome sequence was available, preventing comprehensive comparative genomic analyses. Such comparative analyses are essential in understanding the mechanisms involved in the virulence of pathogens and to identify virulence factors involved in pathogenicity. RESULTS: In this study, we produced high-quality, complete genome sequences of four strains of Xtg, complementing the recently obtained complete genome sequence of the Xtg pathotype strain. These genomic resources allowed for a comprehensive comparative analysis, which revealed a high genomic plasticity with many chromosomal rearrangements, although the strains were highly related. A high number of transposases were exclusively found in Xtg and corresponded to 413 to 457 insertion/excision transposable elements per strain. These mobile genetic elements are likely to be involved in the observed genomic plasticity and may play an important role in the adaptation of Xtg. The pathovar was found to lack a type IV secretion system, and it possessed the smallest set of type III effectors in the species. However, three XopE and XopX family effectors were found, while in the other pathovars of the species two or less were present. Additional genes that were specific to the pathovar were identified, including a unique set of minor pilins of the type IV pilus, 17 TonB-dependent receptors (TBDRs), and 11 plant cell wall degradative enzymes. CONCLUSION: These results suggest a high adaptability of Xtg, conferred by the abundance of mobile genetic elements, which could play a crucial role in pathogen adaptation. The large amount of such elements in Xtg compared to other pathovars of the species could, at least partially, explain its high virulence and broad host range. Conserved features that were specific to Xtg were identified, and further investigation will help to determine genes that are essential to pathogenicity and host adaptation of Xtg.

A genome-wide association study of freezing tolerance in red clover (Trifolium pratense L.) germplasm of European origin.

Improvement of persistency is an important breeding goal in red clover (Trifolium pratense L.). In areas with cold winters, lack of persistency is often due to poor winter survival, of which low freezing tolerance (FT) is an important component. We conducted a genome wide association study (GWAS) to identify loci associated with freezing tolerance in a collection of 393 red clover accessions, mostly of European origin, and performed analyses of linkage disequilibrium and inbreeding. Accessions were genotyped as pools of individuals using genotyping-by-sequencing (pool-GBS), generating both single nucleotide polymorphism (SNP) and haplotype allele frequency data at accession level. Linkage disequilibrium was determined as a squared partial correlation between the allele frequencies of pairs of SNPs and found to decay at extremely short distances (< 1 kb). The level of inbreeding, inferred from the diagonal elements of a genomic relationship matrix, varied considerably between different groups of accessions, with the strongest inbreeding found among ecotypes from Iberia and Great Britain, and the least found among landraces. Considerable variation in FT was found, with LT50-values (temperature at which 50% of the plants are killed) ranging from -6.0°C to -11.5°C. SNP and haplotype-based GWAS identified eight and six loci significantly associated with FT (of which only one was shared), explaining 30% and 26% of the phenotypic variation, respectively. Ten of the loci were found within or at a short distance (<0.5 kb) from genes possibly involved in mechanisms affecting FT. These include a caffeoyl shikimate esterase, an inositol transporter, and other genes involved in signaling, transport, lignin synthesis and amino acid or carbohydrate metabolism. This study paves the way for a better understanding of the genetic control of FT and for the development of molecular tools for the improvement of this trait in red clover through genomics assisted breeding.

Multi-location trials and population-based genotyping reveal high diversity and adaptation to breeding environments in a large collection of red clover.

Red clover (Trifolium pratense L.) is an outcrossing forage legume that has adapted to a wide range of climatic and growing conditions across Europe. Red clover is valued for its high yield potential and its forage quality. The high amount of genetic diversity present in red clover provides an invaluable, but often poorly characterized resource to improve key traits such as yield, quality, and resistance to biotic and abiotic stresses. In this study, we examined the genetic and phenotypic diversity within a diverse set of 395 diploid red clover accessions via genome wide allele frequency fingerprinting and multi-location field trials across Europe. We found that the genetic structure of accessions mostly reflected their geographic origin and only few cases were detected, where breeders integrated foreign genetic resources into their local breeding pools. The mean dry matter yield of the first main harvesting season ranged from 0.74 kg m-2 in Serbia and Norway to 1.34 kg m-2 in Switzerland. Phenotypic performance of accessions in the multi-location field trials revealed a very strong accession x location interaction. Local adaptation was especially prominent in Nordic red clover accessions that showed a distinct adaptation to the growing conditions and cutting regime of the North. The traits vigor, dry matter yield and plant density were negatively correlated between the trial location in Norway and the locations Great Britain, Switzerland, Czech Republic and Serbia. Notably, breeding material and cultivars generally performed well at the location where they were developed. Our results confirmed that red clover cultivars were bred from regional ecotypes and show a narrow adaptation to regional conditions. Our study can serve as a valuable basis for identifying interesting materials that express the desired characteristics and contribute to the adaptation of red clover to future climatic conditions.

Phenotypic variation and quantitative trait loci for resistance to southern anthracnose and clover rot in red clover.

KEY MESSAGE: High variability for and candidate loci associated with resistance to southern anthracnose and clover rot in a worldwide collection of red clover provide a first basis for genomics-assisted breeding. Red clover (Trifolium pratense L.) is an important forage legume of temperate regions, particularly valued for its high yield potential and its high forage quality. Despite substantial breeding progress during the last decades, continuous improvement of cultivars is crucial to ensure yield stability in view of newly emerging diseases or changing climatic conditions. The high amount of genetic diversity present in red clover ecotypes, landraces, and cultivars provides an invaluable, but often unexploited resource for the improvement of key traits such as yield, quality, and resistance to biotic and abiotic stresses. A collection of 397 red clover accessions was genotyped using a pooled genotyping-by-sequencing approach with 200 plants per accession. Resistance to the two most pertinent diseases in red clover production, southern anthracnose caused by Colletotrichum trifolii, and clover rot caused by Sclerotinia trifoliorum, was assessed using spray inoculation. The mean survival rate for southern anthracnose was 22.9% and the mean resistance index for clover rot was 34.0%. Genome-wide association analysis revealed several loci significantly associated with resistance to southern anthracnose and clover rot. Most of these loci are in coding regions. One quantitative trait locus (QTL) on chromosome 1 explained 16.8% of the variation in resistance to southern anthracnose. For clover rot resistance we found eight QTL, explaining together 80.2% of the total phenotypic variation. The SNPs associated with these QTL provide a promising resource for marker-assisted selection in existing breeding programs, facilitating the development of novel cultivars with increased resistance against two devastating fungal diseases of red clover.

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.

Complete Genome Assemblies of All Xanthomonas translucens Pathotype Strains Reveal Three Genetically Distinct Clades.

The Xanthomonas translucens species comprises phytopathogenic bacteria that can cause serious damage to cereals and to forage grasses. So far, the genomic resources for X. translucens were limited, which hindered further understanding of the host-pathogen interactions at the molecular level and the development of disease-resistant cultivars. To this end, we complemented the available complete genome sequence of the X. translucens pv. translucens pathotype strain DSM 18974 by sequencing the genomes of all the other 10 X. translucens pathotype strains using PacBio long-read technology and assembled complete genome sequences. Phylogeny based on average nucleotide identity (ANI) revealed three distinct clades within the species, which we propose to classify as clades Xt-I, Xt-II, and Xt-III. In addition to 2,181 core X. translucens genes, a total of 190, 588, and 168 genes were found to be exclusive to each clade, respectively. Moreover, 29 non-transcription activator-like effector (TALE) and 21 TALE type III effector classes were found, and clade- or strain-specific effectors were identified. Further investigation of these genes could help to identify genes that are critically involved in pathogenicity and/or host adaptation, setting the grounds for the development of new resistant cultivars.

The Complete Genome Sequence of Xanthomonas theicola, the Causal Agent of Canker on Tea Plants, Reveals Novel Secretion Systems in Clade-1 Xanthomonads.

Xanthomonas theicola is the causal agent of bacterial canker on tea plants. There is no complete genome sequence available for X. theicola, a close relative of the species X. translucens and X. hyacinthi, thus limiting basic research for this group of pathogens. Here, we release a high-quality complete genome sequence for the X. theicola type strain, CFBP 4691T. Single-molecule real-time sequencing with a mean coverage of 264× revealed two contigs of 4,744,641 bp (chromosome) and 40,955 bp (plasmid) in size. Genome mining revealed the presence of nonribosomal peptide synthases, two CRISPR systems, the Xps type 2 secretion system, and the Hrp type 3 secretion system. Surprisingly, this strain encodes an additional type 2 secretion system and a novel type 3 secretion system with enigmatic function, hitherto undescribed for xanthomonads. Four type 3 effector genes were found on complete or partial transposons, suggesting a role of transposons in effector gene evolution and spread. This genome sequence fills an important gap to better understand the biology and evolution of the early-branching xanthomonads, also known as clade-1 xanthomonads.

A Non-destructive Method to Quantify Leaf Starch Content in Red Clover.

Grassland-based ruminant livestock production provides a sustainable alternative to intensive production systems relying on concentrated feeds. However, grassland-based roughage often lacks the energy content required to meet the productivity potential of modern livestock breeds. Forage legumes, such as red clover, with increased starch content could partly replace maize and cereal supplements. However, breeding for increased starch content requires efficient phenotyping methods. This study is unique in evaluating a non-destructive hyperspectral imaging approach to estimate leaf starch content in red clover for enabling efficient development of high starch red clover genotypes. We assessed prediction performance of partial least square regression models (PLSR) using cross-validation, and validated model performance with an independent test set under controlled conditions. Starch content of the training set ranged from 0.1 to 120.3 mg g-1 DW. The best cross-validated PLSR model explained 56% of the measured variation and yielded a root mean square error (RMSE) of 17 mg g-1 DW. Model performance decreased when applying the trained model on the independent test set (RMSE = 29 mg g-1 DW, R 2 = 0.36). Different variable selection methods did not increase model performance. Once validated in the field, the non-destructive spectral method presented here has the potential to detect large differences in leaf starch content of red clover genotypes. Breeding material could be sampled and selected according to their starch content without destroying the plant.

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.

Phenotyping a Dynamic Trait: Leaf Growth of Perennial Ryegrass Under Water Limiting Conditions.

Water limitation is one of the major factors reducing crop productivity worldwide. In order to develop efficient breeding strategies to improve drought tolerance, accurate methods to identify when a plant reduces growth as a consequence of water deficit have yet to be established. In perennial ryegrass (Lolium perenne L.), an important forage grass of the Poaceae family, leaf elongation is a key factor determining plant growth and hence forage yield. Although leaf elongation has been shown to be temperature-dependent under non-stress conditions, the impact of water limitation on leaf elongation in perennial ryegrass is poorly understood. We describe a method for quantifying tolerance to water deficit based on leaf elongation in relation to temperature and soil moisture in perennial ryegrass. With decreasing soil moisture, three growth response phases were identified: first, a "normal" phase where growth is mainly determined by temperature, second a "slow" phase where leaf elongation decreases proportionally to soil water potential and third an "arrest" phase where leaf growth terminates. A custom R function was able to quantify the points which demarcate these phases and can be used to describe the response of plants to water deficit. Applied to different perennial ryegrass genotypes, this function revealed significant genotypic variation in the response of leaf growth to temperature and soil moisture. Dynamic phenotyping of leaf elongation can be used as a tool to accurately quantify tolerance to water deficit in perennial ryegrass and to improve this trait by breeding. Moreover, the tools presented here are applicable to study the plant response to other stresses in species with linear, graminoid leaf morphology.

Pooled DNA sequencing to identify SNPs associated with a major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.).

KEY MESSAGE: SNPs and candidate genes associated with bacterial wilt resistance in Italian ryegrass were identified by sequencing the parental plants and pooled F1 progeny of a segregating population. Italian ryegrass (Lolium multiflorum Lam.) is one of the most important forage grass species in temperate regions. Its yield, quality and persistency can significantly be reduced by bacterial wilt, a serious disease caused by Xanthomonas translucens pv. graminis. Although a major QTL for bacterial wilt resistance has previously been reported, detailed knowledge on underlying genes and DNA markers to allow for efficient resistance breeding strategies is currently not available. We used pooled DNA sequencing to characterize a major QTL for bacterial wilt resistance of Italian ryegrass and to develop inexpensive sequence-based markers to efficiently target resistance alleles for marker-assisted recurrent selection. From the mapping population segregating for the QTL, DNA of 44 of the most resistant and 44 of the most susceptible F1 individuals was pooled and sequenced using the Illumina HiSeq 2000 platform. Allele frequencies of 18 × 106 single nucleotide polymorphisms (SNP) were determined in the resistant and susceptible pool. A total of 271 SNPs on 140 scaffold sequences of the reference parental genome showed significantly different allele frequencies in both pools. We converted 44 selected SNPs to KASP™ markers, genetically mapped these proximal to the major QTL and thus validated their association with bacterial wilt resistance. This study highlights the power of pooled DNA sequencing to efficiently target binary traits in biparental mapping populations. It delivers genome sequence data, SNP markers and potential candidate genes which will allow to implement marker-assisted strategies to fix bacterial wilt resistance in outcrossing breeding populations of Italian ryegrass.

Comparative genomics of host adaptive traits in Xanthomonas translucens pv. graminis.

BACKGROUND: Xanthomonas translucens pathovars differ in their individual host ranges among Poaceae. As the causal agent of bacterial wilt in Italian ryegrass (Lolium multiflorum Lam.), X. translucens pv. graminis (Xtg) is one of the most important bacterial pathogens in temperate grassland regions. The genomes of six Xtg strains from Switzerland, Norway, and New Zealand were sequenced in order to gain insight into conserved genomic traits from organisms covering a wide geographical range. Subsequent comparative analysis with previously published genome data of seven non-graminis X. translucens strains including the pathovars arrhenatheri, poae, phlei, cerealis, undulosa, and translucens was conducted to identify candidate genes linked to the host adaptation of Xtg to Italian ryegrass. RESULTS: Phylogenetic analysis revealed a tight clustering of Xtg strains, which were found to share a large core genome. Conserved genomic traits included a non-canonical type III secretion system (T3SS) and a type IV pilus (T4P), which both revealed distinct primary structures of the pilins when compared to the non-graminis X. translucens strains. Xtg-specific traits that had no homologues in the other X. translucens strains were further found to comprise several hypothetical proteins, a TonB-dependent receptor, transporters, and effector proteins as well as toxin-antitoxin systems and DNA methyltransferases. While a nearly complete flagellar gene cluster was identified in one of the sequenced Xtg strains, phenotypic analysis pointed to swimming-deficiency as a common trait of the pathovar graminis. CONCLUSION: Our study suggests that host adaptation of X. translucens pv. graminis may be conferred by a combination of pathovar-specific effector proteins, regulatory mechanisms, and adapted nutrient acquisition. Sequence deviations of pathogen-associated molecular patterns (PAMPs), as observed for the pilins of the T4P and T3SS, are moreover likely to impede perception by the plant defense machinery and thus facilitate successful host colonization of Italian ryegrass.

Drought Effects on Proanthocyanidins in Sainfoin (Onobrychis viciifolia Scop.) Are Dependent on the Plant's Ontogenetic Stage.

Sainfoin (Onobrychis viciifolia Scop.) is a forage legume, which improves animal health and the environmental impact of livestock farming due to its proanthocyanidin content. To identify the impact of drought on acetone/water-extractable proanthocyanidin (PA) concentration and composition in the generative and vegetative stages, a rain exclosure experiment was established. Leaves of 120 plants from 5 different sainfoin accessions were sampled repeatedly and analyzed by UPLC-ESI-MS/MS. The results showed distinct differences in response to drought between vegetative and generative plants. Whereas vegetative plants showed a strong response to drought in growth (-56%) and leaf PA concentration (+46%), generative plants showed no response in growth (-2%) or PA concentration (-9%). The PA composition was stable across environments. The five accessions varied in PA concentrations and composition but showed the same pattern of response to the experimental treatments. These results show that the ontogenetic stage at which drought occurs significantly affects the plant's response.

Complete Genome Sequence of the Barley Pathogen Xanthomonas translucens pv. translucens DSM 18974T (ATCC 19319T).

We report here the complete 4.7-Mb genome sequence of Xanthomonas translucens pv. translucens DSM 18974T, which causes black chaff disease on barley (Hordeum vulgare). Genome data of this X. translucens type strain will improve our understanding of this bacterial species.

Draft Genome Sequence of the Xanthomonas bromi Type Strain LMG 947.

Here, we report the draft genome sequence of the Xanthomonas bromi type strain LMG 947, an important pathogen of bromegrasses (Bromus spp.). Comparative analysis with other Xanthomonas spp. that are pathogenic on forage grasses will assist the analysis of host-plant adaptation at the genome level.

Characterization of novel SSR markers in diverse sainfoin (Onobrychis viciifolia) germplasm.

BACKGROUND: Sainfoin is a perennial forage legume with beneficial properties for animal husbandry due to the presence of secondary metabolites. However, worldwide cultivation of sainfoin is marginal due to the lack of varieties with good agronomic performance, adapted to a broad range of environmental conditions. Little is known about the genetics of sainfoin and only few genetic markers are available to assist breeding and genetic investigations. The objective of this study was to develop a set of SSR markers useful for genetic studies in sainfoin and their characterization in diverse germplasm. RESULTS: A set of 400 SSR primer combinations were tested for amplification and their ability to detect polymorphisms in a set of 32 sainfoin individuals, representing distinct varieties or landraces. Alleles were scored for presence or absence and polymorphism information content of each SSR locus was calculated with an adapted formula taking into account the tetraploid character of sainfoin. Relationships among individuals were visualized using cluster and principle components analysis. Of the 400 primer combinations tested, 101 reliably detected polymorphisms among the 32 sainfoin individuals. Among the 1154 alleles amplified 250 private alleles were observed. The number of alleles per locus ranged from 2 to 24 with an average of 11.4 alleles. The average polymorphism information content reached values of 0.14 to 0.36. The clustering of the 32 individuals suggested a separation into two groups depending on the origin of the accessions. CONCLUSIONS: The SSR markers characterized and tested in this study provide a valuable tool to detect polymorphisms in sainfoin for future genetic studies and breeding programs. As a proof of concept, we showed that these markers can be used to separate sainfoin individuals based on their origin.

Draft genome sequences of three Xanthomonas translucens pathovar reference strains (pv. arrhenatheri, pv. poae and pv. phlei) with different specificities for forage grasses.

As causal agents of bacterial wilt in pastures and meadows, bacteria of the species Xanthomonas translucens are a serious issue in forage grass production. So far, only little is known about host-pathogen interactions at the molecular level and the lack of comprehensive genome data impeded targeted breeding strategies towards resistant forage grass cultivars. Here we announce the draft genome sequences of three grass-pathogenic Xanthomonas translucens pathotype strains, i.e. pv. arrhenatheri LMG 727, pv. poae LMG 728 and pv. phlei LMG 730 isolated from Arrhenatherum elatius (L.) P. Beauv. ex J. Presl & C. Presl (Switzerland), Poa trivialis L. (Switzerland) and Phleum pratense L. (Norway), respectively. The genomes of all three strains revealed a non-canonical type III secretion system and a set of 22 type III effectors as common virulence-related traits. Distinct inter-pathovar differences were observed for the lipopolysaccharide biosynthesis gene cluster and the presence of nonribosomal peptide synthetases.

Evidence and consequences of self-fertilisation in the predominantly outbreeding forage legume Onobrychis viciifolia.

BACKGROUND: Sainfoin (Onobrychis viciifolia) is a promising alternative forage plant of good quality, moderate nutrient demand and a high content of polyphenolic compounds. Its poor adoption is caused by the limited availability of well performing varieties. Sainfoin is characterised as tetraploid and mainly outcrossing, but the extent of self-fertilisation and its consequences was not investigated so far. This study aimed at assessing the rate of self-fertilisation in sainfoin under different pollination regimes and at analysing the consequences on plant performance in order to assist future breeding efforts. METHODS: The self-fertilisation rate was assessed in three sainfoin populations with artificially directed pollination (ADP) and in three populations with non-directed pollination (NDP). Dominant SRAP (sequence-related amplified polymorphism) and codominant SSR (simple sequence repeats) markers were used to detect self-fertilisation in sainfoin for the first time based on molecular marker data. RESULTS: High rates of self-fertilisation of up to 64.8% were observed for ADP populations in contrast to only up to 3.9% for NDP populations. Self-fertilisation in ADP populations led to a reduction in plant height, plant vigour and, most severely, for seed yield. CONCLUSIONS: Although sainfoin is predominantly outcrossing, self-fertilisation can occur to a high degree under conditions of limited pollen availability. These results will influence future breeding efforts because precautions have to be taken when crossing breeding material. The resulting inbreeding depression can lead to reduced performance in self-fertilised offspring. Nevertheless the possibility of self-fertilisation also offers new ways for hybrid breeding based on the development of homogenous inbred lines.