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While shaping of plant microbiome composition through 'host filtering' is well documented in legume-rhizobium symbioses, it is less clear to what extent different varieties and genotypes of the same plant species differentially influence symbiont community diversity and composition. Here, we compared how clover host varieties and genotypes affect the structure of Rhizobium populations in root nodules under conventional field and controlled greenhouse conditions. We first grew four Trifolium repens (white clover) F 2 crosses and one variety in a conventional field trial and compared differences in root nodule Rhizobium leguminosarum symbiovar trifolii (Rlt) genotype diversity using high-throughput amplicon sequencing of chromosomal housekeeping (rpoB and recA) genes and auxiliary plasmid-borne symbiosis genes (nodA and nodD). We found that Rlt nodule diversities significantly differed between clover crosses, potentially due to host filtering. However, variance in Rlt diversity largely overlapped between crosses and was also explained by the spatial distribution of plants in the field, indicative of the role of local environmental conditions for nodule diversity. To test the effect of host filtering, we conducted a controlled greenhouse trial with a diverse Rlt inoculum and several host genotypes. We found that different clover varieties and genotypes of the same variety selected for significantly different Rlt nodule communities and that the strength of host filtering (deviation from the initial Rhizobium inoculant composition) was positively correlated with the efficiency of symbiosis (rate of plant greenness colouration). Together, our results suggest that selection by host genotype and local growth conditions jointly influence white clover Rlt nodule diversity and community composition.

Scaffolds with significant SNPs. (DOCX 14Â kb)

Vernalization, the requirement of a period of low temperature to induce transition from vegetative to reproductive state, is an important trait in grasses. The objective of this study was to identify the causative polymorphism(s) for the variation in vernalization requirement at the VRN1 locus in perennial ryegrass (Lolium perenne). Two allelic BAC clones of the VRN1 locus from the two genotypes Veyo and Falster with contrasting vernalization requirements were identified, sequenced, and characterized. The region surrounding the VRN1 locus in perennial ryegrass showed micro-colinearity to the corresponding region on chromosome 3 in Oryza sativa with conserved gene order and orientation, while the micro-colinearity to the corresponding region in Triticum monococcum was less conserved. Repeat elements constituted 42 and 58% of the Veyo and Falster BAC sequences, respectively, of which the majority showed similarity to the Athila-like Class I LTR retrotransposon Sabrina from Hordeum vulgare. Bioinformatic analysis of the allelic sequences identified a 8.6kb deletion in the first intron of the VRN1 gene in the Veyo genotype, which in a divergent selection experiment was associated with low vernalization requirement. Our findings suggest that the 8.6kb region in the first intron of the VRN1 gene is an important regulatory region for vernalization response in perennial ryegrass.

GRASP was a EU framework V project involving eight public institutions and one commercial partner. The major topic of GRASP was the development of "functional" allele-specific markers associated with relevant traits in L. perenne such as forage quality, nitrogen use efficiency, disease resistance, and abiotic stress tolerance. More than 100 candidate genes for these traits were isolated and the respective alleles of 20 genotypes ( Lolium test set=LTS) were sequenced in order to derive allele-specific DNA markers. In parallel, the LTS was used to develop synthetic populations to be propagated under divergent selection over two generations for the different traits of interest. The frequency shift of DNA marker alleles over generations was monitored and used to validate candidate gene-trait associations. In addition to markers and populations, genomic tools were developed including a BAC library for L. perenne, subtracted cDNA libraries, microarrays for expression profiling, and genetic reference maps. Information generated in GRASP will be made available through the existing forage grass database in the UKCropnet (http://ukcrop.net/grass.html). GRASP is expected to be a platform for modern forage grass breeding but also for international follow-up projects.

Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloë. Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analyzed collected populations of meadow fescue (Schedonorus pratensis) from 135 different locations across Europe, 255 accessions from the United States Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus, S. giganteus, and Lolium perenne. All plants were screened for the presence of Epichloë endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect (r = 0.227) was detected. Concentrations of loline alkaloids were measured in 218 infected meadow fescue plants. Average amount of total loline and the proportions of the single loline alkaloids differed significantly among endophyte haplotypes (P < 0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and alkaloid variation observed in E. uncinata., Frontiers in Plant Science, 10, ISSN:1664-462X