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Both Europe and China have a low level of protein autonomy. They import 30% and 60%, respectively, of the world plant protein production for both human and animal nutrition. However, in both continents, regions with appropriate agro-ecological conditions for the production of plant proteins exist. The project EUCLEG, recently granted by the H2020 programme of European Union, has the ambition to give a new momentum to forage legume (alfalfa and red clover) and grain legume (pea, faba bean and soybean) breeding. The scientific objectives of this project are i) to broaden the genetic base of legume crops, ii) to analyse the genetic diversity of European and Chinese legume accessions using phenotypic traits (yield, quality in a context of climate change) and molecular markers, iii) to analyse the genetic architecture of key breeding traits using association genetics (GWAS) and iv) to evaluate the benefits brought by genomic selection (GS) to create new legume varieties. Comprehensive searchable databases containing passport data, as well as agronomic and genetic features will be developed. The breeders that are members of EUCLEG will exploit the results to create the new varieties needed to improve protein production.

Background Sainfoin (Onobrychis viciifolia) is a highly nutritious tannin-containing forage legume. In the diet of ruminants sainfoin can have anti-parasitic effects and reduce methane emissions under in vitro conditions. Many of these benefits have been attributed to condensed tannins or proanthocyanidins in sainfoin. A combination of increased use of industrially produced nitrogen fertilizer, issues with establishment and productivity in the first year and more reliable alternatives, such as red clover led to a decline in the use of sainfoin since the middle of the last century. In recent years there has been a resurgence of interest in sainfoin due to its potential beneficial nutraceutical and environmental attributes. However, genomic resources are scarce, thus hampering progress in genetic analysis and improvement. To address this we have used next generation RNA sequencing technology to obtain the first transcriptome of sainfoin. We used the library to identify gene-based simple sequence repeats (SSRs) and potential single nucleotide polymorphisms (SNPs). Results One genotype from each of five sainfoin accessions was sequenced. Paired-end (PE) sequences were generated from cDNA libraries of RNA extracted from 7 day old seedlings. A combined assembly of 92,772 transcripts was produced de novo using the Trinity programme. About 18,000 transcripts were annotated with at least one GO (gene ontology) term. A total of 63 transcripts were annotated as involved in the tannin biosynthesis pathway. We identified 3786 potential SSRs. SNPs were identified by mapping the reads of the individual assemblies against the combined assembly. After stringent filtering a total of 77,000 putative SNPs were identified. A phylogenetic analysis of single copy number genes showed that sainfoin was most closely related to red clover and Medicago truncatula, while Lotus japonicus, bean and soybean are more distant relatives. Conclusions This work describes the first transcriptome assembly in sainfoin. The 92 K transcripts provide a rich source of SNP and SSR polymorphisms for future use in genetic studies of this crop. Annotation of genes involved in the condensed tannin biosynthesis pathway has provided the basis for further studies of the genetic control of this important trait in sainfoin. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3083-6) contains supplementary material, which is available to authorized users.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12863-016-0431-0) contains supplementary material, which is available to authorized users.

Relationship between modified Roger’s Distance to Euclidian Distance and to Nei’s Distance. (PDF 319 kb)

The Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) gene and its orthologs in other plant species (e.g. rice [Oryza sativa] OsFTL2/Hd3a) have an established role in the photoperiodic induction of flowering response. The genomic and phenotypic variations associated with the perennial ryegrass (Lolium perenne) ortholog of FT, designated LpFT3, was assessed in a diverse collection of nine European germplasm populations, which together constituted an association panel of 864 plants. Sequencing and genotyping of a series of amplicons derived from the nine populations, containing the complete exon and intron sequences as well as 5' and 3' noncoding sequences of LpFT3, identified a total of seven haplotypes. Genotyping assays designed to detect the genomic variation showed that three haplotypes were present in approximately equal proportions and represented 84% of the total, with a fourth representing a further 11%. Of the three major haplotypes, two were predicted to code for identical protein products and the third contained two amino acid substitutions. Association analysis using either a mixed model with a relationship matrix to correct for population structure and relatedness or structured association with further correction using genomic control indicated significant associations between LpFT3 and variation in flowering time. These associations were corroborated in a validation population segregating for the same major alleles. The most "diagnostic" region of genomic variation was situated 5' of the coding sequence. Analysis of this region identified that the interhaplotype variation was closely associated with sequence motifs that were apparently conserved in the 5' region of orthologs of LpFT3 from other plant species. These may represent cis-regulatory elements involved in influencing the expression of this gene.

The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from Bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria Gluconacetobacter diazotrophicus strain BR11281 and Herbaspirillum seropedicae strain BR11335 were used as models. The cry3A gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the cry gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of delta-endotoxin by the recombinant H. seropedicae strain was detected by dot blot while for G. diazotrophicus the Western-blot technique was used. In both cases, a specific antibody raised against the B. thuringiensis toxin was applied. The delta-endotoxin production showed by the G. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3A gene was fused to a nif promoter. In the case of H. seropedicae the delta-endotoxin expression was not affected by the promoter (rhi) used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests. Este estudo teve como objetivo avaliar o potencial de uso de bactérias diazotróficas endofíticas como vetores para a expressão de genes cry de Bacillus thuringiensis. As bactérias diazotróficas endofíticas Gluconacetobacter diazotrophicus estirpe BR11281 e Herbaspirillum seropedicae estirpe BR11335 foram usadas como modelo. O gene cry3A foi transferido através de um plasmídeo suicída por conjugação e os recombinantes foram selecionados pela sua capacidade de fixar nitrogênio em meio semi-sólido sem N. A presença do gene cry3A no genoma dos transconjugantes foi detectada através da técnica de "Southern blot" utilizando como sonda um fragmento de 1,0 kb, interno ao gene cry3A. A produção de delta-endotoxina pelos transconjugantes foi detectada por "Dot blot" em de H. seropedicae e por "Western blot" em G. diazotrophicus, usando-se o anticorpo específico para a toxina de B. thuringiensis. A avaliação da produção da delta-endotoxina mostrou que a expressão do gene cry3A em G. diazotrophicus é dependente do processo de fixação de nitrogênio por estar fusionado a um promotor nif nesta bactéria. No caso de H. seropedicae a expressão não foi influenciada pelo promotor de rizosfera (rhi) usado. Os resultados obtidos sugerem que estas bactérias diazotróficas endofíticas podem ser usadas como vetores para expressar genes com atividade entomopatogênica, visando o controle de pragas de cana-de-açúcar ou outras plantas de interesse econômico.