Leif Skøt

An association mapping approach to identify flowering time genes in natural populations of Lolium perenne (L.)

We describe an association mapping approach using natural populations of perennial ryegrass (Lolium perenne L.) to identify molecular markers associated with heading date, an important trait affecting seasonal production, tillering, digestibility and grassland management regimes. Twenty-three natural populations originating from throughout Europe, with heading date phenotypes ranging from very early to very late, as well as three synthetic populations (varieties) were used for molecular marker genotyping using AFLP. In total, 589 polymorphic markers were identified. Hierarchical clustering, principal coordinate and other statistical analyses identified four outlying populations forming a clearly distinct sub-group. Removal of those four populations from the subsequent analysis reduced population sub-structure twofold. However, this made relatively little difference to the result of the association analysis. Linear regression identified three markers whose frequency of occurrence correlated with the heading date phenotype. Moreover, these markers were shown to be closely linked to each other within a major QTL on Chromosome 7, explaining 70% of the total variation in heading date. Pairwise linkage disequilibrium among them was also significant. These results suggest that association mapping approaches may be feasible in L. perenne, and that the use of natural populations could provide a useful source of genetic variation in traits of importance in crop improvement.

Synteny between a major heading-date QTL in perennial ryegrass (Lolium perenne L.) and the Hd3 heading-date locus in rice

The genetic control of induction to flowering has been studied extensively in both model and crop species because of its fundamental biological and economic significance. An ultimate aim of many of these studies has been the application of the understanding of control of flowering that can be gained from the study of model species, to the improvement of crop species. The present study identifies a region of genetic synteny between rice and Lolium perenne, which contains the Hd3 heading-date QTL in rice and a major QTL, accounting for up to 70% of the variance associated with heading date in L. perenne. The identification of synteny between rice and L. perenne in this region demonstrates the direct applicability of the rice genome to the understanding of biological processes in other species. Specifically, this syntenic relationship will greatly facilitate the genetic dissection of aspects of heading-date induction by enabling the magnitude of the genetic component of the heading-date QTL in L. perenne to be combined with the sequencing and annotation information from the rice genome.

Association of Candidate Genes With Flowering Time and Water-Soluble Carbohydrate Content in Lolium perenne (L.)

We describe a candidate gene approach for associating SNPs with variation in flowering time and water-soluble carbohydrate (WSC) content and other quality traits in the temperate forage grass species Lolium perenne. Three analysis methods were used, which took the significant population structure into account. First, a linear mixed model was used enabling a structured association analysis to be incorporated with the nine populations identified in the structure analysis as random variables. Second, a within-population analysis of variance was performed. Third, a tree-scanning method was used, in which haplotype trees were associated with phenotypes on the basis of inferred haplotypes. Analysis of variance within populations identified several associations between WSC, nitrogen (N), and dry matter digestibility with allelic variants within an alkaline invertase candidate gene LpcAI. These associations were only detected in material harvested in one of the two years. By contrast, consistent associations between the L. perenne homolog (LpHD1) of the rice photoperiod control gene HD1 and flowering time were identified. One SNP, in the immediate upstream region of the LpHD1 coding sequence (C-4443-A), was significant in the linear mixed model. Within-population analysis of variance and tree-scanning analysis confirmed and extended this result to the 2118 polymorphisms in some of the populations. The merits of the tree-scanning method are compared to the single SNP analysis. The potential usefulness of the 4443 SNP in marker-assisted selection is currently being evaluated in test crosses of genotypes from this work with turf-grass varieties.

De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response, gene discovery and marker identification

BackgroundRed clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress.ResultsPlants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing ‘transcript tags’. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress.ConclusionsThis de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover.

Molecular genecology of temperature response in Lolium perenne: 2. Association of AFLP markers with ecogeography

Improved winter hardiness is an important breeding objective in the forage grass Lolium perenne. This is a complex trait with several components, including the ability to survive and grow at low temperature, to acclimate to cold, tolerate wind, snow cover and ice encasement. Marker‐assisted selection has the potential to increase the efficiency of breeding for improved cold tolerance. Here we describe a genecological approach to identifying molecular markers that are associated with adaptation to low winter temperatures. AFLP was used to assess the genetic diversity in 29 wild populations of ryegrass (Lolium perenne) representing a pan‐European temperature cline in terms of their geographical origin. A further 18 populations from a temperature cline in Bulgaria were also analysed. In addition, two varieties and five populations representing parents of mapping families currently in use at IGER were included in the analysis. Principal coordinate (PCoA) and cluster analyses of the molecular marker data showed that the Bulgarian altitude cline populations could be distinguished clearly from the other populations. Two regression analyses were carried out; one to identify AFLP markers that correlated in frequency with low mean January temperature of the geographical origin of the population, and another to identify AFLP markers correlating in frequency with the cold tolerance phenotype of the populations, as determined by LT50 values in freezing tests. In the first analysis six AFLP markers showed significant type II trends with mean January temperature, and in the second analysis 28 bands had a significant univariate relationship with the LT50 value of the accessions. In steps 2 and 3 of the stepwise analysis a further 4 and 5 bands, respectively, improved the fit significantly. The results of the two types of regression analysis are discussed in relation to ecogeography and cold tolerance phenotype of the populations.

EST-derived SSR markers used as anchor loci for the construction of a consensus linkage map in ryegrass ( Lolium spp.)

BackgroundGenetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm.ResultsA set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2.ConclusionsThe consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.

Expression of insecticidal activity in Rhizobium containing the δ-endotoxin gene cloned from Bacillus thuringiensis subsp. tenebrionis

Bacillus thuringiensis subsp. tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the δ-endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.

Allelic Variation in the Perennial Ryegrass FLOWERING LOCUS T Gene Is Associated with Changes in Flowering Time across a Range of Populations

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.

Genotyping by RAD sequencing enables mapping of fatty acid composition traits in perennial ryegrass (Lolium perenne (L.))

Perennial ryegrass (Lolium perenne L.) is the most important forage crop in temperate livestock agriculture. Its nutritional quality has significant impact on the quality of meat and milk for human consumption. Evidence suggests that higher energy content in forage can assist in reducing greenhouse gas emissions from ruminants. Increasing the fatty acid content (especially α-linolenic acid, an omega-3 fatty acid) may thus contribute to better forage, but little is known about the genetic basis of variation for this trait. To this end, quantitative trait loci (QTLs) were identified associated with major fatty acid content in perennial ryegrass using a population derived from a cross between the heterozygous and outbreeding high-sugar grass variety AberMagic and an older variety, Aurora. A genetic map with 434 restriction-associated DNA (RAD) and SSR markers was generated. Significant QTLs for the content of palmitic (C16:0) on linkage groups (LGs) 2 and 7; stearic (C18:0) on LGs 3, 4 and 7; linoleic (C18:2n-6) on LGs 2 and 5; and α-linolenic acids (C18:3n-3) on LG 1 were identified. Two candidate genes (a lipase and a beta-ketoacyl CoA synthase), both associated with C16:0, and separately with C18:2n-6 and C18:0 contents, were identified. The physical positions of these genes in rice and their genetic positions in perennial ryegrass were consistent with established syntenic relationships between these two species. Validation of these associations is required, but the utility of RAD markers for rapid generation of genetic maps and QTL analysis has been demonstrated for fatty acid composition in a global forage crop.

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses.