Thomas Lübberstedt

QTL mapping of vernalization response in perennial ryegrass (Lolium perenne L.) reveals co-location with an orthologue of wheat VRN1.

The objective of this study was to map quantitative trait loci (QTL) for the vernalization response in perennial ryegrass (Lolium perenne L.). The mapping population consisted of 184 F2 genotypes produced from a cross between one genotype of a synthetic perennial ryegrass variety “Veyo” and one genotype from the perennial ryegrass ecotype “Falster”. Veyo and Falster were chosen among four different populations because of their contrasting vernalization requirements. In total, five QTL for the vernalization response, measured as days to heading, were identified and mapped to linkage groups (LG) LG2, LG4, LG6 and LG7. Individually, these QTL explained between 5.4 and 28.0% of the total phenotypic variation. The overall contribution of these five QTL was 80% of the total phenotypic variation. A putative orthologue of Triticum monococcum VRN1 was amplified from genomic DNA from perennial ryegrass. PCR fragments covering the proximal part of the promoter and the 5′ end of the orthologue were subsequently PCR-amplified from both parents of the mapping population and shown to possess 95% DNA sequence identity to VRN1. Several polymorphisms were identified between Veyo and Falster in this fragment of the putative VRN1 orthologue. A CAPS marker, vrn-1, was developed and found to co-segregate with a major QTL on LG4 for the vernalization response. This indicates that the CAPS marker vrn-1 could be located in an orthologous gene of the wheat VRN1.

Quantitative Trait Loci Mapping of Resistance to Sugarcane Mosaic Virus in Maize

ABSTRACT Sugarcane mosaic virus (SCMV) is an important virus disease of maize (Zea mays) in Europe. In this study, we mapped and characterized quantitative trait loci (QTL) affecting resistance to SCMV in a maize population consisting of 219 F(3) or immortalized F(2) families from the cross of two European maize inbreds, D32 (resistant) x D145 (susceptible). Resistance was evaluated in replicated field trials across two environments under artificial inoculation. The method of composite interval mapping was employed for QTL detection with a linkage map based on 87 restriction fragment length polymorphism and 7 mapped microsatellite markers. Genotypic and genotype x environment interaction variances for SCMV resistance were highly significant in the population. Heritabilities ranged from 0.77 to 0.94 for disease scores recorded on seven consecutive dates. Five QTL for SCMV resistance were identified on chromosomes 1, 3, 5, 6, and 10 in the joint analyses. Two major QTL on chromosomes 3 and 6 were detected consistently in both environments. Significant epistatic effects were found among some of these QTL. A simultaneous fit with all QTL in the joint analyses explained between 70 and 77% of the phenotypic variance observed at various stages of plant development. Resistance to SCMV was correlated with plant height and days to anthesis.

Nucleotide diversity and linkage disequilibrium in 11 expressed resistance candidate genes in Lolium perenne

BackgroundAssociation analysis is an alternative way for QTL mapping in ryegrass. So far, knowledge on nucleotide diversity and linkage disequilibrium in ryegrass is lacking, which is essential for the efficiency of association analyses.Results11 expressed disease resistance candidate (R) genes including 6 nucleotide binding site and leucine rich repeat (NBS-LRR) like genes and 5 non-NBS-LRR genes were analyzed for nucleotide diversity. For each of the genes about 1 kb genomic fragments were isolated from 20 heterozygous genotypes in ryegrass. The number of haplotypes per gene ranged from 9 to 27. On average, one single nucleotide polymorphism (SNP) was present per 33 bp between two randomly sampled sequences for the 11 genes. NBS-LRR like gene fragments showed a high degree of nucleotide diversity, with one SNP every 22 bp between two randomly sampled sequences. NBS-LRR like gene fragments showed very high non-synonymous mutation rates, leading to altered amino acid sequences. Particularly LRR regions showed very high diversity with on average one SNP every 10 bp between two sequences. In contrast, non-NBS LRR resistance candidate genes showed a lower degree of nucleotide diversity, with one SNP every 112 bp. 78% of haplotypes occurred at low frequency (<5%) within the collection of 20 genotypes. Low intragenic LD was detected for most R genes, and rapid LD decay within 500 bp was detected.ConclusionSubstantial LD decay was found within a distance of 500 bp for most resistance candidate genes in this study. Hence, LD based association analysis is feasible and promising for QTL fine mapping of resistance traits in ryegrass.

High levels of linkage disequilibrium and associations with forage quality at a phenylalanine ammonia-lyase locus in European maize (Zea mays L.) inbreds.

Forage quality of maize is influenced by both the content and structure of lignin in the cell wall. Phenylalanine Ammonia-Lyase (PAL) catalyzes the first step in lignin biosynthesis in plants; the deamination of l-phenylalanine to cinnamic acid. Successive enzymatic steps lead to the formation of three monolignols, constituting the complex structure of lignin. We have cloned and sequenced a PAL genomic sequence from 32 maize inbred lines currently employed in forage maize breeding programs in Europe. Low nucleotide diversity and excessive linkage disequilibrium (LD) was identified at this PAL locus, possibly reflecting selective constrains resulting from PAL being the first enzyme in the monolignol, and other, pathways. While the association analysis was affected by extended LD and population structure, several individual polymorphisms were associated with neutral detergent fiber (not considering population structure) and a single polymorphism was associated with in vitro digestibility of organic matter (considering population structure).

Frequency, type, and distribution of EST-SSRs from three genotypes of Lolium perenne, and their conservation across orthologous sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa

BackgroundSimple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies in several plant species. They are used for cultivar identification, variety protection, as anchor markers in genetic mapping, and in marker-assisted breeding. Currently, a limited number of SSR markers are publicly available for perennial ryegrass (Lolium perenne). We report on the exploitation of a comprehensive EST collection in L. perenne for SSR identification. The objectives of this study were 1) to analyse the frequency, type, and distribution of SSR motifs in ESTs derived from three genotypes of L. perenne, 2) to perform a comparative analysis of SSR motif polymorphisms between allelic sequences, 3) to conduct a comparative analysis of SSR motif polymorphisms between orthologous sequences of L. perenne, Festuca arundinacea, Brachypodium distachyon, and O. sativa, 4) to identify functionally associated EST-SSR markers for application in comparative genomics and breeding.ResultsFrom 25,744 ESTs, representing 8.53 megabases of nucleotide information from three genotypes of L. perenne, 1,458 ESTs (5.7%) contained one or more SSRs. Of these SSRs, 955 (3.7%) were non-redundant. Tri-nucleotide repeats were the most abundant type of repeats followed by di- and tetra-nucleotide repeats. The EST-SSRs from the three genotypes were analysed for allelic- and/or genotypic SSR motif polymorphisms. Most of the SSR motifs (97.7%) showed no polymorphisms, whereas 22 EST-SSRs showed allelic- and/or genotypic polymorphisms. All polymorphisms identified were changes in the number of repeat units. Comparative analysis of the L. perenne EST-SSRs with sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa identified 19 clusters of orthologous sequences between these four species. Analysis of the clusters showed that the SSR motif generally is conserved in the closely related species F. arundinacea, but often differs in length of the SSR motif. In contrast, SSR motifs are often lost in the more distant related species B. distachyon and O. sativa.ConclusionThe results indicate that the L. perenne EST-SSR markers are a valuable resource for genetic mapping, as well as evaluation of co-location between QTLs and functionally associated markers.

The Perennial Ryegrass GenomeZipper: Targeted Use of Genome Resources for Comparative Grass Genomics

Summary: The GenomeZipper presented here is a model of the perennial ryegrass genome on the basis of conserved synteny to barley, Brachypodium, rice, and sorghum. Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources have yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne) genome on the basis of conserved synteny to barley (Hordeum vulgare) and the model grass genome Brachypodium (Brachypodium distachyon) as well as rice (Oryza sativa) and sorghum (Sorghum bicolor). A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macrocollinearity and was then utilized to anchor a collection of perennial ryegrass genes in silico to their predicted genome positions. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci, which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species.

Promoters from Genes for Plastid Proteins Possess Regions with Different Sensitivities toward Red and Blue Light

The light-regulated expression of eight nuclear-encoded genes for plastid proteins from spinach (Spinacia oleracea) (RBCS-1 and CAB-1; ATPC and ATPD, encoding the subunits [gamma] and [delta] of the ATP synthase; PC and FNR; PSAD and PSAF, encoding the subunits II and III of photosystem I reaction center) was analyzed with promoter/[beta]-glucuronidase (GUS) gene fusions in transgenic tobacco (Nicotiana tabacum and Nicotiana plumbaginifolia) seedlings and mature plants under standardized light and growth conditions. Unique response patterns were found for each of these promoters. GUS activities differed more than 30-fold. Strong promoters were found for the PC and PSAD genes. On the other hand, the ATPC promoter was relatively weak. Expression of the CAB/GUS gene fusion in etiolated material was at the detection limit; all other chimeric genes were expressed in the dark as well. Light stimulation of GUS activities ranged from 3- (FNR promoter) to more than 100-fold (CAB-1 promoter). The FNR promoter responded only to red light (RL) and not significantly to blue light (BL), whereas the PC promoter contained regions with different sensitivities toward RL and BL. Furthermore, different RNA accumulation kinetics were observed for the PSAF, CAB, FNR, and PC promoter/GUS gene fusions during de-etiolation, which, at least in the case of the PSAF gene, differed from the regulation of the corresponding endogenous genes in spinach and tobacco. The results suggest either that not all cis elements determining light-regulated and quantitative expression are present on the spinach promoter fragments used or that the spinach cis-regulatory elements respond differently to the host (tobacco) regulatory pathway(s). Furthermore, as in tobacco, but not in spinach, the trans-gene hardly responds to single light pulses that operate through phytochrome. Taken together, the results suggest that the genes have been independently translocated from the organelle to the nucleus during phylogeny. Furthermore, each gene seems to have acquired a unique set of regulatory elements.

Identification of miRNAs and their target genes in developing maize ears by combined small RNA and degradome sequencing

BackgroundIn plants, microRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in many aspects of plant biology, including metabolism, hormone response, epigenetic control of transposable elements, and stress response. Extensive studies of miRNAs have been performed in model plants such as rice and Arabidopsis thaliana. In maize, most miRNAs and their target genes were analyzed and identified by clearly different treatments, such as response to low nitrate, salt and drought stress. However, little is known about miRNAs involved in maize ear development. The objective of this study is to identify conserved and novel miRNAs and their target genes by combined small RNA and degradome sequencing at four inflorescence developmental stages.ResultsWe used deep-sequencing, miRNA microarray assays and computational methods to identify, profile, and describe conserved and non-conserved miRNAs at four ear developmental stages, which resulted in identification of 22 conserved and 21-maize-specific miRNA families together with their corresponding miRNA*. Comparison of miRNA expression in these developmental stages revealed 18 differentially expressed miRNA families. Finally, a total of 141 genes (251 transcripts) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs. Moreover, the differentially expressed miRNAs-mediated pathways that regulate the development of ears were discussed.ConclusionsThis study confirmed 22 conserved miRNA families and discovered 26 novel miRNAs in maize. Moreover, we identified 141 target genes of known and new miRNAs and ta-siRNAs. Of these, 72 genes (117 transcripts) targeted by 62 differentially expressed miRNAs may attribute to the development of maize ears. Identification and characterization of these important classes of regulatory genes in maize may improve our understanding of molecular mechanisms controlling ear development.

Genetic characterisation of seed yield and fertility traits in perennial ryegrass (Lolium perenne L.)

Seed yield is a trait of major interest for the key grassland species Lolium perenne L. An F2 mapping population of perennial ryegrass (VrnA), recently characterised for vernalisation response, was assessed in a glasshouse for traits related to seed yield based on a lattice design with four replications over 2 years. The traits heading date, plant height, length of panicles, number of panicles per plant, seed yield per panicle, flag leaf length, flag leaf width and seed yield per plant revealed repeatabilities ranging from 41 to 76% and a considerable amount of genetic variation in the VrnA population. Path analysis partitioned the direct and indirect effects of seed yield components on seed yield per plant. Seed yield per panicle showed the highest effect on total seed yield. The adjusted mean values of each trait and a genetic linkage map consisting of 97 anonymous and 85 gene associated DNA markers were used for quantitative trait loci (QTL) analysis. Of particular interest were two QTL on linkage group (LG) 1 and LG 2, explaining 41 and 18%, respectively, of the observed phenotypic variation for the trait seed yield per panicle. Both QTL co-located with two major QTL for total seed yield per plant possibly representing the S and Z loci of the gametophytic self incompatibility (SI) system of perennial ryegrass. The diversity of SI alleles in mapping parents and the degree of heterozygosity at SI loci in the full sib progeny determines the interference of self incompatibility with seed production.

Development and mapping of DArT markers within the Festuca - Lolium complex.

BackgroundGrasses are among the most important and widely cultivated plants on Earth. They provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Among cultivated grasses, species within the Festuca-Lolium complex predominate, especially in temperate regions. To facilitate high-throughput genome profiling and genetic mapping within the complex, we have developed a Diversity Arrays Technology (DArT) array for five grass species: F. pratensis, F. arundinacea, F. glaucescens, L. perenne and L. multiflorum.ResultsThe DArTFest array contains 7680 probes derived from methyl-filtered genomic representations. In a first marker discovery experiment performed on 40 genotypes from each species (with the exception of F. glaucescens for which only 7 genotypes were used), we identified 3884 polymorphic markers. The number of DArT markers identified in every single genotype varied from 821 to 1852. To test the usefulness of DArTFest array for physical mapping, DArT markers were assigned to each of the seven chromosomes of F. pratensis using single chromosome substitution lines while recombinants of F. pratensis chromosome 3 were used to allocate the markers to seven chromosome bins.ConclusionThe resources developed in this project will facilitate the development of genetic maps in Festuca and Lolium, the analysis on genetic diversity, and the monitoring of the genomic constitution of the Festuca × Lolium hybrids. They will also enable marker-assisted selection for multiple traits or for specific genome regions.