James H. Baird

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.

Flow sorting and sequencing meadow fescue chromosome 4F

Sorting and sequencing of individual chromosomes of forage grasses facilitates the analysis of genome structure and evolution and provides a resource for the community of plant biologists and breeders. The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before.

DArTFest DNA Array—Applications and Perspectives for Grass Genetics, Genomics and Breeding

DArTFest is a DNA microarray developed for the Festuca-Lolium complex, consisting of 7,680 probes. As it offers high-throughput and low-cost screening of thousands of genomic loci, it has been successfully used in a number of projects. These include the analysis of interspecific, intraspecific and intravarietal genetic variation, genetic mapping, assigning markers to chromosomal segments, comparative analysis of the Festuca and Lolium genomes against model plant species rice and Brachypodium, an analysis of genomic constitution of Festulolium cultivars and QTL mapping of agronomically important traits such as resistance against crown rust and frost tolerance. In order to facilitate the use of DArT markers linked to traits of interests, a large number of them has been already sequenced and conversion to PCR-based markers is in progress.

DArTFest – A Platform for High-Throughput Genome Profiling Within the Festuca – Lolium Complex

With the aim to facilitate high-throughput genome profiling and genetic and physical mapping within the Festuca-Lolium complex, we have developed a Diversity Arrays Technology (DArT) array for five important species: Festuca pratensis, Festuca arundinacea, Festuca glaucescens, Lolium perenne and Lolium multiflorum. The DArTFest array contains 7,680 probes derived from methyl-filtered genomic representations. Of 3,884 polymorphic DArT markers identified in the first marker discovery experiment, over 1,000 markers detected a positive allele in each species.