The cereals are of enormous importance to mankind. Many of the major cereal species (wheat, barley, oat, rye, and maize) have large genomes. They have a high percentage of repetitive DNA and, for wheat, a polyploid nature, all of which hamper genomic analyses especially when combined. For example, wheat genome (16.9 Gbp) is more than 5 times bigger human genome.
During the past few years, we have been developing strategies to isolate chromosomes and chromosome arms from the major Triticeae using flow cytometric sorting thereby dissecting the giant genomes into small, manageable parts. Wheat chromosome arms represent only 1 – 3% of the entire wheat genome. The ability to obtain high quality DNA from particular chromosomes and chromosome arms has opened new avenues for Triticeae genomics. Our developed techniques became attractive for cereals research community. The International Wheat Genome Sequencing Consortium (IWGSC; http://www.wheatgenome.org) establish chromosome-based approach as a strategic for sequencing the wheat and current European Seventh Framework Project FP7-212019 ‘Genomics for Triticeae Improvement’ is based on building chromosomal physical maps. Chromosome-based strategy offers a broad portfolio of applications include high-throughput physical mapping of DNA sequences by hybridization to DNA arrays, isolation of markers, shotgun sequencing, development of chromosome-specific BAC resources, and enables development of physical, cytogenetic and integrated maps and positional gene cloning.
Flow cytometryFlow cytometry is a precise and robust technique, which is broadly applied in plant genome analysis as genome size determination, characterization of plant karyotype, analysis and sorting of nuclei or chromosomes. Our laboratory developed unique protocol for chromosome analysing and sorting among 17 plant species and crops. Successful sorting of individual chromosome types suppose multidisciplinary approaches include cell synchronization and accumulation of intact mitotic chromosomes in plant material, preparation of chromosome suspensions, flow karyotyping and discrimination of single chromosome types and their sorting in sufficient purity. Suspensions of wheat mitotic chromosomes are DAPI stained and analysed based on its relative fluorescence intensities using FACS Vantage flow cytometers (Becton Dickinson, Franklin Lakes, NJ, USA).
Flow cytogenetic toolbox for TriticeaeDue to slight differencies in size of cereal chromosomes only rare number of chromosome types could be fully discriminate and sorted with high purity. Fortunately, the existence of cytogenetic stocks, which include chromosome deletion lines, alien chromosome addition lines and substitution lines led to development of platform for chromosome sorting in main cereals crops.
Sequencing of flow-sorted chromosomesFlow sorted chromosomes are very attractive target for application of next generation sequencing (NGS) technology. Flow sorted chromosomes can be used directly (for details see Protocols) or amplified using phi29 polymerase prior sequencing (for details see Protocols). Shot-gun chromosome sequences are convenient for targeted isolation of DNA markers, characterization of gene space. Synteny-based analysis of low-pass shotgun sequenced flow-sorted Triticeae chromosomes can deliver linearly ordered high-resolution gene inventories of individual chromosomes.
Developement of chromosome-specific BAC resourcesOther attractive use of sorted chromosomes is the production of chromosome- and chromosome arm-specific DNA libraries. Such libraries provide a permanent and easily accessible molecular resource specific for a particular part of the genome, is expected to have a major impact on wheat genomics. For construction of chromosome-specific BAC resources details see Protocols. Chromosome-specific BAC resources is suitable substrate for construction physical contig maps, map based cloning, derivation of markers from region of interest, BAC-end sequencing, BAC-FISH etc.
Construction of chromosomal physical mapsPhysical maps of complex cereals genomes are essential for high quality sequence assembly and can speed up positional cloning of traits of interest. The availability of chromosome specific BAC resources opens avenues for the development of a physical maps in a step-wise manner, working with a particular chromosome arm at a time. Recently, our laboratory is building physical contig map of wheat 3DS, 4AS, 7DS.
Map-based cloning of the genesPositional cloning of agronomically important genes is important for accelerating breeding programs and development new varieties of cereals. There are two projects aimed to isolation agronomic improtant genes. Powdery mildew resistance gene QPm.tut-4A (for details see Project 4A) and positional cloning of the Russian wheat aphid resistance gene Dnx (for details see Project 7D) in our laboratory.
Develoment of DNA markersContinuous lack of the DNA markers from particular genome regions can be solved by derivation of markers from flow-sorted chromosomes/chromosome arms. DNA amplified from flow-sorted chromosomes was used for targeted development of molecular markers by Diversity Array Technology (DArT). To date, 1,500 wheat D-genome specific, 353 wheat 3B specific and 59 wheat 1BS specific DART markers were developed. In addition, sequencing of chromosome specific BAC-ends enable development of ISBP (Insertion site based polymorphisms) markers. 711 ISBP markers were developed and placed to wheat 3B consensus map. From short arm of rye chromosome 1 (1RS) were derived 17 ISBP markers. Shot-gun sequences of chromosomes are valuable source of SNPs, microsatellites, EST derived markers.
Chromosomal cytogeneticsCytogenetic approaches help to resolve the order of contigs and clones, confirm the precise physical position of centromere, the distance of markers near the termini of linkage maps from the actual chromosome termini, and estimate the numbers and sizes of gaps. Mitotic chromosomes flow-sorted onto microscopic slides are ideal targets for FISH and PRINS.
