Pavla Suchánková

Unlocking the Barley Genome by Chromosomal and Comparative Genomics

Survey sequence and array hybridization data from flow-sorted barley chromosomes were integrated using a comparative genomics model to define an ordered gene map of the barley genome that contains approximately two-thirds of its estimated 32000 genes. The resulting high-resolution framework facilitated a genome-wide structural analysis of the barley genome and a detailed comparative analysis with wheat. We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley (Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum (Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.

Gene Content and Virtual Gene Order of Barley Chromosome 1H

Chromosome 1H (approximately 622 Mb) of barley (Hordeum vulgare) was isolated by flow sorting and shotgun sequenced by GSFLX pyrosequencing to 1.3-fold coverage. Fluorescence in situ hybridization and stringent sequence comparison against genetically mapped barley genes revealed 95% purity of the sorted chromosome 1H fraction. Sequence comparison against the reference genomes of rice (Oryza sativa) and sorghum (Sorghum bicolor) and against wheat (Triticum aestivum) and barley expressed sequence tag datasets led to the estimation of 4,600 to 5,800 genes on chromosome 1H, and 38,000 to 48,000 genes in the whole barley genome. Conserved gene content between chromosome 1H and known syntenic regions of rice chromosomes 5 and 10, and of sorghum chromosomes 1 and 9 was detected on a per gene resolution. Informed by the syntenic relationships between the two reference genomes, genic barley sequence reads were integrated and ordered to deduce a virtual gene map of barley chromosome 1H. We demonstrate that synteny-based analysis of low-pass shotgun sequenced flow-sorted Triticeae chromosomes can deliver linearly ordered high-resolution gene inventories of individual chromosomes, which complement extensive Triticeae expressed sequence tag datasets. Thus, integration of genomic, transcriptomic, and synteny-derived information represents a major step toward developing reference sequences of chromosomes and complete genomes of the most important plant tribe for mankind.

Development of chromosome-specific BAC resources for genomics of bread wheat.

The large bread wheat genome (1C ∼ 17 Gbp) contains a preponderance of repetitive DNA and the species is polyploid. These characteristics together serve to hamper the molecular analysis of the wheat genome. Its complexity can, however, be reduced by using flow cytometry to isolate individual chromosomes, and these can be exploited to construct chromosome-specific BAC libraries. Such libraries simplify the task of physical map construction, positional cloning and the targeted development of genetic markers. Rapid improvements in the efficiency and cost of DNA sequencing provide an opportunity to contemplate sequencing the wheat genome by preparing sequence-ready physical maps for each chromosome or chromosome arm in turn. The quality of the chromosome-specific libraries depends on their chromosome coverage and the mean insert size. First-generation libraries suffered from a relatively low mean insert size, but improvements to the protocol have generated a second wave of libraries with a significantly increased mean insert size and better chromosome coverage. Each chromosome (arm)-specific library is composed of a manageable number of clones, and so represents a practical tool in the area of wheat genomics.

An Improved Consensus Linkage Map of Barley Based on Flow-Sorted Chromosomes and Single Nucleotide Polymorphism Markers

Recent advances in high‐throughput genotyping have made it easier to combine information from different mapping populations into consensus genetic maps, which provide increased marker density and genome coverage compared to individual maps. Previously, a single nucleotide polymorphism (SNP)‐based genotyping platform was developed and used to genotype 373 individuals in four barley (Hordeum vulgare L.) mapping populations. This led to a 2943 SNP consensus genetic map with 975 unique positions. In this work, we add data from six additional populations and more individuals from one of the original populations to develop an improved consensus map from 1133 individuals. A stringent and systematic analysis of each of the 10 populations was performed to achieve uniformity. This involved reexamination of the four populations included in the previous map. As a consequence, we present a robust consensus genetic map that contains 2994 SNP loci mapped to 1163 unique positions. The map spans 1137.3 cM with an average density of one marker bin per 0.99 cM. A novel application of the genotyping platform for gene detection allowed the assignment of 2930 genes to flow‐sorted chromosomes or arms, confirmed the position of 2545 SNP‐mapped loci, added chromosome or arm allocations to an additional 370 SNP loci, and delineated pericentromeric regions for chromosomes 2H to 7H. Marker order has been improved and map resolution has been increased by almost 20%. These increased precision outcomes enable more optimized SNP selection for marker‐assisted breeding and support association genetic analysis and map‐based cloning. It will also improve the anchoring of DNA sequence scaffolds and the barley physical map to the genetic map.

Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley

BackgroundFlow cytometry facilitates sorting of single chromosomes and chromosome arms which can be used for targeted genome analysis. However, the recovery of microgram amounts of DNA needed for some assays requires sorting of millions of chromosomes which is laborious and time consuming. Yet, many genomic applications such as development of genetic maps or physical mapping do not require large DNA fragments. In such cases time-consuming de novo sorting can be minimized by utilizing whole-genome amplification.ResultsHere we report a protocol optimized in barley including amplification of DNA from only ten thousand chromosomes, which can be isolated in less than one hour. Flow-sorted chromosomes were treated with proteinase K and amplified using Phi29 multiple displacement amplification (MDA). Overnight amplification in a 20-microlitre reaction produced 3.7 – 5.7 micrograms DNA with a majority of products between 5 and 30 kb. To determine the purity of sorted fractions and potential amplification bias we used quantitative PCR for specific genes on each chromosome. To extend the analysis to a whole genome level we performed an oligonucleotide pool assay (OPA) for interrogation of 1524 loci, of which 1153 loci had known genetic map positions. Analysis of unamplified genomic DNA of barley cv. Akcent using this OPA resulted in 1426 markers with present calls. Comparison with three replicates of amplified genomic DNA revealed >99% concordance. DNA samples from amplified chromosome 1H and a fraction containing chromosomes 2H – 7H were examined. In addition to loci with known map positions, 349 loci with unknown map positions were included. Based on this analysis 40 new loci were mapped to 1H.ConclusionThe results indicate a significant potential of using this approach for physical mapping. Moreover, the study showed that multiple displacement amplification of flow-sorted chromosomes is highly efficient and representative which considerably expands the potential of chromosome flow sorting in plant genomics.

A first survey of the rye (Secale cereale) genome composition through BAC end sequencing of the short arm of chromosome 1R

BackgroundRye (Secale cereale L.) belongs to tribe Triticeae and is an important temperate cereal. It is one of the parents of man-made species Triticale and has been used as a source of agronomically important genes for wheat improvement. The short arm of rye chromosome 1 (1RS), in particular is rich in useful genes, and as it may increase yield, protein content and resistance to biotic and abiotic stress, it has been introgressed into wheat as the 1BL.1RS translocation. A better knowledge of the rye genome could facilitate rye improvement and increase the efficiency of utilizing rye genes in wheat breeding.ResultsHere, we report on BAC end sequencing of 1,536 clones from two 1RS-specific BAC libraries. We obtained 2,778 (90.4%) useful sequences with a cumulative length of 2,032,538 bp and an average read length of 732 bp. These sequences represent 0.5% of 1RS arm. The GC content of the sequenced fraction of 1RS is 45.9%, and at least 84% of the 1RS arm consists of repetitive DNA. We identified transposable element junctions in BESs and developed insertion site based polymorphism markers (ISBP). Out of the 64 primer pairs tested, 17 (26.6%) were specific for 1RS. We also identified BESs carrying microsatellites suitable for development of 1RS-specific SSR markers.ConclusionThis work demonstrates the utility of chromosome arm-specific BAC libraries for targeted analysis of large Triticeae genomes and provides new sequence data from the rye genome and molecular markers for the short arm of rye chromosome 1.

A novel resource for genomics of Triticeae: BAC library specific for the short arm of rye (Secale cereale L.) chromosome 1R (1RS)

BackgroundGenomics of rye (Secale cereale L.) is impeded by its large nuclear genome (1C~7,900 Mbp) with prevalence of DNA repeats (> 90%). An attractive possibility is to dissect the genome to small parts after flow sorting particular chromosomes and chromosome arms. To test this approach, we have chosen 1RS chromosome arm, which represents only 5.6% of the total rye genome. The 1RS arm is an attractive target as it carries many important genes and because it became part of the wheat gene pool as the 1BL.1RS translocation.ResultsWe demonstrate that it is possible to sort 1RS arm from wheat-rye ditelosomic addition line. Using this approach, we isolated over 10 million of 1RS arms using flow sorting and used their DNA to construct a 1RS-specific BAC library, which comprises 103,680 clones with average insert size of 73 kb. The library comprises two sublibraries constructed using Hin dIII and Eco RI and provides a deep coverage of about 14-fold of the 1RS arm (442 Mbp). We present preliminary results obtained during positional cloning of the stem rust resistance gene SrR, which confirm a potential of the library to speed up isolation of agronomically important genes by map-based cloning.ConclusionWe present a strategy that enables sorting short arms of several chromosomes of rye. Using flow-sorted chromosomes, we have constructed a deep coverage BAC library specific for the short arm of chromosome 1R (1RS). This is the first subgenomic BAC library available for rye and we demonstrate its potential for positional gene cloning. We expect that the library will facilitate development of a physical contig map of 1RS and comparative genomics of the homoeologous chromosome group 1 of wheat, barley and rye.

BAC Libraries from Wheat Chromosome 7D: Efficient Tool for Positional Cloning of Aphid Resistance Genes

Positional cloning in bread wheat is a tedious task due to its huge genome size and hexaploid character. BAC libraries represent an essential tool for positional cloning. However, wheat BAC libraries comprise more than million clones, which makes their screening very laborious. Here, we present a targeted approach based on chromosome-specific BAC libraries. Such libraries were constructed from flow-sorted arms of wheat chromosome 7D. A library from the short arm (7DS) consisting of 49,152 clones with 113 kb insert size represented 12.1 arm equivalents whereas a library from the long arm (7DL) comprised 50,304 clones of 116 kb providing 14.9x arm coverage. The 7DS library was PCR screened with markers linked to Russian wheat aphid resistance gene DnCI2401, the 7DL library was screened by hybridization with a probe linked to greenbug resistance gene Gb3. The small number of clones combined with high coverage made the screening highly efficient and cost effective.

Molecular Analysis and Genomic Organization of Major DNA Satellites in Banana (Musa spp.)

Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa.

Chromosome Genomics in the Triticeae

The Triticeae species are unique among the important agricultural crops in possessing massive genomes with a prevalence of dispersed DNA repeats. The highest level of complexity is observed in tetraploid and hexaploid wheat whose nuclear genomes comprise two and three homoeologous genomes, respectively. Polyploidy and the presence of repeats make gene cloning and genome sequencing in the Triticeae extremely difficult. Chromosome genomics simplifies these tasks by targeting single chromosomes and chromosome arms, which represent only a few percent of the nuclear genomes. The advantages of this strategy over a whole-genome approach include the avoidance of problems due to the presence of homoeologs in wheat, reduction of work to manageable portions, cost efficiency, and an opportunity to structure collaborative projects where individual laboratories work on particular chromosomes. In this chapter, we describe how chromosomes and chromosome arms can be isolated by flow cytometric sorting and we review development of flow cytogenetics in the Triticeae. We then discuss various applications of flow-sorted chromosomes and assess the potential of chromosome genomics in the Triticeae.