Cytologically integrated physical restriction fragment length polymorphism maps for the barley genome based on translocation breakpoints.
ABSTRACT: We have developed a new technique for the physical mapping of barley chromosomes using microdissected translocation chromosomes for PCR with sequence-tagged site primers derived from >300 genetically mapped RFLP probes. The positions of 240 translocation breakpoints were integrated as physical landmarks into linkage maps of the seven barley chromosomes. This strategy proved to be highly efficient in relating physical to genetic distances. A very heterogeneous distribution of recombination rates was found along individual chromosomes. Recombination is mainly confined to a few relatively small areas spaced by large segments in which recombination is severely suppressed. The regions of highest recombination frequency (
Project description:Comparative mapping of cereals has shown that chromosomes of barley, wheat, and maize can be described in terms of rice "linkage segments." However, little is known about marker order in the junctions between linkage blocks or whether this will impair comparative analysis of major genes that lie in such regions. We used genetic and physical mapping to investigate the relationship between the distal part of rice chromosome 7L, which contains the Hd2 heading date gene, and the region of barley chromosome 2HS containing the Ppd-H1 photoperiod response gene, which lies near the junction between rice 7 and rice 4 linkage segments. RFLP markers were mapped in maize to identify regions that might contain Hd2 or Ppd-H1 orthologs. Rice provided useful markers for the Ppd-H1 region but comparative mapping was complicated by loss of colinearity and sequence duplications that predated the divergence of rice, maize, and barley. The sequences of cDNA markers were used to search for homologs in the Arabidopsis genome. Homologous sequences were found for 13 out of 16 markers but they were dispersed in Arabidopsis and did not identify any candidate equivalent region. The implications of the results for comparative trait mapping in junction regions are discussed.
Project description:Physical mapping and genome sequencing are underway for the ?17 Gb wheat genome. Physical mapping methods independent of meiotic recombination, such as radiation hybrid (RH) mapping, will aid precise anchoring of BAC contigs in the large regions of suppressed recombination in Triticeae genomes. Reports of endosperm development following pollination with irradiated pollen at dosages that cause embryo abortion prompted us to investigate endosperm as a potential source of RH mapping germplasm. Here, we report a novel approach to construct RH based physical maps of all seven D-genome chromosomes of the hexaploid wheat 'Chinese Spring', simultaneously. An 81-member subset of endosperm samples derived from 20-Gy irradiated pollen was genotyped for deletions, and 737 markers were mapped on seven D-genome chromosomes. Analysis of well-defined regions of six chromosomes suggested a map resolution of ?830 kb could be achieved; this estimate was validated with assays of markers from a sequenced contig. We estimate that the panel contains ?6,000 deletion bins for D-genome chromosomes and will require ?18,000 markers for high resolution mapping. Map-based deletion estimates revealed a majority of 1-20 Mb interstitial deletions suggesting mutagenic repair of double-strand breaks in pollen provides a useful resource for RH mapping and map based cloning studies.
Project description:Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.
Project description:<h4>Background</h4>Earlier comparative maps between the genomes of rice (Oryza sativa L.), barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were linkage maps based on cDNA-RFLP markers. The low number of polymorphic RFLP markers has limited the development of dense genetic maps in wheat and the number of available anchor points in comparative maps. Higher density comparative maps using PCR-based anchor markers are necessary to better estimate the conservation of colinearity among cereal genomes. The purposes of this study were to characterize the proportion of transcribed DNA sequences containing simple sequence repeats (SSR or microsatellites) by length and motif for wheat, barley and rice and to determine in-silico rice genome locations for primer sets developed for wheat and barley Expressed Sequence Tags.<h4>Results</h4>The proportions of SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) and motifs varied with the length of the SSRs within and among the three species, with trinucleotide SSRs being the most frequent. Distributions of genomic microsatellites (gSSRs), EST-derived microsatellites (EST-SSRs), and transcribed regions in the contiguous sequence of rice chromosome 1 were highly correlated. More than 13,000 primer pairs were developed for use by the cereal research community as potential markers in wheat, barley and rice.<h4>Conclusion</h4>Trinucleotide SSRs were the most common type in each of the species; however, the relative proportions of SSR types and motifs differed among rice, wheat, and barley. Genomic microsatellites were found to be primarily located in gene-rich regions of the rice genome. Microsatellite markers derived from the use of non-redundant EST-SSRs are an economic and efficient alternative to RFLP for comparative mapping in cereals.
Project description:Meiotic recombination plays a crucial role in the generation of new varieties. The effectiveness of recombination is limited by the distribution of crossover events, which in wheat and many other crops is skewed toward the distal regions of the chromosomes. Whole-genome sequencing of wheat has revealed that there are numerous important genes in the pericentromeric regions, which are inaccessible to manipulation due to the lack of crossover events. Studies in barley have shown that the distribution of recombination events can be shifted toward the centromeres by increasing temperature during meiosis. Here we present an analysis of the effects of temperature on the distribution and frequency of recombination events in wheat. Our data show that although increased temperature during meiosis does cause an inward shift in recombination distribution for some chromosomes, its overall utility is limited, with many genes remaining highly linked.
Project description:We carried out a genome-wide analysis of polymorphism (4,596 SNP loci across 190 elite cultivated accessions) chosen to represent the available genetic variation in current elite North West European and North American barley germplasm. Population sub-structure, patterns of diversity and linkage disequilibrium varied considerably across the seven barley chromosomes. Gene-rich and rarely recombining haplotype blocks that may represent up to 60% of the physical length of barley chromosomes extended across the 'genetic centromeres'. By positioning 2,132 bi-parentally mapped SNP markers with minimum allele frequencies higher than 0.10 by association mapping, 87.3% were located to within 5 cM of their original genetic map position. We show that at this current marker density genetically diverse populations of relatively small size are sufficient to fine map simple traits, providing they are not strongly stratified within the sample, fall outside the genetic centromeres and population sub-structure is effectively controlled in the analysis. Our results have important implications for association mapping, positional cloning, physical mapping and practical plant breeding in barley and other major world cereals including wheat and rye that exhibit comparable genome and genetic features.
Project description:Understanding the genetic architecture of grain size is a prerequisite to manipulating grain development and improving the potential crop yield. In this study, we conducted a whole genome-wide quantitative trait locus (QTL) mapping of grain-size-related traits by constructing a high-density genetic map using 109 recombinant inbred lines of einkorn wheat. We explored the candidate genes underlying QTLs through homologous analysis and RNA sequencing. The high-density genetic map spanned 1873 cM and contained 9937 single nucleotide polymorphism markers assigned to 1551 bins on seven chromosomes. Strong collinearity and high genome coverage of this map were revealed by comparison with physical maps of wheat and barley. Six grain size-related traits were surveyed in five environments. In total, 42 QTLs were identified; these were assigned to 17 genomic regions on six chromosomes and accounted for 52.3-66.7% of the phenotypic variation. Thirty homologous genes involved in grain development were located in 12 regions. RNA sequencing identified 4959 genes differentially expressed between the two parental lines. Twenty differentially expressed genes involved in grain size development and starch biosynthesis were mapped to nine regions that contained 26 QTLs, indicating that the starch biosynthesis pathway plays a vital role in grain development in einkorn wheat. This study provides new insights into the genetic architecture of grain size in einkorn wheat; identification of the underlying genes enables understanding of grain development and wheat genetic improvement. Furthermore, the map facilitates quantitative trait mapping, map-based cloning, genome assembly, and comparative genomics in wheat taxa.
Project description:Identification of causal mutations in barley and wheat is hampered by their large genomes and suppressed recombination. To overcome these obstacles, we have developed MutChromSeq, a complexity reduction approach based on flow sorting and sequencing of mutant chromosomes, to identify induced mutations by comparison to parental chromosomes. We apply MutChromSeq to six mutants each of the barley Eceriferum-q gene and the wheat Pm2 genes. This approach unambiguously identified single candidate genes that were verified by Sanger sequencing of additional mutants. MutChromSeq enables reference-free forward genetics in barley and wheat, thus opening up their pan-genomes to functional genomics.
Project description:The production of balanced fertile haploid gametes requires the faithful separation of paired (synapsed) chromosomes toward the end of meiotic prophase I (desynapsis). This involves the timely dissolution of the synaptonemal complex during the pachytene-diplotene transition, a stage traditionally referred to as the "diffuse stage." In species with large genomes such as, barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) we know most about the early stages of meiotic prophase I. There, synapsis initiates at the telomeric ends of chromosomes and progresses toward the centromeric regions through the ordered assembly of the synaptonemal complex (SC). Synapsis is impacted by recombination (crossing over, CO) which locally modifies the extent of chromatin compaction and extension. CO is uneven along the chromosomes, occurring mainly toward the telomeric regions resulting in a highly skewed distribution of recombination events. However, we know very little about the process of desynapsis which occurs during the "diffuse stage," where the synapsed and recombined chromosomes faithfully desynapse and separate into daughter cells. Here, using 3D-SIM super-resolution immuno-cytology combined with the use of antibodies directed against two crucial SC proteins, ASY1 and ZYP1, we followed the whole of meiosis I (i.e., both synapsis and desynapsis) in both barley and wheat. We showed that synapsis forms a characteristic tri-partite SC structure in zygotene (more clearly seen in barley). Toward the end of meiosis I, as the SC starts to disassemble, we show that extensive chromosome axis remodeling results in the formation of characteristic "tinsel-like" structures in both wheat and barley. By using a mutant (des10) that is severely compromised in polymerization of ZYP1during synapsis, we show that tinsel structure formation during SC dissolution is not dependant on full synapsis and may relate instead to changes in expansion stress. Our observations highlight a potentially new role for ASYNAPSIS1 (ASY1) in desynapsis, in addition to chromosome synapsis and cohesion.
Project description:BACKGROUND: Because of its size, allohexaploid nature and high repeat content, the wheat genome has always been perceived as too complex for efficient molecular studies. We recently constructed the first physical map of a wheat chromosome (3B). However gene mapping is still laborious in wheat because of high redundancy between the three homoeologous genomes. In contrast, in the closely related diploid species, barley, numerous gene-based markers have been developed. This study aims at combining the unique genomic resources developed in wheat and barley to decipher the organisation of gene space on wheat chromosome 3B. RESULTS: Three dimensional pools of the minimal tiling path of wheat chromosome 3B physical map were hybridised to a barley Agilent 15K expression microarray. This led to the fine mapping of 738 barley orthologous genes on wheat chromosome 3B. In addition, comparative analyses revealed that 68% of the genes identified were syntenic between the wheat chromosome 3B and barley chromosome 3 H and 59% between wheat chromosome 3B and rice chromosome 1, together with some wheat-specific rearrangements. Finally, it indicated an increasing gradient of gene density from the centromere to the telomeres positively correlated with the number of genes clustered in islands on wheat chromosome 3B. CONCLUSION: Our study shows that novel structural genomics resources now available in wheat and barley can be combined efficiently to overcome specific problems of genetic anchoring of physical contigs in wheat and to perform high-resolution comparative analyses with rice for deciphering the organisation of the wheat gene space.