Physical mapping integrated with syntenic analysis to characterize the gene space of the long arm of wheat chromosome 1A

Background: Bread wheat (Triticum aestivum L.) is one of the most important crops worldwide and its production faces pressing challenges, the solution of which demands genome information. However, the large, highly repetitive hexaploid wheat genome has been considered intractable to standard sequencing approaches. Therefore the International Wheat Genome Sequencing Consortium (IWGSC) proposes to map and sequence the genome on a chromosome-by-chromosome basis. Methodology/Principal Findings: We have constructed a physical map of the long arm of bread wheat chromosome 1A using chromosome-specific BAC libraries by High Information Content Fingerprinting (HICF). Two alternative methods (FPC and LTC) were used to assemble the fingerprints into a high-resolution physical map of the chromosome arm. A total of 365 molecular markers were added to the map, in addition to 1122 putative unique transcripts that were identified by microarray hybridization. The final map consists of 1180 FPC based or 583 LTC based contigs. Conclusions/Significance: The physical map presented here marks an important step forward in mapping of hexaploid bread wheat. The map is orders of magnitude more detailed than previously available maps of this chromosome, and the assignment of over a thousand putative expressed gene sequences to specific map locations will greatly assist future functional studies. This map will be an essential tool for future sequencing of and positional cloning within chromosome 1A

A Graph-Theoretical Approach to the Selection of the Minimum Tiling Path from a Physical Map

The problem of computing the minimum tiling path (MTP) from a set of clones arranged in a physical map is a cornerstone of hierarchical (clone-by-clone) genome sequencing projects. We formulate this problem in a graph theoretical framework, and then solve by a combination of minimum hitting set and minimum spanning tree algorithms. The tool implementing this strategy, called FMTP, shows improved performance compared to the widely used software FPC. When we execute FMTP and FPC on the same physical map, the MTP produced by FMTP covers a higher portion of the genome, and uses a smaller number of clones. For instance, on the rice genome the MTP produced by our tool would reduce by about 11 percent the cost of a clone-by-clone sequencing project. Source code, benchmark data sets, and documentation of FMTP are freely available at \u3ehttp://code.google.com/p/fingerprint-based-minimal-tiling-path/ under MIT license

Isolation and fine mapping of Rps6: An intermediate host resistance gene in barley to wheat stripe rust

A plant may be considered a nonhost of a pathogen if all known genotypes of a plant species are resistant to all known isolates of a pathogen species. However, if a small number of genotypes are susceptible to some known isolates of a pathogen species this plant maybe considered an intermediate host. Barley (Hordeum vulgare) is an intermediate host for Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust. We wanted to understand the genetic architecture underlying resistance to Pst and to determine whether any overlap exists with resistance to the host pathogen, Puccinia striiformis f. sp. hordei (Psh). We mapped Pst resistance to chromosome 7H and show that host and intermediate host resistance is genetically uncoupled. Therefore, we designate this resistance locus Rps6. We used phenotypic and genotypic selection on F2:3 families to isolate Rps6 and fine mapped the locus to a 0.1 cM region. Anchoring of the Rps6 locus to the barley physical map placed the region on two adjacent fingerprinted contigs. Efforts are now underway to sequence the minimal tiling path and to delimit the physical region harbouring Rps6. This will facilitate additional marker development and permit identification of candidate genes in the region

Genetic and physical mapping of DNA replication origins in Haloferax volcanii

The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle. © 2007 Norais et al

Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders

Background Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families. Methods Here we present data that refine this locus to a 0.5 cM region, flanked by the microsatellite markers D2S2345 and D2S326. The minimal region contains the candidate gene GAD1, which encodes a glutamate decarboxylase isoform (GAD67), involved in conversion of the amino acid and excitatory neurotransmitter glutamate to the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Results A novel amino acid mis-sense mutation in GAD67 was detected, which segregated with CP in affected individuals. Conclusions This result is interesting because auto-antibodies to GAD67 and the more widely studied GAD65 homologue encoded by the GAD2 gene, are described in patients with Stiff-Person Syndrome (SPS), epilepsy, cerebellar ataxia and Batten disease. Further investigation seems merited of the possibility that variation in the GAD1 sequence, potentially affecting glutamate/GABA ratios, may underlie this form of spastic CP, given the presence of anti-GAD antibodies in SPS and the recognised excitotoxicity of glutamate in various contexts

Sequencing the potato genome: outline and first results to come from the elucidation of the sequence of the world's third most important food crop

Potato is a member of the Solanaceae, a plant family that includes several other economically important species, such as tomato, eggplant, petunia, tobacco and pepper. The Potato Genome Sequencing Consortium (PGSC) aims to elucidate the complete genome sequence of potato, the third most important food crop in the world. The PGSC is a collaboration between 13 research groups from China, India, Poland, Russia, the Netherlands, Ireland, Argentina, Brazil, Chile, Peru, USA, New Zealand and the UK. The potato genome consists of 12 chromosomes and has a (haploid) length of approximately 840 million base pairs, making it a medium-sized plant genome. The sequencing project builds on a diploid potato genomic bacterial artificial chromosome (BAC) clone library of 78000 clones, which has been fingerprinted and aligned into ~7000 physical map contigs. In addition, the BAC-ends have been sequenced and are publicly available. Approximately 30000 BACs are anchored to the Ultra High Density genetic map of potato, composed of 10000 unique AFLPTM markers. From this integrated genetic-physical map, between 50 to 150 seed BACs have currently been identified for every chromosome. Fluorescent in situ hybridization experiments on selected BAC clones confirm these anchor points. The seed clones provide the starting point for a BAC-by-BAC sequencing strategy. This strategy is being complemented by whole genome shotgun sequencing approaches using both 454 GS FLX and Illumina GA2 instruments. Assembly and annotation of the sequence data will be performed using publicly available and tailor-made tools. The availability of the annotated data will help to characterize germplasm collections based on allelic variance and to assist potato breeders to more fully exploit the genetic potential of potat

Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome

BACKGROUND There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys. RESULTS A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley. CONCLUSIONS We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes.This work was financially supported by the following grants: project GABI-BARLEX, German Federal Ministry of Education and Research (BMBF), #0314000 to MP, US, KFXM and NS; Triticeae Coordinated Agricultural Project, USDA-NIFA #2011-68002-30029 to GJM; and Agriculture and Food Research Initiative Plant Genome, Genetics and Breeding Program of USDA’s Cooperative State Research and Extension Service, #2009-65300- 05645 to GJM

Identification of candidate genes and molecular markers for heat-induced brown discoloration of seed coats in cowpea [Vigna unguiculata (L.) Walp].

BackgroundHeat-induced browning (Hbs) of seed coats is caused by high temperatures which discolors the seed coats of many legumes, affecting the visual appearance and quality of seeds. The genetic determinants underlying Hbs in cowpea are unknown.ResultsWe identified three QTL associated with the heat-induced browning of seed coats trait, Hbs-1, Hbs-2 and Hbs-3, using cowpea RIL populations IT93K-503-1 (Hbs positive) x CB46 (hbs negative) and IT84S-2246 (Hbs positive) x TVu14676 (hbs negative). Hbs-1 was identified in both populations, accounting for 28.3% -77.3% of the phenotypic variation. SNP markers 1_0032 and 1_1128 co-segregated with the trait. Within the syntenic regions of Hbs-1 in soybean, Medicago and common bean, several ethylene forming enzymes, ethylene responsive element binding factors and an ACC oxidase 2 were observed. Hbs-1 was identified in a BAC clone in contig 217 of the cowpea physical map, where ethylene forming enzymes were present. Hbs-2 was identified in the IT93K-503-1 x CB46 population and accounted for of 9.5 to 12.3% of the phenotypic variance. Hbs-3 was identified in the IT84S-2246 x TVu14676 population and accounted for 6.2 to 6.8% of the phenotypic variance. SNP marker 1_0640 co-segregated with the heat-induced browning phenotype. Hbs-3 was positioned on BAC clones in contig512 of the cowpea physical map, where several ACC synthase 1 genes were present.ConclusionThe identification of loci determining heat-induced browning of seed coats and co-segregating molecular markers will enable transfer of hbs alleles into cowpea varieties, contributing to higher quality seeds