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Background: The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-informationcontent- fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy. Results: The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries. Conclusions: The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes. [ABSTRACT FROM AUTHOR]

Background: Brachypodium distachyon (Brachypodium) has been recognized as a new model species for comparative and functional genomics of cereal and bioenergy crops because it possesses many biological attributes desirable in a model, such as a small genome size, short stature, self-pollinating habit, and short generation cycle. To maximize the utility of Brachypodium as a model for basic and applied research it is necessary to develop genomic resources for it. A BAC-based physical map is one of them. A physical map will facilitate analysis of genome structure, comparative genomics, and assembly of the entire genome sequence. Results: A total of 67,151 Brachypodium BAC clones were fingerprinted with the SNaPshot HICF fingerprinting method and a genome-wide physical map of the Brachypodium genome was constructed. The map consisted of 671 contigs and 2,161 clones remained as singletons. The contigs and singletons spanned 414 Mb. A total of 13,970 gene-related sequences were detected in the BAC end sequences (BES). These gene tags aligned 345 contigs with 336 Mb of rice genome sequence, showing that Brachypodium and rice genomes are generally highly colinear. Divergent regions were mainly in the rice centromeric regions. A dot-plot of Brachypodium contigs against the rice genome sequences revealed remnants of the whole-genome duplication caused by paleotetraploidy, which were previously found in rice and sorghum. Brachypodium contigs were anchored to the wheat deletion bin maps with the BES gene-tags, opening the door to Brachypodium-Triticeae comparative genomics. Conclusion: The construction of the Brachypodium physical map, and its comparison with the rice genome sequence demonstrated the utility of the SNaPshot-HICF method in the construction of BAC-based physical maps. The map represents an important genomic resource for the completion of Brachypodium genome sequence and grass comparative genomics. A draft of the physical map and its comparisons with rice and wheat are available at http://phymap.ucdavis.edu/brachypodium/. [ABSTRACT FROM AUTHOR]

Background: Current techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGate™ assay. Results: To test the efficacy of the Golden Gate assay in BAC library screening, multidimensional pools involving 302976 Aegilops tauschii BAC clones were genotyped for the presence/absence of specific gene sequences with multiplexed Illumina GoldenGate oligonucleotide assays previously used to place single nucleotide polymorphisms on an Ae. tauschii genetic map. Of 1384 alleleinformative oligonucleotide assays, 87.6% successfully clustered BAC pools into those positive for a BAC clone harboring a specific gene locus and those negative for it. The location of the positive BAC clones within contigs assembled from 199190 fingerprinted Ae. tauschii BAC clones was used to evaluate the precision of anchoring of BAC clones and contigs on the Ae. tauschii genetic map. For 41 (95%) assays, positive BAC clones were neighbors in single contigs. Those contigs could be unequivocally assigned to loci on the genetic map. For two (5%) assays, positive clones were in two different contigs and the relationships of these contigs to loci on the Ae. tauschii genetic map were equivocal. Screening of BAC libraries with a simple five-dimensional BAC pooling strategy was evaluated and shown to allow direct detection of positive BAC clones without the need for manual deconvolution of BAC clone pools. Conclusion: The highly parallel Illumina oligonucleotide assay is shown here to be an efficient tool for screening BAC libraries and a strategy for high-throughput anchoring of BAC contigs on genetic maps during the construction of physical maps of eukaryotic genomes. In most cases, screening of BAC libraries with Illumina oligonucleotide assays results in the unequivocal relationship of BAC clones with loci on the genetic map. [ABSTRACT FROM AUTHOR]

The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions. [ABSTRACT FROM AUTHOR]

Cycles of whole-genome duplication (WGD) and diploidization are hallmarks of eukaryotic genome evolution and speciation. Polyploid wheat (Triticum aestivum) has had a massive increase in genome size largely due to recent WGDs. How these processes may impact the dynamics of gene evolution was studied by comparing the patterns of gene structure changes, alternative splicing (AS), and codon substitution rates among wheat and model grass genomes. In orthologous gene sets, significantly more acquired and lost exonic sequences were detected in wheat than in model grasses. In wheat, 35% of these gene structure rearrangements resulted in frame-shift mutations and premature termination codons. An increased codon mutation rate in the wheat lineage compared with Brachypodium distachyon was found for 17% of orthologs. The discovery of premature termination codons in 38% of expressed genes was consistent with ongoing pseudogenization of the wheat genome. The rates of AS within the individual wheat subgenomes (21%-25%) were similar to diploid plants. However, we uncovered a high level of AS pattern divergence between the duplicated homeologous copies of genes. Our results are consistent with the accelerated accumulation of AS isoforms, nonsynonymous mutations, and gene structure rearrangements in the wheat lineage, likely due to genetic redundancy created by WGDs. Whereas these processes mostly contribute to the degeneration of a duplicated genome and its diploidization, they have the potential to facilitate the origin of new functional variations, which, upon selection in the evolutionary lineage, may play an important role in the origin of novel traits. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this study, we developed 359 detection primers for single nucleotide polymorphisms (SNPs) previously discovered within intron sequences of wheat genes and used them to evaluate SNP polymorphism in common wheat ( Triticum aestivum L.). These SNPs showed an average polymorphism information content (PIC) of 0.18 among 20 US elite wheat cultivars, representing seven market classes. This value increased to 0.23 when SNPs were pre-selected for polymorphisms among a diverse set of 13 hexaploid wheat accessions (excluding synthetic wheats) used in the wheat SNP discovery project (). PIC values for SNP markers in the D genome were approximately half of those for the A and B genomes. D genome SNPs also showed a larger PIC reduction relative to the other genomes ( P < 0.05) when US cultivars were compared with the more diverse set of 13 wheat accessions. Within those accessions, D genome SNPs show a higher proportion of alleles with low minor allele frequencies (<0.125) than found in the other two genomes. These data suggest that the reduction of PIC values in the D genome was caused by differential loss of low frequency alleles during the population size bottleneck that accompanied the development of modern commercial cultivars. Additional SNP discovery efforts targeted to the D genome in elite wheat germplasm will likely be required to offset the lower diversity of this genome. With increasing SNP discovery projects and the development of high-throughput SNP assay technologies, it is anticipated that SNP markers will play an increasingly important role in wheat genetics and breeding applications. [ABSTRACT FROM AUTHOR]

Background: Microsatellite (simple sequence repeat -- SSR) and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping. Often, large-scale genomic research projects require high-throughput computer-assisted primer design. Numerous such web-based or standard-alone programs for PCR primer design are available but vary in quality and functionality. In particular, most programs lack batch primer design capability. Such a high-throughput software tool for designing SSR flanking primers and SNP genotyping primers is increasingly demanded. Results: A new web primer design program, BatchPrimer3, is developed based on Primer3. BatchPrimer3 adopted the Primer3 core program as a major primer design engine to choose the best primer pairs. A new scorebased primer picking module is incorporated into BatchPrimer3 and used to pick position-restricted primers. BatchPrimer3 v1.0 implements several types of primer designs including generic primers, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. DNA sequences in FASTA format can be batch read into the program. The basic information of input sequences, as a reference of parameter setting of primer design, can be obtained by pre-analysis of sequences. The input sequences can be pre-processed and masked to exclude and/or include specific regions, or set targets for different primer design purposes as in Primer3Web and primer3Plus. A tab-delimited or Excel-formatted primer output also greatly facilitates the subsequent primer-ordering process. Thousands of primers, including wheat conserved intron-flanking primers, wheat genome-specific SNP genotyping primers, and Brachypodium SSR flanking primers in several genome projects have been designed using the program and validated in several laboratories. Conclusion: BatchPrimer3 is a comprehensive web primer design program to develop different types of primers in a high-throughput manner. Additional methods of primer design can be easily integrated into future versions of BatchPrimer3. The program with source code and thousands of PCR and sequencing primers designed for wheat and Brachypodium are accessible at http://wheat.pw.usda.gov/demos/BatchPrimer3/. [ABSTRACT FROM AUTHOR]