Fast-forward genetics by radiation hybrids to saturate the locus regulating nuclear-cytoplasmic compatibility in Triticum.

The nuclear-encoded species cytoplasm specific (scs) genes control nuclear-cytoplasmic compatibility in wheat (genus Triticum). Alloplasmic cells, which have nucleus and cytoplasm derived from different species, produce vigorous and vital organisms only when the correct version of scs is present in their nucleus. In this study, bulks of in vivo radiation hybrids segregating for the scs phenotype have been genotyped by sequencing with over 1.9 million markers. The high marker saturation obtained for a critical region of chromosome 1D allowed identification of 3318 reads that mapped in close proximity of the scs. A novel in silico approach was deployed to extend these short reads to sequences of up to 70 Kb in length and identify candidate open reading frames (ORFs). Markers were developed to anchor the short contigs containing ORFs to a radiation hybrid map of 650 individuals with resolution of 288 Kb. The region containing the scs locus was narrowed to a single Bacterial Artificial Chromosome (BAC) contig of Aegilops tauschii. Its sequencing and assembly by nano-mapping allowed rapid identification of a rhomboid gene as the only ORF existing within the refined scs locus. Resequencing of this gene from multiple germplasm sources identified a single nucleotide mutation, which gives rise to a functional amino acid change. Gene expression characterization revealed that an active copy of this rhomboid exists on all homoeologous chromosomes of wheat, and depending on the specific cytoplasm each copy is preferentially expressed. Therefore, a new methodology was applied to unique genetic stocks to rapidly identify a strong candidate gene for the control of nuclear-cytoplasmic compatibility in wheat.

Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars.

Domesticated crops experience strong human-mediated selection aimed at developing high-yielding varieties adapted to local conditions. To detect regions of the wheat genome subject to selection during improvement, we developed a high-throughput array to interrogate 9,000 gene-associated single-nucleotide polymorphisms (SNP) in a worldwide sample of 2,994 accessions of hexaploid wheat including landraces and modern cultivars. Using a SNP-based diversity map we characterized the impact of crop improvement on genomic and geographic patterns of genetic diversity. We found evidence of a small population bottleneck and extensive use of ancestral variation often traceable to founders of cultivars from diverse geographic regions. Analyzing genetic differentiation among populations and the extent of haplotype sharing, we identified allelic variants subjected to selection during improvement. Selective sweeps were found around genes involved in the regulation of flowering time and phenology. An introgression of a wild relative-derived gene conferring resistance to a fungal pathogen was detected by haplotype-based analysis. Comparing selective sweeps identified in different populations, we show that selection likely acts on distinct targets or multiple functionally equivalent alleles in different portions of the geographic range of wheat. The majority of the selected alleles were present at low frequency in local populations, suggesting either weak selection pressure or temporal variation in the targets of directional selection during breeding probably associated with changing agricultural practices or environmental conditions. The developed SNP chip and map of genetic variation provide a resource for advancing wheat breeding and supporting future population genomic and genome-wide association studies in wheat.

Transcriptome-scale homoeolog-specific transcript assemblies of bread wheat.

BACKGROUND: Bread wheat is one of the world's most important food crops and considerable efforts have been made to develop genomic resources for this species. This includes an on-going project by the International Wheat Genome Sequencing Consortium to assemble its large and complex genome, which is hexaploid and contains three closely related 'homoeologous' copies for each chromosome. This multi-national effort avoids the complications polyploidy entails for correct assembly of the genome by sequencing flow-sorted chromosome arms one at a time. Here we report on an alternate approach, a direct homoeolog-specific assembly of the expressed portion of the genome, the transcriptome. RESULTS: After assessment of the ability of various assemblers to generate homoeolog-specific assemblies, we employed a two-stage assembly process to produce a high-quality assembly of the transcriptome of hexaploid wheat from Roche-454 and Illumina GAIIx paired-end sequence reads. The assembly process made use of a rapid partitioning of expressed sequences into homoeologous clusters, followed by a parallel high-fidelity assembly of each cluster on a 1150-processor compute cloud. We assessed assembly quality through comparison to known wheat gene sequences and found that in ca. 98.5% of cases the assembly was sufficiently accurate for homoeologous triplets to be cleanly separated into either two or three separate contigs. Comparison to publicly available transcript collections suggests that the assembly covers ~75-80% of the complete transcriptome. CONCLUSIONS: This work therefore describes the first homoeolog-specific sequence assembly of the wheat transcriptome and provides a reference transcriptome for future wheat research. Furthermore, our assembly methodology is transferable to other polyploid organisms.

Effectiveness of an innovative prototype subtracted diversity array (SDA) for fingerprinting plant species of medicinal importance.

The accurate identification of medicinal plants is becoming increasingly important due to reported concerns about purity, quality and safety. The previously developed prototype subtracted diversity array (SDA) had been validated for the ability to distinguish clade-level targets in a phylogenetically accurate manner. This study represents the rigorous investigation of the SDA for genotyping capabilities, including the genotyping of plant species not included during the construction of the SDA, as well as to lower classification levels including family and species. The results show that the SDA, in its current form, has the ability to accurately genotype species not included during SDA development to clade level. Additionally, for those species that were included during SDA development, genotyping is successful to the family level, and to the species level with minor exceptions. Twenty polymorphic SDA features were sequenced in a first attempt to characterize the polymorphic DNA between species, which showed that transposon-like sequences may be valuable as polymorphic features to differentiate angiosperm families and species. Future refinements of the SDA to allow more sensitive genotyping are discussed with the overall goal of accurate medicinal plant identification in mind.

Construction and validation of a prototype microarray for efficient and high-throughput genotyping of angiosperms.

Until recently, the identification of plants relied on conventional techniques, such as morphological, anatomical and chemical profiling, that are often inefficient or unfeasible in certain situations. Extensive literature exists describing the use of polymerase chain reaction (PCR) DNA-based identification techniques, which offer a reliable platform, but their broad application is often limited by a low throughput. However, hybridization-based microarray technology represents a rapid and high-throughput tool for genotype identification at a molecular level. Using an innovative technique, a 'Subtracted Diversity Array' (SDA) of 376 features was constructed from a pooled genomic DNA library of 49 angiosperm species, from which pooled non-angiosperm genomic DNA was subtracted. Although not the first use of a subtraction technique for genotyping, the SDA method was superior in accuracy, sensitivity and efficiency, and showed high-throughput capacity and broad application. The SDA technique was validated for potential genotyping use, and the results indicated a successful subtraction of non-angiosperm DNA. Statistical analysis of the polymorphic features from the pilot study enabled the establishment of accurate phylogenetic relationships, confirming the potential use of the SDA technique for genotyping. Further, the technique substantially enriched the presence of polymorphic sequences; 68% were polymorphic when using the array to differentiate six angiosperm clades (Asterids, Rosids, Caryophyllids, Ranunculids, Monocots and Eumagnoliids). The 'proof of concept' experiments demonstrate the potential of establishing a highly informative, reliable and high-throughput microarray-based technique for novel application to sequence independent genotyping of major angiosperm clades.