RNA-seq analysis reveals considerable genetic diversity and provides genetic markers saturating all chromosomes in the diploid wild wheat relative Aegilops umbellulata.

BACKGROUND: Aegilops umbellulata Zhuk. (2n = 14), a wild diploid wheat relative, has been the source of trait improvement in wheat breeding. Intraspecific genetic variation of Ae. umbellulata, however, has not been well studied and the genomic information in this species is limited. RESULTS: To develop novel genetic markers distributed over all chromosomes of Ae. umbellulata and to evaluate its genetic diversity, we performed RNA sequencing of 12 representative accessions and reconstructed transcripts by de novo assembly of reads for each accession. A large number of single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were obtained and anchored to the pseudomolecules of Ae. tauschii and barley (Hordeum vulgare L.), which were regarded as virtual chromosomes of Ae. umbellulata. Interestingly, genetic diversity in Ae. umbellulata was higher than in Ae. tauschii, despite the narrow habitat of Ae. umbellulata. Comparative analyses of nucleotide polymorphisms between Ae. umbellulata and Ae. tauschii revealed no clear lineage differentiation and existence of alleles with rarer frequencies predominantly in Ae. umbellulata, with patterns clearly distinct from those in Ae. tauschii. CONCLUSIONS: The anchored SNPs, covering all chromosomes, provide sufficient genetic markers between Ae. umbellulata accessions. The alleles with rarer frequencies might be the main source of the high genetic diversity in Ae. umbellulata.

Genome-wide identification of novel genetic markers from RNA sequencing assembly of diverse Aegilops tauschii accessions.

The wild species in the Triticeae tribe are tremendous resources for crop breeding due to their abundant natural variation. However, their huge and highly repetitive genomes have hindered the establishment of physical maps and the completeness of their genome sequences. To develop molecular markers for the efficient utilization of their valuable traits while avoiding their genome complexity, we assembled RNA sequences of ten representative accessions of Aegilops tauschii, the progenitor of the wheat D genome, and estimated single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). The deduced unigenes were anchored to the chromosomes of Ae. tauschii and barley. The SNPs and indels in the anchored unigenes, covering entire chromosomes, were sufficient for linkage map construction, even in combinations between the genetically closest accessions. Interestingly, the resolution of SNP and indel distribution on barley chromosomes was slightly higher than on Ae. tauschii chromosomes. Since barley chromosomes are regarded as virtual chromosomes of Triticeae species, our strategy allows capture of genetic markers arranged on the chromosomes in order based on the conserved synteny. The resolution of these genetic markers will be comparable to that of the Ae. tauschii whose draft genome sequence is available. Our procedure should be applicable to marker development for Triticeae species, which have no draft sequences available.

Improvement of barley genome annotations by deciphering the Haruna Nijo genome.

Full-length (FL) cDNA sequences provide the most reliable evidence for the presence of genes in genomes. In this report, detailed gene structures of barley, whole genome shotgun (WGS) and additional transcript data of the cultivar Haruna Nijo were quality controlled and compared with the published Morex genome information. Haruna Nijo scaffolds have longer total sequence length with much higher N50 and fewer sequences than those in Morex WGS contigs. The longer Haruna Nijo scaffolds provided efficient FLcDNA mapping, resulting in high coverage and detection of the transcription start sites. In combination with FLcDNAs and RNA-Seq data from four different tissue samples of Haruna Nijo, we identified 51,249 gene models on 30,606 loci. Overall sequence similarity between Haruna Nijo and Morex genome was 95.99%, while that of exon regions was higher (99.71%). These sequence and annotation data of Haruna Nijo are combined with Morex genome data and released from a genome browser. The genome sequence of Haruna Nijo may provide detailed gene structures in addition to the current Morex barley genome information.

454 sequencing of pooled BAC clones on chromosome 3H of barley.

BACKGROUND: Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000 Mbp). Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H. RESULTS: We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1. CONCLUSIONS: We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.