Next-generation sequencing and physical mapping of wheat chromosome 5D and comparison with its wild progenitor

Wheat is a staple grain crop, essential to human nutrition and animal feed. Despite its agronomic importance, wheat genomics research has long lagged behind its counterparts, due to its genome attributes. Bread wheat genome is almost six times as large as the human genome at a size of ~17 Gigabases, and is composed of >80% repetitive elements. The hexaploid genome is organized into three related sub-genomes, giving rise to numerous paralogous and homeologous loci. In this study, we characterized the flow-sorted 5D chromosome of bread wheat, Triticum aestivum, through survey sequencing and physical mapping, including its repeat landscape, gene content and conservation and putative tRNA repertoire. The virtual gene order of 5D chromosome suggested several perturbations in synteny, in addition to a number of putatively wheat-specific genome rearrangements. The 5DS physical map revealed that its gene space is largely organized into gene islands with an increasing gradient towards the telomere. Physical size estimates on the physical map indicated that cytogenetic estimates may considerably underestimate the 0.63-0.67 deletion bin interval. Comparative analyses of its wild progenitor, Aegilops tauschii 5D chromosome shed light into wheat genome evolution. The high density 5DS physical map at ~10.5 markers/Mb and 1.34x-1.61x survey sequences of the entire chromosome provides the foundation of the reference sequencing of this chromosome and presents a valuable genomics resource that the breeders and the researchers should benefit from

Molecular organization and comparative analysis of chromosome 5B of the wild wheat ancestor Triticum dicoccoides

Wild emmer wheat, Triticum turgidum ssp. dicoccoides is the wild relative of Triticum turgidum, the progenitor of durum and bread wheat, and maintains a rich allelic diversity among its wild populations. The lack of adequate genetic and genomic resources, however, restricts its exploitation in wheat improvement. Here, we report next-generation sequencing of the flow-sorted chromosome 5B of T. dicoccoides to shed light into its genome structure, function and organization by exploring the repetitive elements, protein-encoding genes and putative microRNA and tRNA coding sequences. Comparative analyses with its counterparts in modern and wild wheats suggest clues into the B-genome evolution. Syntenic relationships of chromosome 5B with the model grasses can facilitate further efforts for fine-mapping of traits of interest. Mapping of 5B sequences onto the root transcriptomes of two additional T. dicoccoides genotypes, with contrasting drought tolerances, revealed several thousands of single nucleotide polymorphisms, of which 584 shared polymorphisms on 228 transcripts were specific to the drought-tolerant genotype. To our knowledge, this study presents the largest genomics resource currently available for T. dicoccoides, which, we believe, will encourage the exploitation of its genetic and genomic potential for wheat improvement to meet the increasing demand to feed the world

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

Plant miRNAs: biogenesis, organization and origins

MicroRNAs, or miRNAs, are posttranscriptional regulators of gene expression. A wealth of observations and findings suggest highly complex, multicomponent, and intermingled pathways governing miRNA biogenesis and miRNA-mediated gene silencing. Plant miRNA genes are usually found as individual entities scattered around the intergenic and-to a much lesser extent-intragenic space, while miRNA gene clusters, formed by tandem or segmental duplications, also exist in plant genomes. Genome duplications are proposed to contribute to miRNA family expansions, as well. Evolutionarily young miRNAs retaining extensive homology to their loci of origin deliver important clues into miRNA origins and evolution. Additionally, imprecisely processed miRNAs evidence noncanonical routes of biogenesis, which may affect miRNA expression levels or targeting capabilities. Majority of the knowledge regarding miRNAs comes from model plant species. As ongoing research progressively expands into nonmodel systems, our understanding of miRNAs and miRNA-related pathways changes which opens up new perspectives and frontiers in miRNA research

Dissecting miRNAs in wheat d genome progenitor, aegilops tauschii

As the post-transcriptional regulators of gene expression, microRNAs or miRNAs comprise an integral part of understanding how genomes function. Although miRNAs have been a major focus of recent efforts, miRNA research is still in its infancy in most plant species. Aegilops tauschii, the D genome progenitor of bread wheat, is a wild diploid grass exhibiting remarkable population diversity. Due to the direct ancestry and the diverse gene pool, A. tauschii is a promising source for bread wheat improvement. In this study, a total of 87 Aegilops miRNA families, including 51 previously unknown, were computationally identified both at the subgenomic level, using flow-sorted A. tauschii 5D chromosome, and at the whole genome level. Predictions at the genomic and subgenomic levels suggested A. tauschii 5D chromosome as rich in pre-miRNAs that are highly associated with Class II DNA transposons. In order to gain insights into miRNA evolution, putative 5D chromosome miRNAs were compared to its modern ortholog, Triticum aestivum 5D chromosome, revealing that 48 of the 58 A. tauschii 5D miRNAs were conserved in orthologous T. aestivum 5D chromosome. The expression profiles of selected miRNAs (miR167, miR5205, miR5175, miR5523) provided the first experimental evidence for miR5175, miR5205 and miR5523, and revealed differential expressional changes in response to drought in different genetic backgrounds for miR167 and miR5175. Interestingly, while miR5523 coding regions were present and expressed as pre-miR5523 in both T. aestivum and A. tauschii, the expression of mature miR5523 was observed only in A. tauschii under normal conditions, pointing out to an interference at the downstream processing of pre-miR5523 in T. aestivum. Overall, this study expands our knowledge on the miRNA catalog of A. tauschii, locating a subset specifically to the 5D chromosome, with ample functional and comparative insight which should contribute to and complement efforts to develop drought tolerant wheat varieties

Dehydration stress-responsive miRNA in brachypodium distachyon: evident by genome-wide screening of microRNAs expression

There is a lack of knowledge on the tissue-specific expression of miRNAs in response to dehydration stress in Brachypodium (Brachypodium distachyon (L.) Beauv), a model for temperate grass species. In this study, miRNA expression patterns of drought-tolerant Brachypodium were investigated using the miRNA microarray platform. A total of 205 miRNAs in control and 438 miRNAs in both drought-treated leaf and root tissues were expressed. Seven of the detected Brachypodium miRNAs were dehydration stress responsive. Expression levels of known drought-responsive miRNAs, miR896, and miR1867 were quantified by qRT-PCR in Brachypodium upon 4 h and 8 h dehydration stress applications. This was performed to compare drought responsiveness of miRNAs in closely related species. Target transcripts of selected drought responsive miRNAs, miR170, miR1850, miR896, miR406, miR528, miR390, were computationally predicted. Target transcript of miR896 was verified by retrieving a cleaved miR896 transcript from drought stress-treated leaf samples using a modified 5' RLM-RACE. Brachypodium dehydration responsive miRNA were also detected in barley and wild emmer wheat. Hence, the outcomes highlighted the conserved features of miRNA upon dehydration stress in Triticeae

A large-scale chromosome-specific SNP discovery guideline

Single-nucleotide polymorphisms (SNPs) are the most prevalent type of variation in genomes that are increasingly being used as molecular markers in diversity analyses, mapping and cloning of genes, and germplasm characterization. However, only a few studies reported large-scale SNP discovery in Aegilops tauschii, restricting their potential use as markers for the low-polymorphic D genome. Here, we report 68,592 SNPs found on the gene-related sequences of the 5D chromosome of Ae. tauschii genotype MvGB589 using genomic and transcriptomic sequences from seven Ae. tauschii accessions, including AL8/78, the only genotype for which a draft genome sequence is available at present. We also suggest a workflow to compare SNP positions in homologous regions on the 5D chromosome of Triticum aestivum, bread wheat, to mark single nucleotide variations between these closely related species. Overall, the identified SNPs define a density of 4.49 SNPs per kilobyte, among the highest reported for the genic regions of Ae. tauschii so far. To our knowledge, this study also presents the first chromosome-specific SNP catalog in Ae. tauschii that should facilitate the association of these SNPs with morphological traits on chromosome 5D to be ultimately targeted for wheat improvement

Root precursors of microRNAs in wild emmer and modern wheats show major differences in response to drought stress

MicroRNAs, small regulatory molecules with significant impacts on the transcriptional network of all living organisms, have been the focus of several studies conducted mostly on modern wheat cultivars. In this study, we investigated miRNA repertoires of modern durum wheat and its wild relatives, with differing degrees of drought tolerance, to identify miRNA candidates and their targets involved in drought stress response. Root transcriptomes of Triticum turgidum ssp. durum variety Kızıltan and two Triticum turgidum ssp. dicoccoides genotypes TR39477 and TTD-22 under control and drought conditions were assembled from individual RNA-Seq reads and used for in silico identification of miRNAs. A total of 66 miRNAs were identified from all species, across all conditions, of which 46 and 38 of the miRNAs identified from modern durum wheat and wild genotypes, respectively, had not been previously reported. Genotype- and/or stress-specific miRNAs provide insights into our understanding of the complex drought response. Particularly, miR1435, miR5024, and miR7714, identified only from drought-stress roots of drought-tolerant genotype TR39477, can be candidates for future studies to explore and exploit the drought response to develop tolerant varieties