Unique and conserved MicroRNAs in wheat chromosome 5D revealed by next-generation sequencing

MicroRNAs are a class of short, non-coding, single-stranded RNAs that act as post-transcriptional regulators in gene expression. miRNA analysis of Triticum aestivum chromosome 5D was performed on 454 GS FLX Titanium sequences of flow sorted chromosome 5D with a total of 3,208,630 good quality reads representing 1.34x and 1.61x coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. In silico and structural analyses revealed a total of 55 miRNAs; 48 and 42 miRNAs were found to be present on 5DL and 5DS respectively, of which 35 were common to both chromosome arms, while 13 miRNAs were specific to 5DL and 7 miRNAs were specific to 5DS. In total, 14 of the predicted miRNAs were identified in wheat for the first time. Representation (the copy number of each miRNA) was also found to be higher in 5DL (1,949) compared to 5DS (1,191). Targets were predicted for each miRNA, while expression analysis gave evidence of expression for 6 out of 55 miRNAs. Occurrences of the same miRNAs were also found in Brachypodium distachyon and Oryza sativa genome sequences to identify syntenic miRNA coding sequences. Based on this analysis, two other miRNAs: miR1133 and miR167 were detected in B. distachyon syntenic region of wheat 5DS. Five of the predicted miRNA coding regions (miR6220, miR5070, miR169, miR5085, miR2118) were experimentally verified to be located to the 5D chromosome and three of them : miR2118, miR169 and miR5085, were shown to be 5D specific. Furthermore miR2118 was shown to be expressed in Chinese Spring adult leaves. miRNA genes identified in this study will expand our understanding of gene regulation in bread wheat

Chromosome Bin Map of Expressed Sequence Tags in Homoeologous Group 1 of Hexaploid Wheat and Homoeology With Rice and Arabidopsis

A total of 944 expressed sequence tags (ESTs) generated 2212 EST loci mapped to homoeologous group 1 chromosomes in hexaploid wheat (Triticum aestivum L.). EST deletion maps and the consensus map of group 1 chromosomes were constructed to show EST distribution. EST loci were unevenly distributed among chromosomes 1A, 1B, and 1D with 660, 826, and 726, respectively. The number of EST loci was greater on the long arms than on the short arms for all three chromosomes. The distribution of ESTs along chromosome arms was nonrandom with EST clusters occurring in the distal regions of short arms and middle regions of long arms. Duplications of group 1 ESTs in other homoeologous groups occurred at a rate of 35.5%. Seventy-five percent of wheat chromosome 1 ESTs had significant matches with rice sequences (E ≤ e(−10)), where large regions of conservation occurred between wheat consensus chromosome 1 and rice chromosome 5 and between the proximal portion of the long arm of wheat consensus chromosome 1 and rice chromosome 10. Only 9.5% of group 1 ESTs showed significant matches to Arabidopsis genome sequences. The results presented are useful for gene mapping and evolutionary and comparative genomics of grasses

Control of flowering time and spike development in cereals: the earliness per se Eps-1 region in wheat, rice, and Brachypodium

The earliness per se gene Eps-Am1 from diploid wheat Triticum monococcum affects heading time, spike development, and spikelet number. In this study, the Eps1 orthologous regions from rice, Aegilops tauschii, and Brachypodium distachyon were compared as part of current efforts to clone this gene. A single Brachypodium BAC clone spanned the Eps-Am1 region, but a gap was detected in the A. tauschii physical map. Sequencing of the Brachypodium and A. tauschii BAC clones revealed three genes shared by the three species, which showed higher identity between wheat and Brachypodium than between them and rice. However, most of the structural changes were detected in the wheat lineage. These included an inversion encompassing the wg241-VatpC region and the presence of six unique genes. In contrast, only one unique gene (and one pseudogene) was found in Brachypodium and none in rice. Three genes were present in both Brachypodium and wheat but were absent in rice. Two of these genes, Mot1 and FtsH4, were completely linked to the earliness per se phenotype in the T. monococcum high-density genetic map and are candidates for Eps-Am1. Both genes were expressed in apices and developing spikes, as expected for Eps-Am1 candidates. The predicted MOT1 protein showed amino acid differences between the parental T. monococcum lines, but its effect is difficult to predict. Future steps to clone the Eps-Am1 gene include the generation of mot1 and ftsh4 mutants and the completion of the T. monococcum physical map to test for the presence of additional candidate genes

COMPARISON BETWEEN TWO GENERIC 3D BUILDING RECONSTRUCTION APPROACHES – POINT CLOUD BASED VS. IMAGE PROCESSING BASED

This paper compares two generic approaches for the reconstruction of buildings. Synthesized and real oblique and vertical aerial imagery is transformed on the one hand into a dense photogrammetric 3D point cloud and on the other hand into photogrammetric 2.5D surface models depicting a scene from different cardinal directions. One approach evaluates the 3D point cloud statistically in order to extract the hull of structures, while the other approach makes use of salient line segments in 2.5D surface models, so that the hull of 3D structures can be recovered. With orders of magnitudes more analyzed 3D points, the point cloud based approach is an order of magnitude more accurate for the synthetic dataset compared to the lower dimensioned, but therefor orders of magnitude faster, image processing based approach. For real world data the difference in accuracy between both approaches is not significant anymore. In both cases the reconstructed polyhedra supply information about their inherent semantic and can be used for subsequent and more differentiated semantic annotations through exploitation of texture information

A microcolinearity study at the earliness per se gene Eps-A(m)1 region reveals an ancient duplication that preceded the wheat-rice divergence.

Wheat flowering is controlled by numerous genes, which respond to environmental signals such as photoperiod and vernalization. Earliness per se (Eps) genes control flowering time independently of these environmental cues and are responsible for the fine tuning of flowering time. We recently mapped the Eps-A(m)1 gene on the end of Triticum monococcum chromosome arm 1A(m)L. As a part of our efforts to clone Eps-A(m)1 we developed PCR markers flanking this gene within a 2.7 cM interval. We screened more than one thousand gametes with these markers and identified 27 lines with recombination between them. Recombinant lines were used to generate a high-density map and to investigate the microcolinearity between wheat and rice in this region. We mapped ten genes from a 149 kb region located at the distal part of rice chromosome 5 (cdo393 - Ndk3) on a 3.7 cM region on wheat chromosome one. This region is part of an ancient duplication between rice chromosomes 5 and 1. Genes present in both rice chromosomes were less similar to each other than to the closest wheat orthologues, suggesting that this duplication preceded the divergence between wheat and rice. This hypothesis was supported by the presence of 18 loci duplicated both in rice chromosomes 5 and 1 and in the colinear wheat chromosomes from homologous groups 1 and 3. Independent gene deletions in wheat and rice lineages explain the alternations of colinearity between rice chromosome 5 and wheat chromosomes 1 and 3. Colinearity between the end of rice chromosome 5 and wheat chromosome 1 was also interrupted by a small inversion, and several non-colinear genes. These results suggest that the distal region of the long arm of wheat chromosome 1 was involved in numerous changes that differentiated wheat and rice genomes. This comparative study provided sufficient markers to saturate the Eps-A(m)1 gene region and to precisely map this gene within a 0.9 cM interval flanked by the VatpC and Smp loci

INLINING 3D RECONSTRUCTION, MULTI-SOURCE TEXTURE MAPPING AND SEMANTIC ANALYSIS USING OBLIQUE AERIAL IMAGERY

This paper proposes an in-line method for the simplified reconstruction of city buildings from nadir and oblique aerial images that at the same time are being used for multi-source texture mapping with minimal resampling. Further, the resulting unrectified texture atlases are analyzed for fac¸ade elements like windows to be reintegrated into the original 3D models. Tests on real-world data of Heligoland/ Germany comprising more than 800 buildings exposed a median positional deviation of 0.31 m at the fac¸ades compared to the cadastral map, a correctness of 67% for the detected windows and good visual quality when being rendered with GPU-based perspective correction. As part of the process building reconstruction takes the oriented input images and transforms them into dense point clouds by semi-global matching (SGM). The point sets undergo local RANSAC-based regression and topology analysis to detect adjacent planar surfaces and determine their semantics. Based on this information the roof, wall and ground surfaces found get intersected and limited in their extension to form a closed 3D building hull. For texture mapping the hull polygons are projected into each possible input bitmap to find suitable color sources regarding the coverage and resolution. Occlusions are detected by ray-casting a full-scale digital surface model (DSM) of the scene and stored in pixel-precise visibility maps. These maps are used to derive overlap statistics and radiometric adjustment coefficients to be applied when the visible image parts for each building polygon are being copied into a compact texture atlas without resampling whenever possible. The atlas bitmap is passed to a commercial object-based image analysis (OBIA) tool running a custom rule set to identify windows on the contained fac¸ade patches. Following multi-resolution segmentation and classification based on brightness and contrast differences potential window objects are evaluated against geometric constraints and conditionally grown, fused and filtered morphologically. The output polygons are vectorized and reintegrated into the previously reconstructed buildings by sparsely ray-tracing their vertices. Finally the enhanced 3D models get stored as textured geometry for visualization and semantically annotated ”LOD-2.5” CityGML objects for GIS applications

Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures

Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8′-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches

Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures

Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8′-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches