Cereal Genomics II

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The book should prove useful to students, teachers and young research workers as a ready reference to the latest information on cereal genomics. Table of contents Preface.

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Gupta, and Rajeev K. Gupta, Sachin Rustgi, Reyazul R. Application of next-generation sequencing technologies for genetic diversity analysis in cereals: Seifollah Kiani, Alina Akhunova, Eduard Akhunov. Transposons in cereals: shaping genomes and driving their evolution: Jan P. Buchmann, Beat Keller, Thomas Wicker.

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Gupta, Pawan L. Kulwal, Reyazul R. High-throughput and precision phenotyping for cereal breeding programs: Boddupalli M. Learn about new offers and get more deals by joining our newsletter.

Applications of Genetic and Genomic Research in Cereals - 1st Edition

Sign up now. Scaffold lengths varied from 3 kb to kb, and they comprised from one to eleven complete TaeCsTr99 units. Altogether, we identified in unassigned scaffolds 36 complete tandemly organised units totalling 98 kb of length, accompanied by several incomplete units and unit fragments.

Based on a high sequence homology Figure S3 , some of the scaffolds could be overlapping. Out of the twelve scaffolds, only ChrUn carried an OM contig 77 placed the ChrUn to position Organization of the TaeCsTr99 region. Numbers at the 7D pseudomolecules indicate assembly coordinates. Illumina assemblies of these BAC clones were rather fragmented and did not allow reconstructing the entire TaeCsTr99 array. This suggested the presence of two separate TaeCsTr99 arrays in a close proximity, which we termed distal covered by clones K16 and 28N04 and proximal covered by ChrUn, G18 and 30G22 , respectively.

We hypothesized that the difficulties in assembling and incorporating of the TaeCsTr99 arrays into the pseudomolecule could be overcome by employing longer reads, such as those generated by SMRT sequencing PacBio technology. To verify this, we explored bread wheat Triticum 3. The blastn search revealed in the position 9. On the contrary, we did not find any evidence of the proximal array in the Triticum 3. To resolve the discrepancies in the location and organization of the TaeCsTr99 arrays identified in various assemblies, we made use of the long-read platform of Oxford Nanopore Technologies ONT and generated nanopore reads from BAC clones 28N04 and G18, which cover the distal and proximal array, respectively.

For each of the clones, we obtained two reads that spanned over the entire insert and showed a consistent array structure. ONT read 51ef File S2 of 99, bp covering clone 28N04 confirmed the complex structure of the distal array composed of two sub-arrays with differently organised units Figure 2 D, Figure S4B.

Sequence based DNA markers and genotyping for cereal genomics and breeding

The distally located sub-array, approximately 27 kb in size, comprised of 10 complete units of the TaeCsTr The proximal sub-array was of similar length and consisted of 12 incomplete units, bearing a distinct deletion between and bp of the TaeCsTr99 sequence. Except for the variation in the number of units, the overall structure of the distal array looked highly similar in the BAC Illumina assembly and the ONT read.

On the contrary, the corresponding array in whole-genome Triticum 3. The total number of TaeCsTr99 units in the ONT reads covering the distal and the proximal array 18 was smaller than that identified in unassigned scaffolds This could be due to non-recognized overlaps between the scaffolds, which may have resulted in overestimating the number of the repeats. Alternatively, we cannot exclude the presence of additional TaeCsTr99 array s , missing both in the pseudomolecules and in the 7DS BAC assemblies that might be located in proximity of the confirmed ones.

The data obtained in our study suggest that tandemly organised repeats with unit size of 1—3 kb are not the major contributor to the missing part of the wheat IWGSC RefSeq v1.

Cereal Genomics

Nevertheless, a more detailed analysis of a repeat with unit size of bp revealed that it was completely missing from the 7D pseudomolecule and was found in unassigned scaffolds ChrUn of the RefSeq v1. Thus, we concluded that this type of repeats may cause shrinking of pseudomolecules without impacting size of the entire assembly. This indicates that advanced assemblers can to some extent assemble shorter arrays of tandemly organised repeats but integration of these arrays in the pseudomolecule context may still pose a substantial challenge.

The TaeCsTr99 repeat was also found underrepresented and likely misassembled in the 7D pseudomolecule of Triticum v3. Moreover, both tested wheat whole-genome assemblies failed in discriminating two similar arrays located kb apart, which could only be resolved after nanopore sequencing of BAC clones.

propdobowolfketk.tk This approach was successful not only because it employed a technology that provides reads exceeding the length of the whole array, but also because it leveraged the separation of the two arrays into the individually sequenced BAC clones. Interestingly, the identification of relatively long arrays of tandem repeats in BAC clones contradicts the finding of [ 11 ] that the tandem repeats are underrepresented in BAC libraries because of their toxicity for bacteria.

The organisation of the whole tandem arranged region was resolved thanks to the application of the OM of the 7DS arm, which provided a reference for alignment of various sequences and revealed existing gaps and misassemblies. Consequently, the repeat arrays could not be recognised in the map through a specific labelling pattern, but appeared as longer regions devoid of labels. This shortage of the method might be overcome by the application of a new approach based on CRISPR-mediated labelling of specific sequences in the context of the optical map [ 35 ], which may facilitate straightforward mapping and quantifying of any repeat of interest.

Our second target was a minor 45S ribosomal DNA locus in barley chromosome 1H, identified by in situ hybridisation in various barley cultivars [ 32 , 33 ]. Fragments of the unit were found between To investigate completeness of the sequence in this region, we aligned it to available OM of barley cv. Morex [ 2 ], which identified OM contig spanning over the region Figure 4.

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Central part of the contig did not align to the pseudomolecule and showed a regular labelling pattern with label spacing of approximately 5 kb. We compared it with the label pattern predicted for tandemly organised rDNA units. The reconstructed rDNA unit sequence comprised of three Bsp QI sites File S3 , but two of them were located just bp apart, which is too close for them to be discriminated in optical maps generated on the Bionano Genomics Irys platform.

This fragment covers the intergenic spacer IGS that comprises of two types of shorter tandem repeats with and bp unit length, respectively. It is likely that these repeats are collapsed in our consensus sequence and their real number is larger, extending the proposed IGS size by as much as 1. This hypothesis was supported by the analysis of several partial rDNA units found in the 1H pseudomolecule, which were showing for both spacer repeats a higher number than included in our rDNA consensus sequence. Our copy number estimate is close to that of [ 32 ] who quantified rRNA genes in the 1H chromosomes by in situ hybridization and proposed copies in this locus.

The slight discrepancy could be due to using a different barley cultivar Morex vs.

Co-localisation with a cluster of 45S rDNA fragments in the sequence assembly highlighted by yellow bar indicates that the array represents the 1H rDNA locus. Using the optical map, we identified a kb segment that is absent from the Morex 1H pseudomolecule. This indicates that the missing rDNA loci contribute significantly to the dark matter of the cereal genomes. In order to identify new tandem repeats specific for the short arm of wheat chromosome 7D 7DS , we randomly selected 3.

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Raw reads were examined and filtered by quality using FastQC and Trimmomatic tool. The pipeline employs graph representation of read similarities to find clusters of frequently overlapping reads corresponding to various repetitive elements or their parts.

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Putative tandem repeats were identified based on circular topology of their graphs [ 22 ] and tandem structure of contigs assembled from the reads within individual clusters. Sequences of the assembled contigs were then used to design PCR primers to verify the presence of corresponding sequences in the wheat genome Table S1. For the in situ hybridisation experiment, we employed seeds of Triticum aestivum L.

Chinese Spring, kindly provided by Dr. Seed germination, cell cycle synchronisation, metaphase accumulation and squash preparations were performed from wheat root tip meristems according to [ 38 ] with minor modifications. Metaphase accumulation was done by incubating root tips in 2.

GAA microsatellite, used for identification of wheat chromosomes, was labelled by digoxigenin. Morex SRR The graph-based clustering resulted in five clusters homologous to the barley consensus 45S rDNA. To validate sequences and analyse repeats in wheat 7DS and barley 1H chromosome, we employed available optical BNG maps constructed from 7DS chromosome arm of wheat cv. Chinese Spring [ 26 ] and the whole genome of barley cv. Morex [ 31 ], respectively. Both maps were assembled from single molecule data obtained after labelling molecules at Nt.

Comparison of the optical maps with sequences was carried out using the IrysView 2. For the alignment, cmap files were generated from fasta files of particular sequences.