Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication-related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.

• Klíčová slova
• 7DL chromosome arm, BAC by BAC, domestication, gene loss, physical mapping, wheat,
• MeSH
• Aegilops genetika   MeSH
• biologická evoluce * MeSH
• chromozomy rostlin genetika   MeSH
• genom rostlinný * MeSH
• lokus kvantitativního znaku MeSH
• pšenice genetika   MeSH
• srovnávací genomová hybridizace MeSH
• syntenie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Reference genomes of important cereals, including barley, emmer wheat and bread wheat, were released recently. Their comparison with genome size estimates obtained by flow cytometry indicated that the assemblies represent not more than 88-98% of the complete genome. This work is aimed at identifying the missing parts in two cereal genomes and proposing techniques to make the assemblies more complete. We focused on tandemly organised repetitive sequences, known to be underrepresented in genome assemblies generated from short-read sequence data. Our study found arrays of three tandem repeats with unit sizes of 1242 to 2726 bp present in the bread wheat reference genome generated from short reads. However, this and another wheat genome assembly employing long PacBio reads failed in integrating correctly the 2726-bp repeat in the pseudomolecule context. This suggests that tandem repeats of this size, frequently incorporated in unassigned scaffolds, may contribute to shrinking of pseudomolecules without reducing size of the entire assembly. We demonstrate how this missing information may be added to the pseudomolecules with the aid of nanopore sequencing of individual BAC clones and optical mapping. Using the latter technique, we identified and localised a 470-kb long array of 45S ribosomal DNA absent from the reference genome of barley.

• Klíčová slova
• BAC, barley, bread wheat, genome assembly, optical mapping, ribosomal DNA,
• MeSH
• chromozomy rostlin genetika   MeSH
• genom rostlinný * MeSH
• ječmen (rod) genetika   MeSH
• pšenice genetika   MeSH
• tandemové repetitivní sekvence * MeSH
• Publikační typ
• časopisecké články MeSH

A segment of Triticum militinae chromosome 7G harbors a gene(s) conferring powdery mildew resistance which is effective at both the seedling and the adult plant stages when transferred into bread wheat (T. aestivum). The introgressed segment replaces a piece of wheat chromosome arm 4AL. An analysis of segregating materials generated to positionally clone the gene highlighted that in a plant heterozygous for the introgression segment, only limited recombination occurs between the introgressed region and bread wheat 4A. Nevertheless, 75 genetic markers were successfully placed within the region, thereby confining the gene to a 0.012 cM window along the 4AL arm. In a background lacking the Ph1 locus, the localized rate of recombination was raised 33-fold, enabling the reduction in the length of the region containing the resistance gene to a 480 kbp stretch harboring 12 predicted genes. The substituted segment in the reference sequence of bread wheat cv. Chinese Spring is longer (640 kbp) and harbors 16 genes. A comparison of the segments' sequences revealed a high degree of divergence with respect to both their gene content and nucleotide sequence. Of the 12 T. militinae genes, only four have a homolog in cv. Chinese Spring. Possible candidate genes for the resistance have been identified based on function predicted from their sequence.

• MeSH
• anotace sekvence MeSH
• Ascomycota fyziologie   MeSH
• chléb MeSH
• chromozomy rostlin genetika   MeSH
• genetická variace * MeSH
• genetické lokusy * MeSH
• klonování DNA MeSH
• mapování chromozomů MeSH
• nemoci rostlin genetika   imunologie   mikrobiologie   MeSH
• odolnost vůči nemocem genetika   MeSH
• pšenice genetika   imunologie   mikrobiologie   MeSH
• rostlinné geny * MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The IWGSC strategy for construction of the reference sequence of the bread wheat genome is based on first obtaining physical maps of the individual chromosomes. Our aim is to develop and use the physical map for analysis of the organization of the short arm of wheat chromosome 5B (5BS) which bears a number of agronomically important genes, including genes conferring resistance to fungal diseases. RESULTS: A physical map of the 5BS arm (290 Mbp) was constructed using restriction fingerprinting and LTC software for contig assembly of 43,776 BAC clones. The resulting physical map covered ~ 99% of the 5BS chromosome arm (111 scaffolds, N50 = 3.078 Mb). SSR, ISBP and zipper markers were employed for anchoring the BAC clones, and from these 722 novel markers were developed based on previously obtained data from partial sequencing of 5BS. The markers were mapped using a set of Chinese Spring (CS) deletion lines, and F2 and RICL populations from a cross of CS and CS-5B dicoccoides. Three approaches have been used for anchoring BAC contigs on the 5BS chromosome, including clone-by-clone screening of BACs, GenomeZipper analysis, and comparison of BAC-fingerprints with in silico fingerprinting of 5B pseudomolecules of T. dicoccoides. These approaches allowed us to reach a high level of BAC contig anchoring: 96% of 5BS BAC contigs were located on 5BS. An interesting pattern was revealed in the distribution of contigs along the chromosome. Short contigs (200-999 kb) containing markers for the regions interrupted by tandem repeats, were mainly localized to the 5BS subtelomeric block; whereas the distribution of larger 1000-3500 kb contigs along the chromosome better correlated with the distribution of the regions syntenic to rice, Brachypodium, and sorghum, as detected by the Zipper approach. CONCLUSION: The high fingerprinting quality, LTC software and large number of BAC clones selected by the informative markers in screening of the 43,776 clones allowed us to significantly increase the BAC scaffold length when compared with the published physical maps for other wheat chromosomes. The genetic and bioinformatics resources developed in this study provide new possibilities for exploring chromosome organization and for breeding applications.

• Klíčová slova
• Chromosome 5BS, Genetic markers, Hexaploid wheat, Physical mapping, Sequencing, Synteny, Triticum aestivum,
• MeSH
• chléb * MeSH
• chromozomy rostlin genetika   MeSH
• fyzikální mapování chromozomů * MeSH
• polymerázová řetězová reakce MeSH
• pšenice genetika   MeSH
• umělé bakteriální chromozomy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules.

• Klíčová slova
• chromosomes, flow sorting, optical mapping, physical map, sequencing, wheat,
• MeSH
• biotechnologie metody   MeSH
• chromozomy rostlin genetika   MeSH
• genom rostlinný * MeSH
• mapování chromozomů metody   MeSH
• pšenice genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• tandemové repetitivní sekvence MeSH
• umělé bakteriální chromozomy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. RESULTS: We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. CONCLUSIONS: We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

• Klíčová slova
• 7DS, Assembly, BAC, Next-generation sequencing, SASSY, Saccharum spp, Triticum aestivum,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

• MeSH
• chromozomy rostlin MeSH
• fyzikální mapování chromozomů metody   MeSH
• genetické markery MeSH
• genová přestavba MeSH
• molekulární evoluce MeSH
• organizátor jadérka MeSH
• pořadí genů MeSH
• pšenice genetika   MeSH
• umělé bakteriální chromozomy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genetické markery MeSH

Making use of wheat chromosomal resources, we developed 11 gene-associated markers for the region of interest, which allowed reducing gene interval and spanning it by four BAC clones. Positional gene cloning and targeted marker development in bread wheat are hampered by high complexity and polyploidy of its nuclear genome. Aiming to clone a Russian wheat aphid resistance gene Dn2401 located on wheat chromosome arm 7DS, we have developed a strategy overcoming problems due to polyploidy and enabling efficient development of gene-associated markers from the region of interest. We employed information gathered by GenomeZipper, a synteny-based tool combining sequence data of rice, Brachypodium, sorghum and barley, and took advantage of a high-density linkage map of Aegilops tauschii. To ensure genome- and locus-specificity of markers, we made use of survey sequence assemblies of isolated wheat chromosomes 7A, 7B and 7D. Despite the low level of polymorphism of the wheat D subgenome, our approach allowed us to add in an efficient and cost-effective manner 11 new gene-associated markers in the Dn2401 region and narrow down the target interval to 0.83 cM. Screening 7DS-specific BAC library with the flanking markers revealed a contig of four BAC clones that span the Dn2401 region in wheat cultivar 'Chinese Spring'. With the availability of sequence assemblies and GenomeZippers for each of the wheat chromosome arms, the proposed strategy can be applied for focused marker development in any region of the wheat genome.

• MeSH
• býložravci MeSH
• chromozomy rostlin MeSH
• DNA primery MeSH
• DNA rostlinná genetika   MeSH
• genetická vazba MeSH
• genetické markery MeSH
• genomika MeSH
• jednonukleotidový polymorfismus MeSH
• mapování chromozomů * MeSH
• mikrosatelitní repetice MeSH
• mšice * MeSH
• pšenice genetika   MeSH
• rostlinné geny * MeSH
• syntenie MeSH
• umělé bakteriální chromozomy MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Rusko MeSH
• Názvy látek
• DNA primery MeSH
• DNA rostlinná MeSH
• genetické markery MeSH

BACKGROUND: The substantially large bread wheat genome, organized into highly similar three sub-genomes, renders genomic research challenging. The construction of BAC-based physical maps of individual chromosomes reduces the complexity of this allohexaploid genome, enables elucidation of gene space and evolutionary relationships, provides tools for map-based cloning, and serves as a framework for reference sequencing efforts. In this study, we constructed the first comprehensive physical map of wheat chromosome arm 5DS, thereby exploring its gene space organization and evolution. RESULTS: The physical map of 5DS was comprised of 164 contigs, of which 45 were organized into 21 supercontigs, covering 176 Mb with an N50 value of 2,173 kb. Fifty-eight of the contigs were larger than 1 Mb, with the largest contig spanning 6,649 kb. A total of 1,864 molecular markers were assigned to the map at a density of 10.5 markers/Mb, anchoring 100 of the 120 contigs (>5 clones) that constitute ~95 % of the cumulative length of the map. Ordering of 80 contigs along the deletion bins of chromosome arm 5DS revealed small-scale breaks in syntenic blocks. Analysis of the gene space of 5DS suggested an increasing gradient of genes organized in islands towards the telomere, with the highest gene density of 5.17 genes/Mb in the 0.67-0.78 deletion bin, 1.4 to 1.6 times that of all other bins. CONCLUSIONS: Here, we provide a chromosome-specific view into the organization and evolution of the D genome of bread wheat, in comparison to one of its ancestors, revealing recent genome rearrangements. The high-quality physical map constructed in this study paves the way for the assembly of a reference sequence, from which breeding efforts will greatly benefit.

• MeSH
• chromozomy rostlin MeSH
• DNA rostlinná analýza   MeSH
• duplikace genu * MeSH
• fyzikální mapování chromozomů metody   MeSH
• genová přestavba * MeSH
• kontigové mapování metody   MeSH
• molekulární evoluce MeSH
• pšenice genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH

Improved knowledge of genome composition, especially of its repetitive component, generates important information for both theoretical and applied research. The olive repetitive component is made up of two main classes of sequences: tandem repeats and retrotransposons (REs). In this study, we provide characterization of a sample of 254 unique full-length long terminal repeat (LTR) REs. In the sample, Ty1-Copia elements were more numerous than Ty3-Gypsy elements. Mapping a large set of Illumina whole-genome shotgun reads onto the identified retroelement set revealed that Gypsy elements are more redundant than Copia elements. The insertion time of intact retroelements was estimated based on sister LTR's divergence. Although some elements inserted relatively recently, the mean insertion age of the isolated retroelements is around 18 million yrs. Gypsy and Copia retroelements showed different waves of transposition, with Gypsy elements especially active between 10 and 25 million yrs ago and nearly inactive in the last 7 million yrs. The occurrence of numerous solo-LTRs related to isolated full-length retroelements was ascertained for two Gypsy elements and one Copia element. Overall, the results reported in this study show that RE activity (both retrotransposition and DNA loss) has impacted the olive genome structure in more ancient times than in other angiosperms.

• Klíčová slova
• BAC sequencing, LTR retrotransposons, insertion age, next-generation sequencing, olive,
• MeSH
• celogenomová asociační studie * MeSH
• koncové repetice * MeSH
• mapování chromozomů * MeSH
• Olea genetika   MeSH
• retroelementy * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• retroelementy * MeSH