BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes

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.     

SPC-P1: a pathogenicity-associated prophage of Salmonella paratyphi C

Background

The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy.

Results

The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries.

Conclusions

The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.     

The physical map of wheat chromosome 1BS provides insights into its gene space organization and evolution

Background

The wheat genome sequence is an essential tool for advanced genomic research and improvements. The generation of a high-quality wheat genome sequence is challenging due to its complex 17 Gb polyploid genome. To overcome these difficulties, sequencing through the construction of BAC-based physical maps of individual chromosomes is employed by the wheat genomics community. Here, we present the construction of the first comprehensive physical map of chromosome 1BS, and illustrate its unique gene space organization and evolution.

Results

Fingerprinted BAC clones were assembled into 57 long scaffolds, anchored and ordered with 2,438 markers, covering 83% of chromosome 1BS. The BAC-based chromosome 1BS physical map and gene order of the orthologous regions of model grass species were consistent, providing strong support for the reliability of the chromosome 1BS assembly. The gene space for chromosome 1BS spans the entire length of the chromosome arm, with 76% of the genes organized in small gene islands, accompanied by a two-fold increase in gene density from the centromere to the telomere.

Conclusions

This study provides new evidence on common and chromosome-specific features in the organization and evolution of the wheat genome, including a non-uniform distribution of gene density along the centromere-telomere axis, abundance of non-syntenic genes, the degree of colinearity with other grass genomes and a non-uniform size expansion along the centromere-telomere axis compared with other model cereal genomes. The high-quality physical map constructed in this study provides a solid basis for the assembly of a reference sequence of chromosome 1BS and for breeding applications.     

NBS-LRR sequence family is associated with leaf and stripe rust resistance on the end of homoeologous chromosome group 1S of wheat

A detailed RFLP map was constructed of the distal end of the short arm of chromosome 1D of Aegilops tauschii and wheat. At least two unrelated resistance-gene analogs (RGAs) mapped close to known leaf rust resistance genes (Lr21 and Lr40) located distal to seed storage protein genes on chromosome 1DS. One of the two RGA clones, which was previously shown to be part of a candidate gene for stripe rust resistance (Yr10) located within the homoeologous region on 1BS, identified at least three gene family members on chromosome 1DS of Ae. tauschii. One of the gene members co-segregated with the leaf rust resistance genes, Lr21 and Lr40, in Ae. tauschii and wheat segregating families. Hence, a RGA clone derived from a candidate gene for stripe rust resistance located on chromosome 1BS detected candidate genes for leaf rust resistance located in the corresponding region on 1DS of wheat. Received: 10 January 2000 / Accepted: 25 March 2000     

Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.     

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.     

Sequencing and assembly of low copy and genic regions of isolated Triticum aestivum chromosome arm 7DS

The genome of bread wheat (Triticum aestivum) is predicted to be greater than 16 Gbp in size and consist predominantly of repetitive elements, making the sequencing and assembly of this genome a major challenge. We have reduced genome sequence complexity by isolating chromosome arm 7DS and applied second‐generation technology and appropriate algorithmic analysis to sequence and assemble low copy and genic regions of this chromosome arm. The assembly represents approximately 40% of the chromosome arm and all known 7DS genes. Comparison of the 7DS assembly with the sequenced genomes of rice (Oryza sativa) and Brachypodium distachyon identified large regions of conservation. The syntenic relationship between wheat, B. distachyon and O. sativa, along with available genetic mapping data, has been used to produce an annotated draft 7DS syntenic build, which is publicly available at http://www.wheatgenome.info . Our results suggest that the sequencing of isolated chromosome arms can provide valuable information of the gene content of wheat and is a step towards whole‐genome sequencing and variation discovery in this important crop.     

A Physical Map of the Short Arm of Wheat Chromosome 1A

Bread wheat (Triticum aestivum) has a large and highly repetitive genome which poses major technical challenges for its study. To aid map-based cloning and future genome sequencing projects, we constructed a BAC-based physical map of the short arm of wheat chromosome 1A (1AS). From the assembly of 25,918 high information content (HICF) fingerprints from a 1AS-specific BAC library, 715 physical contigs were produced that cover almost 99% of the estimated size of the chromosome arm. The 3,414 BAC clones constituting the minimum tiling path were end-sequenced. Using a gene microarray containing ∼40 K NCBI UniGene EST clusters, PCR marker screening and BAC end sequences, we arranged 160 physical contigs (97 Mb or 35.3% of the chromosome arm) in a virtual order based on synteny with Brachypodium, rice and sorghum. BAC end sequences and information from microarray hybridisation was used to anchor 3.8 Mbp of Illumina sequences from flow-sorted chromosome 1AS to BAC contigs. Comparison of genetic and synteny-based physical maps indicated that ∼50% of all genetic recombination is confined to 14% of the physical length of the chromosome arm in the distal region. The 1AS physical map provides a framework for future genetic mapping projects as well as the basis for complete sequencing of chromosome arm 1AS.     

Reassessment of the evolution of wheat chromosomes 4A, 5A,and 7B

Key message

Comparison of genome sequences of wild emmer wheat and Aegilops tauschii suggests a novel scenario of the evolution of rearranged wheat chromosomes 4A, 5A, and 7B.

Abstract

Past research suggested that wheat chromosome 4A was subjected to a reciprocal translocation T(4AL;5AL)1 that occurred in the diploid progenitor of the wheat A subgenome and to three major rearrangements that occurred in polyploid wheat: pericentric inversion Inv(4AS;4AL)1, paracentric inversion Inv(4AL;4AL)1, and reciprocal translocation T(4AL;7BS)1. Gene collinearity along the pseudomolecules of tetraploid wild emmer wheat (Triticum turgidum ssp. dicoccoides, subgenomes AABB) and diploid Aegilops tauschii (genomes DD) was employed to confirm these rearrangements and to analyze the breakpoints. The exchange of distal regions of chromosome arms 4AS and 4AL due to pericentric inversion Inv(4AS;4AL)1 was detected, and breakpoints were validated with an optical Bionano genome map. Both breakpoints contained satellite DNA. The breakpoints of reciprocal translocation T(4AL;7BS)1 were also found. However, the breakpoints that generated paracentric inversion Inv(4AL;4AL)1 appeared to be collocated with the 4AL breakpoints that had produced Inv(4AS;4AL)1 and T(4AL;7BS)1. Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 either originated sequentially, and Inv(4AL;4AL)1 was produced by recurrent chromosome breaks at the same breakpoints that generated Inv(4AS;4AL)1 and T(4AL;7BS)1, or Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 originated simultaneously. We prefer the latter hypothesis since it makes fewer assumptions about the sequence of events that produced these chromosome rearrangements.

     

Whole‐genome profiling and shotgun sequencing delivers an anchored,gene‐decorated,physical map assembly of bread wheat chromosome 6A

Bread wheat (Triticum aestivum L.) is the most important staple food crop for 35% of the world's population. International efforts are underway to facilitate an increase in wheat production, of which the International Wheat Genome Sequencing Consortium (IWGSC) plays an important role. As part of this effort, we have developed a sequence‐based physical map of wheat chromosome 6A using whole‐genome profiling (WGP^?). The bacterial artificial chromosome (BAC) contig assembly tools fingerprinted contig (fpc ) and linear topological contig (ltc ) were used and their contig assemblies were compared. A detailed investigation of the contigs structure revealed that ltc created a highly robust assembly compared with those formed by fpc . The ltc assemblies contained 1217 contigs for the short arm and 1113 contigs for the long arm, with an L₅₀ of 1 Mb. To facilitate in silico anchoring, WGP^? tags underlying BAC contigs were extended by wheat and wheat progenitor genome sequence information. Sequence data were used for in silico anchoring against genetic markers with known sequences, of which almost 79% of the physical map could be anchored. Moreover, the assigned sequence information led to the ‘decoration’ of the respective physical map with 3359 anchored genes. Thus, this robust and genetically anchored physical map will serve as a framework for the sequencing of wheat chromosome 6A, and is of immediate use for map‐based isolation of agronomically important genes/quantitative trait loci located on this chromosome.     

Genetic mapping of a novel recessive allele for non-glaucousness in wild diploid wheat <Emphasis Type="Italic">Aegilops tauschii</Emphasis>: implications for the evolution of common wheat

Cuticular wax on the aerial surface of plants has a protective function against many environmental stresses. The bluish–whitish appearance of wheat leaves and stems is called glaucousness. Most modern cultivars of polyploid wheat species exhibit the glaucous phenotype, while in a wild wheat progenitor, Ae. tauschii, both glaucous and non-glaucous accessions exist. Iw2, a wax inhibitor locus on the short arm of chromosome 2D, is the main contributor to this phenotypic variation in Ae. tauschii, and the glaucous/non-glaucous phenotype of Ae. tauschii is usually inherited by synthetic hexaploid wheat. However, a few synthetic lines show the glaucous phenotype although the parental Ae. tauschii accessions are non-glaucous. Molecular marker genotypes indicate that the exceptional non-glaucous Ae. tauschii accessions share the same genotype in the Iw2 chromosomal region as glaucous accessions, suggesting that these accessions have a different causal locus for their phenotype. This locus was assigned to the long arm of chromosome 3D using an F₂ mapping population and designated W4, a novel glaucous locus in Ae. tauschii. The dominant W4 allele confers glaucousness, consistent with phenotypic observation of Ae. tauschii accessions and the derived synthetic lines. These results implied that glaucous accessions of Ae. tauschii with the W2W2iw2iw2W4W4 genotype could have been the D-genome donor of common wheat.     

A chromosome-specific sequence common to the B genome of polyploid wheat and Aegilops searsii

A low-copy, non-coding chromosome-specific DNA sequence, isolated from common wheat, was physically mapped to the distal 19% region of the long arm of chromosome 3B (3BL) of common wheat. This sequence, designated WPG118, was then characterized by Southern hybridization, PCR amplification and sequence comparison using a large collection of polyploid wheats and diploid Triticum and Aegilops species. The data show that the sequence exists in all polyploid wheats containing the B genome and absent from those containing the G genome. At the diploid level, it exists only in Ae. searsii, a diploid species of section Sitopsis, and not in other diploids including Ae. speltoides, the closest extant relative to the donor of the B genome of polyploid wheat. This finding may support the hypothesis that the B-genome of polyploid wheat is of a polyphyletic origin, i.e. it is a recombined genome derived from two or more diploid Aegilops species.     

Chromosome assignment of four photosynthesis-related genes and their variability in wheat species

Copy numbers of four photosynthesis-related genes, PhyA, Ppc, RbcS and Lhcb1 ^*1, in wheat genomes were estimated by slot-blot analysis, and these genes were assigned to the chromosome arms of common wheat by Southern hybridization of DNA from an aneuploid series of the cultivar Chinese Spring. The copy number of PhyA was estimated to be one locus per haploid genome, and this gene was assigned to chromosomes 4AL, 4BS and 4DS. The Ppc gene showed a low copy number of small multigenes, and was located on the short arm of homoeologous group 3 chromosomes and the long arm of chromosomes of homoeologous group 7. RbcS consisted of a multigene family, with approximately 100 copies in the common wheat genome, and was located on the short arm of group 2 chromosomes and the long arm of group 5 chromosomes. Lhcb1 ^*1 also consisted of a multigene family with about 50 copies in common wheat. Only a limited number of restriction fragments (approximately 15%) were used to determine the locations of members of this family on the long arm of group 1 chromosomes owing to the multiplicity of DNA bands. The variability of hybridized bands with the four genes was less in polyploids, but was more in the case of multigene families. RFLP analysis of polyploid wheats and their presumed ancestors was carried out with probes of the oat PhyA gene, the maize Ppc gene, the wheat RbcS gene and the wheat Lhcb1 ^*1 gene. The RFLP patterns of common wheat most closely resembled those of T. Dicoccum (Emmer wheat), T. urartu (A genome), Ae. speltoides (S genome) and Ae. squarrosa (D genome). Diversification of genes in the wheat complex appear to have occurred mainly at the diploid level. Based on RFLP patterns, B and S genomes were clustered into two major groups. The fragment numbers per genome were reduced in proportion to the increase of ploidy level for all four genes, suggesting that some mechanism(s) might operate to restrict, and so keep to a minimum, the gene numbers in the polyploid genomes. However, the RbcS genes, located on 2BS, were more conserved (double dosage), indicating that the above mechanism(s) does not operate equally on individual genes.     

A ‘Chinese Spring’ wheat (Triticum aestivum L.) bacterial artificial chromosome library and its use in the isolation of SSR markers for targeted genome regions

A bacterial artificial chromosome (BAC) library was constructed from the bread wheat (Triticum aestivum L.) genotype ‘Chinese Spring’ (‘CS’). The library consists of 395,136 clones with an estimated average insert size of 157 kb. This library provides an estimated 3.4-fold genome coverage for this hexaploid species. The genome coverage was confirmed by RFLP analysis of single-copy RFLP clones. The CS BAC library was used to develop simple sequence repeat (SSR) markers for targeted genome regions using five sequence-tagged-site (STS) markers designed from the chromosome arm of 3BS. The SSR markers for the targeted genome region were successfully obtained. However, similar numbers of new SSR markers were also generated for the other two homoeologous group 3 chromosomes. This data suggests that BAC clones belonging to all three chromosomes of homoeologous group 3 were isolated using the five STS primers. The potential impacts of these results on marker isolation in wheat and on library screening in general are discussed.     

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat

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.     

Mapping of loci affecting copper tolerance in wheat—The possible impact of the vernalization gene Vrn-A1

Pot experiments with copper-treated soil and a control were performed in a greenhouse to determine QTLs for copper tolerance in wheat, using deletion, introgression and single chromosome recombinant lines. Genetic and physical mapping identified loci for copper tolerance on the long arm of chromosomes 5A and 5D, while loci with minor effects were found on the long and short arms of chromosome 5B. Tests on ‘Chinese Spring’–Aegilops tauschii introgression lines revealed a locus influencing copper tolerance on chromosome 3DS. QTLs for copper tolerance on chromosome 5A were mapped genetically and physically to exactly the same position as the gene for vernalization requirement (Vrn-A1). It is therefore suggested that Vrn-A1 may have a pleiotropic effect on copper tolerance may be due to the control of Cbf genes.     

Homoeologous set of NBS-LRR genes located at leaf and stripe rust resistance loci on short arms of chromosome 1 of wheat

Homoeologous group 1 chromosomes of wheat contain important genes that confer resistance to leaf, stem and stripe rusts, powdery mildew and Russian wheat aphid. A disease resistance gene analog encoding nucleotide binding site-leucine rich repeat (NBS-LRR), designated RgaYr10, was previously identified at the stripe rust resistant locus, Yr10, located on chromosome 1BS distal to the storage protein, Gli-B1 locus. RgaYr10 identified gene members in the homoeologous region of chromosome 1DS cosegregating with the leaf rust resistance gene, Lr21, which originally was transferred from a diploid D genome progenitor. Four RgaYr10 gene members were isolated from chromosome 1DS and compared to two gene members previously isolated from the chromosome 1BS homeologue. NBS-LRR genes tightly linked to stripe rust resistance gene Yr10 on chromosome 1BS were closely related in sequence and structure to NBS-LRR genes tightly linked to leaf rust resistance gene Lr21 located within the homoeologous region on chromosome 1DS. The level of sequence homology was similar between NBS-LRR genes that were isolated from different genomes as compared to genes from the same genome. Electronic Publication     

BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements

Fluorescence in situ hybridization (FISH) has been widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts making them amenable for FISH mapping. We used BAC-FISH to study genome organization and evolution in hexaploid wheat and its relatives. We selected 56 restriction fragment length polymorphism (RFLP) locus-specific BAC clones from libraries of Aegilops tauschii (the D-genome donor of hexaploid wheat) and A-genome diploid Triticum monococcum. Different types of repetitive sequences were identified using BAC-FISH. Two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to the repetitive DNA family pAs1. In addition, we identified several novel classes of repetitive sequences: one BAC clone hybridized to the centromeric regions of wheat and other cereal species, except rice; one BAC clone hybridized to all subtelomeric chromosome regions in wheat, rye, barley and oat; one BAC clone contained a localized tandem repeat and hybridized to five D-genome chromosome pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These repeats are valuable markers for defined chromosome regions and can also be used for chromosome identification. Sequencing results revealed that all these repeats are transposable elements (TEs), indicating the important role of TEs, especially retrotransposons, in genome evolution of wheat.Communicated by P.B. Moens