Assembly Of Whole-Chromosome Pseudomolecules For Polyploid Plant Genomes Using Outcrossed Mapping Populations (original) (raw)
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Accurate and in-phase de novo assembly of highly polymorphic diploid and polyploid plant genomes remains a critical yet unsolved problem. “Out-of-the-box” assemblies on such data can produce numerous small con- tigs, at lower than expected coverage, which are hypothe- sized to represent sequences that are not uniformly present on all copies of a homologous set of chromosomes. Such “heterotigs” are not routinely identified in current assem- bly algorithms and could be used for haplotype phasing and other assembly improvements for such genomes. We introduce an algorithm which attempts to robustly identify heterotigs present in the assembly of a highly polymorphic diploid organism. The algorithm presented is for use with the 454 platform and for diploid assembly, but is readily adaptable to other sequencing platforms and to polyploid assembly.
Plant biotechnology journal, 2016
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 a...
The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker2based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new 936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal "pseudomolecules".
BMC Genomics, 2010
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-informationcontent-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.
Strategies for Sequence Assembly of Plant Genomes
Plant Genomics, 2016
The field of plant genome assembly has greatly benefited from the development and widespread adoption of next-generation DNA sequencing platforms. Very high sequencing throughputs and low costs per nucleotide have considerably reduced the technical and budgetary constraints associated with early assembly projects done primarily with a traditional Sanger-based approach. Those improvements led to a sharp increase in the number of plant genomes being sequenced, including large and complex genomes of economically important crops. Although next-generation DNA sequencing has considerably improved our understanding of the overall structure and dynamics of many plant genomes, severe limitations still remain because next-generation DNA sequencing reads typically are shorter than Sanger reads. In addition, the software tools used to de novo assemble sequences are not necessarily designed to optimize the use of short reads. These cause challenges, common to many plant species with large genome sizes, high repeat contents, polyploidy and genome-wide duplications. This chapter provides an overview of historical and current methods used to sequence and assemble plant genomes, along with new solutions offered by the emergence of technologies such as single molecule sequencing and optical mapping to address the limitations of current sequence assemblies.
Chromosome-scale reference genome assembly of a diploid potato clone derived from an elite variety
G3 Genes|Genomes|Genetics, 2021
Potato (Solanum tuberosum L.) is one of the most important crops with a worldwide production of 370 million metric tons. The objectives of this study were (1) to create a high-quality consensus sequence across the two haplotypes of a diploid clone derived from a tetraploid elite variety and assess the sequence divergence from the available potato genome assemblies, as well as among the two haplotypes; (2) to evaluate the new assembly’s usefulness for various genomic methods; and (3) to assess the performance of phasing in diploid and tetraploid clones, using linked-read sequencing technology. We used PacBio long reads coupled with 10x Genomics reads and proximity ligation scaffolding to create the dAg1_v1.0 reference genome sequence. With a final assembly size of 812 Mb, where 750 Mb are anchored to 12 chromosomes, our assembly is larger than other available potato reference sequences and high proportions of properly paired reads were observed for clones unrelated by pedigree to dAg...
A sequence-based genetic linkage map as a reference for Brassica rapa pseudochromosome assembly
BMC Genomics, 2011
Background: Brassica rapa is an economically important crop and a model plant for studies concerning polyploidization and the evolution of extreme morphology. The multinational B. rapa Genome Sequencing Project (BrGSP) was launched in 2003. In 2008, next generation sequencing technology was used to sequence the B. rapa genome. Several maps concerning B. rapa pseudochromosome assembly have been published but their coverage of the genome is incomplete, anchoring approximately 73.6% of the scaffolds on to chromosomes. Therefore, a new genetic map to aid pseudochromosome assembly is required. Results: This study concerns the construction of a reference genetic linkage map for Brassica rapa, forming the backbone for anchoring sequence scaffolds of the B. rapa genome resulting from recent sequencing efforts. One hundred and nineteen doubled haploid (DH) lines derived from microspore cultures of an F1 cross between a Chinese cabbage (B. rapa ssp. pekinensis) DH line (Z16) and a rapid cycling inbred line (L144) were used to construct the linkage map. PCR-based insertion/deletion (InDel) markers were developed by re-sequencing the two parental lines. The map comprises a total of 507 markers including 415 InDels and 92 SSRs. Alignment and orientation using SSR markers in common with existing B. rapa linkage maps allowed ten linkage groups to be identified, designated A01-A10. The total length of the linkage map was 1234.2 cM, with an average distance of 2.43 cM between adjacent marker loci. The lengths of linkage groups ranged from 71.5 cM to 188.5 cM for A08 and A09, respectively. Using the developed linkage map, 152 scaffolds were anchored on to the chromosomes, encompassing more than 82.9% of the B. rapa genome. Taken together with the previously available linkage maps, 183 scaffolds were anchored on to the chromosomes and the total coverage of the genome was 88.9%. Conclusions: The development of this linkage map is vital for the integration of genome sequences and genetic information, and provides a useful resource for the international Brassica research community.
Sweetpotato (Ipomoea batatas(L.) Lam) is the world’s seventh most important food crop by production quantity. Cultivated sweetpotato is a hexaploid (2n = 6x = 90), and its genome (B1B1B2B2B2B2) is quite complex due to polyploidy, self-incompatibility, and high heterozygosity. Here we established a haploid-resolved and chromosome-scalede novoassembly of autohexaploid sweetpotato genome sequences. Before constructing the genome, we created chromosome-scale genome sequences inI. trifidausing a highly homozygous accession, Mx23Hm, with PacBio RSII and Hi-C reads. Haploid-resolved genome assembly was performed for a sweetpotato cultivar, Xushu18 by hybrid assembly with Illumina paired-end (PE) and mate-pair (MP) reads, 10X genomics reads, and PacBio RSII reads. Then, 90 chromosome-scale pseudomolecules were generated by aligning the scaffolds onto a sweetpotato linkage map.De novoassemblies were also performed for chloroplast and mitochondrial genomes inI. trifidaand sweetpotato. In tota...