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Assembling allopolyploid genomes: no longer formidable
© Ming and Man Wai; licensee BioMed Central. 2015
- Published: 31 January 2015
A combined approach of whole genome shotgun sequencing and ultra-high density linkage mapping using skim sequencing of a segregating population is effective for assembling allopolyploid genomes.
- Double Haploid
- Genome Assembly
- Oilseed Rape
- Sequencing Depth
- Wheat Genome
Polyploidy has contributed substantially to the diversification of seed plants and to the extraordinary success of angiosperms, which dominate the earth . Among seed plant species, 35% are polyploids . The ratio of allopolyploidy (from two species) and autopolyploidy (from the same species) is unknown because chromosome counting is insufficient for distinguishing these polyploid cytotypes. Autopolyploid formation is thought to be more frequent than that of allopolyploids , but allopolyploids have more advantages during the establishment phase owing to their potential for heterosis; autopolyploids also suffer from reduced fertility . This is consistent with the observation that there are more allopolyploid crops (including wheat, cotton and tobacco, strawberry, and oilseed rape) than there are autopolyploid crops (such as potato, sugarcane and banana).
Allopolyploids are formed by the hybridization of two closely related species, primarily by fertilization of two unreduced gametes or, to a lesser extent, by genome doubling after fertilization of two reduced gametes . The first challenge for assembling allopolyploid genomes is to distinguish between two closely related subgenomes of a species. A second challenge is that, although rare, translocations, duplication and crossovers between homeologous chromosomes (that is, partially homologous chromosomes) do occur. This embedding of fragments of one subgenome into another could result in misassembly of different homeologous chromosomes into one mosaic artificial chromosome. Third, a common feature of plant genomes is that there is often an over-dominance of retrotransposons, which could copy and paste among homeologous chromosomes and potentially cause misassembly.
In this issue of Genome Biology, Chapman and colleagues  have presented a novel integrative approach to generate a genome assembly of the hexaploid bread wheat genome. Their method combines whole genome shotgun sequencing with ultra-high density linkage mapping achieved by skim sequencing, and the resulting genome quality exceeds that of chromosome arms-based assembly .
In light of the challenges described above, the whole genome shotgun approach appears not to be applicable for assembling allopolyploid genomes. Traditionally, the approach for sequencing allopolyploid genomes of crop plants has been to sequence progenitor diploid genomes, as done for cotton, strawberry, coffee and oilseed rape. However, the progenitor genomes of the 17 gigabase (Gb) wheat genome are larger than any of the allopolyploid genomes mentioned above, and sequencing any of the three 5.5 Gb progenitor genomes requires substantial investment . The wheat genome comprises 21 large and distinguishable chromosomes, and the wheat community took an approach of sorting each chromosome or chromosome arm for sequencing and assembly . Using this approach it is possible to eliminate misassembly of homeologous chromosomes. However, chromosome sorting does not yield sufficient quantities of DNA for high-throughput sequencing using Illumina technology. It is therefore necessary to amplify each chromosome or arm in short fragments, making it impossible to construct large-insert jumping libraries for scaffolding, which results in short contigs in the assembled genome . This makes subsequent genomic research less efficient.
These challenges demonstrate the significance of the Genome Biology study by Chapman and colleagues, which combined whole genome shotgun sequencing and ultra-high density linkage mapping to assemble an allopolyploid genome. ‘Synthetic W7984’ was generated by crossing a tetraploid wheat AABB genome with the diploid DD genome, followed by chromosome doubling, resulting in a contemporary reconstitution of hexaploid wheat. This homozygous line was sequenced at 30× coverage using a whole genome shotgun approach with 2 × 150 base pair (bp) sequences of Illumina TruSeq libraries in paired-end and mate pairs of 250 bp to 4.5 kb in size . The genome of ‘W7984’ was assembled using an enhanced version of Meraculous, a new algorithm for de novo genome assembly with deep paired-end short reads . Analysis of 51-mers revealed that no genomic features were present in double or triple copy, indicating the three sets of homeologous chromosomes were separated in the genome assembly. Simulation of 81-mer sequences yielded substantially higher fractions of unique sequences in the genome than that of 51-mers, implying that increasing the sequencing depth further improved the quality of the assembled genome. Identical exons were assembled into the correct subgenomes using information from more divergent flanking intronic and intergenic sequences, a key feature of allopolyploid genomes.
The combination of whole genome shotgun sequencing and linkage mapping by skim sequencing produced a better genome assembly than both the chromosome arm-based assembly and a previously described whole genome shotgun sequencing assembly approach with 5× 454 sequences (limited by low sequencing depth) . Increasing sequencing depth from the current 30× to 90× that is normally practiced for whole genome sequencing using short reads would further improve the assembly. Even with the current fragmented assembly, it serves as a working reference for mining target genes and for genome-wide association studies through re-sequencing of a collection of diverse germplasm.
This method is not only applicable and beneficial for other large allopolyploid genomes like rye and oats. It should also be applied to draft genome sequencing of all species, both diploid and polyploid. Genotyping by skim sequencing of individual genomes is affordable and the sequencing depth could be increased from 0.2× in rice and 1.4× in wheat to 2× to 4× in any genome [4,10]. The increased depth would not enhance the map resolution, but it would increase power and accuracy for the correction of misassembled scaffolds.
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