Paul Visendi

Discovery of Single Nucleotide Polymorphisms in Complex Genomes Using SGSautoSNP

Single nucleotide polymorphisms (SNPs) are becoming the dominant form of molecular marker for genetic and genomic analysis. The advances in second generation DNA sequencing provide opportunities to identify very large numbers of SNPs in a range of species. However, SNP identification remains a challenge for large and polyploid genomes due to their size and complexity. We have developed a pipeline for the robust identification of SNPs in large and complex genomes using Illumina second generation DNA sequence data and demonstrated this by the discovery of SNPs in the hexaploid wheat genome. We have developed a SNP discovery pipeline called SGSautoSNP (Second-Generation Sequencing AutoSNP) and applied this to discover more than 800,000 SNPs between four hexaploid wheat cultivars across chromosomes 7A, 7B and 7D. All SNPs are presented for download and viewing within a public GBrowse database. Validation suggests an accuracy of greater than 93% of SNPs represent polymorphisms between wheat cultivars and hence are valuable for detailed diversity analysis, marker assisted selection and genotyping by sequencing. The pipeline produces output in GFF3, VCF, Flapjack or Illumina Infinium design format for further genotyping diverse populations. As well as providing an unprecedented resource for wheat diversity analysis, the method establishes a foundation for high resolution SNP discovery in other large and complex genomes.

Dispersion and domestication shaped the genome of bread wheat

Despite the international significance of wheat, its large and complex genome hinders genome sequencing efforts. To assess the impact of selection on this genome, we have assembled genomic regions representing genes for chromosomes 7A, 7B and 7D. We demonstrate that the dispersion of wheat to new environments has shaped the modern wheat genome. Most genes are conserved between the three homoeologous chromosomes. We found differential gene loss that supports current theories on the evolution of wheat, with greater loss observed in the A and B genomes compared with the D. Analysis of intervarietal polymorphisms identified fewer polymorphisms in the D genome, supporting the hypothesis of early gene flow between the tetraploid and hexaploid. The enrichment for genes on the D genome that confer environmental adaptation may be associated with dispersion following wheat domestication. Our results demonstrate the value of applying next-generation sequencing technologies to assemble gene-rich regions of complex genomes and investigate polyploid genome evolution. We anticipate the genome-wide application of this reduced-complexity syntenic assembly approach will accelerate crop improvement efforts not only in wheat, but also in other polyploid crops of significance.

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

Summary 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.

A chromosomal genomics approach to assess and validate the desi and kabuli draft chickpea genome assemblies

With the expansion of next-generation sequencing technology and advanced bioinformatics, there has been a rapid growth of genome sequencing projects. However, while this technology enables the rapid and cost-effective assembly of draft genomes, the quality of these assemblies usually falls short of gold standard genome assemblies produced using the more traditional BAC by BAC and Sanger sequencing approaches. Assembly validation is often performed by the physical anchoring of genetically mapped markers, but this is prone to errors and the resolution is usually low, especially towards centromeric regions where recombination is limited. New approaches are required to validate reference genome assemblies. The ability to isolate individual chromosomes combined with next-generation sequencing permits the validation of genome assemblies at the chromosome level. We demonstrate this approach by the assessment of the recently published chickpea kabuli and desi genomes. While previous genetic analysis suggests that these genomes should be very similar, a comparison of their chromosome sizes and published assemblies highlights significant differences. Our chromosomal genomics analysis highlights short defined regions that appear to have been misassembled in the kabuli genome and identifies large-scale misassembly in the draft desi genome. The integration of chromosomal genomics tools within genome sequencing projects has the potential to significantly improve the construction and validation of genome assemblies. The approach could be applied both for new genome assemblies as well as published assemblies, and complements currently applied genome assembly strategies.

High-resolution skim genotyping by sequencing reveals the distribution of crossovers and gene conversions in Cicer arietinum and Brassica napus

Key messageWe characterise the distribution of crossover and non-crossover recombination inBrassica napusandCicer arietinumusing a low-coverage genotyping by sequencing pipeline SkimGBS.AbstractThe growth of next-generation DNA sequencing technologies has led to a rapid increase in sequence-based genotyping for applications including diversity assessment, genome structure validation and gene–trait association. We have established a skim-based genotyping by sequencing method for crop plants and applied this approach to genotype-segregating populations of Brassica napus and Cicer arietinum. Comparison of progeny genotypes with those of the parental individuals allowed the identification of crossover and non-crossover (gene conversion) events. Our results identify the positions of recombination events with high resolution, permitting the mapping and frequency assessment of recombination in segregating populations.

Identification and characterization of more than 4 million intervarietal SNPs across the group 7 chromosomes of bread wheat

Despite being a major international crop, our understanding of the wheat genome is relatively poor due to its large size and complexity. To gain a greater understanding of wheat genome diversity, we have identified single nucleotide polymorphisms between 16 Australian bread wheat varieties. Whole-genome shotgun Illumina paired read sequence data were mapped to the draft assemblies of chromosomes 7A, 7B and 7D to identify more than 4 million intervarietal SNPs. SNP density varied between the three genomes, with much greater density observed on the A and B genomes than the D genome. This variation may be a result of substantial gene flow from the tetraploid Triticum turgidum, which possesses A and B genomes, during early co-cultivation of tetraploid and hexaploid wheat. In addition, we examined SNP density variation along the chromosome syntenic builds and identified genes in low-density regions which may have been selected during domestication and breeding. This study highlights the impact of evolution and breeding on the bread wheat genome and provides a substantial resource for trait association and crop improvement. All SNP data are publically available on a generic genome browser GBrowse at www.wheatgenome.info.

Gene loss in the fungal canola pathogen Leptosphaeria maculans

Recent comparisons of the increasing number of genome sequences have revealed that variation in gene content is considerably more prevalent than previously thought. This variation is likely to have a pronounced effect on phenotypic diversity and represents a crucial target for the assessment of genomic diversity. Leptosphaeria maculans, a causative agent of phoma stem canker, is the most devastating fungal pathogen of Brassica napus (oilseed rape/canola). A number of L. maculans genes are known to be present in some isolates but lost in the others. We analyse gene content variation within three L. maculans isolates using a hybrid mapping and genome assembly approach and identify genes which are present in one of the isolates but missing in the others. In total, 57 genes are shown to be missing in at least one isolate. The genes encode proteins involved in a range of processes including oxidative processes, DNA maintenance, cell signalling and sexual reproduction. The results demonstrate the effectiveness of the method and provide new insight into genomic diversity in L. maculans.

Next Generation Characterisation of Cereal Genomes for Marker Discovery

Cereal crops form the bulk of the world’s food sources, and thus their importance cannot be understated. Crop breeding programs increasingly rely on high-resolution molecular genetic markers to accelerate the breeding process. The development of these markers is hampered by the complexity of some of the major cereal crop genomes, as well as the time and cost required. In this review, we address current and future methods available for the characterisation of cereal genomes, with an emphasis on faster and more cost effective approaches for genome sequencing and the development of markers for trait association and marker assisted selection (MAS) in crop breeding programs.

Complete genome sequence of a new member of the genus Badnavirus, Dioscorea bacilliform RT virus 3, reveals the first evidence of recombination in yam badnaviruses

Yams (Dioscorea spp.) host a diverse range of badnaviruses (genus Badnavirus, family Caulimoviridae). The first complete genome sequence of Dioscorea bacilliform RT virus 3 (DBRTV3), which belongs to the monophyletic species group K5, is described. This virus is most closely related to Dioscorea bacilliform SN virus (DBSNV, group K4) based on a comparison of genome sequences. Recombination analysis identified a unique recombination event in DBRTV3, with DBSNV likely to be the major parent and Dioscorea bacilliform AL virus (DBALV) the minor parent, providing the first evidence for recombination in yam badnaviruses. This has important implications for yam breeding programmes globally.

An efficient approach to BAC based assembly of complex genomes

BackgroundThere 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.ResultsWe 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.ConclusionsWe demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.