Paul J. Berkman

Single nucleotide polymorphism discovery from wheat next-generation sequence data.

Single nucleotide polymorphisms (SNPs) are the most abundant type of molecular genetic marker and can be used for producing high-resolution genetic maps, marker-trait association studies and marker-assisted breeding. Large polyploid genomes such as wheat present a challenge for SNP discovery because of the potential presence of multiple homoeologs for each gene. AutoSNPdb has been successfully applied to identify SNPs from Sanger sequence data for several species, including barley, rice and Brassica, but the volume of data required to accurately call SNPs in the complex genome of wheat has prevented its application to this important crop. DNA sequencing technology has been revolutionized by the introduction of next-generation sequencing, and it is now possible to generate several million sequence reads in a timely and cost-effective manner. We have produced wheat transcriptome sequence data using 454 sequencing technology and applied this for SNP discovery using a modified autoSNPdb method, which integrates SNP and gene annotation information with a graphical viewer. A total of 4,694,141 sequence reads from three bread wheat varieties were assembled to identify a total of 38 928 candidate SNPs. Each SNP is within an assembly complete with annotation, enabling the selection of polymorphism within genes of interest.

Sequencing wheat chromosome arm 7BS delimits the 7BS/4AL translocation and reveals homoeologous gene conservation

Complex Triticeae genomes pose a challenge to genome sequencing efforts due to their size and repetitive nature. Genome sequencing can reveal details of conservation and rearrangements between related genomes. We have applied Illumina second generation sequencing technology to sequence and assemble the low copy and unique regions of Triticum aestivum chromosome arm 7BS, followed by the construction of a syntenic build based on gene order in Brachypodium. We have delimited the position of a previously reported translocation between 7BS and 4AL with a resolution of one or a few genes and report approximately 13% genes from 7BS having been translocated to 4AL. An additional 13 genes are found on 7BS which appear to have originated from 4AL. The gene content of the 7DS and 7BS syntenic builds indicate a total of ~77,000 genes in wheat. Within wheat syntenic regions, 7BS and 7DS share 740 genes and a common gene conservation rate of ~39% of the genes from the corresponding regions in Brachypodium, as well as a common rate of colinearity with Brachypodium of ~60%. Comparison of wheat homoeologues revealed ~84% of genes previously identified in 7DS have a homoeologue on 7BS or 4AL. The conservation rates we have identified among wheat homoeologues and with Brachypodium provide a benchmark of homoeologous gene conservation levels for future comparative genomic analysis. The syntenic build of 7BS is publicly available at http://www.wheatgenome.info.

Next-generation sequencing applications for wheat crop improvement

Bread wheat (Triticum aestivum; Poaceae) is a crop plant of great importance. It provides nearly 20% of the worlds daily food supply measured by calorie intake, similar to that provided by rice. The yield of wheat has doubled over the last 40 years due to a combination of advanced agronomic practice and improved germplasm through selective breeding. More recently, yield growth has been less dramatic, and a significant improvement in wheat production will be required if demand from the growing human population is to be met. Next-generation sequencing (NGS) technologies are revolutionizing biology and can be applied to address critical issues in plant biology. Technologies can produce draft sequences of genomes with a significant reduction to the cost and timeframe of traditional technologies. In addition, NGS technologies can be used to assess gene structure and expression, and importantly, to identify heritable genome variation underlying important agronomic traits. This review provides an overview of the wheat genome and NGS technologies, details some of the problems in applying NGS technology to wheat, and describes how NGS technologies are starting to impact wheat crop improvement.

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.

Future tools for association mapping in crop plants.

Association mapping currently relies on the identification of genetic markers. Several technologies have been adopted for genetic marker analysis, with single nucleotide polymorphisms (SNPs) being the most popular where a reasonable quantity of genome sequence data are available. We describe several tools we have developed for the discovery, annotation, and visualization of molecular markers for association mapping. These include autoSNPdb for SNP discovery from assembled sequence data; TAGdb for the identification of gene specific paired read Illumina GAII data; CMap3D for the comparison of mapped genetic and physical markers; and BAC and Gene Annotator for the online annotation of genes and genomic sequences.

Complete genome sequence of sporisorium scitamineum and biotrophic interaction transcriptome with sugarcane

Sporisorium scitamineum is a biotrophic fungus responsible for the sugarcane smut, a worldwide spread disease. This study provides the complete sequence of individual chromosomes of S. scitamineum from telomere to telomere achieved by a combination of PacBio long reads and Illumina short reads sequence data, as well as a draft sequence of a second fungal strain. Comparative analysis to previous available sequences of another strain detected few polymorphisms among the three genomes. The novel complete sequence described herein allowed us to identify and annotate extended subtelomeric regions, repetitive elements and the mitochondrial DNA sequence. The genome comprises 19,979,571 bases, 6,677 genes encoding proteins, 111 tRNAs and 3 assembled copies of rDNA, out of our estimated number of copies as 130. Chromosomal reorganizations were detected when comparing to sequences of S. reilianum, the closest smut relative, potentially influenced by repeats of transposable elements. Repetitive elements may have also directed the linkage of the two mating-type loci. The fungal transcriptome profiling from in vitro and from interaction with sugarcane at two time points (early infection and whip emergence) revealed that 13.5% of the genes were differentially expressed in planta and particular to each developmental stage. Among them are plant cell wall degrading enzymes, proteases, lipases, chitin modification and lignin degradation enzymes, sugar transporters and transcriptional factors. The fungus also modulates transcription of genes related to surviving against reactive oxygen species and other toxic metabolites produced by the plant. Previously described effectors in smut/plant interactions were detected but some new candidates are proposed. Ten genomic islands harboring some of the candidate genes unique to S. scitamineum were expressed only in planta. RNAseq data was also used to reassure gene predictions.

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.

WheatGenome.info: An Integrated Database and Portal for Wheat Genome Information

Bread wheat (Triticum aestivum) is one of the most important crop plants, globally providing staple food for a large proportion of the human population. However, improvement of this crop has been limited due to its large and complex genome. Advances in genomics are supporting wheat crop improvement. We provide a variety of web-based systems hosting wheat genome and genomic data to support wheat research and crop improvement. WheatGenome.info is an integrated database resource which includes multiple web-based applications. These include a GBrowse2-based wheat genome viewer with BLAST search portal, TAGdb for searching wheat second-generation genome sequence data, wheat autoSNPdb, links to wheat genetic maps using CMap and CMap3D, and a wheat genome Wiki to allow interaction between diverse wheat genome sequencing activities. This system includes links to a variety of wheat genome resources hosted at other research organizations. This integrated database aims to accelerate wheat genome research and is freely accessible via the web interface at http://www.wheatgenome.info/.

Sequence-Based Analysis of Translocations and Inversions in Bread Wheat (Triticum aestivum L.)

Structural changes of chromosomes are a primary mechanism of genome rearrangement over the course of evolution and detailed knowledge of such changes in a given species and its close relatives should increase the efficiency and precision of chromosome engineering in crop improvement. We have identified sequences bordering each of the main translocation and inversion breakpoints on chromosomes 4A, 5A and 7B of the modern bread wheat genome. The locations of these breakpoints allow, for the first time, a detailed description of the evolutionary origins of these chromosomes at the gene level. Results from this study also demonstrate that, although the strategy of exploiting sorted chromosome arms has dramatically simplified the efforts of wheat genome sequencing, simultaneous analysis of sequences from homoeologous and non-homoeologous chromosomes is essential in understanding the origins of DNA sequences in polyploid species.

Targeted identification of genomic regions using TAGdb

BackgroundThe introduction of second generation sequencing technology has enabled the cost effective sequencing of genomes and the identification of large numbers of genes and gene promoters. However, the assembly of DNA sequences to create a representation of the complete genome sequence remains costly, especially for the larger and more complex plant genomes.ResultsWe have developed an online database, TAGdb, that enables researchers to identify paired read sequences that share identity with a submitted query sequence. These tags can be used to design oligonucleotide primers for the PCR amplification of the region in the target genome.ConclusionsThe ability to produce large numbers of paired read genome tags using second generation sequencing provides a cost effective method for the identification of genes and promoters in large, complex or orphan species without the need for whole genome assembly.