A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology.

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

A differential k-mer analysis pipeline for comparing RNA-Seq transcriptome and meta-transcriptome datasets without a reference.

Next-generation DNA sequencing technologies, such as RNA-Seq, currently dominate genome-wide gene expression studies. A standard approach to analyse this data requires mapping sequence reads to a reference and counting the number of reads which map to each gene. However, for many transcriptome studies, a suitable reference genome is unavailable, especially for meta-transcriptome studies which assay gene expression from mixed populations of organisms. Where a reference is unavailable, it is possible to generate a reference by the de novo assembly of the sequence reads. However, the high cost of generating high-coverage data for de novo assembly hinders this approach and more importantly the accurate assembly of such data is challenging, especially for meta-transcriptome data, and resulting assemblies frequently suffer from collapsed regions or chimeric sequences. As an alternative to the standard reference mapping approach, we have developed a k-mer-based analysis pipeline (DiffKAP) to identify differentially expressed reads between RNA-Seq datasets without the requirement for a reference. We compared the DiffKAP approach with the traditional Tophat/Cuffdiff method using RNA-Seq data from soybean, which has a suitable reference genome. We subsequently examined differential gene expression for a coral meta-transcriptome where no reference is available, and validated the results using qRT-PCR. We conclude that DiffKAP is an accurate method to study differential gene expression in complex meta-transcriptomes without the requirement of a reference genome.

Chromatin immunoprecipitation (ChIP) method for non-model fruit flies (Diptera: Tephritidae) and evidence of histone modifications.

Interactions between DNA and proteins located in the cell nucleus play an important role in controlling physiological processes by specifying, augmenting and regulating context-specific transcription events. Chromatin immunoprecipitation (ChIP) is a widely used methodology to study DNA-protein interactions and has been successfully used in various cell types for over three decades. More recently, by combining ChIP with genomic screening technologies and Next Generation Sequencing (e.g. ChIP-seq), it has become possible to profile DNA-protein interactions (including covalent histone modifications) across entire genomes. However, the applicability of ChIP-chip and ChIP-seq has rarely been extended to non-model species because of a number of technical challenges. Here we report a method that can be used to identify genome wide covalent histone modifications in a group of non-model fruit fly species (Diptera: Tephritidae). The method was developed by testing and refining protocols that have been used in model organisms, including Drosophila melanogaster. We demonstrate that this method is suitable for a group of economically important pest fruit fly species, viz., Bactrocera dorsalis, Ceratitis capitata, Zeugodacus cucurbitae and Bactrocera tryoni. We also report an example ChIP-seq dataset for B. tryoni, providing evidence for histone modifications in the genome of a tephritid fruit fly for the first time. Since tephritids are major agricultural pests globally, this methodology will be a valuable resource to study taxa-specific evolutionary questions and to assist with pest management. It also provides a basis for researchers working with other non-model species to undertake genome wide DNA-protein interaction studies.

The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence.

Nicotiana benthamiana is employed around the world for many types of research and one transgenic line has been used more extensively than any other. This line, 16c, expresses the Aequorea victoria green fluorescent protein (GFP), highly and constitutively, and has been a major resource for visualising the mobility and actions of small RNAs. Insights into the mechanisms studied at a molecular level in N. benthamiana 16c are likely to be deeper and more accurate with a greater knowledge of the GFP gene integration site. Therefore, using next generation sequencing, genome mapping and local alignment, we identified the location and characteristics of the integrated T-DNA. As suggested from previous molecular hybridisation and inheritance data, the transgenic line contains a single GFP-expressing locus. However, the GFP coding sequence differs from that originally reported. Furthermore, a 3.2 kb portion of a transposon, appears to have co-integrated with the T-DNA. The location of the integration mapped to a region of the genome represented by Nbv0.5scaffold4905 in the www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the www.solgenomics.net assembly. The transposon is not endogenous to laboratory strains of N. benthamiana or Agrobacterium tumefaciens strain GV3101 (MP90), which was reportedly used in the generation of line 16c. However, it is present in the popular LBA4404 strain. The integrated transposon sequence includes its 5' terminal repeat and a transposase gene, and is immediately adjacent to the GFP gene. This unexpected genetic arrangement may contribute to the characteristics that have made the 16c line such a popular research tool and alerts researchers, taking transgenic plants to commercial release, to be aware of this genomic hitchhiker.

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.

A high-throughput SNP array in the amphidiploid species Brassica napus shows diversity in resistance genes.

Single-nucleotide polymorphisms (SNPs)are molecular markers based on nucleotide variation and can be used for genotyping assays across populations and to track genomic inheritance. SNPs offer a comprehensive genotyping alternative to whole-genome sequencing for both agricultural and research purposes including molecular breeding and diagnostics, genome evolution and genetic diversity analyses, genetic mapping, and trait association studies. Here genomic SNPs were discovered between four cultivars of the important amphidiploid oilseed species Brassica napus and used to develop a B. napus Infinium™ array containing 5,306 SNPs randomly dispersed across the genome. Assay success was high, with >94 % of these producing a reproducible, polymorphic genotype in the 1,070 samples screened. Although the assay was designed to B. napus, successful SNP amplification was achieved in the B. napus progenitor species, Brassica rapa and Brassica oleracea, and to a lesser extent in the related species Brassica nigra. Phylogenetic analysis was consistent with the expected relationships between B. napus individuals. This study presents an efficient custom SNP assay development pipeline in the complex polyploid Brassica genome and demonstrates the utility of the array for high-throughput genotyping in a number of related Brassica species. It also demonstrates the utility of this assay in genotyping resistance genes on chromosome A7, which segregate amongst the 1,070 samples.

Identifying genetic diversity of avirulence genes in Leptosphaeria maculans using whole genome sequencing.

Next generation sequencing technology allows rapid re-sequencing of individuals, as well as the discovery of single nucleotide polymorphisms (SNPs), for genomic diversity and evolutionary analyses. By sequencing two isolates of the fungal plant pathogen Leptosphaeria maculans, the causal agent of blackleg disease in Brassica crops, we have generated a resource of over 76 million sequence reads aligned to the reference genome. We identified over 21,000 SNPs with an overall SNP frequency of one SNP every 2,065 bp. Sequence validation of a selection of these SNPs in additional isolates collected throughout Australia indicates a high degree of polymorphism in the Australian population. In preliminary phylogenetic analysis, isolates from Western Australia clustered together and those collected from Brassica juncea stubble were identical. These SNPs provide a novel marker resource to study the genetic diversity of this pathogen. We demonstrate that re-sequencing provides a method of validating previously characterised SNPs and analysing differences in important genes, such as the disease related avirulence genes of L. maculans. Understanding the genetic characteristics of this devastating pathogen is vital in developing long-term solutions to managing blackleg disease in Brassica crops.

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.

Transient Nod factor-dependent gene expression in the nodulation-competent zone of soybean (Glycine max [L.] Merr.) roots.

All lateral organ development in plants, such as nodulation in legumes, requires the temporal and spatial regulation of genes and gene networks. A total mRNA profiling approach using RNA-seq to target the specific soybean (Glycine max) root tissues responding to compatible rhizobia [i.e. the Zone Of Nodulation (ZON)] revealed a large number of novel, often transient, mRNA changes occurring during the early stages of nodulation. Focusing on the ZON enabled us to discard the majority of root tissues and their developmentally diverse gene transcripts, thereby highlighting the lowly and transiently expressed nodulation-specific genes. It also enabled us to concentrate on a precise moment in early nodule development at each sampling time. We focused on discovering genes regulated specifically by the Bradyrhizobium-produced Nod factor signal, by inoculating roots with either a competent wild-type or incompetent mutant (nodC(-) ) strain of Bradyrhizobium japonicum. Collectively, 2915 genes were identified as being differentially expressed, including many known soybean nodulation genes. A number of unknown nodulation gene candidates and soybean orthologues of nodulation genes previously reported in other legume species were also identified. The differential expression of several candidates was confirmed and further characterized via inoculation time-course studies and qRT-PCR. The expression of many genes, including an endo-1,4-ß-glucanase, a cytochrome P450 and a TIR-LRR-NBS receptor kinase, was transient, peaking quickly during the initiation of nodule ontogeny. Additional genes were found to be down-regulated. Significantly, a set of differentially regulated genes acting in the gibberellic acid (GA) biosynthesis pathway was discovered, suggesting a novel role of GAs in nodulation.

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.

Identification and characterization of candidate Rlm4 blackleg resistance genes in Brassica napus using next-generation sequencing.

A thorough understanding of the relationships between plants and pathogens is essential if we are to continue to meet the agricultural needs of the world's growing population. The identification of genes underlying important quantitative trait loci is extremely challenging in complex genomes such as Brassica napus (canola, oilseed rape or rapeseed). However, recent advances in next-generation sequencing (NGS) enable much quicker identification of candidate genes for traits of interest. Here, we demonstrate this with the identification of candidate disease resistance genes from B. napus for its most devastating fungal pathogen, Leptosphaeria maculans (blackleg fungus). These two species are locked in an evolutionary arms race whereby a gene-for-gene interaction confers either resistance or susceptibility in the plant depending on the genotype of the plant and pathogen. Preliminary analysis of the complete genome sequence of Brassica rapa, the diploid progenitor of B. napus, identified numerous candidate genes with disease resistance characteristics, several of which were clustered around a region syntenic with a major locus (Rlm4) for blackleg resistance on A7 of B. napus. Molecular analyses of the candidate genes using B. napus NGS data are presented, and the difficulties associated with identifying functional gene copies within the highly duplicated Brassica genome are discussed.

Bread matters: a national initiative to profile the genetic diversity of Australian wheat.

The large and complex genome of wheat makes genetic and genomic analysis in this important species both expensive and resource intensive. The application of next-generation sequencing technologies is particularly resource intensive, with at least 17 Gbp of sequence data required to obtain minimal (1×) coverage of the genome. A similar volume of data would represent almost 40× coverage of the rice genome. Progress can be made through the establishment of consortia to produce shared genomic resources. Australian wheat genome researchers, working with Bioplatforms Australia, have collaborated in a national initiative to establish a genetic diversity dataset representing Australian wheat germplasm based on whole genome next-generation sequencing data. Here, we describe the establishment and validation of this resource which can provide a model for broader international initiatives for the analysis of large and complex genomes.

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 world's 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.

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.

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.

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.