Genome assembly of the Australian black tiger shrimp (Penaeus monodon) reveals a novel fragmented IHHNV EVE sequence.

Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such endogenous viral elements and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of endogenous viral elements. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for 1 generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific endogenous viral elements identified an element comprised of a 9,045-bp stretch of repeated, inverted, and jumbled genome fragments of infectious hypodermal and hematopoietic necrosis virus bounded by a repeated 591/590 bp host sequence. As only near complete linear ∼4 kb infectious hypodermal and hematopoietic necrosis virus genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear endogenous viral element types. The existence of joined inverted infectious hypodermal and hematopoietic necrosis virus genome fragments also provides a means by which hairpin double-stranded RNA could be expressed and processed by the shrimp RNA interference machinery.

Progress in single-access information systems for wheat and rice crop improvement.

Improving productivity of the staple crops wheat and rice is essential to feed the growing global population, particularly in the context of a changing climate. However, current rates of yield gain are insufficient to support the predicted population growth. New approaches are required to accelerate the breeding process, and many of these are driven by the application of large-scale crop data. To leverage the substantial volumes and types of data that can be applied for precision breeding, the wheat and rice research communities are working towards the development of integrated systems to access and standardize the dispersed, heterogeneous available data. Here, we outline the initiatives of the International Wheat Information System (WheatIS) and the International Rice Informatics Consortium (IRIC) to establish Web-based single-access systems and data mining tools to make the available resources more accessible, drive discovery and accelerate the production of new crop varieties. We discuss the progress of WheatIS and IRIC towards unifying specialized wheat and rice databases and building custom software platforms to manage and interrogate these data. Single-access crop information systems will strengthen scientific collaboration, optimize the use of public research funds and help achieve the required yield gains in the two most important global food crops.

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.

Variation in abundance of predicted resistance genes in the Brassica oleracea pangenome.

Brassica oleracea is an important agricultural species encompassing many vegetable crops including cabbage, cauliflower, broccoli and kale; however, it can be susceptible to a variety of fungal diseases such as clubroot, blackleg, leaf spot and downy mildew. Resistance to these diseases is meditated by specific disease resistance genes analogs (RGAs) which are differently distributed across B. oleracea lines. The sequenced reference cultivar does not contain all B. oleracea genes due to gene presence/absence variation between individuals, which makes it necessary to search for RGA candidates in the B. oleracea pangenome. Here we present a comparative analysis of RGA candidates in the pangenome of B. oleracea. We show that the presence of RGA candidates differs between lines and suggests that in B. oleracea, SNPs and presence/absence variation drive RGA diversity using separate mechanisms. We identified 59 RGA candidates linked to Sclerotinia, clubroot, and Fusarium wilt resistance QTL, and these findings have implications for crop breeding in B. oleracea, which may also be applicable in other crops species.

Genomic comparison of two independent seagrass lineages reveals habitat-driven convergent evolution.

Seagrasses are marine angiosperms that live fully submerged in the sea. They evolved from land plant ancestors, with multiple species representing at least three independent return-to-the-sea events. This raises the question of whether these marine angiosperms followed the same adaptation pathway to allow them to live and reproduce under the hostile marine conditions. To compare the basis of marine adaptation between seagrass lineages, we generated genomic data for Halophila ovalis and compared this with recently published genomes for two members of Zosteraceae, as well as genomes of five non-marine plant species (Arabidopsis, Oryza sativa, Phoenix dactylifera, Musa acuminata, and Spirodela polyrhiza). Halophila and Zosteraceae represent two independent seagrass lineages separated by around 30 million years. Genes that were lost or conserved in both lineages were identified. All three species lost genes associated with ethylene and terpenoid biosynthesis, and retained genes related to salinity adaptation, such as those for osmoregulation. In contrast, the loss of the NADH dehydrogenase-like complex is unique to H. ovalis. Through comparison of two independent return-to-the-sea events, this study further describes marine adaptation characteristics common to seagrass families, identifies species-specific gene loss, and provides molecular evidence for convergent evolution in seagrass lineages.

Homoeologous exchange is a major cause of gene presence/absence variation in the amphidiploid Brassica napus.

Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.

Databases for Wheat Genomics and Crop Improvement.

The genomics revolution brought on by advances in high-throughput sequencing has led to the production of vast amounts of data. Databases play an essential role in storing and managing this information to make it available to researchers and crop breeders. This chapter provides an outline of how to use databases and tools for wheat genome research.

BioNanoAnalyst: a visualisation tool to assess genome assembly quality using BioNano data.

BACKGROUND: Reference genome assemblies are valuable, as they provide insights into gene content, genetic evolution and domestication. The higher the quality of a reference genome assembly the more accurate the downstream analysis will be. During the last few years, major efforts have been made towards improving the quality of genome assemblies. However, erroneous and incomplete assemblies are still common. Complementary to DNA sequencing technologies, optical mapping has advanced genomic studies by facilitating the production of genome scaffolds and assessing structural variation. However, there are few tools available to comprehensively examine misassemblies in reference genome sequences using optical map data. RESULTS: We present BioNanoAnalyst, a software package to examine genome assemblies based on restriction endonuclease cut sites and optical map data. A graphical user interface (GUI) allows users to assess reference genome sequences on different computer platforms without the requirement of programming knowledge. The zoom function makes visualisation convenient, while a GFF3 format output file gives an option to directly visualise questionable assembly regions by location and nucleotides following import into a local genome browser. CONCLUSIONS: BioNanoAnalyst is a tool to identify misassemblies in a reference genome sequence using optical map data. With the reported information, users can rapidly identify assembly errors and correct them using other software tools, which could facilitate an accurate downstream analysis.

Assembly and comparison of two closely related Brassica napus genomes.

As an increasing number of plant genome sequences become available, it is clear that gene content varies between individuals, and the challenge arises to predict the gene content of a species. However, genome comparison is often confounded by variation in assembly and annotation. Differentiating between true gene absence and variation in assembly or annotation is essential for the accurate identification of conserved and variable genes in a species. Here, we present the de novo assembly of the B. napus cultivar Tapidor and comparison with an improved assembly of the Brassica napus cultivar Darmor-bzh. Both cultivars were annotated using the same method to allow comparison of gene content. We identified genes unique to each cultivar and differentiate these from artefacts due to variation in the assembly and annotation. We demonstrate that using a common annotation pipeline can result in different gene predictions, even for closely related cultivars, and repeat regions which collapse during assembly impact whole genome comparison. After accounting for differences in assembly and annotation, we demonstrate that the genome of Darmor-bzh contains a greater number of genes than the genome of Tapidor. Our results are the first step towards comparison of the true differences between B. napus genomes and highlight the potential sources of error in future production of a B. napus pangenome.

The pangenome of hexaploid bread wheat.

There is an increasing understanding that variation in gene presence-absence plays an important role in the heritability of agronomic traits; however, there have been relatively few studies on variation in gene presence-absence in crop species. Hexaploid wheat is one of the most important food crops in the world and intensive breeding has reduced the genetic diversity of elite cultivars. Major efforts have produced draft genome assemblies for the cultivar Chinese Spring, but it is unknown how well this represents the genome diversity found in current modern elite cultivars. In this study we build an improved reference for Chinese Spring and explore gene diversity across 18 wheat cultivars. We predict a pangenome size of 140 500 ± 102 genes, a core genome of 81 070 ± 1631 genes and an average of 128 656 genes in each cultivar. Functional annotation of the variable gene set suggests that it is enriched for genes that may be associated with important agronomic traits. In addition to variation in gene presence, more than 36 million intervarietal single nucleotide polymorphisms were identified across the pangenome. This study of the wheat pangenome provides insight into genome diversity in elite wheat as a basis for genomics-based improvement of this important crop. A wheat pangenome, GBrowse, is available at http://appliedbioinformatics.com.au/cgi-bin/gb2/gbrowse/WheatPan/, and data are available to download from http://wheatgenome.info/wheat_genome_databases.php.

The pangenome of an agronomically important crop plant Brassica oleracea.

There is an increasing awareness that as a result of structural variation, a reference sequence representing a genome of a single individual is unable to capture all of the gene repertoire found in the species. A large number of genes affected by presence/absence and copy number variation suggest that it may contribute to phenotypic and agronomic trait diversity. Here we show by analysis of the Brassica oleracea pangenome that nearly 20% of genes are affected by presence/absence variation. Several genes displaying presence/absence variation are annotated with functions related to major agronomic traits, including disease resistance, flowering time, glucosinolate metabolism and vitamin biosynthesis.

The Genome of a Southern Hemisphere Seagrass Species (Zostera muelleri).

Seagrasses are marine angiosperms that evolved from land plants but returned to the sea around 140 million years ago during the early evolution of monocotyledonous plants. They successfully adapted to abiotic stresses associated with growth in the marine environment, and today, seagrasses are distributed in coastal waters worldwide. Seagrass meadows are an important oceanic carbon sink and provide food and breeding grounds for diverse marine species. Here, we report the assembly and characterization of the Zostera muelleri genome, a southern hemisphere temperate species. Multiple genes were lost or modified in Z. muelleri compared with terrestrial or floating aquatic plants that are associated with their adaptation to life in the ocean. These include genes for hormone biosynthesis and signaling and cell wall catabolism. There is evidence of whole-genome duplication in Z. muelleri; however, an ancient pan-commelinid duplication event is absent, highlighting the early divergence of this species from the main monocot lineages.

An efficient approach to BAC based assembly of complex genomes.

BACKGROUND: There 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. RESULTS: We 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. CONCLUSIONS: We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

Analysis of RNA-Seq Data Using TopHat and Cufflinks.

The recent advances in high throughput RNA sequencing (RNA-Seq) have generated huge amounts of data in a very short span of time for a single sample. These data have required the parallel advancement of computing tools to organize and interpret them meaningfully in terms of biological implications, at the same time using minimum computing resources to reduce computation costs. Here we describe the method of analyzing RNA-seq data using the set of open source software programs of the Tuxedo suite: TopHat and Cufflinks. TopHat is designed to align RNA-seq reads to a reference genome, while Cufflinks assembles these mapped reads into possible transcripts and then generates a final transcriptome assembly. Cufflinks also includes Cuffdiff, which accepts the reads assembled from two or more biological conditions and analyzes their differential expression of genes and transcripts, thus aiding in the investigation of their transcriptional and post transcriptional regulation under different conditions. We also describe the use of an accessory tool called CummeRbund, which processes the output files of Cuffdiff and gives an output of publication quality plots and figures of the user's choice. We demonstrate the effectiveness of the Tuxedo suite by analyzing RNA-Seq datasets of Arabidopsis thaliana root subjected to two different conditions.

Transcriptomic Changes in Coral Holobionts Provide Insights into Physiological Challenges of Future Climate and Ocean Change.

Tropical reef-building coral stress levels will intensify with the predicted rising atmospheric CO2 resulting in ocean temperature and acidification increase. Most studies to date have focused on the destabilization of coral-dinoflagellate symbioses due to warming oceans, or declining calcification due to ocean acidification. In our study, pH and temperature conditions consistent with the end-of-century scenarios of the Intergovernmental Panel on Climate Change (IPCC) caused major changes in photosynthesis and respiration, in addition to decreased calcification rates in the coral Acropora millepora. Population density of symbiotic dinoflagellates (Symbiodinium) under high levels of ocean acidification and temperature (Representative Concentration Pathway, RCP8.5) decreased to half of that found under present day conditions, with photosynthetic and respiratory rates also being reduced by 40%. These physiological changes were accompanied by evidence for gene regulation of calcium and bicarbonate transporters along with components of the organic matrix. Metatranscriptomic RNA-Seq data analyses showed an overall down regulation of metabolic transcripts, and an increased abundance of transcripts involved in circadian clock control, controlling the damage of oxidative stress, calcium signaling/homeostasis, cytoskeletal interactions, transcription regulation, DNA repair, Wnt signaling and apoptosis/immunity/ toxins. We suggest that increased maintenance costs under ocean acidification and warming, and diversion of cellular ATP to pH homeostasis, oxidative stress response, UPR and DNA repair, along with metabolic suppression, may underpin why Acroporid species tend not to thrive under future environmental stress. Our study highlights the potential increased energy demand when the coral holobiont is exposed to high levels of ocean warming and acidification.

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

KEY MESSAGE: We characterise the distribution of crossover and non-crossover recombination in Brassica napus and Cicer arietinum using a low-coverage genotyping by sequencing pipeline SkimGBS. The 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.

Genome-wide survey of the seagrass Zostera muelleri suggests modification of the ethylene signalling network.

Seagrasses are flowering plants which grow fully submerged in the marine environment. They have evolved a range of adaptations to environmental challenges including light attenuation through water, the physical stress of wave action and tidal currents, high concentrations of salt, oxygen deficiency in marine sediment, and water-borne pollination. Although, seagrasses are a key stone species of the costal ecosystems, many questions regarding seagrass biology and evolution remain unanswered. Genome sequence data for the widespread Australian seagrass species Zostera muelleri were generated and the unassembled data were compared with the annotated genes of five sequenced plant species (Arabidopsis thaliana, Oryza sativa, Phoenix dactylifera, Musa acuminata, and Spirodela polyrhiza). Genes which are conserved between Z. muelleri and the five plant species were identified, together with genes that have been lost in Z. muelleri. The effect of gene loss on biological processes was assessed on the gene ontology classification level. Gene loss in Z. muelleri appears to influence some core biological processes such as ethylene biosynthesis. This study provides a foundation for further studies of seagrass evolution as well as the hormonal regulation of plant growth and development.

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.

Unfolding the secrets of coral-algal symbiosis.

Dinoflagellates from the genus Symbiodinium form a mutualistic symbiotic relationship with reef-building corals. Here we applied massively parallel Illumina sequencing to assess genetic similarity and diversity among four phylogenetically diverse dinoflagellate clades (A, B, C and D) that are commonly associated with corals. We obtained more than 30,000 predicted genes for each Symbiodinium clade, with a majority of the aligned transcripts corresponding to sequence data sets of symbiotic dinoflagellates and <2% of sequences having bacterial or other foreign origin. We report 1053 genes, orthologous among four Symbiodinium clades, that share a high level of sequence identity to known proteins from the SwissProt (SP) database. Approximately 80% of the transcripts aligning to the 1053 SP genes were unique to Symbiodinium species and did not align to other dinoflagellates and unrelated eukaryotic transcriptomes/genomes. Six pathways were common to all four Symbiodinium clades including the phosphatidylinositol signaling system and inositol phosphate metabolism pathways. The list of Symbiodinium transcripts common to all four clades included conserved genes such as heat shock proteins (Hsp70 and Hsp90), calmodulin, actin and tubulin, several ribosomal, photosynthetic and cytochrome genes and chloroplast-based heme-containing cytochrome P450, involved in the biosynthesis of xanthophylls. Antioxidant genes, which are important in stress responses, were also preserved, as were a number of calcium-dependent and calcium/calmodulin-dependent protein kinases that may play a role in the establishment of symbiosis. Our findings disclose new knowledge about the genetic uniqueness of symbiotic dinoflagellates and provide a list of homologous genes important for the foundation of coral-algal symbiosis.

Early transcriptional changes in the reef-building coral Acropora aspera in response to thermal and nutrient stress.

BACKGROUND: Changes to the environment as a result of human activities can result in a range of impacts on reef building corals that include coral bleaching (reduced concentrations of algal symbionts), decreased coral growth and calcification, and increased incidence of diseases and mortality. Understanding how elevated temperatures and nutrient concentration affect early transcriptional changes in corals and their algal endosymbionts is critically important for evaluating the responses of coral reefs to global changes happening in the environment. Here, we investigated the expression of genes in colonies of the reef-building coral Acropora aspera exposed to short-term sub-lethal levels of thermal (+6°C) and nutrient stress (ammonium-enrichment: 20 µM). RESULTS: The RNA-Seq data provided hundreds of differentially expressed genes (DEGs) corresponding to various stress regimes, with 115 up- and 78 down-regulated genes common to all stress regimes. A list of DEGs included up-regulated coral genes like cytochrome c oxidase and NADH-ubiquinone oxidoreductase and up-regulated photosynthetic genes of algal origin, whereas coral GFP-like fluorescent chromoprotein and sodium/potassium-transporting ATPase showed reduced transcript levels. Taxonomic analyses of the coral holobiont disclosed the dominant presence of transcripts from coral (~70%) and Symbiodinium (~10-12%), as well as ~15-20% of unknown sequences which lacked sequence identity to known genes. Gene ontology analyses revealed enriched pathways, which led to changes in the dynamics of protein networks affecting growth, cellular processes, and energy requirement. CONCLUSIONS: In corals with preserved symbiont physiological performance (based on Fv/Fm, photo-pigment and symbiont density), transcriptomic changes and DEGs provided important insight into early stages of the stress response in the coral holobiont. Although there were no signs of coral bleaching after exposure to short-term thermal and nutrient stress conditions, we managed to detect oxidative stress and apoptotic changes on a molecular level and provide a list of prospective stress biomarkers for both partners in symbiosis. Consequently, our findings are important for understanding and anticipating impacts of anthropogenic global climate change on coral reefs.