Plant Genomics

Abstract (provisional)Background

Visualization plays an essential role in genomics research by making it possible to observe correlations and trends in large datasets as well as communicate findings to others. Visual analysis, which combines visualization with analysis tools to enable seamless use of both approaches for scientific investigation, offers a powerful method for performing complex genomic analyses. However, there are numerous challenges that arise when creating rich, interactive Web-based visualizations/visual analysis applications for high-throughput genomics. These challenges include managing data flow from Web server to Web browser, integrating analysis tools and visualizations, and sharing visualizations with colleagues.

Results

We have created a platform simplifies the creation of Web-based visualization/visual analysis applications for high-throughput genomics. This platform provides components that make it simple to efficiently query very large datasets, draw common representations of genomic data, integrate with analysis tools, and share or publish fully interactive visualizations. Using this platform, we have created a Circos-style genome-wide viewer, a generic scatter plot for correlation analysis, an interactive phylogenetic tree, a scalable genome browser for next-generation sequencing data, and an application for systematically exploring tool parameter spaces to find good parameter values. All visualizations are interactive and fully customizable. The platform is integrated with the Galaxy (http://galaxyproject.org) genomics workbench, making it easy to integrate new visual applications into Galaxy.

Conclusions

Visualization and visual analysis play an important role in high-throughput genomics experiments, and approaches are needed to make it easier to create applications for these activities. Our framework provides a foundation for creating Web-based visualizations and integrating them into Galaxy. Finally, the visualizations we have created using the framework are useful tools for high-throughput genomics experiments.

Abstract (provisional)Background

Understanding the genetic architecture of quantitative traits is important for developing genome-based crop improvement methods. Genome-wide association study (GWAS) is a powerful technique for mining novel functional variants. Using a family-based design involving 1,200 apple (Malus x domestica Borkh.) seedlings genotyped for an 8K SNP array, we report the first systematic evaluation of the relative contributions of different genomic regions to various traits related to eating quality and susceptibility to some physiological disorders. Single-SNP analyses models that accounted for population structure, or not, were compared with models fitting all markers simultaneously. The patterns of linkage disequilibrium (LD) were also investigated.

Results

A high degree of LD even at longer distances between markers was observed, and the patterns of LD decay were similar across successive generations. Genomic regions were identified, some of which coincided with known candidate genes, with significant effects on various traits. Phenotypic variation explained by the loci identified through a whole-genome scan ranged from 3% to 25% across different traits, while fitting all markers simultaneously generally provided heritability estimates close to those from pedigree-based analysis. Results from 'Q+K' and 'K' models were very similar, suggesting that the SNP-based kinship matrix captures most of the underlying population structure. Correlations between allele substitution effects obtained from single-marker and all-marker analyses were about 0.90 for all traits. Use of SNP-derived realized relationships in linear mixed models provided a better goodness-of-fit than pedigree-based expected relationships. Genomic regions with probable pleiotropic effects were supported by the corresponding higher linkage group (LG) level estimated genetic correlations.

Conclusions

The accuracy of artificial selection in plants species can be increased by using more precise marker-derived estimates of realized coefficients of relationships. All-marker analyses that indirectly account for population- and pedigree structure will be a credible alternative to single-SNP analyses in GWAS. This study revealed large differences in the genetic architecture of apple fruit traits, and the marker-trait associations identified here will help develop genome-based breeding methods for apple cultivar development.

The gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways. - See more at: http://elife.elifesciences.org/content/2/e00675#sthash.I8JTZch1.dpufThe gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways. - See more at: http://elife.elifesciences.org/content/2/e00675#sthash.I8JTZch1.dpufThe gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways. - See more at: http://elife.elifesciences.org/content/2/e00675#sthash.I8JTZch1.dpuf

Abstract (provisional)Background

Ophiostoma piceae is a wood-staining fungus that grows in the sapwood of conifer logs and lumber. We sequenced its genome and analyzed its transcriptomes under a range of growth conditions. A comparison with the genome and transcriptomes of the mountain pine beetle-associated pathogen Grosmannia clavigera highlights differences between a pathogen that colonizes and kills living pine trees and a saprophyte that colonizes wood and the inner bark of dead trees.

Results

We assembled a 33 Mbp genome in 45 scaffolds, and predicted approximately 8,884 genes. The genome size and gene content were similar to those of other ascomycetes. Despite having similar ecological niches, O. piceae and G. clavigera showed no large-scale synteny. We identified O. piceae genes involved in the biosynthesis of melanin, which causes wood discoloration and reduces the commercial value of wood products. We also identified genes and pathways involved in growth on simple carbon sources and in sapwood, O. piceae's natural substrate. Like the pathogen, the saprophyte is able to tolerate terpenes, which are a major class of pine tree defense compounds; unlike the pathogen, it cannot utilize monoterpenes as a carbon source.

Conclusions

This work makes available the second annotated genome of a softwood ophiostomatoid fungus, and suggests that O. piceae's tolerance to terpenes may be due in part to these chemicals being removed from the cells by an ABC transporter that is highly induced by terpenes. The data generated will provide the research community with resources for work on host-vector-fungus interactions for wood-inhabiting, beetle-associated saprophytes and pathogens.

Abstract (provisional)Background

Hop (Humulus lupulus L.) is cultivated for its cones, the secondary metabolites of which contribute bitterness, flavour and aroma to beer. Molecular breeding methods, such as marker assisted selection (MAS), have great potential for improving the efficiency of hop breeding. The success of MAS is reliant on the identification of reliable marker-trait associations. This study used quantitative trait loci (QTL) analysis to identify marker-trait associations for hop, focusing on traits related to expediting plant sex identification, increasing yield capacity and improving bittering, flavour and aroma chemistry.

Results

QTL analysis was performed on two new linkage maps incorporating transferable Diversity Arrays Technology (DArT) markers. Sixty-three QTL were identified, influencing 36 of the 50 traits examined. A putative sex-linked marker was validated in a different pedigree, confirming the potential of this marker as a screening tool in hop breeding programs. An ontogenetically stable QTL was identified for the yield trait dry cone weight; and a QTL was identified for essential oil content, which verified the genetic basis for variation in secondary metabolite accumulation in hop cones. A total of 60 QTL were identified for 33 secondary metabolite traits. Of these, 51 were pleiotropic/linked, affecting a substantial number of secondary metabolites; nine were specific to individual secondary metabolites.

Conclusions

Pleiotropy and linkage, found for the first time to influence multiple hop secondary metabolites, have important implications for molecular selection methods. The selection of particular secondary metabolite profiles using pleiotropic/linked QTL will be challenging because of the difficulty of selecting for specific traits without adversely changing others. QTL specific to individual secondary metabolites, however, offer unequalled value to selection programs. In addition to their potential for selection, the QTL identified in this study advance our understanding of the genetic control of traits of current economic and breeding significance in hop and demonstrate the complex genetic architecture underlying variation in these traits. The linkage information obtained in this study, based on transferable markers, can be used to facilitate the validation of QTL, crucial to the success of MAS.

 

Abstract (provisional)Background

Paris polyphylla var. yunnanensis is an important medicinal plant. Seed dormancy is one of the main factors restricting artificial cultivation. The molecular mechanisms of seed dormancy remain unclear, and little genomic or transcriptome data are available for this plant.

Results

In this study, massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform was used to generate a substantial sequence dataset for the P. polyphylla embryo. 369,496 high quality reads were obtained, ranging from 50 to 1146 bp, with a mean of 219 bp. These reads were assembled into 47,768 unigenes, which included 16,069 contigs and 31,699 singletons. Using BLASTX searches of public databases, 15,757 (32.3%) unique transcripts were identified. Gene Ontology and Cluster of Orthologous Groups of proteins annotations revealed that these transcripts were broadly representative of the P. polyphylla embryo transcriptome. The Kyoto Encyclopedia of Genes and Genomes assigned 5961 of the unique sequences to specific metabolic pathways. Relative expression levels analysis showed that eleven phytohormone-related genes and five other genes have different expression patterns in the embryo and endosperm in the seed stratification process.

Conclusions

Gene annotation and quantitative RT-PCR expression analysis identified 464 transcripts that may be involved in phytohormone catabolism and biosynthesis, hormone signal, seed dormancy, seed maturation, cell wall growth and circadian rhythms. In particular, the relative expression analysis of sixteen genes (CYP707A, NCED, GA20ox2, GA20ox3, ABI2, PP2C, ARP3, ARP7, IAAH, IAAS, BRRK, DRM, ELF1, ELF2, SFR6, and SUS) in embryo and endosperm and at two temperatures indicated that these related genes may be candidates for clarifying the molecular basis of seed dormancy in P. polyphlla var. yunnanensis.

 

Abstract (provisional)Background

The major bottle neck in genetic and linkage studies in tomato has been the lack of a sufficient number of molecular markers. This has radically changed with the application of next generation sequencing and high throughput genotyping. A set of 6000 SNPs was identified and 5528 of them were used to evaluate tomato germplasm at the level of species, varieties and segregating populations.

Results

From the 5528 SNPs, 1980 originated from 454-sequencing, 3495 from Illumina Solexa sequencing and 53 were additional known markers. Genotyping different tomato samples allowed the evaluation of the level of heterozygosity and introgressions among commercial varieties. Cherry tomatoes were especially different from round/beefs in chromosomes 4, 5 and 12. We were able to identify a set of 750 unique markers distinguishing S. lycopersicum 'Moneymaker' from all its distantly related wild relatives. Clustering and neighbour joining analysis among varieties and species showed expected grouping patterns, with S. pimpinellifolium as the most closely related to commercial tomatoesearlier results.

Conclusions

Our results show that a SNP search in only a few breeding lines already provides generally applicable markers in tomato and its wild relatives. It also shows that the Illumina bead array generated data are highly reproducible. Our SNPs can roughly be divided in two categories: SNPs of which both forms are present in the wild relatives and in domesticated tomatoes (originating from common ancestors) and SNPs unique for the domesticated tomato (originating from after the domestication event). The SNPs can be used for genotyping, identification of varieties, comparison of genetic and physical linkage maps and to confirm (phylogenetic) relations. In the SNPs used for the array there is hardly any overlap with the SolCAP array and it is strongly recommended to combine both SNP sets and to select a core collection of robust SNPs completely covering the entire tomato genome.

Abstract (provisional)Background

Autotoxicity plays an important role in regulating crop yield and quality. To help characterize the autotoxicity mechanism of rice, we performed a large-scale, transcriptomic analysis of the rice root response to ferulic acid, an autotoxin from rice straw.

Results

Root growth rate was decreased and reactive oxygen species, calcium content and lipoxygenase activity were increased with increasing ferulic acid concentration in roots. Transcriptome analysis revealed more transcripts responsive to short ferulic-acid exposure (1- and 3-h treatments, 1,204 genes) than long exposure (24 h, 176 genes). Induced genes were involved in cell wall formation, chemical detoxification, secondary metabolism, signal transduction, and abiotic stress response. Genes associated with signaling and biosynthesis for ethylene and jasmonic acid were upregulated with ferulic acid. Ferulic acid upregulated ATP-binding cassette and amino acid/auxin permease transporters as well as genes encoding signaling components such as leucine-rich repeat VIII and receptor-like cytoplasmic kinases VII protein kinases, APETALA2/ethylene response factor, WRKY, MYB and Zinc-finger protein expressed in inflorescence meristem transcription factors.

Conclusions

The results of a transcriptome analysis suggest the molecular mechanisms of plants in response to FA, including toxicity, detoxicification and signaling machinery. FA may have a significant effect on inhibiting rice root elongation through modulating ET and JA hormone homeostasis. FA-induced gene expression of AAAP transporters may contribute to detoxicification of the autotoxin. Moreover, the WRKY and Myb TFs and LRR-VIII and SD-2b kinases might regulate downstream genes under FA stress but not general allelochemical stress. This comprehensive description of gene expression information could greatly facilitate our understanding of the mechanisms of autotoxicity in plants.

Abstract (provisional)Background

Several beta-galactosidases of the Glycosyl Hydrolase 35 (GH35) family have been characterized, and many of these modify cell wall components, including pectins, xyloglucans, and arabinogalactan proteins. The phloem fibres of flax (Linum usitatissimum) have gelatinous-type cell walls that are rich in crystalline cellulose and depend on beta-galactosidase activity for their normal development. In this study, we investigate the transcript expression patterns and inferred evolutionary relationships of the complete set of flax GH35 genes, to better understand the functions of these genes in flax and other species.

Results

Using the recently published flax genome assembly, we identified 43 beta-galactosidase-like (BGAL) genes, based on the presence of a GH35 domain. Phylogenetic analyses of their protein sequences clustered them into eight sub-families. Sub-family B, whose members in other species were known to be expressed in developing flowers and pollen, was greatly under represented in flax (p-value < 0.01). Sub-family A5, whose sole member from arabidopsis has been described as its primary xyloglucan BGAL, was greatly expanded in flax (p-value < 0.01). A number of flax BGALs were also observed to contain non-consensus GH35 active sites. Expression patterns of the flax BGALs were investigated using qRT-PCR and publicly available microarray data. All predicted flax BGALs showed evidence of expression in at least one tissue.

Conclusion

Flax has a large number of BGAL genes, which display a distinct distribution among the BGAL sub-families, in comparison to other closely related species with available whole genome assemblies. Almost every flax BGAL was expressed in fibres, the majority of which expressed predominately in fibres as compared to other tissues, suggesting an important role for the expansion of this gene family in the development of this species as a fibre crop. Variations displayed in the canonical GH35 active site suggest a variety of roles unique to flax, which will require further characterization.

Abstract (provisional)Background

Cotton fiber length is very important to the quality of textiles. Understanding the genetics and physiology of cotton fiber elongation can provide valuable tools to the cotton industry by targeting genes or other molecules responsible for fiber elongation. Ligon Lintless-1 (Li1) is a monogenic mutant in Upland cotton (Gossypium hirsutum) which exhibits an early cessation of fiber elongation resulting in very short fibers (< 6mm) at maturity. This presents an excellent model system for studying the underlying molecular and cellular processes involved with cotton fiber elongation. Previous reports have characterized Li1 at early cell wall elongation and during later secondary cell wall synthesis, however there has been very limited analysis of the transition period between these developmental time points.

Results

Physical and morphological measurements of the Li1 mutant fibers were conducted, including measurement of the cellulose content during development. Affymetrix microarrays were used to analyze transcript profiles at the critical developmental time points of 3 days post anthesis (DPA), the late elongation stage of 12 DPA and the early secondary cell wall synthesis stage of 16 DPA. The results indicated severe disruption to key hormonal and other pathways related to fiber development, especially pertaining to the transition stage from elongation to secondary cell wall synthesis. Gene Ontology enrichment analysis identified several key pathways at the transition stage that exhibited altered regulation. Genes involved in ethylene biosynthesis and primary cell wall rearrangement were affected, and a primary cell wall-related cellulose synthase was transcriptionally repressed. Linkage mapping using a population of 2,553 F2 individuals identified SSR markers associated with the Li1 genetic locus on chromosome 22. Linkage mapping in combination with utilizing the diploid G. raimondii genome sequences permitted additional analysis of the region containing the Li1 gene.

Conclusions

The early termination of fiber elongation in the Li1 mutant is likely controlled by an early upstream regulatory factor resulting in the altered regulation of hundreds of downstream genes. Several elongation-related genes that exhibited altered expression profiles in the Li1 mutant were identified. Molecular markers closely associated with the Li1 locus were developed. Results presented here will lay the foundation for further investigation of the genetic and molecular mechanisms of fiber elongation.

Abstract (provisional)Background

Arachidonic acid (ArA) is important for human health because it is one of the major components of mammalian brain membrane phospholipids. The interest in ArA inspired the search for a new sustainable source, and the green microalga Myrmecia incisa Reisigl H4301 has been found a potential ArA-producer due to a high content of intracellular ArA. To gain more molecular information about metabolism pathways, including the biosynthesis of ArA in the non-model microalga, a transcriptomic analysis was performed.

Results

The 454 pyrosequencing generated 371,740 high-quality reads, which were assembled into 51,908 unique sequences consisting of 22,749 contigs and 29,159 singletons. A total of 11,873 unique sequences were annotated through BLAST analysis, and 3,733 were assigned to Gene Ontology (GO) categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered a C4-like photosynthesis pathway in M. incisa. The biosynthesis pathways of lipid particularly those of ArA and triacylglycerol (TAG) were analyzed in detail, and TAG was proposed to be accumulated in oil bodies in the cytosol with the help of caleosin or oil globule-associated proteins. In addition, the carotenoid biosynthesis pathways are discussed.

Conclusion

This transcriptomic analysis of M. incisa enabled a global understanding of mechanisms involved in photosynthesis, de novo biosynthesis of ArA, metabolism of carotenoids, and accumulation of TAG in M. incisa. These findings provided a molecular basis for the research and possibly economic exploitation of this ArA-rich microalga.

Abstract (provisional)Background

White clover (Trifolium repens L.) is a temperate forage legume with an allotetraploid genome (2n=4x=32) estimated at 1093 Mb. Several linkage maps of various sizes, marker sources and completeness are available, however, no integrated map and marker set has explored consistency of linkage analysis among unrelated mapping populations. Such integrative analysis requires tools for homoeologue matching among populations. Development of these tools provides for a consistent framework map of the white clover genome, and facilitates in silico alignment with the model forage legume, Medicago truncatula.

Results

This is the first report of integration of independent linkage maps in white clover, and adds to the literature on methyl filtered GeneThresher(R)-derived microsatellite (simple sequence repeat; SSR) markers for linkage mapping. Gene-targeted SSR markers were discovered in a GeneThresher(R) (TrGT) methyl-filtered database of 364,539 sequences, which yielded 15,647 SSR arrays. Primers were designed for 4,038 arrays and of these, 465 TrGT-SSR markers were used for parental consensus genetic linkage analysis in an F1 mapping population (MP2). This was merged with an EST-SSR consensus genetic map of an independent population (MP1), using markers to match homoeologues and develop a multi-population integrated map of the white clover genome. This integrated map (IM) includes 1109 loci based on 804 SSRs over 1274 cM, covering 97% of the genome at a moderate density of one locus per 1.2 cM. Eighteen candidate genes and one morphological marker were also placed on the IM. Despite being derived from disparate populations and marker sources, the component maps and the derived IM had consistent representations of the white clover genome for marker order and genetic length. In silico analysis at an E-value threshold of 1e-20 revealed substantial co-linearity with the Medicago truncatula genome, and indicates a translocation between T. repens groups 2 and 6 relative to M. truncatula.

Conclusions

This integrated genetic linkage analysis provides a consistent and comprehensive linkage analysis of the white clover genome, with alignment to a model forage legume. Associated marker locus information, particularly the homoeologue-specific markers, offers a new resource for forage legume research to enable genetic analysis and improvement of this forage and grassland species.

One of the most powerful drivers of speciation in plants is pollinator-mediated disruptive selection, which leads to the divergence of floral traits adapted to the morphology and behavior of different pollinators. Despite the widespread importance of this speciation mechanism, its genetic basis has been explored in only a few groups. Here, we characterize the genetic basis of pollinator-mediated divergence of two species in genus Ipomopsis, I. guttata and I. tenuifolia, using quantitative trait locus (QTL) analyses of floral traits and other variable phenotypes. We detected one to six QTLs per trait, with each QTL generally explaining small to modest amounts of the phenotypic variance of a backcross hybrid population. In contrast, flowering time and anthocyanin abundance (a metric of color variation) were controlled by a few QTLs of relatively large effect. QTLs were strongly clustered within linkage groups, with 26 of 37 QTLs localized to six marker-interval ‘hotspots,’ all of which harbored pleiotropic QTLs. In contrast to other studies that have examined the genetic basis of pollinator shifts, our results indicate that, in general, mutations of small to modest effect on phenotype were involved. Thus, the evolutionary transition between the distinct pollination modes of I. guttata and I. tenuifolia likely proceeded incrementally, rather than saltationally.

Abstract (provisional)Background

Alternative splicing (AS) of genes is an efficient means of generating variation in protein structure and function. AS variation has been observed between tissues, cell types, and different treatments in non-woody plants such as Arabidopsis thaliana (Arabidopsis) and rice. However, little is known about AS patterns in wood-forming tissues and how much AS variation exists within plant populations.

Results

Here we used high-throughput RNA sequencing to analyze the Populus trichocarpa (P. trichocarpa) xylem transcriptome in 20 individuals from different populations across much of its range in western North America. Deep transcriptome sequencing and mapping of reads to the P. trichocarpa reference genome identified a suite of xylem-expressed genes common to all accessions. Our analysis suggests that at least 36% of the xylem-expressed genes in P. trichocarpa are alternatively spliced. Extensive AS was observed in cell-wall biosynthesis related genes such as glycosyl transferases and C2H2 transcription factors. 27902 AS events were documented and most of these events were not conserved across individuals. Differences in isoform-specific read densities indicated that 7% and 13% of AS events showed significant differences between individuals within geographically separated southern and northern populations, a level that is in general agreement with AS variation in human populations.

Conclusions

This genome-wide analysis of alternative splicing reveals high levels of AS in P. trichocarpa and extensive inter-individual AS variation. We provide the most comprehensive analysis of AS in P. trichocarpa to date, which will serve as a valuable resource for the plant community to study transcriptome complexity and AS regulation during wood formation.

Abstract (provisional)Background

Solanum dulcamara (bittersweet, climbing nightshade) is one of the few species of the Solanaceae family native to Europe. As a common weed it is adapted to a wide range of ecological niches and it has long been recognized as one of the alternative hosts for pathogens and pests responsible for many important diseases in potato, such as Phytophthora. At the same time, it may represent an alternative source of resistance genes against these diseases. Despite its unique ecology and potential as a genetic resource, genomic research tools are lacking for S. dulcamara. We have taken advantage of next-generation sequencing to speed up research on and use of this non-model species.

Results

In this work, we present the first large-scale characterization of the S. dulcamara transcriptome. Through comparison of RNAseq reads from two different accessions, we were able to predict transcript-based SNP and SSR markers. Using the SNP markers in combination with genomic AFLP and CAPS markers, the first genome-wide genetic linkage map of bittersweet was generated. Based on gene orthology, the markers were anchored to the genome of related Solanum species (tomato, potato and eggplant), revealing both conserved and novel chromosomal rearrangements. This allowed a better estimation of the evolutionary moment of rearrangements in a number of cases and showed that chromosomal breakpoints are regularly re-used.

Conclusion

Knowledge and tools developed as part of this study pave the way for future genomic research and exploitation of this wild Solanum species. The transcriptome assembly represents a resource for functional analysis of genes underlying interesting biological and agronomical traits and, in the absence of the full genome, provides a reference for RNAseq gene expression profiling aimed at understanding the unique biology of S. dulcamara. Cross-species orthology-based marker selection is shown to be a powerful tool to quickly generate a comparative genetic map, which may speed up gene mapping and contribute to the understanding of genome evolution within the Solanaceae family.

Abstract (provisional)Background

The presence of homoeologous sequences and absence of a reference genome sequence make discovery and genotyping of single nucleotide polymorphisms (SNPs) more challenging in polyploid crops.

Results

To address this challenge, we constructed reduced representation libraries (RRLs) for two Brassica napus inbred lines and their 91 doubled haploid (DH) progenies using a modified ddRADseq technique. A bioinformatics pipeline termed RFAPtools was developed to discover and genotype SNPs and presence/absence variations (PAVs). Using this pipeline, a pseudo-reference sequence (PRF) containing 180,991 sequence tags was constructed. By aligning sequence reads to the pseudo-reference sequence, allelic SNPs as well as PAVs were identified and genotyped with RFAPtools. Two parallel linkage maps, one SNP bin map containing 8,780 SNP loci and one PAV linkage map containing 12,423 dominant loci, were constructed. By aligning marker sequences to B. rapa sequence scaffolds, whose genome is available, we assigned 44 unassembled sequence scaffolds comprising 8.15 Mb onto the B. rapa chromosomes, and also identified 14 instances of misassembly and eight instances of mis-ordering sequence scaffolds.

Conclusions

These results indicate that the modified ddRADseq approach is a cost-effective and simple method to genotype tens of thousands SNPs and PAV markers in a polyploidy plant species. The results also demonstrated that RFAPtools developed in this study are powerful to mine allelic SNPs from homoeologous sequences in polyploids, therefore they are generally applicable in either diploid or polyploid species with or without a reference genome sequence.

Abstract (provisional)Background

Phenomena such as incomplete lineage sorting, horizontal gene transfer, gene duplication and subsequent sub- and neo-functionalisation can result in distinct local phylogenetic relationships that are discordant with species phylogeny. In order to assess the possible biological roles for these subdivisions, they must first be identified and characterised, preferably on a large scale and in an automated fashion.

Results

We developed Saguaro, a combination of a Hidden Markov Model (HMM) and a Self Organising Map (SOM), to characterise local phylogenetic relationships among aligned sequences using cacti, matrices of pair-wise distance measures. While the HMM determines the genomic boundaries from aligned sequences, the SOM hypothesises new cacti in an unsupervised and iterative fashion based on the regions that were modelled least well by existing cacti. After testing the software on simulated data, we demonstrate the utility of Saguaro by testing two different data sets: (i) 181 Dengue virus strains, and (ii) 5 primate genomes. Saguaro identifies regions under lineage-specific constraint for the first set, and genomic segments that we attribute to incomplete lineage sorting in the second dataset. Intriguingly for the primate data, Saguaro also classified an additional ~3% of the genome as most incompatible with the expected species phylogeny. A substantial fraction of these regions was found to overlap genes associated with both the innate and adaptive immune systems.

Conclusions

Saguaro detects distinct cacti describing local phylogenetic relationships without requiring any a priori hypotheses. We have successfully demonstrated Saguaro's utility with two contrasting data sets, one containing many members with short sequences (Dengue viral strains: n = 181, genome size = 10,700 nt), and the other with few members but complex genomes (related primate species: n = 5, genome size = 3 Gb), suggesting that the software is applicable to a wide variety of experimental populations. Saguaro is written in C++, runs on the Linux operating system, and can be downloaded from http://saguarogw.sourceforge.net/.

Abstract (provisional)Background

Ever since the recent completion of the peach genome, the focus of genetic research in this area has turned to the identification of genes related to important traits, such as fruit aroma volatiles. Of the over 100 volatile compounds described in peach, lactones most likely have the strongest effect on fruit aroma, while esters, terpenoids, and aldehydes have minor, yet significant effects. The identification of key genes underlying the production of aroma compounds is of interest for any fruit-quality improvement strategy.

Results

Volatile (52 compounds) and gene expression (4348 genes) levels were profiled in peach fruit from a maturity time-course series belonging to two peach genotypes that showed considerable differences in maturation characteristics and postharvest ripening. This data set was analyzed by complementary correlation-based approaches to discover the genes related to the main aroma-contributing compounds: lactones, esters, and phenolic volatiles, among others. As a case study, one of the candidate genes was cloned and expressed in yeast to show specificity as an omega-6 Oleate desaturase, which may be involved in the production of a precursor of lactones/esters.

Conclusions

Our approach revealed a set of genes (an alcohol acyl transferase, fatty acid desaturases, transcription factors, protein kinases, cytochromes, etc.) that are highly associated with peach fruit volatiles, and which could prove useful in breeding or for biotechnological purposes.