Assembly and Annotation of Red Spruce (Picea rubens) Chloroplast Genome, Identification of Simple Sequence Repeats, and Phylogenetic Analysis in Picea.

We have sequenced the chloroplast genome of red spruce (Picea rubens) for the first time using the single-end, short-reads (44 bp) Illumina sequences, assembled and functionally annotated it, and identified simple sequence repeats (SSRs). The contigs were assembled using SOAPdenovo2 following the retrieval of chloroplast genome sequences using the black spruce (Picea mariana) chloroplast genome as the reference. The assembled genome length was 122,115 bp (gaps included). Comparatively, the P. rubens chloroplast genome reported here may be considered a near-complete draft. Global genome alignment and phylogenetic analysis based on the whole chloroplast genome sequences of Picea rubens and 10 other Picea species revealed high sequence synteny and conservation among 11 Picea species and phylogenetic relationships consistent with their known classical interrelationships and published molecular phylogeny. The P. rubens chloroplast genome sequence showed the highest similarity with that of P. mariana and the lowest with that of P. sitchensis. We have annotated 107 genes including 69 protein-coding genes, 28 tRNAs, 4 rRNAs, few pseudogenes, identified 42 SSRs, and successfully designed primers for 26 SSRs. Mononucleotide A/T repeats were the most common followed by dinucleotide AT repeats. A similar pattern of microsatellite repeats occurrence was found in the chloroplast genomes of 11 Picea species.

Genomic tools for durum wheat breeding: de novo assembly of Svevo transcriptome and SNP discovery in elite germplasm.

BACKGROUND: The tetraploid durum wheat (Triticum turgidum L. ssp. durum Desf. Husnot) is an important crop which provides the raw material for pasta production and a valuable source of genetic diversity for breeding hexaploid wheat (Triticum aestivum L.). Future breeding efforts to enhance yield potential and climate resilience will increasingly rely on genomics-based approaches to identify and select beneficial alleles. A deeper characterisation of the molecular and functional diversity of the durum wheat transcriptome will be instrumental to more effectively harness its genetic diversity. RESULTS: We report on the de novo transcriptome assembly of durum wheat cultivar 'Svevo'. The transcriptome of four tissues/organs (shoots and roots at the seedling stage, reproductive organs and developing grains) was assembled de novo, yielding 180,108 contigs, with a N50 length of 1121 bp and mean contig length of 883 bp. Alignment against the transcriptome of nine plant species identified 43% of transcripts with homology to at least one reference transcriptome. The functional annotation was completed by means of a combination of complementary software. The presence of differential expression between the A- and B-homoeolog copies of the durum wheat tetraploid genome was ascertained by phase reconstruction of polymorphic sites based on the T. urartu transcripts and inferring homoeolog-specific sequences. We observed greater expression divergence between A and B homoeologs in grains rather than in leaves and roots. The transcriptomes of 13 durum wheat cultivars spanning the breeding period from 1969 to 2005 were analysed for SNP diversity, leading to 95,358 non-rare, hemi-SNPs shared among two or more cultivars and 33,747 locus-specific (diploid inheritance) SNPs. CONCLUSIONS: Our study updates and expands the de novo transcriptome reference assembly available for durum wheat. Out of 180,108 assembled transcripts, 13,636 were specific to the Svevo cultivar as compared to the only other reference transcriptome available for durum, thus contributing to the identification of the tetraploid wheat pan-transcriptome. Additionally, the analysis of 13 historically relevant hallmark varieties produced a SNP dataset that could successfully validate the genotyping in tetraploid wheat and provide a valuable resource for genomics-assisted breeding of both tetraploid and hexaploid wheats.

Single primer enrichment technology as a tool for massive genotyping: a benchmark on black poplar and maize.

BACKGROUND AND AIMS: The advent of molecular breeding is advocated to improve the productivity and sustainability of second-generation bioenergy crops. Advanced molecular breeding in bioenergy crops relies on the ability to massively sample the genetic diversity. Genotyping-by-sequencing has become a widely adopted method for cost-effective genotyping. It basically requires no initial investment for design as compared with array-based platforms which have been shown to offer very robust assays. The latter, however, has the drawback of being limited to analyse only the genetic diversity accounted during selection of a set of polymorphisms and design of the assay. In contrast, genotyping-by-sequencing with random sampling of genomic loci via restriction enzymes or random priming has been shown to be fast and convenient but lacks the ability to target specific regions of the genome and to maintain high reproducibility across laboratories. METHODS: Here we present a first adoption of single-primer enrichment technology (SPET) which provides a highly efficient and scalable system to obtain targeted sequence-based large genotyping data sets, bridging the gaps between array-based systems and traditional sequencing-based protocols. To fully explore SPET performance, we conducted a benchmark study in ten Zea mays lines and a large-scale study of a natural black poplar population of 540 individuals with the aim of discovering polymorphisms associated with biomass-related traits. KEY RESULTS: Our results showed the ability of this technology to provide dense genotype information on a customized panel of selected polymorphisms, while yielding hundreds of thousands of untargeted variable sites. This provided an ideal resource for association analysis of natural populations harbouring unexplored allelic diversities and structure such as in black poplar. CONCLUSION: The improvement of sequencing throughput and the development of efficient library preparation methods has made it feasible to carry out targeted genotyping-by-sequencing experiments cost-competitively with either random complexity reduction systems or traditional array-based platforms, while maintaining the key advantages of both technologies.

Metagenomic profiles of different types of Italian high-moisture Mozzarella cheese.

The microbiota of different types of Italian high-moisture Mozzarella cheese produced using cow or buffalo milk, acidified with natural or selected cultures, and sampled at the dairy or at the mass market, was evaluated using a Next Generation Sequencing approach, in order to identify possible drivers of the bacterial diversity. Cow Mozzarella and buffalo Mozzarella acidified with commercial cultures were dominated by Streptococcus thermophilus, while buffalo samples acidified with natural whey cultures showed similar prevalence of L. delbrueckii subsp. bulgaricus, L. helveticus and S. thermophilus. Moreover, several species of non-starter lactic acid bacteria were frequently detected. The diversity in cow Mozzarella microbiota was much higher than that of water buffalo samples. Cluster analysis clearly separated cow's cheeses from buffalo's ones, the former having a higher prevalence of psychrophilic taxa, and the latter of Lactobacillus and Streptococcus. A higher prevalence of psychrophilic species and potential spoilers was observed in samples collected at the mass retail, suggesting that longer exposures to cooling temperatures and longer production-to-consumption times could significantly affect microbiota diversity. Our results could help in detecting some kind of thermal abuse during the production or storage of mozzarella cheese.

Characterization of the Poplar Pan-Genome by Genome-Wide Identification of Structural Variation.

Many recent studies have emphasized the important role of structural variation (SV) in determining human genetic and phenotypic variation. In plants, studies aimed at elucidating the extent of SV are still in their infancy. Evidence has indicated a high presence and an active role of SV in driving plant genome evolution in different plant species.With the aim of characterizing the size and the composition of the poplar pan-genome, we performed a genome-wide analysis of structural variation in three intercrossable poplar species: Populus nigra, Populus deltoides, and Populus trichocarpa We detected a total of 7,889 deletions and 10,586 insertions relative to the P. trichocarpa reference genome, covering respectively 33.2 Mb and 62.9 Mb of genomic sequence, and 3,230 genes affected by copy number variation (CNV). The majority of the detected variants are inter-specific in agreement with a recent origin following separation of species.Insertions and deletions (INDELs) were preferentially located in low-gene density regions of the poplar genome and were, for the majority, associated with the activity of transposable elements. Genes affected by SV showed lower-than-average expression levels and higher levels of dN/dS, suggesting that they are subject to relaxed selective pressure or correspond to pseudogenes.Functional annotation of genes affected by INDELs showed over-representation of categories associated with transposable elements activity, while genes affected by genic CNVs showed enrichment in categories related to resistance to stress and pathogens. This study provides a genome-wide catalogue of SV and the first insight on functional and structural properties of the poplar pan-genome.

The build-up of osmotic stress responses within the growing root apex using kinematics and RNA-sequencing.

Molecular regulation of growth must include spatial and temporal coupling of cell production and cell expansion. The underlying mechanisms, especially under environmental challenge, remain obscure. Spatial patterns of cell processes make the root apex well suited to deciphering stress signaling pathways, and to investigating both processes. Kinematics and RNA-sequencing were used to analyze the immediate growth response of hydroponically grown Populus nigra cuttings submitted to osmotic stress. About 7400 genes and unannotated transcriptionally active regions were differentially expressed between the division and elongation zones. Following the onset of stress, growth decreased sharply, probably due to mechanical effects, before recovering partially. Stress impaired cell expansion over the apex, progressively shortened the elongation zone, and reduced the cell production rate. Changes in gene expression revealed that growth reduction was mediated by a shift in hormone homeostasis. Osmotic stress rapidly elicited auxin, ethylene, and abscisic acid. When growth restabilized, transcriptome remodeling became complex and zone specific, with the deployment of hormone signaling cascades, transcriptional regulators, and stress-responsive genes. Most transcriptional regulations fit growth reduction, but stress also promoted expression of some growth effectors, including aquaporins and expansins Together, osmotic stress interfered with growth by activating regulatory proteins rather than by repressing the machinery of expansive growth.

The physical map of wheat chromosome 5DS revealed gene duplications and small rearrangements.

BACKGROUND: The substantially large bread wheat genome, organized into highly similar three sub-genomes, renders genomic research challenging. The construction of BAC-based physical maps of individual chromosomes reduces the complexity of this allohexaploid genome, enables elucidation of gene space and evolutionary relationships, provides tools for map-based cloning, and serves as a framework for reference sequencing efforts. In this study, we constructed the first comprehensive physical map of wheat chromosome arm 5DS, thereby exploring its gene space organization and evolution. RESULTS: The physical map of 5DS was comprised of 164 contigs, of which 45 were organized into 21 supercontigs, covering 176 Mb with an N50 value of 2,173 kb. Fifty-eight of the contigs were larger than 1 Mb, with the largest contig spanning 6,649 kb. A total of 1,864 molecular markers were assigned to the map at a density of 10.5 markers/Mb, anchoring 100 of the 120 contigs (>5 clones) that constitute ~95 % of the cumulative length of the map. Ordering of 80 contigs along the deletion bins of chromosome arm 5DS revealed small-scale breaks in syntenic blocks. Analysis of the gene space of 5DS suggested an increasing gradient of genes organized in islands towards the telomere, with the highest gene density of 5.17 genes/Mb in the 0.67-0.78 deletion bin, 1.4 to 1.6 times that of all other bins. CONCLUSIONS: Here, we provide a chromosome-specific view into the organization and evolution of the D genome of bread wheat, in comparison to one of its ancestors, revealing recent genome rearrangements. The high-quality physical map constructed in this study paves the way for the assembly of a reference sequence, from which breeding efforts will greatly benefit.

High-throughput physical map anchoring via BAC-pool sequencing.

BACKGROUND: Physical maps created from large insert DNA libraries, typically cloned in BAC vector, are valuable resources for map-based cloning and de novo genome sequencing. The maps are most useful if contigs of overlapping DNA clones are anchored to chromosome(s), and ordered along them using molecular markers. Here we present a novel approach for anchoring physical maps, based on sequencing three-dimensional pools of BAC clones from minimum tilling path. RESULTS: We used physical map of wheat chromosome arm 3DS to validate the method with two different DNA sequence datasets. The first comprised 567 genes ordered along the chromosome arm based on syntenic relationship of wheat with the sequenced genomes of Brachypodium, rice and sorghum. The second dataset consisted of 7,136 SNP-containing sequences, which were mapped genetically in Aegilops tauschii, the donor of the wheat D genome. Mapping of sequence reads from individual BAC pools to the first and the second datasets enabled unambiguous anchoring 447 and 311 3DS-specific sequences, respectively, or 758 in total. CONCLUSIONS: We demonstrate the utility of the novel approach for BAC contig anchoring based on mass parallel sequencing of three-dimensional pools prepared from minimum tilling path of physical map. The existing genetic markers as well as any other DNA sequence could be mapped to BAC clones in a single in silico experiment. The approach reduces significantly the cost and time needed for anchoring and is applicable to any genomic project involving the construction of anchored physical map.

Synergistic parasite-pathogen interactions mediated by host immunity can drive the collapse of honeybee colonies.

The health of the honeybee and, indirectly, global crop production are threatened by several biotic and abiotic factors, which play a poorly defined role in the induction of widespread colony losses. Recent descriptive studies suggest that colony losses are often related to the interaction between pathogens and other stress factors, including parasites. Through an integrated analysis of the population and molecular changes associated with the collapse of honeybee colonies infested by the parasitic mite Varroa destructor, we show that this parasite can de-stabilise the within-host dynamics of Deformed wing virus (DWV), transforming a cryptic and vertically transmitted virus into a rapidly replicating killer, which attains lethal levels late in the season. The de-stabilisation of DWV infection is associated with an immunosuppression syndrome, characterized by a strong down-regulation of the transcription factor NF-κB. The centrality of NF-κB in host responses to a range of environmental challenges suggests that this transcription factor can act as a common currency underlying colony collapse that may be triggered by different causes. Our results offer an integrated account for the multifactorial origin of honeybee losses and a new framework for assessing, and possibly mitigating, the impact of environmental challenges on honeybee health.

Flow sorting and sequencing meadow fescue chromosome 4F.

The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before.

Odorant-binding proteins and olfactory coding in the solitary bee Osmia cornuta.

Solitary bees are major pollinators but their chemical communication system has been poorly studied. We investigated olfactory coding in Osmia cornuta from two perspectives, chemical and biochemical. We identified (E)-geranyl acetone and 2-hexyl-1,3-dioxolane, specifically secreted by females and males, respectively. A transcriptome analysis of antennae revealed 48 ORs (olfactory receptors), six OBPs (odorant-binding proteins), five CSPs (chemosensory proteins), and a single SNMP (sensory neuron membrane protein). The numbers of ORs and OBPs are much lower than in the honeybee, in particular, C-minus OBPs are lacking in the antennae of O. cornuta. We have expressed all six OBPs of O. cornuta and studied their binding specificities. The best ligands are common terpene plant odorants and both volatiles produced by the bee and identified in this work.

The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.

The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.

Draft genome sequence of Lactobacillus rossiae DSM 15814(T).

The draft genome sequence of Lactobacillus rossiae DSM 15814(T) (CS1, ATCC BAA-88) was determined by a whole-genome shotgun approach. Reads were assembled to a 2.9-Mb draft version. RAST genome annotation evidenced 2,723 predicted coding sequences. Many carbohydrate, amino acid, and amino acid derivative subsystem features were found.

Large-scale detection of rare variants via pooled multiplexed next-generation sequencing: towards next-generation Ecotilling.

Common variants, such as those identified by genome-wide association scans, explain only a small proportion of trait variation. Growing evidence suggests that rare functional variants, which are usually missed by genome-wide association scans, play an important role in determining the phenotype. We used pooled multiplexed next-generation sequencing and a customized analysis workflow to detect mutations in five candidate genes for lignin biosynthesis in 768 pooled Populus nigra accessions. We identified a total of 36 non-synonymous single nucleotide polymorphisms, one of which causes a premature stop codon. The most common variant was estimated to be present in 672 of the 1536 tested chromosomes, while the rarest was estimated to occur only once in 1536 chromosomes. Comparison with individual Sanger sequencing in a selected sub-sample confirmed that variants are identified with high sensitivity and specificity, and that the variant frequency was estimated accurately. This proposed method for identification of rare polymorphisms allows accurate detection of variation in many individuals, and is cost-effective compared to individual sequencing.

The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis.

BACKGROUND: Plant microRNAs (miRNAs) are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA) libraries can detect even poorly expressed miRNAs.No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots. RESULTS: Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families) of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs) identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1. CONCLUSIONS: The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.

Functional features of a single chromosome arm in wheat (1AL) determined from its structure.

Bread wheat (Triticum aestivum L.) is one of the most important crops globally and a high priority for genetic improvement, but its large and complex genome has been seen as intractable to whole genome sequencing. Isolation of individual wheat chromosome arms has facilitated large-scale sequence analyses. However, so far there is no such survey of sequences from the A genome of wheat. Greater understanding of an A chromosome could facilitate wheat improvement and future sequencing of the entire genome. We have constructed BAC library from the long arm of T. aestivum chromosome 1A (1AL) and obtained BAC end sequences from 7,470 clones encompassing the arm. We obtained 13,445 (89.99%) useful sequences with a cumulative length of 7.57 Mb, representing 1.43% of 1AL and about 0.14% of the entire A genome. The GC content of the sequences was 44.7%, and 90% of the chromosome was estimated to comprise repeat sequences, while just over 1% encoded expressed genes. From the sequence data, we identified a large number of sites suitable for development of molecular markers (362 SSR and 6,948 ISBP) which will have utility for mapping this chromosome and for marker assisted breeding. From 44 putative ISBP markers tested 23 (52.3%) were found to be useful. The BAC end sequence data also enabled the identification of genes and syntenic blocks specific to chromosome 1AL, suggesting regions of particular functional interest and targets for future research.

Intraspecific sequence comparisons reveal similar rates of non-collinear gene insertion in the B and D genomes of bread wheat.

BACKGROUND: Polyploidization is considered one of the main mechanisms of plant genome evolution. The presence of multiple copies of the same gene reduces selection pressure and permits sub-functionalization and neo-functionalization leading to plant diversification, adaptation and speciation. In bread wheat, polyploidization and the prevalence of transposable elements resulted in massive gene duplication and movement. As a result, the number of genes which are non-collinear to genomes of related species seems markedly increased in wheat. RESULTS: We used new-generation sequencing (NGS) to generate sequence of a Mb-sized region from wheat chromosome arm 3DS. Sequence assembly of 24 BAC clones resulted in two scaffolds of 1,264,820 and 333,768 bases. The sequence was annotated and compared to the homoeologous region on wheat chromosome 3B and orthologous loci of Brachypodium distachyon and rice. Among 39 coding sequences in the 3DS scaffolds, 32 have a homoeolog on chromosome 3B. In contrast, only fifteen and fourteen orthologs were identified in the corresponding regions in rice and Brachypodium, respectively. Interestingly, five pseudogenes were identified among the non-collinear coding sequences at the 3B locus, while none was found at the 3DS locus. CONCLUSION: Direct comparison of two Mb-sized regions of the B and D genomes of bread wheat revealed similar rates of non-collinear gene insertion in both genomes with a majority of gene duplications occurring before their divergence. Relatively low proportion of pseudogenes was identified among non-collinear coding sequences. Our data suggest that the pseudogenes did not originate from insertion of non-functional copies, but were formed later during the evolution of hexaploid wheat. Some evidence was found for gene erosion along the B genome locus.

A 3,000-loci transcription map of chromosome 3B unravels the structural and functional features of gene islands in hexaploid wheat.

To improve our understanding of the organization and regulation of the wheat (Triticum aestivum) gene space, we established a transcription map of a wheat chromosome (3B) by hybridizing a newly developed wheat expression microarray with bacterial artificial chromosome pools from a new version of the 3B physical map as well as with cDNA probes derived from 15 RNA samples. Mapping data for almost 3,000 genes showed that the gene space spans the whole chromosome 3B with a 2-fold increase of gene density toward the telomeres due to an increase in the number of genes in islands. Comparative analyses with rice (Oryza sativa) and Brachypodium distachyon revealed that these gene islands are composed mainly of genes likely originating from interchromosomal gene duplications. Gene Ontology and expression profile analyses for the 3,000 genes located along the chromosome revealed that the gene islands are enriched significantly in genes sharing the same function or expression profile, thereby suggesting that genes in islands acquired shared regulation during evolution. Only a small fraction of these clusters of cofunctional and coexpressed genes was conserved with rice and B. distachyon, indicating a recent origin. Finally, genes with the same expression profiles in remote islands (coregulation islands) were identified suggesting long-distance regulation of gene expression along the chromosomes in wheat.

Evaluation of sensitivity and specificity in RNA-Seq-based detection of grapevine viral pathogens.

Virus detection is a crucial step for the implementation of clean stock programs that preserve healthy crop species. Viral infections in grapevine, a vegetatively propagated perennial crop, cannot be eradicated from the vineyards by the application of agrochemicals and must be curtailed at the stage of nursery production during the propagation of planting material. Viral detection is routinely performed using enzyme-linked immunosorbent assays (ELISA) or Reverse Transcription-quantitative Polymerase Chain Reactions (RT-qPCR). High throughput sequencing (HTS) approaches have the potential to detect all viral pathogens in a plant specimen. However, to date, no published HTS-based study has used threshold selection based on ROC curves for discriminating positive from negative samples. To fill this gap, we assessed the specificity and sensitivity of different sequencing and bioinformatics approaches for nine common viruses, which were tested in the same specimens using ELISA and/or RT-qPCR. The normalized detection thresholds giving the best results were 19.28 Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for alignment-based total RNA-Seq approaches, 386 Reads Per Million mapped reads (RPM) for metagenomics-based total RNA-Seq, 1572 FPKM for alignment-based small RNA-Seq analysis and 0.97 % of contigs for de novo analysis of small RNA-Seq data. Validation of the proposed thresholds using independent specimens collected over time from the same stocks and other specimens collected from nearby stocks that had derived from the same propagating material showed that HTS approaches are accurate, with RNA-Seq approaches showing better performance than small RNA-Seq.

Analysis of transposons and repeat composition of the sunflower (Helianthus annuus L.) genome.

A sample-sequencing strategy combined with slot-blot hybridization and FISH was used to study the composition of the repetitive component of the sunflower genome. One thousand six hundred thirty-eight sequences for a total of 954,517 bp were analyzed. The fraction of sequences that can be classified as repetitive using computational and hybridization approaches amounts to 62% in total. Almost two thirds remain as yet uncharacterized in nature. Of those characterized, most belong to the gypsy superfamily of LTR-retrotransposons. Unlike in other species, where single families can account for large fractions of the genome, it appears that no transposon family has been amplified to very high levels in sunflower. All other known classes of transposable elements were also found. One family of unknown nature (contig 61) was the most repeated in the sunflower genome. The evolution of the repetitive component in the Helianthus genus and in other Asteraceae was studied by comparative analysis of the hybridization of total genomic DNAs from these species to the sunflower small-insert library and compared to gene-based phylogeny. Very little similarity is observed between Helianthus species and two related Asteraceae species outside of the genus. Most repetitive elements are similar in annual and perennial Helianthus species indicating that sequence amplification largely predates such divergence. Gypsy-like elements are more represented in the annuals than in the perennials, while copia-like elements are similarly represented, attesting a different amplification history of the two superfamilies of LTR-retrotransposons in the Helianthus genus.