Oxford Nanopore and Bionano Genomics technologies evaluation for plant structural variation detection.

BACKGROUND: Structural Variations (SVs) are genomic rearrangements derived from duplication, deletion, insertion, inversion, and translocation events. In the past, SVs detection was limited to cytological approaches, then to Next-Generation Sequencing (NGS) short reads and partitioned assemblies. Nowadays, technologies such as DNA long read sequencing and optical mapping have revolutionized the understanding of SVs in genomes, due to the enhancement of the power of SVs detection. This study aims to investigate performance of two techniques, 1) long-read sequencing obtained with the MinION device (Oxford Nanopore Technologies) and 2) optical mapping obtained with Saphyr device (Bionano Genomics) to detect and characterize SVs in the genomes of the two ecotypes of Arabidopsis thaliana, Columbia-0 (Col-0) and Landsberg erecta 1 (Ler-1). RESULTS: We described the SVs detected from the alignment of the best ONT assembly and DLE-1 optical maps of A. thaliana Ler-1 against the public reference genome Col-0 TAIR10.1. After filtering (SV > 1 kb), 1184 and 591 Ler-1 SVs were retained from ONT and Bionano technologies respectively. A total of 948 Ler-1 ONT SVs (80.1%) corresponded to 563 Bionano SVs (95.3%) leading to 563 common locations. The specific locations were scrutinized to assess improvement in SV detection by either technology. The ONT SVs were mostly detected near TE and gene features, and resistance genes seemed particularly impacted. CONCLUSIONS: Structural variations linked to ONT sequencing error were removed and false positives limited, with high quality Bionano SVs being conserved. When compared with the Col-0 TAIR10.1 reference genome, most of the detected SVs discovered by both technologies were found in the same locations. ONT assembly sequence leads to more specific SVs than Bionano one, the latter being more efficient to characterize large SVs. Even if both technologies are complementary approaches, ONT data appears to be more adapted to large scale populations studies, while Bionano performs better in improving assembly and describing specificity of a genome compared to a reference.

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.

Dissecting genetic architecture of grape proanthocyanidin composition through quantitative trait locus mapping.

BACKGROUND: Proanthocyanidins (PAs), or condensed tannins, are flavonoid polymers, widespread throughout the plant kingdom, which provide protection against herbivores while conferring organoleptic and nutritive values to plant-derived foods, such as wine. However, the genetic basis of qualitative and quantitative PA composition variation is still poorly understood. To elucidate the genetic architecture of the complex grape PA composition, we first carried out quantitative trait locus (QTL) analysis on a 191-individual pseudo-F1 progeny. Three categories of PA variables were assessed: total content, percentages of constitutive subunits and composite ratio variables. For nine functional candidate genes, among which eight co-located with QTLs, we performed association analyses using a diversity panel of 141 grapevine cultivars in order to identify causal SNPs. RESULTS: Multiple QTL analysis revealed a total of 103 and 43 QTLs, respectively for seed and skin PA variables. Loci were mainly of additive effect while some loci were primarily of dominant effect. Results also showed a large involvement of pairwise epistatic interactions in shaping PA composition. QTLs for PA variables in skin and seeds differed in number, position, involvement of epistatic interaction and allelic effect, thus revealing different genetic determinisms for grape PA composition in seeds and skin. Association results were consistent with QTL analyses in most cases: four out of nine tested candidate genes (VvLAR1, VvMYBPA2, VvCHI1, VvMYBPA1) showed at least one significant association with PA variables, especially VvLAR1 revealed as of great interest for further functional investigation. Some SNP-phenotype associations were observed only in the diversity panel. CONCLUSIONS: This study presents the first QTL analysis on grape berry PA composition with a comparison between skin and seeds, together with an association study. Our results suggest a complex genetic control for PA traits and different genetic architectures for grape PA composition between berry skin and seeds. This work also uncovers novel genomic regions for further investigation in order to increase our knowledge of the genetic basis of PA composition.

An efficient and rapid protocol for plant nuclear DNA preparation suitable for next generation sequencing methods.

PREMISE OF THE STUDY: In this study, we developed a nuclear DNA extraction protocol for Next Generation Sequencers (NGS). METHODS AND RESULTS: We applied this extraction method to grapevines and coffee trees, which are known to contain many secondary metabolites. The nuclear DNA obtained was sequenced by the 454/GS-FLX method. We obtained excellent results, with less than 4% cytoplasmic DNA, in a similar way to a BAC (Bacterial Artificial Chromosome)-building protocol. We also compared our protocol with a classic DNA extraction using specific cytoplasmic DNA amplification. Results showed a lower cytoplasmic DNA contamination with the new protocol. CONCLUSIONS: The method presented here is fast and economical. The DNA obtained is of high quality, with a low level of cytoplasmic DNA contamination, and very efficient for the construction of sequencing libraries.

Patterns of sequence polymorphism in the fleshless berry locus in cultivated and wild Vitis vinifera accessions.

BACKGROUND: Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (Vitis vinifera L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the fleshless berry mutation (flb) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication. RESULTS: In total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both V. vinifera cv. Chardonnay and a genotype carrying the flb mutation, V. vinifera cv. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that flb could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the flb region, representing 48,874 bp in a highly diverse set of cultivated and wild V. vinifera genotypes, to identify possible signatures of domestication in the cultivated V. vinifera compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the flb region. CONCLUSIONS: We observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the flb region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the flb gene in a F2 population when available.

A physical map of the heterozygous grapevine 'Cabernet Sauvignon' allows mapping candidate genes for disease resistance.

BACKGROUND: Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed. RESULTS: The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome. CONCLUSION: Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.

Decoding the oak genome: public release of sequence data, assembly, annotation and publication strategies.

The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole-genome shotgun (WGS) approach, without the use of costly and time-consuming methods, such as fosmid or BAC clone-based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS-FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired-end and mate-pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired-end reads and contaminants detected, resulting in a total of 17,910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.

Production of taxoids with biological activity by plants and callus culture from selected Taxus genotypes.

Twenty seven different yew trees belonging to various genotypes and hybrids have been screened for their capacity to produce significant amounts of taxoids provided with biological activity in the tubulin test. From the three best genotypes selected, Taxus x media "Sargentii" proved to be able to produce viable calluses from excised roots placed in vitro. Taxoid composition at various times of the in vitro culture was determined and the carcinostatic efficiency of the extracts was established using the KB cell cytotoxicity test. In leaves and calluses, respectively, 0.069 and 0.032% paclitaxel (taxol) contents were found. These contents were significantly higher than those previously reported for other genotypes.

Neutral invertases in grapevine and comparative analysis with Arabidopsis, poplar and rice.

Neutral invertases (NIs, EC 3.2.1.26) cleave sucrose to glucose and fructose. They are encoded by a small gene family of 9 members in the Arabidopsis genome, 8 in rice, 16 in poplar and 9 in Vitis vinifera (L.). The grapevine NIs were identified in the 8.4X genome assembly of the quasi-homozygous line PN40024. In addition, alleles of three NIs were sequenced in the heterozygous cultivar 'Cabernet Sauvignon'. Analyses of sequence variation between alleles, homoeologous and paralogous copies in grapevine and their orthologues in Arabidopsis, poplar and rice are provided. In grapevine, NIs were classified into four alpha NIs and five beta NIs and subsequently grouped into hierarchical clades using a combination of evidence including amino acid identity, exon/intron structure, rate of synonymous substitutions (K (s)) and chromosomal distribution. Estimation of K (s) proved the ancient origin of all NIs and the lack of expansion by gene duplication past the event of polyploidisation. We then focused on transcription analysis of five NIs for which evidence of expression was available from expressed sequence tag databases. Among these, four NIs consisted of pairs of homoeologous copies, each pair lying on a pair of chromosomes duplicated by polyploidy. Unequal expression of homoeologous genes was observed by quantitative RT-PCR in leaf, flower, seed and root tissues. Since NIs might play significant roles in fruit and wine quality, NIs expression was monitored in flesh and skin of 'Merlot' berries and shown in parallel with the suite of changes that accompany fruit ripening, including glucose and fructose accumulation.

Molecular characterization and expression analysis of the Rop GTPase family in Vitis vinifera.

Rop/Rac GTPases are plant-specific signalling proteins with multiple roles, some of which have implications in plant development and in hormone signal transduction. Using expressed sequence tag (EST) and gene database analyses, members of the Rop family were characterized for the first time in a perennial species (Vitis vinifera). The grapevine genome was found to contain seven expressed VvRops. The phylogenetic analyses indicated that VvRops could be distributed into four groups, as described in the literature for model plants. Genetic mapping was successfully performed for five VvRops, which were localized on independent linkage groups. Conserved and divergent regions were identified on the protein sequences. The results of VvRop expression obtained by real-time quantitative reverse transcription-PCR analyses indicated that all the organs investigated displayed VvRop expression, however with different patterns. Whereas no total organ specificity for VvRop expression could be evidenced, VvRop9 displayed high expression in developing berries only. During berry development, the transcript profile was generally similar for all the VvRops, i.e. displaying a peak early in the herbaceous phase followed by a decline towards veraison and thereafter. Western blotting gave a similar expression profile for VvRop proteins. Response to growth regulators was generally specific to each VvRop. The potential involvement of specific VvRops in grapevine development is discussed.

Variation in crossing-over rates across chromosome 4 of Arabidopsis thaliana reveals the presence of meiotic recombination "hot spots".

Crossover (CO) is a key process for the accurate segregation of homologous chromosomes during the first meiotic division. In most eukaryotes, meiotic recombination is not homogeneous along the chromosomes, suggesting a tight control of the location of recombination events. We genotyped 71 single nucleotide polymorphisms (SNPs) covering the entire chromosome 4 of Arabidopsis thaliana on 702 F2 plants, representing 1404 meioses and allowing the detection of 1171 COs, to study CO localization in a higher plant. The genetic recombination rates varied along the chromosome from 0 cM/Mb near the centromere to 20 cM/Mb on the short arm next to the NOR region, with a chromosome average of 4.6 cM/Mb. Principal component analysis showed that CO rates negatively correlate with the G+C content (P = 3x10(-4)), in contrast to that reported in other eukaryotes. COs also significantly correlate with the density of single repeats and the CpG ratio, but not with genes, pseudogenes, transposable elements, or dispersed repeats. Chromosome 4 has, on average, 1.6 COs per meiosis, and these COs are subjected to interference. A detailed analysis of several regions having high CO rates revealed "hot spots" of meiotic recombination contained in small fragments of a few kilobases. Both the intensity and the density of these hot spots explain the variation of CO rates along the chromosome.

Single-nucleotide polymorphism frequency in a set of selected lines of bread wheat (Triticum aestivum L.).

Information on single-nucleotide polymorphisms (SNPs) in hexaploid bread wheat is still scarce. The goal of this study was to detect SNPs in wheat and examine their frequency. Twenty-six bread wheat lines from different origins worldwide were used. Specific PCR-products were obtained from 21 genes and directly sequenced. SNPs were discovered from the alignment of these sequences. The overall sequence polymorphism observed in this sample appears to be low; 64 single-base polymorphisms were detected in approximately 21.5 kb (i.e., 1 SNP every 335 bp). The level of polymorphism is highly variable among the different genes studied. Fifty percent of the genes studied contained no sequence polymorphism, whereas most SNPs detected were located in only 2 genes. As expected, taking into account a synthetic line created with a wild Triticum tauschii parent increases the level of polymorphism (101 SNPs; 1 SNP every 212 bp). The detected SNPs are available at http://urgi.versailles.inra.fr/GnpSNP">http://urgi.versailles.inra.fr/GnpSNP. Data on linkage disequilibrium (LD) are still preliminary. They showed a significant level of LD in the 2 most polymorphic genes. To conclude, the genome size of hexaploid wheat and its low level of polymorphism complicate SNP discovery in this species.