Chimeras in Merlot grapevine revealed by phased assembly.

Chimerism is the phenomenon when several genotypes coexist in a single individual. Used to understand plant ontogenesis they also have been valorised through new cultivar breeding. Viticulture has been taking economic advantage out of chimeras when the variant induced an important modification of wine type such as berry skin colour. Crucial agronomic characters may also be impacted by chimeras that aren't identified yet. Periclinal chimera where the variant has entirely colonised a cell layer is the most stable and can be propagated through cuttings. In grapevine, leaves are derived from both meristem layers, L1 and L2. However, lateral roots are formed from the L2 cell layer only. Thus, comparing DNA sequences of roots and leaves allows chimera detection. In this study we used new generation Hifi long reads sequencing, recent bioinformatics tools and trio-binning with parental sequences to detect periclinal chimeras on 'Merlot' grapevine cultivar. Sequencing of cv. 'Magdeleine Noire des Charentes' and 'Cabernet Franc', the parents of cv. 'Merlot', allowed haplotype resolved assembly. Pseudomolecules were built with a total of 33 to 47 contigs and in few occasions a unique contig for one chromosome. This high resolution allowed haplotype comparison. Annotation was transferred from PN40024 VCost.v3 to all pseudomolecules. After strong selection of variants, 51 and 53 'Merlot' specific periclinal chimeras were found on the Merlot-haplotype-CF and Merlot-haplotype-MG respectively, 9 and 7 been located in a coding region. A subset of positions was analysed using Molecular Inversion Probes (MIPseq) and 69% were unambiguously validated, 25% are doubtful because of technological noise or weak depth and 6% invalidated. These results open new perspectives on chimera detection as an important resource to improve cultivars through clonal selection or breeding.

Complete genome sequence of a novel grapevine-infecting member of the genus Polerovirus, grapevine polerovirus 1.

Double-stranded RNAs and total RNAs purified from grapevine (Vitis vinifera) phloem scrapings of two varieties held in the INRAE (France) grapevine germplasm collection were analyzed by high-throughput sequencing. BLAST annotation revealed contigs with homology to Polerovirus genus members. The full genome sequence of one isolate (KT) was determined (5651 nucleotides [nt]), and a partial sequence representing about half of the genome was assembled for a second isolate (KS) that was found to share 95% nt sequence identity with the KT isolate. The genome has a typical polerovirus organization, containing six open reading frames (ORFs) as well as a putative additional ORF3a. Based on genome organization and phylogenetic relationships, the new virus belongs to the genus Polerovirus but, similar to the recently described persimmon polerovirus 1, is characterized by a highly divergent coat-protein/readthrough domain. Considering the species demarcation criteria for the family Luteoviridae, these two isolates, together with a closely related sequence recently deposited in the GenBank database (LC507098), represent a new Polerovirus species for which the name "Grapevine polerovirus 1" is proposed.

1.15 PW-850 J compressed beam demonstration using the PETAL facility.

The Petawatt Aquitaine Laser (PETAL) facility was designed and constructed by the French Commissariat à l'énergie atomique et aux énergies alternatives (CEA) as an additional PW beamline to the Laser MegaJoule (LMJ) facility. PETAL energy is limited to 1 kJ at the beginning due to the damage threshold of the final optics. In this paper, we present the commissioning of the PW PETAL beamline. The first kJ shots in the amplifier section with a large spectrum front end, the alignment of the synthetic aperture compression stage and the initial demonstration of the 1.15 PW @ 850 J operations in the compression stage are detailed. Issues encountered relating to damage to optics are also addressed.

Complete Nucleotide Sequence of an Isolate of Grapevine Satellite Virus and Evidence for the Presence of Multimeric Forms in an Infected Grapevine.

The complete nucleotide sequence of an isolate of grapevine satellite virus (GV-Sat) was determined by next-generation sequencing (NGS) and compared with the single available complete sequence. The NGS data unexpectedly provided evidence for the existence of multimeric forms of GV-Sat, which were experimentally confirmed, allowing the redefinition of GV-Sat genomic ends.

High throughput analysis of grape genetic diversity as a tool for germplasm collection management.

Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI = 0.839 and 0.865, respectively) than V. vinifera cultivars (GDI = 0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI = 0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management.

Evolution of the VvMybA gene family, the major determinant of berry colour in cultivated grapevine (Vitis vinifera L.).

Polymorphisms in the grape transcription factor family VvMybA are responsible for variation in anthocyanin content in the berries of cultivated grapevine (Vitis vinifera L. subsp. sativa). Previous study has shown that white grapes arose through the mutation of two adjacent genes: a retroelement insertion in VvMybA1 and a single-nucleotide polymorphism mutation in VvMybA2. The purpose of this study was to understand how these mutations emerged and affected genetic diversity at neighbouring sites and how they structured the genetic diversity of cultivated grapevines. We sequenced a total of 3225 bp of these genes in a core collection of genetic resources, and carried out empirical selection tests, phylogenetic- and coalescence-based demographic analyses. The insertion in the VvMybA1 promoter was shown to have occurred recently, after the mutation of VvMybA2, both mutations followed by a selective sweep. The mutational pattern for these colour genes is consistent with progressively relaxed selection from constrained ancestral coloured haplotypes to light coloured and finally white haplotypes. Dynamics of population size in the VvMybA genes showed an initial exponential growth, followed by population size stabilization. Most ancestral haplotypes are found in cultivars from western region, whereas recent haplotypes are essentially present in table cultivars from eastern regions where intense breeding practices may have replaced the original diversity. Finally, the emergence of the white allele was followed by a recent strong exponential growth, showing a very fast diffusion of the initial white allele.

Linkage disequilibrium in wild French grapevine, Vitis vinifera L. subsp. silvestris.

Association mapping based on linkage disequilibrium (LD) can provide high resolution for whole-genome mapping of genes underlying phenotypic variation. This field has received considerable attention over the last decade. We present here the first characterization of LD in wild French grapevine, Vitis vinifera L. subsp. silvestris. To assess the pattern and extent of LD, we used a sample of 85 plants from southern France and 36 microsatellite markers distributed over 5 linkage groups. LD was evaluated with independence tests and multiallelic r(2), using both unphased genotypic data and reconstructed haplotypic data. LD decayed rapidly, with r(2) values decreasing to 0.1 within 2.7 cM for genotypic data and within 1.4 cM for haplotypic data. Compared to the results of a previous study on cultivated grapevine subsp. sativa, where significant LD was found up to 16.8 cM, LD in subsp. silvestris was no longer significant past 1.4 cM. LD was therefore 12 times further extended in cultivated than wild grapevine, even though LD in wild grapevine seemed to extend slightly further than in wild relatives of other crops. Domestication bottlenecks and vegetative propagation are the primary factors responsible for this difference between cultivated and wild grapevine. The rapid decay of LD observed in this study seems promising for future association mapping studies of functional variation in wild V. vinifera grapevine.

Development of a standard set of microsatellite reference alleles for identification of grape cultivars.

In order to investigate the comparability of microsatellite profiles obtained in different laboratories, ten partners in seven countries analyzed 46 grape cultivars at six loci (VVMD5, VVMD7, VVMD27, VVS2, VrZAG62, and VrZAG79). No effort was made to standardize equipment or protocols. Although some partners obtained very similar results, in other cases different absolute allele sizes and, sometimes, different relative allele sizes were obtained. A strategy for data comparison by means of reference to the alleles detected in well-known cultivars was proposed. For each marker, each allele was designated by a code based on the name of the reference cultivar carrying that allele. Thirty-three cultivars, representing from 13 to 23 alleles per marker, were chosen as references. After the raw data obtained by the different partners were coded, more than 97% of the data were in agreement. Minor discrepancies were attributed to errors, suboptimal amplification and visualization, and misscoring of heterozygous versus homozygous allele pairs. We have shown that coded microsatellite data produced in different laboratories with different protocols and conditions can be compared, and that it is suitable for the identification and SSR allele characterization of cultivars. It is proposed that the six markers employed here, already widely used, be adopted as a minimal standard marker set for future grapevine cultivar analyses, and that additional cultivars be characterized by means of the coded reference alleles presented here. The complete database is available at http://www.genres.de/eccdb/vitis/ Cuttings of the 33 reference cultivars are available on request from the Institut National de la Recherche Agronomique Vassal collection (didier.vares@ensam.inra.fr).

Subversion of the T/B lineage decision in the thymus by lunatic fringe-mediated inhibition of Notch-1.

Notch-1 signaling is essential for lymphoid progenitors to undergo T cell commitment, but the mechanism has not been defined. Here we show that thymocytes ectopically expressing Lunatic Fringe, a modifier of Notch-1 signaling, induce lymphoid progenitors to develop into B cells in the thymus. This cell fate switch resulted from Lunatic Fringe-mediated inhibition of Notch-1 function, as revealed by experiments utilizing lymphoid progenitors in which Notch-1 activity was genetically manipulated. These data identify Lunatic Fringe as a potent regulator of Notch-1 during the T/B lineage decision and show that an important function of Notch-1 in T cell commitment is to suppress B cell development in the thymus.

Monocyte chemoattractant protein-1 production by intestinal epithelial cells in vitro: a role for p38 in epithelial chemokine expression.

The intestinal epithelial cell (IEC) represents the first cellular barrier to infection. Consistent with this sentinel role, IEC are known to produce a variety of chemokines in response to bacterial infection or proinflammatory cytokines. These chemokines act as potent leukocyte activators and chemoattractants in vivo. In this report, we begin to characterize the regulation of expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in the rat small intestinal IEC-18 line. Following stimulation with either interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS), IEC-18 cells produced MCP-1, with IL-1 proving a more effective stimulus than LPS at both the mRNA and protein levels. Expression of MCP-1 due to either stimulus was inhibited by tyrosine kinase inhibitors, prompting us to investigate potential phosphotyrosine-dependent targets responsible for MCP-1 expression. We detected activation of p38, a member of the mitogen-activated protein kinase family, following either IL-1 or LPS treatment. Specific inhibition of this kinase using the compound SB203580 caused a destabilization of MCP-1 mRNA. These data point to a role for p38 in the regulation of MCP-1 mRNA expression by the IEC.

Species-specific alternative splicing generates a catalytically inactive form of human hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The enzyme is also expressed in steroidogenic tissues, mammary gland, muscle tissues and macrophages. A novel HSL mRNA termed hHSL-S, 228 bp shorter than the full-length HSL mRNA, was detected in human adipocytes. hHSL-S mRNA results from the in-frame skipping of exon 6, which encodes the serine residue of the catalytic triad. The corresponding 80 kDa protein was identified in human adipocytes after immunoprecipitation. The truncated protein expressed in COS cells showed neither lipase nor esterase activity but was phosphorylated by cAMP-dependent protein kinase. hHSL-S mRNA was found in all human tissues expressing HSL, except brown adipose tissue from newborns. It represented approx. 20% of total HSL transcripts in human subcutaneous adipocytes. No alternative splicing was detected in other mammals. Human and mouse three-exon HSL minigenes transfected into primate and rodent cell lines reproduced the splicing pattern of the endogenous HSL genes. Analysis of hybrid human/mouse minigenes transfected into human cell lines showed that cis-acting elements responsible for the skipping of human exon 6 were restricted to a 247 bp region including exon 6 and the first 19 nt of intron 6. Moreover, divergence in exonic splicing elements between mouse and human was shown to be critical for the species-specific alternative splicing.