High quality phased assembly of grape genome offer new opportunities in chimera detection

In perennial plants and especially those propagated through cuttings, several genotypes can coexist in a single individual, thus leading to chimeras. When the variant induces a noticeable phenotype modification, it can lead to a new cultivar. Viticulture already took economic advantage of this natural phenomenon: for instance, the berry skin of ‘Pinot gris’ derived from ‘Pinot noir’ by the selection of a chimera. Chimeras could also impact other crucial traits without being visually identified. Periclinal chimera where the variant has entirely colonized a cell layer is the most stable and can be propagated through cuttings. In grapevine, two functional cell layers are present in leaves, L1 and L2. However, lateral roots are formed from the L2 cell layer only. Thus, comparing DNA sequences of roots and leaves could allow chimera detection. In this study we used new generation Hifi long reads sequencing and recent bioinformatics tools applied to ‘Merlot’ to detect periclinal chimeras. Sequencing of ‘Magdeleine Noire des Charentes’ and ‘Cabernet franc’, the parents of ‘Merlot’, allowed haplotype resolved assembly. Pseudomolecules were built with few contigs, in some occasions only one per chromosome. This high resolution allowed haplotype comparison. Annotation from PN40024 was transferred to all pseudomolecules. Through variant detection, periclinal chimeras were found on both haplotypes. These results open new perspectives on chimera detection, which is an important resource to improve cultivars through clonal selection or breed new ones. Detailed results will be presented and discussed

Genetic diversity of wild and cultivated grapevine accessions from southeast Turkey

INRA UMR 1334 AGAP, Equipe DAVEM = Diversité et Adaptation de la Vigne et des Espèces MéditerranéennesWild grapevine genetic diversity in southeast Turkey has not been documented to date. In the present work, in order to clarify the relationships between wild and cultivated grape accessions from southeastern Turkey, 22 nuclear and three chloroplast microsatellite loci were used on 21 wild grapevine Vitis vinifera L. ssp. sylvestris (Gmelin) and 13 cultivated grapevine Vitis vinifera ssp. sativa accessions. The number of alleles per SSR locus ranged from 4 (VVIn16) to 20 (VVIv67) and the mean allele number per locus was 10.09. Expected locus heterozygosity ranged from 0.586 (locus VVIb01) to 0.898 (locus (VVIv67)). The three cpSSR molecular markers presented variation in size both in cultivars and in wild Turkish accessions. Two size variants were detected for cpSSR3 (106 and 107 bp) for cpSSR5 (104 and 105 bp), and for cpSSR10 (115 and 116 bp). The six alleles in wild grapevines fell into three haplotypes B, C and D. A genetic structure according to accessions taxonomic status (wild or cultivated) was revealed by UPGMA analysis. This highlighted a clear separation between domesticated and wild accessions in Turkish germplasm. The results pointed out the need to further collect and characterize this wild and cultivated grapevine germplasm

Genetic diversity, linkage disequilibrium and power of a large grapevine (Vitis vinifera L) diversity panel newly designed for association studies

UMR-AGAP Equipe DAVV (Diversité, adaptation et amélioration de la vigne) ; équipe ID (Intégration de Données)International audienceAbstractBackgroundAs for many crops, new high-quality grapevine varieties requiring less pesticide and adapted to climate change are needed. In perennial species, breeding is a long process which can be speeded up by gaining knowledge about quantitative trait loci linked to agronomic traits variation. However, due to the long juvenile period of these species, establishing numerous highly recombinant populations for high resolution mapping is both costly and time-consuming. Genome wide association studies in germplasm panels is an alternative method of choice, since it allows identifying the main quantitative trait loci with high resolution by exploiting past recombination events between cultivars. Such studies require adequate panel design to represent most of the available genetic and phenotypic diversity. Assessing linkage disequilibrium extent and panel power is also needed to determine the marker density required for association studies.ResultsStarting from the largest grapevine collection worldwide maintained in Vassal (France), we designed a diversity panel of 279 cultivars with limited relatedness, reflecting the low structuration in three genetic pools resulting from different uses (table vs wine) and geographical origin (East vs West), and including the major founders of modern cultivars. With 20 simple sequence repeat markers and five quantitative traits, we showed that our panel adequately captured most of the genetic and phenotypic diversity existing within the entire Vassal collection. To assess linkage disequilibrium extent and panel power, we genotyped single nucleotide polymorphisms: 372 over four genomic regions and 129 distributed over the whole genome. Linkage disequilibrium, measured by correlation corrected for kinship, reached 0.2 for a physical distance between 9 and 458 Kb depending on genetic pool and genomic region, with varying size of linkage disequilibrium blocks. This panel achieved reasonable power to detect associations between traits with high broad-sense heritability (> 0.7) and causal loci with intermediate allelic frequency and strong effect (explaining > 10 % of total variance).ConclusionsOur association panel constitutes a new, highly valuable resource for genetic association studies in grapevine, and deserves dissemination to diverse field and greenhouse trials to gain more insight into the genetic control of many agronomic traits and their interaction with the environment

Les marqueurs AFLP : révélation a l'aide d'un séquenceur automatique et mise au point sur Arabidopsis thaliana et Pisum sativum

National audienc

The potential of HTS approaches for accurate genotyping in grapevine (Vitis vinifera L.)

The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price

The Potential of HTS Approaches for Accurate Genotyping in Grapevine (Vitis vinifera L.)

The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price

Characterization of Vitis vinifera L. somatic variants exhibiting abnormal flower development patterns

Mutants have proven to be a key resource for functional genomic studies in model annual plant species. In perennial plant species where mutants are difficult to generate and to screen, spontaneous somatic variants represent a unique resource to understand the genetic control of complex developmental patterns. The morphological and histological characterization of six Vitis vinifera L. somatic variants that display four different abnormal phenotypes of flower development are described here. A phenotype of reiterated reproductive meristems (RRM), with both flower and petal reiteration, was observed in a somatic variant of the cultivar Carignan. An abnormal development of reproductive organs was displayed by the unfused carpels (UFC) somatic variant of cv. Bouchalès, while a somatic variant of cv. Mourvèdre named carpel-less (CLS) developed abnormal ovules in the absence of carpels. Finally, three independent somatic variants in cvs Gamay, Morrastel, and Pinot displayed a phenotype of multiple perianth whorls (MPW). Gene expression studies showed that the expression profiles of VvMADS-box 1, 2, and 3 (putative orthologues of Arabidopsis flowering genes AG, SEP, and AGL13), were altered during grapevine flower development in the somatic variants, whereas the corresponding original cultivars displayed similar VvMADS-box gene expression profiles. Phenotypic and molecular characterization of these variants allowed the development of hypotheses on genetic functions that might be altered in most of the variants in light of the current ABCDE flower modelPeer reviewe

Silver staining and recovery of AFLP amplification products on large denaturing polyacrylamide gels

International audienc

Silver staining and recovery of AFLP amplification products on large denaturing polyacrylamide gels

International audienc