High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology

Background: Single-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes. Results: In order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape ( = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlex genotyping technology in a sample of grapevine genotypes and segregating progenies. Conclusion: These results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlex genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlex high throughput genotyping technology provide a powerful tool for grapevine genetic analysis. © 2007 Lijavetzky et al; licensee BioMed Central Ltd

Development and evaluation of a 9K SNP array for peach by internationally coordinated SNP detection and validation in breeding germplasm

Although a large number of single nucleotide polymorphism (SNP) markers covering the entire genome are needed to enable molecular breeding efforts such as genome wide association studies, fine mapping, genomic selection and marker-assisted selection in peach [Prunus persica (L.) Batsch] and related Prunus species, only a limited number of genetic markers, including simple sequence repeats (SSRs), have been available to date. To address this need, an international consortium (The International Peach SNP Consortium; IPSC) has pursued a coordinated effort to perform genome-scale SNP discovery in peach using next generation sequencing platforms to develop and characterize a high-throughput Illumina Infinium® SNP genotyping array platform. We performed whole genome re-sequencing of 56 peach breeding accessions using the Illumina and Roche/454 sequencing technologies. Polymorphism detection algorithms identified a total of 1,022,354 SNPs. Validation with the Illumina GoldenGate® assay was performed on a subset of the predicted SNPs, verifying ∼75% of genic (exonic and intronic) SNPs, whereas only about a third of intergenic SNPs were verified. Conservative filtering was applied to arrive at a set of 8,144 SNPs that were included on the IPSC peach SNP array v1, distributed over all eight peach chromosomes with an average spacing of 26.7 kb between SNPs. Use of this platform to screen a total of 709 accessions of peach in two separate evaluation panels identified a total of 6,869 (84.3%) polymorphic SNPs.The almost 7,000 SNPs verified as polymorphic through extensive empirical evaluation represent an excellent source of markers for future studies in genetic relatedness, genetic mapping, and dissecting the genetic architecture of complex agricultural traits. The IPSC peach SNP array v1 is commercially available and we expect that it will be used worldwide for genetic studies in peach and related stone fruit and nut species

High-throughput 18K SNP array to assess genetic variability of the main grapevine cultivars from Sicily

The viticulture of Sicily, for its vocation, is one of the most important and ancient forms in Italy. Autochthonous grapevine cultivars, many of which known throughout the world, have always been cultivated in the island from many centuries. With the aim to preserve this large grapevine diversity, previous studies have already started to assess the genetic variability among the Sicilian cultivars by using morphological and microsatellite markers. In this study, simple sequence repeat (SSR) were utilized to verify the true-to-typeness of a large clone collection (101) belonging to 21 biotypes of the most 10 cultivated Sicilian cultivars. Afterwards, 42 Organization Internationale de la Vigne et du Vin (OIV) descriptors and a high-throughput single nucleotide polymorphism (SNP) genotyping array (Vitis18kSNP) were applied to assess genetic variability among cultivars and biotypes of the same cultivar. Ampelographic traits and high-throughput SNP genotyping platforms provided an accuracy estimation of genetic diversity in the Sicilian germplasm, showing the relationships among cultivars by cluster and multivariate analyses. The large SNP panel defined sub-clusters unable to discern among biotypes, previously classified by ampelographic analysis, belonging to each cultivar. These results suggested that a very large number of SNP did not cover the genome regions harboring few morphological traits. Genetic structure of the collection revealed a clear optimum number of groups for K = 3, clustering in the same group a significant portion of family-related genotypes. Parentage analysis highlighted significant relationships among Sicilian grape cultivars and Sangiovese, as already reported, but also the first evidences of the relationships between Nero d’Avola and both Inzolia and Catarratto. Finally, a small panel of highly informative markers (12 SNPs) allowed us to isolate a private profile for each Sicilian cultivar, providing a new tool for cultivar identification

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

Plant cell culture technology in the cosmetics and food industries : current state and future trends

The production of drugs, cosmetics, and food which are derived from plant cell and tissue cultures has a long tradition. The emerging trend of manufacturing cosmetics and food products in a natural and sustainable manner has brought a new wave in plant cell culture technology over the past 10 years. More than 50 products based on extracts from plant cell cultures have made their way into the cosmetics industry during this time, whereby the majority is produced with plant cell suspension cultures. In addition, the first plant cell culture-based food supplement ingredients, such as Echigena Plus and Teoside 10, are now produced at production scale. In this mini review, we discuss the reasons for and the characteristics as well as the challenges of plant cell culture-based productions for the cosmetics and food industries. It focuses on the current state of the art in this field. In addition, two examples of the latest developments in plant cell culture-based food production are presented, that is, superfood which boosts health and food that can be produced in the lab or at home

A completely-phased diploid genome assembly for ‘Malbec’ cultivar (Vitis vinifera L.)

Poster. Publicado en: BAG Journal of Basic and Applied Genetics, 32 (1 suppl), 2021Most grapevine cultivars originated from the outcrossing of two genetically diverse parents, and are clonally propagated to preserve phenotypes of productive interest. Hence, cultivars are first filial generations (F1) with highly heterozygous diploid genomes, that turn challenging to assemble. ‘Malbec’ is the main cultivar for the Argentine wine industry and it originated in France, from the outcrossing of ‘Magdeleine Noir des Charentes’ and ‘Prunelard’ cultivars. Based on that mother-father-offspring relationship, here we followed the algorithm implemented in the software CanuTrio to produce a phased assembly of ‘Malbec’ genome. For this aim, parental cultivars’ Illumina short-reads were used to sort ‘Malbec’ PacBio long-reads into its haploid complements, to be assembled separately. Postassembly, bioinformatic procedures were employed to reduce the number of duplicated regions and perform sequence error corrections (using ‘Malbec’ Illumina short-reads). We obtained two highly complete and contiguous haploid assemblies for ‘Malbec’, Haplotype-Prunelard (482.4 Mb size; contig N50=7.7 Mb) and Haplotype-Magdeleine (479.4 Mb size; contig N50=6.6 Mb), with 96.1 and 95.8% of BUSCO genes, respectively. We tested for the composition of both haplophases with the tool Merqury, and observed 15% of both assemblies affected by structural variations, along with 3.2 million SNPs and 0.6 million InDels. Our results indicate that this is a valid approach to assemble highly heterozygous and complex diploid genomes in a completely-phased way.EEA MendozaFil: Calderón, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Carbonell-Bejerano, P. Max Planck Institute for Developmental Biology; AlemaniaFil: Mauri, Nuria. Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de La Rioja). Finca La Grajera; ArgentinaFil: Muñoz, C. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; ArgentinaFil: Bree, Laura. Vivero Mercier; ArgentinaFil: Sola, Cristóbal. Vivero Mercier; ArgentinaFil: Bergamin, Daniel. Vivero Mercier; ArgentinaFil: Gomez Talquenca, Gonzalo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Martínez Zapater, José Miguel. Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de La Rioja). Finca La Grajera; ArgentinaFil: Weigel, D. Max Planck Institute for Developmental Biology; AlemaniaFil: Lijavetzky, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentin

A completely phased diploid genome assembly for "Malbec" cultivar (Vitis vinifera L.)

Most grapevine cultivars originated from the outcrossing of two genetically diverse parents, and are clonally propagated to preserve phenotypes of productive interest. Hence, cultivars are first filial generations (F1) with highly heterozygous diploid genomes, that turn challenging to assemble. "Malbec" is the main cultivar for the Argentine wine industry and it originated in France, from the outcrossing of Magdeleine Noir des Charentes and Prunelard cultivars. Based on that mother-father-offspring relationship, here we followed the algorithm implemented in the software CanuTrio to produce a phased assembly of Malbec genome. For this aim, parental cultivars? Illumina short-reads were used to sort ?Malbec? PacBio long-reads into its haploid complements, to be assembled separately. Post- assembly, bioinformatic procedures were employed to reduce the number of duplicated regions and perform sequence error corrections (using Malbec Illumina short-reads). We obtained two highly complete and contiguous haploid assemblies for Malbec, Haplotype- Prunelard (482.4 Mb size; contig N50=7.7 Mb) and Haplotype-Magdeleine (479.4 Mb size; contig N50=6.6 Mb), with 96.1 and 95.8% of BUSCO genes, respectively. We tested for the composition of both haplophases with the tool Merqury, and observed 15% of both assemblies affected by structural variations, along with 3.2 million SNPs and 0.6 million InDels. Our results indicate that this is a valid approach to assemble highly heterozygous and complex diploid genomes in a completely-phased way.Fil: Calderón, Pablo Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Carbonell Bejerano, P.. Max Planck Institute for Developmental Biology; AlemaniaFil: Mauri, N. Instituto de Ciencias de la Vid y del Vino; EspañaFil: Muñoz, Claudio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Bree, Laura. No especifíca;Fil: Sola, C. No especifíca;Fil: Bergamin, D. No especifíca;Fil: Gómez Talquenca, Sebastián. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Ibañez, J. Instituto de Ciencias de la Vid y del Vino; EspañaFil: Martinez-Zapater, JM. Instituto de Ciencias de la Vid y del Vino; EspañaFil: Lijavetzky, Diego Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaXVIII Congreso Latinoamericano de Genética; LIV Reunión Anual de la Sociedad de Genética de Chile; XLIX Congreso Argentino de Genética; VIII Congreso de la Sociedad Uruguaya de Genética; I Congreso Paraguayo de Genética y V Congreso Latinoamericano de Genética HumanaChileSociedad Argentina de Genétic