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

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

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

Pink-Colored Grape Berry Is the Result of Short Insertion in Intron of Color Regulatory Gene

We report here that pink grape berries were obtained by a short insertion in the intron of the MybA1 gene, a gene that regulates grape berry color. Genetic variation was detected among the MybA1 genes from grapes cultivated worldwide. PCR analysis of the MybA1 gene demonstrated that the size of the MybA1 gene in the red allele differs among grapes. Oriental V. vinifera bearing pink berries has the longest MybA1 gene among grapes, whereas the shortest MybA1 gene was detected in occidental V. vinifera grapes. The nucleotide sequences of the MybA1 genes demonstrated that oriental V. vinifera has two additional gene fragments (44 bp and 111 bp) in the promoter region of the MybA1 gene in the red allele and another 33 bp fragment in the second intron of the MybA1 gene in the red allele. The short insertion in the intron decreased the transcription activity in the model system and retained MybA1 transcripts with unspliced intron in the total RNA. From the experiments using deletion mutants of the 33 bp short insertion, 16 bp of the 3′ end in the insertion is a key structure for a defect in splicing of MybA1 transcripts. Thus, a weakly colored grape berry might be a result of the short insertion in the intron of a color regulatory gene. This is new evidence concerning the molecular mechanism of the fate of grape berry color. These findings are expected to contribute to the further understanding of the color variation in grape berries, which is correlated with the evolutional events occurring in the MybA1 gene of grapes

High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

<p>Abstract</p> <p>Background</p> <p>High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera.</p> <p>Results</p> <p>We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of <it>Eucalyptus </it>from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for <it>E. grandis</it>. A systematic assessment of <it>in silico </it>SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous <it>in silico </it>constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species.</p> <p>SNP reliability was high across nine <it>Eucalyptus </it>species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased.</p> <p>Conclusions</p> <p>This study indicates that the GGGT performs well both within and across species of <it>Eucalyptus </it>notwithstanding its nucleotide diversity ≥2%. The development of a much larger array of informative SNPs across multiple <it>Eucalyptus </it>species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in <it>Eucalyptus</it>.</p

Genome-Wide Analysis of the “Cut-and-Paste” Transposons of Grapevine

Background: The grapevine is a widely cultivated crop and a high number of different varieties have been selected since its domestication in the Neolithic period. Although sexual crossing has been a major driver of grapevine evolution, its vegetative propagation enhanced the impact of somatic mutations and has been important for grapevine diversity. Transposable elements are known to be major contributors to genome variability and, in particular, to somatic mutations. Thus, transposable elements have probably played a major role in grapevine domestication and evolution. The recent publication of the complete grapevine genome opens the possibility for an in deep analysis of its transposon content. Principal Findings: We present here a detailed analysis of the ‘‘cut-and-paste’ ’ class II transposons present in the genome of grapevine. We characterized 1160 potentially complete grapevine transposons as well as 2086 defective copies. We report on the structure of each element, their potentiality to encode a functional transposase, and the existence of matching ESTs that could suggest their transcription. Conclusions: Our results show that these elements have transduplicated and amplified cellular sequences and some of them have been domesticated and probably fulfill cellular functions. In addition, we provide evidences that the mobility o

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