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A high quality draft consensus sequence of the genome of a heterozygous grapevine variety.

Velasco R, Zharkikh A, Troggio M, Cartwright DA, Cestaro A, Pruss D, Pindo M, Fitzgerald LM, Vezzulli S, Reid J, Malacarne G, Iliev D, Coppola G, Wardell B, Micheletti D, Macalma T, Facci M, Mitchell JT, Perazzolli M, Eldredge G, Gatto P, Oyzerski R, Moretto M, Gutin N, Stefanini M, Chen Y, Segala C, Davenport C, Demattè L, Mraz A, Battilana J, Stormo K, Costa F, Tao Q, Si-Ammour A, Harkins T, Lackey A, Perbost C, Taillon B, Stella A, Solovyev V, Fawcett JA, Sterck L, Vandepoele K, Grando SM, Toppo S, Moser C, Lanchbury J, Bogden R, Skolnick M, Sgaramella V, Bhatnagar SK, Fontana P, Gutin A, Van de Peer Y, Salamini F, Viola R - PLoS ONE (2007)

Bottom Line: A consensus sequence of the genome and a set of mapped marker loci were generated.Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps).SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

View Article: PubMed Central - PubMed

Affiliation: IASMA Research Center, San Michele all'Adige, Trento, Italy. riccardo.velasco@iasma.it

ABSTRACT

Background: Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal findings: We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions: Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

Show MeSH
V. vinifera pathways for phenolic and terpenoid biosynthesis.A) V. vinifera general pathway for phenolics biosynthesis leading to stilbenes (C6-C2-C6) and flavonoids (C6-C3-C6). For each enzyme, the gene copy number is reported in brackets. Genes were identified by similarity search using BLAST where the references were the sequences of phenolic biosynthetic genes previously characterized in grape and in other plant species. Putative homologues and gene copy numbers were determined by comparing aligned amino acid sequences based on a threshold of 80% similarity between the grape sequences, and 60% similarity between grape and other species. For the large StSy, PAL and F3'5'H families, phylogenetic analysis was performed with MEGA4 package [126] after aligning with ClustalW [127]. The following enzymes involved in the pathway are shown: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; StSy, stilbene synthase; RSGT, resveratrol glucosyltransferase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3′-hydroxylase; F3'5'H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol-4-reductase; FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; OMT, O-methyltransferase; ACCase, acetyl CoA carboxylase. PA refers to proanthocyanidins. Enzymatic steps that have not been experimentally confirmed are marked with an asterisk (*). B) Steps of plastidic isoprenoid pathway and monoterpenoids biosynthesis. For each enzyme, the gene copy number is reported in brackets. Gene annotation was performed as described in Material and methods. Abbreviations: G3P, glyceraldehyde 3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2C-methyl-D-erythritol; CDP-MEP, 4-diphosphocytidyl-2Cmethyl-D-erythritol 2-phosphate; ME-cPP, 2C-ethyl-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate. The enzymes in the pathway are indicated in blue: DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR 1-deoxy-D-xylulose 5-phosphate reductase; ISPD, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; ISPE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; ISPF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; ISPG, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and ISPH 2-C-methyl-D-erythritol 2,4-cyclodiphosphate reductase (ISPG and ISPH are probably the same enzyme and convert directly MEcPP in IPP and DMAPP); ISPA, geranyltransferase; IDI, isopentenyl diphosphate delta-isomerase; PT, prenyltransferase; LIMS, limonene synthase; LIS, linalool synthase; GES, geraniol synthase; TES, α-terpineol synthase; TPS-CIN, myrcene/(E)-beta-ocimene synthase; CYTP450, cytochrome P450 hydroxylase; ALDH, aldehyde dehydrogenase; NADPDH, NADP dehydrogenase. Enzymatic steps that have not been experimentally confirmed are marked with an asterisk (*).
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pone-0001326-g004: V. vinifera pathways for phenolic and terpenoid biosynthesis.A) V. vinifera general pathway for phenolics biosynthesis leading to stilbenes (C6-C2-C6) and flavonoids (C6-C3-C6). For each enzyme, the gene copy number is reported in brackets. Genes were identified by similarity search using BLAST where the references were the sequences of phenolic biosynthetic genes previously characterized in grape and in other plant species. Putative homologues and gene copy numbers were determined by comparing aligned amino acid sequences based on a threshold of 80% similarity between the grape sequences, and 60% similarity between grape and other species. For the large StSy, PAL and F3'5'H families, phylogenetic analysis was performed with MEGA4 package [126] after aligning with ClustalW [127]. The following enzymes involved in the pathway are shown: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; StSy, stilbene synthase; RSGT, resveratrol glucosyltransferase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3′-hydroxylase; F3'5'H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol-4-reductase; FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; OMT, O-methyltransferase; ACCase, acetyl CoA carboxylase. PA refers to proanthocyanidins. Enzymatic steps that have not been experimentally confirmed are marked with an asterisk (*). B) Steps of plastidic isoprenoid pathway and monoterpenoids biosynthesis. For each enzyme, the gene copy number is reported in brackets. Gene annotation was performed as described in Material and methods. Abbreviations: G3P, glyceraldehyde 3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2C-methyl-D-erythritol; CDP-MEP, 4-diphosphocytidyl-2Cmethyl-D-erythritol 2-phosphate; ME-cPP, 2C-ethyl-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate. The enzymes in the pathway are indicated in blue: DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR 1-deoxy-D-xylulose 5-phosphate reductase; ISPD, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; ISPE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; ISPF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; ISPG, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and ISPH 2-C-methyl-D-erythritol 2,4-cyclodiphosphate reductase (ISPG and ISPH are probably the same enzyme and convert directly MEcPP in IPP and DMAPP); ISPA, geranyltransferase; IDI, isopentenyl diphosphate delta-isomerase; PT, prenyltransferase; LIMS, limonene synthase; LIS, linalool synthase; GES, geraniol synthase; TES, α-terpineol synthase; TPS-CIN, myrcene/(E)-beta-ocimene synthase; CYTP450, cytochrome P450 hydroxylase; ALDH, aldehyde dehydrogenase; NADPDH, NADP dehydrogenase. Enzymatic steps that have not been experimentally confirmed are marked with an asterisk (*).

Mentions: Gene predictions corresponding to all those genes known to encode enzymes of the pathway could now be found. These include C4H and 4CL (acronyms are explicated in note 1 of Figure 4A) which were not previously identified in grape. The majority of genes were organized in large (PAL, F3'5'H) or small (CHS, F3H, FLS, LAR) gene families, the remainder consisting of single copy genes (C4H, 4CL, CHI, F3'H, DFR, LDOX, ANR, UFGT) (Figure 4A; Table S4).


A high quality draft consensus sequence of the genome of a heterozygous grapevine variety.

Velasco R, Zharkikh A, Troggio M, Cartwright DA, Cestaro A, Pruss D, Pindo M, Fitzgerald LM, Vezzulli S, Reid J, Malacarne G, Iliev D, Coppola G, Wardell B, Micheletti D, Macalma T, Facci M, Mitchell JT, Perazzolli M, Eldredge G, Gatto P, Oyzerski R, Moretto M, Gutin N, Stefanini M, Chen Y, Segala C, Davenport C, Demattè L, Mraz A, Battilana J, Stormo K, Costa F, Tao Q, Si-Ammour A, Harkins T, Lackey A, Perbost C, Taillon B, Stella A, Solovyev V, Fawcett JA, Sterck L, Vandepoele K, Grando SM, Toppo S, Moser C, Lanchbury J, Bogden R, Skolnick M, Sgaramella V, Bhatnagar SK, Fontana P, Gutin A, Van de Peer Y, Salamini F, Viola R - PLoS ONE (2007)

V. vinifera pathways for phenolic and terpenoid biosynthesis.A) V. vinifera general pathway for phenolics biosynthesis leading to stilbenes (C6-C2-C6) and flavonoids (C6-C3-C6). For each enzyme, the gene copy number is reported in brackets. Genes were identified by similarity search using BLAST where the references were the sequences of phenolic biosynthetic genes previously characterized in grape and in other plant species. Putative homologues and gene copy numbers were determined by comparing aligned amino acid sequences based on a threshold of 80% similarity between the grape sequences, and 60% similarity between grape and other species. For the large StSy, PAL and F3'5'H families, phylogenetic analysis was performed with MEGA4 package [126] after aligning with ClustalW [127]. The following enzymes involved in the pathway are shown: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; StSy, stilbene synthase; RSGT, resveratrol glucosyltransferase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3′-hydroxylase; F3'5'H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol-4-reductase; FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; OMT, O-methyltransferase; ACCase, acetyl CoA carboxylase. PA refers to proanthocyanidins. Enzymatic steps that have not been experimentally confirmed are marked with an asterisk (*). B) Steps of plastidic isoprenoid pathway and monoterpenoids biosynthesis. For each enzyme, the gene copy number is reported in brackets. Gene annotation was performed as described in Material and methods. Abbreviations: G3P, glyceraldehyde 3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2C-methyl-D-erythritol; CDP-MEP, 4-diphosphocytidyl-2Cmethyl-D-erythritol 2-phosphate; ME-cPP, 2C-ethyl-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate. The enzymes in the pathway are indicated in blue: DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR 1-deoxy-D-xylulose 5-phosphate reductase; ISPD, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; ISPE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; ISPF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; ISPG, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and ISPH 2-C-methyl-D-erythritol 2,4-cyclodiphosphate reductase (ISPG and ISPH are probably the same enzyme and convert directly MEcPP in IPP and DMAPP); ISPA, geranyltransferase; IDI, isopentenyl diphosphate delta-isomerase; PT, prenyltransferase; LIMS, limonene synthase; LIS, linalool synthase; GES, geraniol synthase; TES, α-terpineol synthase; TPS-CIN, myrcene/(E)-beta-ocimene synthase; CYTP450, cytochrome P450 hydroxylase; ALDH, aldehyde dehydrogenase; NADPDH, NADP dehydrogenase. Enzymatic steps that have not been experimentally confirmed are marked with an asterisk (*).
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Related In: Results  -  Collection

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pone-0001326-g004: V. vinifera pathways for phenolic and terpenoid biosynthesis.A) V. vinifera general pathway for phenolics biosynthesis leading to stilbenes (C6-C2-C6) and flavonoids (C6-C3-C6). For each enzyme, the gene copy number is reported in brackets. Genes were identified by similarity search using BLAST where the references were the sequences of phenolic biosynthetic genes previously characterized in grape and in other plant species. Putative homologues and gene copy numbers were determined by comparing aligned amino acid sequences based on a threshold of 80% similarity between the grape sequences, and 60% similarity between grape and other species. For the large StSy, PAL and F3'5'H families, phylogenetic analysis was performed with MEGA4 package [126] after aligning with ClustalW [127]. The following enzymes involved in the pathway are shown: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; StSy, stilbene synthase; RSGT, resveratrol glucosyltransferase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3′-hydroxylase; F3'5'H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol-4-reductase; FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; OMT, O-methyltransferase; ACCase, acetyl CoA carboxylase. PA refers to proanthocyanidins. Enzymatic steps that have not been experimentally confirmed are marked with an asterisk (*). B) Steps of plastidic isoprenoid pathway and monoterpenoids biosynthesis. For each enzyme, the gene copy number is reported in brackets. Gene annotation was performed as described in Material and methods. Abbreviations: G3P, glyceraldehyde 3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2C-methyl-D-erythritol; CDP-MEP, 4-diphosphocytidyl-2Cmethyl-D-erythritol 2-phosphate; ME-cPP, 2C-ethyl-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate. The enzymes in the pathway are indicated in blue: DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR 1-deoxy-D-xylulose 5-phosphate reductase; ISPD, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; ISPE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; ISPF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; ISPG, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and ISPH 2-C-methyl-D-erythritol 2,4-cyclodiphosphate reductase (ISPG and ISPH are probably the same enzyme and convert directly MEcPP in IPP and DMAPP); ISPA, geranyltransferase; IDI, isopentenyl diphosphate delta-isomerase; PT, prenyltransferase; LIMS, limonene synthase; LIS, linalool synthase; GES, geraniol synthase; TES, α-terpineol synthase; TPS-CIN, myrcene/(E)-beta-ocimene synthase; CYTP450, cytochrome P450 hydroxylase; ALDH, aldehyde dehydrogenase; NADPDH, NADP dehydrogenase. Enzymatic steps that have not been experimentally confirmed are marked with an asterisk (*).
Mentions: Gene predictions corresponding to all those genes known to encode enzymes of the pathway could now be found. These include C4H and 4CL (acronyms are explicated in note 1 of Figure 4A) which were not previously identified in grape. The majority of genes were organized in large (PAL, F3'5'H) or small (CHS, F3H, FLS, LAR) gene families, the remainder consisting of single copy genes (C4H, 4CL, CHI, F3'H, DFR, LDOX, ANR, UFGT) (Figure 4A; Table S4).

Bottom Line: A consensus sequence of the genome and a set of mapped marker loci were generated.Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps).SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

View Article: PubMed Central - PubMed

Affiliation: IASMA Research Center, San Michele all'Adige, Trento, Italy. riccardo.velasco@iasma.it

ABSTRACT

Background: Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal findings: We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions: Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

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