Grapevine (Vitis vinifera L.) is one of the major fruit crop worldwide with thousands of cultivated varieties. The economical success of the grape and wine related agribusiness depends on the quantity and quality of the grapes produced, which are heavily influenced by genotype and environment. Among the important Italian varieties, 'Nebbiolo' is a valuable cultivar from Piedmont renowned for its use in producing high quality red wines (Barolo, Barbaresco and others). 'Nebbiolo' is characterized by a wide genetic variability expressed by a high number of clones (i.e. vegetatively propagated lines of selected mother plants) registered in the National Catalogue. However, little morphological differences exist between clones and often it is very difficult to visually distinguish the different clones. Furthermore, an important point is to ensure traceability of the vine supply chain, from grape nurseries up to wine industry, in order to provide a product of sure origin to the consumers. To study clonal diversity, simple sequence repeats (SSRs) have been used as markers, however with low success rates in discriminating clones. Recently, studies reported that allelic variations within the genome of a plant species could be identified by using single nucleotide polymorphisms (SNPs) as high-throughput sequence-based markers. The aim of this on-going study is to identify a set of SNPs able to discriminate the registered 'Nebbiolo' clones, setting up an effective and rapid SNP genotyping protocol based on the TaqMan® probes technology. Three 'Nebbiolo' clones were sequenced by Illumina technology (CVT 71, CVT 185 and CVT 423) and processed reads were mapped against the PN40024 reference genome. SNPs were first detected using the GATK-UnifiedGenotyper tool with default parameters followed by filtering and by a calibration step. Moreover, for the allelic variations identified in coding regions, prediction of changes in the amino acid sequence was performed using SNPeff tool. Then, SNPs were validated as true positive by Sanger sequencing and divided in: i) clone specific SNPs, ii) clones common SNPs, iii) SNPs in protein coding region by affecting genes. To date, Sanger sequencing confirmed 11 SNPs (29%), and nine of these were successfully used to perform TaqMan® genotyping assays, amplifying and detecting specific SNP alleles in genomic DNA from 37 'Nebbiolo' clones. Combining results obtained from each single SNP assay, we classified the 'Nebbiolo' clones in six main groups linked to the geographical origin of accessions. This work is in progress to implement the set of SNPs and to improve 'Nebbiolo' discrimination from other grape cultivars. Furthermore, SNPs validation of allelic variations in coding regions involved in relevant biochemical pathways (i.e., flavonoid biosynthesis) is in progress as well.
SNP genotyping as powerful tool to discriminate clones of Vitis vinifera cv Nebbiolo
Chitarra W;Boccacci P;Perrone I;Mannini F;Gribaudo I;Gambino G
2015
Abstract
Grapevine (Vitis vinifera L.) is one of the major fruit crop worldwide with thousands of cultivated varieties. The economical success of the grape and wine related agribusiness depends on the quantity and quality of the grapes produced, which are heavily influenced by genotype and environment. Among the important Italian varieties, 'Nebbiolo' is a valuable cultivar from Piedmont renowned for its use in producing high quality red wines (Barolo, Barbaresco and others). 'Nebbiolo' is characterized by a wide genetic variability expressed by a high number of clones (i.e. vegetatively propagated lines of selected mother plants) registered in the National Catalogue. However, little morphological differences exist between clones and often it is very difficult to visually distinguish the different clones. Furthermore, an important point is to ensure traceability of the vine supply chain, from grape nurseries up to wine industry, in order to provide a product of sure origin to the consumers. To study clonal diversity, simple sequence repeats (SSRs) have been used as markers, however with low success rates in discriminating clones. Recently, studies reported that allelic variations within the genome of a plant species could be identified by using single nucleotide polymorphisms (SNPs) as high-throughput sequence-based markers. The aim of this on-going study is to identify a set of SNPs able to discriminate the registered 'Nebbiolo' clones, setting up an effective and rapid SNP genotyping protocol based on the TaqMan® probes technology. Three 'Nebbiolo' clones were sequenced by Illumina technology (CVT 71, CVT 185 and CVT 423) and processed reads were mapped against the PN40024 reference genome. SNPs were first detected using the GATK-UnifiedGenotyper tool with default parameters followed by filtering and by a calibration step. Moreover, for the allelic variations identified in coding regions, prediction of changes in the amino acid sequence was performed using SNPeff tool. Then, SNPs were validated as true positive by Sanger sequencing and divided in: i) clone specific SNPs, ii) clones common SNPs, iii) SNPs in protein coding region by affecting genes. To date, Sanger sequencing confirmed 11 SNPs (29%), and nine of these were successfully used to perform TaqMan® genotyping assays, amplifying and detecting specific SNP alleles in genomic DNA from 37 'Nebbiolo' clones. Combining results obtained from each single SNP assay, we classified the 'Nebbiolo' clones in six main groups linked to the geographical origin of accessions. This work is in progress to implement the set of SNPs and to improve 'Nebbiolo' discrimination from other grape cultivars. Furthermore, SNPs validation of allelic variations in coding regions involved in relevant biochemical pathways (i.e., flavonoid biosynthesis) is in progress as well.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
