Abstract
Whole-genome comparisons of Vitis vinifera subsp. sativa and V. vinifera subsp. sylvestris are expected to provide a better estimate of the valuable genetic diversity still present in grapevine, and help to reconstruct the evolutionary history of a major crop worldwide. To this aim, the increase of molecular marker density across the grapevine genome is fundamental. Here we describe the SNP discovery in a grapevine germplasm collection of 51 cultivars and 44 wild accessions through a novel protocol of restriction-site associated DNA (RAD) sequencing. By resequencing 1.1% of the grapevine genome at a high coverage, we recovered 34K BamHI unique restriction sites, of which 6.8% were absent in the ‘PN40024’ reference genome. Moreover, we identified 37,748 single nucleotide polymorphisms (SNPs), 93% of which belonged to the 19 assembled chromosomes with an average of 1.8K SNPs per chromosome. Nearly half of the SNPs fell in genic regions mostly assigned to the functional categories of metabolism and regulation, whereas some nonsynonymous variants were identified in genes related with the detection and response to environmental stimuli. SNP validation was carried-out, showing the ability of RAD-seq to accurately determine genotypes in a highly heterozygous species. To test the usefulness of our SNP panel, the main diversity statistics were evaluated, highlighting how the wild grapevine retained less genetic variability than the cultivated form. Furthermore, the analysis of Linkage Disequilibrium (LD) in the two subspecies separately revealed how the LD decays faster within the domesticated grapevine compared to its wild relative. Being the first application of RAD-seq in a diverse grapevine germplasm collection, our approach holds great promise for exploiting the genetic resources available in one of the most economically important fruit crops.
Highlights
The introduction of molecular markers in plant breeding has enabled remarkable advances in agricultural production thanks to the discovery of genes associated to major agronomic traits, the study of species diversity and evolution, and the characterization of plant genetic resources [1]
In this study we describe the Single Nucleotide Polymorphisms (SNP) discovery carried out for the first time in a diverse set of cultivated and wild forms of V. vinifera through a novel protocol of restriction-site associated DNA (RAD) sequencing based on the 5500 SOLiDTM System
We selected BamHI as candidate restriction enzyme to construct RAD-seq libraries. It showed almost a constant and high number of recovered RE sites at different levels of coverage, compared to the other two REs used to test the technical performance of the novel RADseq protocol (S2 Table)
Summary
The introduction of molecular markers in plant breeding has enabled remarkable advances in agricultural production thanks to the discovery of genes associated to major agronomic traits, the study of species diversity and evolution, and the characterization of plant genetic resources [1]. During the last ten years, Single Nucleotide Polymorphisms (SNP) have become the most widely used markers due to their abundance in genomes. They compensate the biallelic nature by being ubiquitous and amenable to high-throughput automation [2]. Huge progress has been achieved for high throughput SNP genotyping thanks to the introduction of array-based technologies, able to screen several thousand SNPs per assay [3]. Myles et al [5] designed the first SNP array for grape (Illumina Vitis9KSNP chip) by using a discovering panel of 17 genomic DNA samples from V. vinifera cultivars and wild Vitis species. Many experiments have shown how the application of these array-based technologies to population genetic studies may underestimate the real genetic diversity of the investigated populations, especially when the discovery panel is evolutionary divergent from the studied accessions [7,8]
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