Genome-wide SNP detection, validation, and development of an 8K SNP array for apple

Abstract

As high-throughput genetic marker screening systems are essential for a range of genetics studies and plant breeding applications, the International RosBREED SNP Consortium (IRSC) has utilized the Illumina Infinium® II system to develop a medium- to high-throughput SNP screening tool for genome-wide evaluation of allelic variation in apple (Malus×domestica) breeding germplasm. For genome-wide SNP discovery, 27 apple cultivars were chosen to represent worldwide breeding germplasm and re-sequenced at low coverage with the Illumina Genome Analyzer II. Following alignment of these sequences to the whole genome sequence of 'Golden Delicious', SNPs were identified using SoapSNP. A total of 2,113,120 SNPs were detected, corresponding to one SNP to every 288 bp of the genome. The Illumina GoldenGate® assay was then used to validate a subset of 144 SNPs with a range of characteristics, using a set of 160 apple accessions. This validation assay enabled fine-tuning of the final subset of SNPs for the Illumina Infinium® II system. The set of stringent filtering criteria developed allowed choice of a set of SNPs that not only exhibited an even distribution across the apple genome and a range of minor allele frequencies to ensure utility across germplasm, but also were located in putative exonic regions to maximize genotyping success rate. A total of 7867 apple SNPs was established for the IRSC apple 8K SNP array v1, of which 5554 were polymorphic after evaluation in segregating families and a germplasm collection. This publicly available genomics resource will provide an unprecedented resolution of SNP haplotypes, which will enable marker-locus-trait association discovery, description of the genetic architecture of quantitative traits, investigation of genetic variation (neutral and functional), and genomic selection in apple.

Figure 1. Workflow for single nucleotide polymorphism (SNP) detection, validation, and final choice employed for development of the IRSC apple 8K SNP array v1.

GDsnp: ‘Golden Delicious’-validated SNP; RosCOS: Rosaceae Conserved Orthologous Set; MAF: Minor Allele Frequency; ADT: Assay Design Tool.

Figure 2. Classification of the 144 apple single nucleotide polymorphisms (SNPs) used for validation using the Illumina GoldenGate® assay.

Figure 3. Detailed view of a genomic region at the top of Linkage Group 1 showing SNPs chosen for the International RosBREED SNP Consortium (IRSC) apple 8K SNP array v1.

Physical map locations of SNPs (left; in megabases) are compared with known genetic map locations of SNP markers developed from ‘Golden Delicious’ (GDsnp in centiMorgans). SNP clusters around focal points are boxed, with green boxes denoting GDsnps, blue boxes denoting Rosaceae Conserved Orthologous Set markers (RosCOS), and orange boxes denoting candidate genes.

Figure 4. Genetic maps of linkage group (LG) 1 constructed using the ‘Royal Gala’בGranny Smith’ segregating population.

Marker names indicate the type of SNP, physical location on the ‘Golden Delicious’ genome assembly (in base pair), cluster marker, minor allelic frequency (MAF) in the 27 re-sequenced accessions, gene model, position in the predicted gene model, and NCBI dbSNP accession number (in parentheses).

Figure 5. Example of the usefulness of the International RosBREED SNP Consortium (IRSC) apple 8K SNP array v1 for developing haplotypes.

SNP markers were the same as represented in the example of Figure 3. Haplotypes inferred using FlexQTL™ for each cluster of SNPs are numbered from H1 to H4). Haplotypes inherited by the progeny from the parents are boxed and colored coded for each parent. A putative recombination is indicated by a cross for the ‘(Royal) Gala’בSplendour’−>‘Sciros’ trio.