Development and validation of a 20K single nucleotide polymorphism (SNP) whole genome genotyping array for apple (Malus × domestica Borkh)

Abstract

High-density SNP arrays for genome-wide assessment of allelic variation have made high resolution genetic characterization of crop germplasm feasible. A medium density array for apple, the IRSC 8K SNP array, has been successfully developed and used for screens of bi-parental populations. However, the number of robust and well-distributed markers contained on this array was not sufficient to perform genome-wide association analyses in wider germplasm sets, or Pedigree-Based Analysis at high precision, because of rapid decay of linkage disequilibrium. We describe the development of an Illumina Infinium array targeting 20K SNPs. The SNPs were predicted from re-sequencing data derived from the genomes of 13 Malus × domestica apple cultivars and one accession belonging to a crab apple species (M. micromalus). A pipeline for SNP selection was devised that avoided the pitfalls associated with the inclusion of paralogous sequence variants, supported the construction of robust multi-allelic SNP haploblocks and selected up to 11 entries within narrow genomic regions of ±5 kb, termed focal points (FPs). Broad genome coverage was attained by placing FPs at 1 cM intervals on a consensus genetic map, complementing them with FPs to enrich the ends of each of the chromosomes, and by bridging physical intervals greater than 400 Kbps. The selection also included ∼3.7K validated SNPs from the IRSC 8K array. The array has already been used in other studies where ∼15.8K SNP markers were mapped with an average of ∼6.8K SNPs per full-sib family. The newly developed array with its high density of polymorphic validated SNPs is expected to be of great utility for Pedigree-Based Analysis and Genomic Selection. It will also be a valuable tool to help dissect the genetic mechanisms controlling important fruit quality traits, and to aid the identification of marker-trait associations suitable for the application of Marker Assisted Selection in apple breeding programs.

Figure 1. The SNP selection workflow.

The SNP selection process was a cascade involving four steps: Detection, Quality Filtering, Illumina specific Filtering and SNP Selection, with the number of SNPs reduced after each step. The specific filtering criteria have been reported for each filtering stage, as well as the number of resulting SNPs, with the corresponding percentage survival, relative to the total number of SNP markers after the previous step. From the 20,000 selected, Illumina successfully incorporated a total of 18,019 SNP probes into the new array, 14,714 of which were newly discovered and 3,305 previously validated.

Figure 2. SNP selection within a single focal point (FP) of chromosomes (Chrs) 1 and 9.

Genotype calls are presented for the discovery panel members: 0/1 and 1/2 indicates heterozygosity, all other codes indicate absence of polymorphism within an individual. Green, blue and orange filled cells relate to selected SNPs, having 5–8, 2–4, or just 1 heterozygous panel member/s, respectively. The physical positions at the extremities of the FP are in bold and italics (most proximal) or underlined (most distal). Within each FP, SNPs are sorted firstly by being selected or not, and secondly according to the number of heterozygous members (HET). In the Chr1-FP example, SNP-6 was not selected, as its heterozygosity pattern is identical to that of SNP-2. The single-member-heterozygote SNP-5 was included because no other more polymorphic SNP markers were available to reach the target of 11 SNPs and no nearby FPs had a specific SNP for the same panel member. In the Chr9-FP example all highly specific SNPs were ignored, as there were sufficient markers of higher heterozygosity available. The selected SNPs show a homogeneous representation of the diploid panel members.

Figure 3. Distribution of focal points (FPs) (black lines) and IRSC SNPs (red lines) in the apple physical map v2.

All positions are in Mbps. The average distance between FPs is 311 Kbps. Only two regions longer than 1 Mbps located at the distal end of Chrs 9 and 13 are not covered by FPs and SNPs from the 8K IRSC SNP array. Scale bars = 5 Mbps.

Figure 4. GenomeStudio cluster plot for the 21 F₁ full-sib families used for the construction of genetic maps.

Yellow dots indicate panel members that were re-sequenced and used as parents. Genotypes are called for each sample (dot) by their signal intensity (norm R) and Allele Frequency (Norm Theta) relative to canonical cluster positions (dark shading) for a given SNP marker (red = AA, purple = AB, blue = BB) a) GenomeStudio cluster plot of a newly developed robust SNP marker. b) GenomeStudio cluster plot of a failed/difficult to score SNP marker.