Development and evaluation of a genome-wide 6K SNP array for diploid sweet cherry and tetraploid sour cherry

Abstract

High-throughput genome scans are important tools for genetic studies and breeding applications. Here, a 6K SNP array for use with the Illumina Infinium® system was developed for diploid sweet cherry (Prunus avium) and allotetraploid sour cherry (P. cerasus). This effort was led by RosBREED, a community initiative to enable marker-assisted breeding for rosaceous crops. Next-generation sequencing in diverse breeding germplasm provided 25 billion basepairs (Gb) of cherry DNA sequence from which were identified genome-wide SNPs for sweet cherry and for the two sour cherry subgenomes derived from sweet cherry (avium subgenome) and P. fruticosa (fruticosa subgenome). Anchoring to the peach genome sequence, recently released by the International Peach Genome Initiative, predicted relative physical locations of the 1.9 million putative SNPs detected, preliminarily filtered to 368,943 SNPs. Further filtering was guided by results of a 144-SNP subset examined with the Illumina GoldenGate® assay on 160 accessions. A 6K Infinium® II array was designed with SNPs evenly spaced genetically across the sweet and sour cherry genomes. SNPs were developed for each sour cherry subgenome by using minor allele frequency in the sour cherry detection panel to enrich for subgenome-specific SNPs followed by targeting to either subgenome according to alleles observed in sweet cherry. The array was evaluated using panels of sweet (n = 269) and sour (n = 330) cherry breeding germplasm. Approximately one third of array SNPs were informative for each crop. A total of 1825 polymorphic SNPs were verified in sweet cherry, 13% of these originally developed for sour cherry. Allele dosage was resolved for 2058 polymorphic SNPs in sour cherry, one third of these being originally developed for sweet cherry. This publicly available genomics resource represents a significant advance in cherry genome-scanning capability that will accelerate marker-locus-trait association discovery, genome structure investigation, and genetic diversity assessment in this diploid-tetraploid crop group.

Figure 1. Workflow for SNP detection, validation, and final choice in development of the RosBREED 6K cherry SNP array v1.

Stage 1 filtered 1.9 million cherry SNPs anchored to the peach genome to almost 40K SNPs. More stringent filtering criteria in Stage 2, guided by a prior validation step with a small SNP subset examined for a range of potential filters, putatively enriched the quality of the remaining 32K SNP pool. Finally, the 6K array SNPs were chosen from among stage 2 SNPs by attempting to achieve even genetic spacing over species genomes and subgenomes with pre-determined proportional allocations, after preferential inclusion of certain SNPs. ADT = Illumina's Assay Design Tool. MAF = minor allele frequency.

Figure 2. Performance of the RosBREED cherry 6K SNP array v1 for a sample region of the cherry genome.

SNPs are shown in a 8.2 cM/2.1 Mb region around the S locus at the distal end of Prunus LG/chromosome/scaffold 6. Positions of SSR and RFLP markers positioned in the Prunus reference map and physical map are indicated. SNP positions are scaled genetically to the 8.2 cM window between reference-mapped markers UDP98-412 and CPPCT030. The S locus and Ma040a are also physically positioned, with their genetic locations in the sweet cherry genetic map indicated in parentheses. SNPs designed for sweet cherry that were observed to be polymorphic in sour cherry are assumed here to be polymorphic within the avium subgenome of sour cherry except where haplotypes determined for Figure 3 identified polymorphism in both subgenomes (ss490556251) or in fruticosa (ss490556263).

Figure 3. Minor allele frequency (MAF) of polymorphic cherry SNPs.

MAF was determined in sweet cherry (n = 50) and sour cherry (n = 37) evaluation panels for 1825 and 2058 polymorphic SNPs, respectively.

Figure 4. Reconstruction of a ∼1.2 Mb region spanning the self-incompatibility S locus and its inheritance in cherry.

(a) Sweet cherry, with four parental haplotypes (1–4). (b) Sour cherry, with eight parental haplotypes (1–8). Identical haplotypes have the same background colors. Haplotypes are shown for five sweet cherry and two sour cherry seedlings. Monomorphic SNPs within cross-over regions are highlighted in grey. Genotypes indicated as “u” are for an unresolved polymorphic SNP in sour cherry.