Date Published: December 20, 2012
Publisher: Public Library of Science
Author(s): Cameron Peace, Nahla Bassil, Dorrie Main, Stephen Ficklin, Umesh R. Rosyara, Travis Stegmeir, Audrey Sebolt, Barbara Gilmore, Cindy Lawley, Todd C. Mockler, Douglas W. Bryant, Larry Wilhelm, Amy Iezzoni, Rongling Wu. http://doi.org/10.1371/journal.pone.0048305
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.
Within Prunus (Rosaceae), two cherry species, sweet (P. avium) and sour cherry (P. cerasus), are highly valued for their excellent quality fruit. These two species represent a natural diploid-tetraploid series with the tetraploid sour cherry (2n = 4x = 32) arising through natural hybridization between sweet cherry (2n = 2x = 16) and the wild tetraploid ground cherry (P. fruticosa) , . In cherry, linkage maps constructed for the genetically less complex sweet cherry are primarily based on simple sequence repeat (SSR) markers , , , . The application of these linkage maps for other studies such as quantitative trait locus (QTL) discovery is limited by the low-throughput and, in many cases, low density and low levels of polymorphism in cultivated sweet cherry germplasm for the SSR markers.
The workflow and design parameters described below are summarized in Figure 1.
The RosBREED cherry 6K SNP array v1was determined to be useful for various genetics studies of cultivated cherry, despite the use of a relatively low number of detection panel accessions each sequenced at low depth. The number of SNP detection panel accessions, 16 for sweet cherry and eight for sour cherry, achieved a total depth of genome coverage (46.3× for sweet cherry and 15× for sour cherry) that was less than that of recent SNP detection and array development for peach and apple, two of cherry’s rosaceous relatives. The recently developed International Peach SNP Consortium peach 9K SNP array v1 used 56 accessions that achieved 118× total genome coverage and for which 84.3% of included SNPs were informative when evaluated on diverse breeding germplasm . The 27 accessions used for the International RosBREED SNP Consortium apple 8K SNP array v1 achieved 89× total genome coverage and 70.6% of SNPs were informative . However, sequencing depth per accession was similar among all these efforts, averaging 3.1× genome coverage per accession for sweet cherry and 2.0× for sour cherry (Table 1), 2.2× for peach , and 3.3× for apple . Sour cherry coverage was comparable because the sequencing was performed at twice the depth of sweet cherry. As the proportion of informative cherry SNPs was only half that of peach and apple (only 38% and 46% of those targeted to sweet cherry and sour cherry breeding germplasm, respectively), the relative limitation of the cherry SNP detection panel was either the number or relevance of detection panel accessions. The number of accessions was a compromise based on the available budget and intended allocation of array attention of 75% to sweet cherry and 25% to sour cherry, utilizing state-of-the-art NGS technology (Illumina GA II). Because of the intended use of the array in characterizing genetic variation in cultivated cherry, the few accessions of the detection panel were carefully chosen to efficiently represent cherry breeding germplasm. For both sweet and sour cherry, choices were based on prior knowledge of geographic origin, pedigree, SSR and RosCOS SNP diversity (sweet cherry, ) or isozyme diversity (sour cherry, ), and the potential to confer disease resistance , . The detection panel choice appears to have been suitable because the array performed as well on the original detection panel accessions as on other breeding germplasm, including on material unconnected by pedigree. With the large number of SNPs on the array, the observed levels of polymorphism and heterozygosity translate to an unprecedented resolution for assaying genetic variation in cherry. In sweet cherry, for example, the array is expected to reveal 1500–2000 polymorphic genome-wide SNPs for any given set of cultivars, and 400–700 SNPs heterozygous within any given cultivar, from our observations of polymorphism and heterozygosity in the sweet cherry evaluation panel. This estimate is supported by  who reported 515 to 634 SNPs heterozygous for each of four cultivars when this 6K array was used as the basis for the two highest density genetic maps to date of a Prunus species. Therefore, the set of SNPs on the array developed from a small but diverse set of breeding-relevant accessions is expected to be generally informative for cherry germplasm in cultivation and in breeding programs.
The cherry SNP array described here will foster genetics studies in the Rosaceae and help bridge the gap between genomics and breeding in cherry because breeding germplasm was the basis of detected SNPs and SNP choices of the final array. The RosBREED cherry 6K SNP array v1 is commercially available from Illumina and we expect that it will be used worldwide for genetic studies in cherry and related species. The SNP markers included in the cherry 6K Illumina arrays are available for download in Excel format and viewable in GBrowse at the Genome Database for Rosaceae (GDR; http://www.rosaceae.org).