A New One-Tube Reaction Assay for the Universal Determination of Sweet Cherry (Prunus avium L.) Self-(In)Compatible MGST- and S-Alleles Using Capillary Fragment Analysis.

The sweet cherry plant (Prunus avium L.) is primarily self-incompatible, with so-called S-alleles responsible for the inability of flowers to be pollinated not only by their own pollen grains but also by pollen from other cherries having the same S-alleles. This characteristic has wide-ranging impacts on commercial growing, harvesting, and breeding. However, mutations in S-alleles as well as changes in the expression of M locus-encoded glutathione-S-transferase (MGST) can lead to complete or partial self-compatibility, simplifying orchard management and reducing possible crop losses. Knowledge of S-alleles is important for growers and breeders, but current determination methods are challenging, requiring several PCR runs. Here we present a system for the identification of multiple S-alleles and MGST promoter variants in one-tube PCR, with subsequent fragment analysis on a capillary genetic analyzer. The assay was shown to unequivocally determine three MGST alleles, 14 self-incompatible S-alleles, and all three known self-compatible S-alleles (S3', S4', S5') in 55 combinations tested, and thus it is especially suitable for routine S-allele diagnostics and molecular marker-assisted breeding for self-compatible sweet cherries. In addition, we identified a previously unknown S-allele in the 'Techlovicka´ genotype (S54) and a new variant of the MGST promoter with an 8-bp deletion in the ´Kronio´ cultivar.

High-resolution genome-wide association study of a large Czech collection of sweet cherry (Prunus avium L.) on fruit maturity and quality traits.

In sweet cherry (Prunus avium L.), quantitative trait loci have been identified for fruit maturity, colour, firmness, and size to develop markers for marker-assisted selection. However, resolution is usually too low in those analyses to directly target candidate genes, and some associations are missed. In contrast, genome-wide association studies are performed on broad collections of accessions, and assemblies of reference sequences from Tieton and Satonishiki cultivars enable identification of single nucleotide polymorphisms after whole-genome sequencing, providing high marker density. Two hundred and thirty-five sweet cherry accessions were sequenced and phenotyped for harvest time and fruit colour, firmness, and size. Genome-wide association studies were used to identify single nucleotide polymorphisms associated with each trait, which were verified in breeding material consisting of 64 additional accessions. A total of 1 767 106 single nucleotide polymorphisms were identified. At that density, significant single nucleotide polymorphisms could be linked to co-inherited haplotype blocks (median size ~10 kb). Thus, markers were tightly associated with respective phenotypes, and individual allelic combinations of particular single nucleotide polymorphisms provided links to distinct phenotypes. In addition, yellow-fruit accessions were sequenced, and a ~ 90-kb-deletion on chromosome 3 that included five MYB10 transcription factors was associated with the phenotype. Overall, the study confirmed numerous quantitative trait loci from bi-parental populations using high-diversity accession populations, identified novel associations, and genome-wide association studies reduced the size of trait-associated loci from megabases to kilobases and to a few candidate genes per locus. Thus, a framework is provided to develop molecular markers and evaluate and characterize genes underlying important agronomic traits.