Synergistic approach of PCR-based fragment length analysis and amplicon deep sequencing reveals rich diversity of S- alleles in sweet cherries from the Caucasian region of origin

Abstract

Introduction: The self-incompatibility system in sweet cherry (Prunus avium L.) prevents fertilization with own or genetically related pollen, and is genetically determined by the multi-allelic S-locus. Therefore, determining S-alleles is crucial for plant breeding and fruit production, as it enables the selection of compatible combinations of S-genotypes for successful pollination.

Methods: In this study, S-alleles were identified in a total of 260 genotypes from the Caucasian region, the species' center of origin. S-allele genotyping was conducted using PCR fragment length analysis with the standard marker PaConsI-F/R2 and reference genotypes, complemented by sequence analysis through amplicon deep sequencing.

Results and discussion: The genotypes collected from Azerbaijan and Turkey exhibit a high allelic richness at the S-locus, particularly compared to modern sweet cherry cultivars worldwide. Nine previously undescribed S-alleles were identified and designated as S₄₅, S₄₆, S₄₇, S₄₈, S₄₉, S₅₀, S₅₁, S₅₂ and S₅₃. Given the expected high diversity for other traits, this plant material represents a valuable resource for further breeding research and introgression of new traits in future breeding programs. Furthermore, our results underscore that fragment length alone may not be sufficient for unambiguous assignment of S-alleles due to minimal length differences between different alleles. To address this issue, an S-allele reference ladder was developed using the rich diversity for precise assignment of the S-alleles. This tool can be applied in future experiments as a robust and cost-effective method for accurate S-genotyping across different runs and laboratories. Additionally, several selected S-genotypes were planted in a trial field and will be maintained as an S-allele reference collection.

Keywords: Caucasus; S-allele reference collection; S-alleles; amplicon-deep sequencing; diversity; fragment length analysis; genotyping; sweet cherry.