Exploring S-RNase diversity in the Andean black cherry (Prunus serotina) using MinION sequencing: a cost-effective approach with increased genotyping resolution

The Andean black cherry (P. serotina) is an underutilized fruit species that could contribute to the development of sustainable food systems in the Andean region. The species displays gametophytic self-incompatibility (GSI), a mechanism controlled by the multiallelic S-locus which prevents crossbreeding between genetically related individuals and hinders breeding efforts. To design effective crosses, breeders require accurate knowledge of the S-haplotypes of parental lines. However, S-haplotype diversity is commonly evaluated using PCR-based methods that fail to accurately discriminate alleles. To address this limitation, we developed a new method to identify S-alleles in P. serotina using nanopore sequencing technology. Our method uses the Native Barcoding protocol and MinION sequencer from Oxford Nanopore Technologies to enable scalable, multiplex amplicon sequencing. For sequence analysis, we developed a bioinformatic pipeline that uses Porechop for sample demultiplexing, MeshClust for sequence alignment and clustering, and the Ugene Consensus algorithm to determine allelic variants. In this study, we evaluated the S-RNase gene of 24 P. serotina accessions using our nanopore sequencing and bioinformatic workflow. Among these accessions, we identified 12 previously reported and 6 putative new S-alleles that could not be identified with existing S-genotyping methods. Five accessions were classified as homozygous, while the other 19 were heterozygous with two or three alleles. Our results demonstrate that nanopore sequencing provides a cost-effective alternative for S-allele profiling that improves on the accuracy of existing PCR-based methods. Because of the versatility of MinION sequencing, the reported workflow can be used to characterize the diversity of other useful genes in the species, which are of relevance for conservation and breeding efforts.