This study aims to: (i) identify the Rosa S-locus controlling self-incompatibility (SI); (ii) test the genetic linkage of the S-locus with other loci controlling important ornamental traits, such as the continuous-flowering (CF) characteristic; (iii) identify the S-alleles (S_C) of old Chinese CF cultivars (e.g, Old Blush, Slater’s Crimson China) and examine the changes in the frequency of cultivars with Sc through the history of breeding; (iv) identify wild species carrying the Sc-alleles to infer wild origins of CF cultivars. We identified a new S-RNase (S_C2) of Rosa chinensis in a contig from a genome database that has not been integrated into one of the seven chromosomes yet. Genetic mapping indicated that S_C2 is allelic to the previously-identified S-RNase (S_C1) in chromosome 3. Pollination experiments with half-compatible pairs of roses confirmed that they are the pistil-determinant of SI. The segregation analysis of an F₁-population indicated genetic linkage between the S-locus and the floral repressor gene KSN. The non-functional allele ksn is responsible for the CF characteristic. A total of five S-alleles (S_C1–5) were identified from old CF cultivars. The frequency of cultivars with S_C dramatically increased after the introgression of ksn from Chinese to European cultivars and remains high (80%) in modern cultivars, suggesting that S-genotyping is helpful for effective breeding. Wild individuals carrying S_C were found in Rosa multiflora (S_C1), Rosa chinensis var. spontanea (S_C3), and Rosa gigantea (S_C2, S_C4), supporting the hypothesis of hybrid origins of CF cultivars and providing a new evidence for the involvement of Rosa multiflora.

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