Development and Validation of KASP and InDel Markers Cosegregating With the Fertility Restoring Gene for Ogura Cytoplasmic Male Sterility in Rapeseed ( <scp> <i>Brassica napus</i> </scp> L.)

ABSTRACT Ogura cytoplasmic male sterility (Ogu CMS) is a widely utilized in the production of hybrid rapeseed ( Brassica napus ). Nevertheless, the development of codominant molecular markers associated with restorer genes has consistently presented a formidable challenge, attributable to the fact that these restorer genes are derived from exogenous sequences. In the present study, we have developed four cosegregating markers. Through sequence alignment, we identified rapeseed sequences that are highly homologous to the flanking sequences of the restorer gene Rfo and developed markers based on the differences between these homologous sequences. Based on this method, an Insertion–Deletion (InDel) marker and Kompetitive Allele Specific PCR (KASP) were developed. Restorer lines with different backgrounds, such as CLR650 and R2000, were utilized for marker validation, and the results demonstrated that the InDel marker (CIN6) and the KASP marker (BN9000018_K01) are widely adaptable and can accurately identify the restorer lines. Furthermore, F 2 and BC 1 offspring carrying the Rfo gene were selected using the allele‐specific Rfo marker and exhibited restored fertility. Additionally, the BN9000018_K01 marker is codominant and can distinguish between heterozygous and homozygous genotypes. These molecular markers can accurately assess the genetic purity of Ogu CMS hybrid cultivars, with the results showing a match with grow‐out trials exceeding 97%. Overall, the markers developed in this study cosegregate with the Ogu CMS restorer gene, making them suitable for molecular marker‐assisted breeding of Ogu CMS restorer lines and for assessing the genetic purity of Ogu CMS hybrid cultivars.