Abstract
Distant hybridization between Brassica napus and related genera serves as an effective approach for rapeseed germplasm innovation. Isatis indigotica, a wild relative of Brassica, has emerged as a valuable genetic resource for rapeseed improvement due to its medicinal properties. This study employed anchor mapping of alien chromosomal fragment localization (AMAC) method to efficiently identify alien chromosomal fragments in the progeny derived from distant hybridization between I. indigotica and Brassica napus, 'Songyou No. 1'. Based on the AMAC method, we developed 193,101 IP and SSR markers utilizing the I. indigotica reference genome (Woad-v1.0). Through Electronic-PCR analysis against the Brassica and I. indigotica pan-genome, 27,820 specific single-locus (SSL) IP and SSR markers were obtained. Subsequently, 205 pairs of IP primers and 50 pairs of SSR primers were synthesized randomly, among which 148 pairs of IP markers (72.20%) and 45 pairs of SSR markers (90%) were verified as SSL molecular markers for the I. indigotica genome with no amplification product in four Brassica crops. These 193 SSL markers enable precise identification of one complete I6 chromosome and three chromosomal fragments (I1:1.17 Mb, I5:2.61 Mb, I7:1.11 Mb) in 'Songyou No. 1'. Furthermore, we traced 32 genes involved in bioactive compound biosynthesis within/near these alien segments in 'Songyou No. 1' and developed seven functional markers. This study not only validates the efficacy of SSL markers for detecting exogenous chromatin in intergeneric hybrids but also provides valuable insights for the precise identification and mapping of desired chromosomal fragments or genes embedded in the derivatives from distant hybridization and potential applications in marker-assisted breeding for medicinal plant via distant hybridization strategy between I. indigotica and Brassica crops.