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Transferring desired genes from wild species to cultivars through alien addition lines (AALs) has been shown to be an effective method for genetic improvement. Cucumis hystrix Chakr. (HH, 2n = 24) is a wild species of Cucumis that possesses many resistant genes. A synthetic allotetraploid species, C. hytivus (HHCC, 2n = 38), was obtained from the cross between cultivated cucumber, C. sativus (CC, 2n = 14), and C. hystrix followed by chromosome doubling. Cucumis sativus – C. hystrix AALs were developed by continuous backcrossing to the cultivated cucumbers. In this study, 10 different types of AALs (CC-H01, CC-H06, CC-H08, CC-H10, CC-H12, CC-H06+H09, CC-H06+H10, CC-H06+H12, CC-H08+H10, CC-H01+H06+H10) were identified based on the analysis of fluorescence in situ hybridization (FISH) and molecular markers specific to C. hystrix chromosomes. And the behavior of the alien chromosomes in three AALs (CC-H01, CC-H06+H10, CC-H01+H06+H10) at meiosis was investigated. The results showed that alien chromosomes paired with C. sativus chromosome in few pollen mother cells (PMCs). Further, disomic alien addition lines (DAALs) carrying a pair of C. hystrix chromosome H10 were screened from the selfed progenies of CC-H10. Chromosome pairing between genomes provides cytological evidence for the possible introgression of alien chromosome segments. The development of AALs could serve as a key step for exploiting and utilizing valuable genes from C. hystrix. [ABSTRACT FROM AUTHOR]