High-performance liquid chromatography (HPLC) analyses using a series of alien monosomic addition lines (AMALs) of Japanese bunching onion (Allium fistulosum L.) having extra chromosomes from shallot (A. cepa L. Aggregatum group) were performed to determine the chromosomal locations of the genes for flavonoid and anthocyanin production in leaf sheaths of A. cepa Aggregatum group. In HPLC profiles both at 360 and 520 nm, several peaks were observed in A. cepa Aggregatum group and AMAL with chromosome 5A from A. cepa Aggregatum group but no peak was observed in A. fistulosum and other AMALs. Four of the compounds observed at 360 nm were identified as known flavonoids, i.e., apigenin, kaempferol, quercetin, and rutin. Five out of the total 18 compounds at 520 nm were identified as known anthocyanins, i.e., cyanidin-3-glucoside, cyanidin-3-laminariobioside, peonidin-3-glucoside, cyanidin-3-malonylglucoside, and cyanidin-3-malonyllaminariobioside. These results reveal that a group of the genes related to the flavonoid and anthocyanin production in the leaf sheath of A. cepa Aggregatum group are located on the chromosome 5A.

To study the effect of the extra chromosomes on morphological characters both in vegetative and reproductive stages, a series of alien monosomic addition lines of Japanese bunching onion (Allium fistulosum L.) with extra chromosomes from shallot (A. cepa L. Aggregatum group) were grown in the experimental farm of Saga University, Saga, Japan. Several morphological characters of the alien monosomic addition lines were found to be specific for the respective alien chromosomes from A. cepa Aggregatum group. The most distinctive characteristics in each alien monosomic addition line were as follows; spheroidal spathe in FF+1A, bloom-less leaf blade in FF+2A, slow expansion of leaf in FF+3A, acuminate spathe in FF+4A, reddishyellow leaf sheath in FF+5A, arch-like leaf brade in FF+6A, fast expansion of leaf in FF+7A, and intensely yellow anther in FF+8A. The results indicate that these character expressions are deeply related to alien genes on extra chromosomes from A. cepa Aggregatum group.

Genomic in situ hybridization (GISH) was used to identify alien chromosomes in a series of eight different Allium fistulosum -A. cepa monosomic addition lines. Biotin-labeled total genomic DNA from shallot (A. cepa L. Aggregatum group) was used as a probe together with an excess amount of unlabeled blocking DNA from the recipient plant, Japanese bunching onion (A. fistulosum L.). Probe hybridization sites were detected by FITC-conjugated avidin and anti-avidin antibody using an epifluorescence microscope. In each mitotic metaphase cell of all the eight types of monosomic addition lines, the alien chromosomes were successfully discriminated from other 16 A. fistulosum chromosomes. Furthermore, no clear exchanges of chromosome segments between A. cepa and A. fistulosum were observed. This finding indicates that in each addition line an entire (unrecombined) A. cepa chromosome is present in an integral diploid background of A. fistulosum.

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