Plant & Animal Genome VI Conference

P90

DIG-RAPD AND DIG-AFLP ANALYSIS OF JAPANESE CITRUS CULTIVARS

1. Wakayama Pref. Agri. Exp. Stn., Gobou, Wakayama, Japan
2. Natl. Inst. Agro. Biol. Resour.

AFLP has recently been used for DNA fingerprinting. It is a very powerful technique to distinguish very closely related cultivars. However, it requires a radioisotope compound or fluorescent dye primer. In order to simplify an ALFP method, we have modified the method to establish a DIG-based AFLP procedure. There are three varieties in Citrus kinokuni: Hira-kishu and Mukaku-kishu in addition to the original kishu. Mukaku-kishu is unique in that it is female-sterile and has no seeds. Finding of DNA markers specific for this trait should be important for molecular screening. We first applied DIG-RAPD to distinguish the three different cultivars. Using four different primers, we obtained 18 polymorphic DNA bands. However, no DNA markers were obtained for Mukaku-kishu. For DIG-AFLP, total DNA was prepared from the three citrus cultivars by the CTAB method and digested with a combination of MspI and EcoRI or MspI and PstI. The primers of EcoRI and PstI were labeled with DIG at 5'-terminal. Other conditions were essentially the same as the standard method except that PCR products were electrophoresed on a 8% non-denaturing polyacrylamide gel, blotted onto nylon membrane and stained by immunochemical method. The combination of MspI and EcoRI gave 247 DNA bands including 69 polymorphic bands, and 13 bands appeared only in Mukaku-kishu. In the case of MspI and PstI restriction, 6 out of 123 bands were found specific to Mukaku-kishu. These results indicate that DIG-AFLP is a simple and powerful method for DNA fingerprinting.