IDENTIFICATION OF CLOSELY-RELATED CITRUS CULTIVARS WITH INTER-SIMPLE SEQUENCE REPEAT MARKERS

Inter-simple sequence repeat (ISSR) markers generated by 22 primers we re tested for their ability to distinguish among samples from 94 trees of 68 citrus cultivars. Within each of the six cultivar groups studie d, most of these cultivars are so closely related that they are diffic ult to distinguish by other molecular-marker techniques. ISSR markers involve PCR amplification of DNA using a single primer composed of a m icrosatellite sequence anchored at the 3' or 5' end by 2-4 arbitrary, often degenerate, nucleotides. The amplification products were separat ed on non-denaturing polyacrylamide gels and detected by silver staini ng. ISSR banding profiles were very repeatable on duplicate samples. D ifferent citrus species had very different fingerprint patterns. Withi n Citrus sinensis (L.) Osbeck and C. paradisi Macf., in which all cult ivars have originated by the selection of mutants, ISSR markers distin guished 14 of 33 sweet orange and 1 of 7 grapefruit cultivars. Five of six lemon cultivars were discriminated by ISSR markers. Many differen ces were found among mandarin cultivars; however, all five satsuma cul tivars analyzed had identical ISSR fingerprints. Four of five citrange cultivars were distinguishable, but 'Troyer' and 'Carrizo' had identi cal ISSR fingerprints. 'Kuharske Carrizo' citrange, which has better c itrus nematode resistance than other 'Carrizo' citrange accessions, ha d unique ISSR fingerprints. Three ISSR markers that differentiated cer tain sweet orange cultivars were hybridized to Southern blots of sweet orange DNA digested with different restriction endonucleases. The swe et orange cultivars tested could be distinguished by these ISSR-derive d RFLP markers. Moreover, one ISSR marker unique to 'Ruby' blood orang e was observed in its progeny trees.