FINGERPRINTING TRIFOLIATE ORANGE GERM PLASM ACCESSIONS WITH ISOZYMES,RFLPS, AND INTER-SIMPLE SEQUENCE REPEAT MARKERS

Trifoliate orange [Poncirus trifoliata (L.) Raf.] is frequently used a s a parent in citrus rootstock breeding, but the origin and amount of genetic diversity in germ plasm collections are poorly understood. Mos t accessions are self-compatible, but produce a mixture of sexual and apomictic seedlings. Variation among 48 vegetatively propagated trifol iate orange accessions was assessed at seven isozyme loci, together wi th the restriction fragment length polymorphisms (RFLPs) detected by 3 8 probe-enzyme combinations and the inter-simple sequence repeat (ISSR ) markers generated by II primers. Isozymes and RFLPs detected few pol ymorphisms among accessions, although genetic analysis has shown that the common phenotype is heterozygous for four isozyme and at least fou r RFLP loci. ISSR amplification generated multiple banding profiles wi th an average of 58 fragments/primer/accession. These fragments were r epeatable across DNA samples extracted from different trees of the sam e accession or extracted at different times, and across separate PCR r uns. Seventeen unique marker phenotypes were identified. The 48 trifol iate orange accessions were classified into four major groups based on polymorphic ISSR markers. All large-flowered accessions are in group 4, while small-flowered accessions are in group 3. Many ISSR markers s egregated in progeny derived by open-pollination (probably mostly self ing) of a common accession, indicating that these ISSR markers are als o heterozygous. Accessions having identical genotypes for a large numb er of heterozygous markers are unlikely to have diverged by recombinat ion. Thus the limited divergence we detected among most accessions mos t likely originated by mutation. 'Monoembryonic' and 'Simmons' differe d from other accessions only in the loss of specific markers, indicati ng that they originated as zygotic seedlings of individuals similar to the common genotype. Three accessions recently introduced from China have relatively different fingerprints with 3-14 unique ISSR markers, and probably represent a much more divergent germ plasm that may be a valuable breeding resource.