Submitted to: Journal of Theoretical and Applied Genetics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/15/1997
Publication Date: N/A
Citation: FANG, D., ROOSE, M.L., KRUEGER, R., FEDERICI, C.T. FINGERPRINTING TRIFOLIATE ORANGE GERMPLASM ACCESSIONS WITH ISOZYMES, RFLPS,AND INTER-SIMPLE SEQUENCE REPEAT MARKERS. JOURNAL OF THEORETICAL AND APPLIED GENETICS. 1997. Interpretive Summary: Trifoliate 'orange' (Poncirus trifoliata) is an important taxa in citrus germplasm collections. Poncirus hybridizes readily with Citrus, is a source of disease resistance, cold hardiness, etc for breeding purposes, and can be used as a rootstock. Most trifoliates are self compatible and produce zygotic and nucellar seedlings, and most accessions in germplasm collections are propagated vegetatively. For these reasons, the amount of genetic diversity present in trifoliate accessions is unclear. Isozymes, restriction fragment length polymorphisms (RFLP), and inter-simple sequence repeat (ISSR) markers were used to assess the genetic variability of 48 trifoliate accessions in the USDA-ARS-NCGRCD/UCR Citrus Variety Collection. Isozymes and RFLP detected few polymorphisms, while ISSR showed 17 different patterns. The trifoliate accessions fell in 4 major groups. The two types of trifoliates, 'large flower' and 'small flower', clustered into two distinct groups. Two accessions, 'Monoembryonic' and 'Simmons', differed from other accessions and clustered together in another group; they apparently arose by hybridization from the common genotype. The last group consisted of recently introduced accessions from China that diverged more from the other types; they thus probably represent more genetic variation that is potentially useful in breeding. This is the first report of the use of ISSR markers for analysis of Citrus and related genera. ISSR markers have several advantages: they generate large amounts of bands and thus can distinguish closely related accessions more powerfully than the other techniques, and are less expensive than RFLP or RAPD.
Technical Abstract: Trifoliate orange (Poncirus trifoliata) is frequently used as a parent in citrus rootstock breeding, but the amount of genetic diversity in germplasm collections is poorly understood. Most accessions are self- compatible, but produce a mixture of sexual and apomictic seedlings. Variation among 48 vegetatively propagated trifoliate orange accessions was assessed at seven isozyme loci, The restriction fragment length polymorphisms (RFLPs) detected by 38 probe-enzyme combinations and the inter-simple sequence repeat (ISSR) markers generated by 11 primers. Isozymes and RFLPs detected few polymorphisms among accessions. ISSR amplification generated multiple banding profiles with an average of 58 fragments/primer/accession. Seventeen unique marker phenotypes were identified. The 48 trifoliate accessions were classified into four 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 segregated in progeny derived by open-pollination (probably mostly selfing) of a common accession, indicating that these ISSR markers are also heterozygous. Accessions having identical genotypes for a large number of heterozygous markers are unlikely to have diverged by recombination. Therefore variation among accessions most likely originated by mutation. 'Monoembryonic' and 'Simmons' differed from other accessions only in the loss of specific markers, indicating that they originated as zygotic seedlings of individuals similar to the common genotype. Three accessions recently introduced from China have fingerprints with 3-14 unique ISSR markers, and probably represent a more divergent germplasm that may be a valuable breeding resource.