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Meryem Ipek, Ahmet Ipek, and Philipp W. Simon

Garlic (Allium sativum L.) is an asexually propagated crop that displays much morphological diversity. Studies which have assessed garlic diversity with isozymes and randomly amplified polymorphic DNA (RAPD) markers generally agreed with the morphological observations but sometimes failed to discriminate clones. To discriminate among closely related garlic clones in more detail, we introduced amplified fragment-length polymorphism (AFLPs) to evaluate the genetic diversity and phenetic relatedness of 45 garlic clones and three A. longicuspis clones and we compared AFLP results with RAPD markers and isozymes. Three AFLP primer combinations generated a total of 183 polymorphic fragments. Although similarities between the clusters were low (≥0.30), some clones within the clusters were very similar (>0.95) with AFLP analysis. Sixteen clones represented only six different banding patterns, within which they shared 100% polymorphic AFLPs and RAPD markers, and likely are duplicates. In agreement with the results of other investigators, A. longicuspis and A. sativum clones were clustered together with no clear separation, suggesting these species are not genetically or specifically distinct. The topology of AFLP, RAPD, and isozyme dendrograms were similar, but RAPD and isozyme dendrograms reflected less and much less polymorphism, respectively. Comparison of unweighted pair group method with arithmetic averaging (UPGMA) dendrograms of AFLP, RAPD, and isozyme cluster analyses using the Mantel test indicated a correlation of 0.96, 0.55, and 0.57 between AFLP and RAPD, AFLP and isozyme, and RAPD and isozyme, respectively. Polymorphic AFLPs are abundant in garlic and demonstrated genetic diversity among closely related clones which could not be differentiated with RAPD markers and isozymes. Therefore, AFLP is an additional tool for fingerprinting and detailed assessment of genetic relationships in garlic.

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Ahmet Ipek and Philipp W. Simon

Maize transposable elements, Activator (Ac) and Ds transformed into several heterologous plant species for transposon tagging of genes. Several genes in Arabidopsis, flax, petunia, tobacco, and tomato have been tagged and cloned by using Ac and Ds. We have double transformed carrot lines, B493 and B7262 with stabilized autonomous Ac and non-autonomous Ds element to develop a two-element based transposon tagging system. PCR and Southern hybridization indicated that Ds element transposed from T-DNA in calli, somatic embryos and transgenic plants. The insertion of Ds element into new sites in carrot genome after excision verified by GUS assay, Southern hybridization and inverse-PCR. Currently, the behavior of non-autonomous Ds element is being studied. Ds induced mutation will be screened in transgenic plants. These initial results demonstrate that the Ac/Ds-based transposon tagging system may work in carrot.

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Meryem Ipek, Ahmet Ipek, Douglas Senalik, and Philipp W. Simon

Production of a visible flower stalk, or bolting, has been used as a major trait to categorize garlic (Allium sativum L.) clones. Analysis of mitochondrial genome variation with polymerase chain reaction (PCR) revealed differences between bolting and nonbolting clones of garlic. Screening 333 garlic accessions from diverse geographic origins revealed a 1403-bp mitochondrial DNA marker associated with bolting that the authors call “Bolt Marker” (BltM). Bolt Marker did not amplify in any of the 131 nonbolting clones, whereas amplification of this marker was observed in 127 of 130 (97.7%) garlic clones that bolted completely in Wisconsin. Seventy-two garlic clones bolted incompletely (clones in which some but not all of the plants bolted), and this marker was not amplified in 69 (95.8%) of these clones. Because of the significant association of BltM with bolting, this PCR-based marker can be used to discriminate complete-bolting garlic clones reliably from nonbolting and incomplete-bolting ones. Sequence characterization of this marker revealed that BltM is a chimera involving both mitochondrial and chloroplast DNA. The DNA sequences including and flanking both the 5′ and 3′ ends of this marker are consistent with an ≈4.8-kbp chloroplast DNA fragment having been inserted into the mitochondrial genome downstream from the mitochondrial cox3 gene. Sequence alignment of the chloroplast genes in this chimeric region with the homologous sequences in GenBank indicate the presence of deletions, insertions, and single nucleotide polymorphisms in the coding sequences, resulting in putative, incomplete open reading frames or frame shift mutations. Hence, the authors speculate that this insertion may have occurred long ago in the evolution of garlic.