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Sriyani Rajapakse, Mark Hubbard, Albert Abbott, Robert Ballard, and John Kelly

Restriction Fragment Length Polymorphisms (RFLPs) were investigated in rose cultivars as a means of reliable cultivar identification. A random genomic DNA library was generated by shotgun cloning HindIII digested fragments of DNA extracted from rose cultivar Confection into pUC8 plasmid of Escherichia coli strain JM 83. Compared to genomic clones carrying low or highly repeated sequences, clones with moderately repeated sequences were most effective in cultivar identification. These clones were identified by hybridizing rose DNA fragments from the library with genomic DNA from `Confection'. Clones with moderately repeated copy sequences were used as probes to detect the presence of RFLPs by Southern hybridization of EcoRI digested genomic DNA of various rose cultivars. Several of these probes have revealed RFLPs useful in cultivar identification. By using a combination of two or more of these probes most of the rose cultivars compared at this time can be identified. A dichotomous key useful in identification of rose cultivars was prepared from RFLPs displayed by 3A9 probe.

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Mark Hubbard, John Kelly, Albert Abbott, and Robert Ballard

To protect plant patents, rose breeders would benefit from a reliable and sensitive method for differentiating cultivars at the genetic level. Rcombinant DNA technologies are being employed to characterize individual DNA structure of numerous rose cultivars. Restriction fragment length polymorphisms (RFLPs) are being studied to develop a characteristic pattern, or fingerprint for each cultivar. DNA from various cultivars is restriction enzyme digested and the fragments separated by agarose gel electrophoresis. The gel is Southern blotted and hybridized with probes from the rose DNA library to yield RFLPs. RFLPs are being located and will eventually result in a characteristic fingerprint for each cultivar.

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Sriyani Rajapakse, Mark Hubbard, Albert Abbott, John Kelly, and Robert Ballard

Restricted Fragment Length Polymorphisms (RFLPs) were investigated in closely and distantly related rose cultivars as means of identifying those cultivars for the purpose of patent protection. A random genomic DNA library was constructed using the cultivar `Confection' and the Escherichia Coli strain JM83 plasmid vector pUC8. Clones with interspersed repeat sequences were then identified by hybridizing restriction digested cloned DNA fragments with nick translated genomic DNA of the rose cultivar `Confection'. Hybridization positive clones were screened for polymorphism by Southern hybridization on restriction digested genomic DNA of various rose cultivars. About 75% of the interspersed repeat copy probes screened revealed polymorphisms. We have identified probes that give fingerprint patterns for rose cultivars. From this information, a dichotomous key which differentiates the rose cultivars examined was prepared. Current research involves screening more probes and rose cultivars for polymorphisms, and examining single copy probes for potential use in RFLP genetic linkage map construction in roses.

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Mark Hubbard, John Kelly, Sriyani Rajapakse, Robert Ballard, and Albert Abbott

We have initiated a phylogenetic study using restriction fragment length polymorphisms to examine nuclear DNA variation in a number of Rosa species. Random genomic clones were isolated from the cultivar `Confection'. To generate these clones, genomic DNA was digested with the restriction enzymes Hind III and Eco RI and the resulting fragments cloned into a pUC8 plasmid and transformed into the E. coli bacterial strain JM83. Individual clones from the DNA library were screened for polymorphism by Southern hybridization methods. Those clones displaying polymorphisms were used in combination with one, two, or three restriction enzymes to identify different size restriction fragments. Each fragment was treated as a unit character and was used to generate a phylogenetic tree using the computer program “Phylogenetic Analysis Using Parsimony” (PAUP version 3.0). Results of the studies on the amount of genetic diversity and phylogenetic affinities of Rosa species among the different sections of the subgenus Rosa will be presented.

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Sriyani Rajapakse, Albert Abbott, John Kelly, and Robert Ballard

The feasibility of using RFLP to distinguish genetically related Hybrid Tea rose cultivars for DNA `fingerprinting' was examined with a group of cultivars related to `Peace'. The following cultivars used in this study, `Chicago Peace', `Flaming Peace', `Climbing Peace' and `Lucky Piece', were derived from bud mutations (sports) of `Peace'. We also investigated two additional cultivars, `Perfume Delight' and `Garden Party', in which one of the parents for each was `Peace'. Genomic rose DNA probes, cloned in pUC8 plasmid of Escherichia coli, were hybridized with genomic DNA of these cultivars digested with different restriction enzymes. Although polymorphisms were observed among these related cultivars, only a few probe/enzyme combinations screened produced RFLPs due to the high degree of genetic relatedness of these cultivars. We have identified probes that can distinguish all of these related rose cultivars. This study demonstrates that RFLP markers can be used effectively in DNA `fingerprinting' of genetically related rose cultivars, eventhough the level of detectable polymorphism is quite low.

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Ying Wang, Gregory L. Reighard, Desmond R. Layne, Albert G. Abbott, and Hongwen Huang

Pawpaw (Asimina triloba) produces the largest fruit native to the United States. Six linkage groups were identified for A. triloba using the interspecific cross [PPF1-5 (A. triloba) × RET (A. reticulata Shuttlw. ex Chapman)], covering 206 centimorgans (cM). A total of 134 dominant amplification fragment length polymorphism (AFLP) markers (37 polymorphic and 97 monomorphic) were employed for estimating the genetic diversity of eight wild populations and 31 cultivars and advanced selections. For the wild populations, the percentage of polymorphic loci over all populations was 28.1% for dominant markers and Nei's genetic diversity (He) were 0.077 estimated by 134 dominant markers. Genetic diversity and the percentage of polymorphic loci estimated using only polymorphic dominant AFLPs were 0.245 and 79%, respectively, which are comparable with other plant species having the same characteristics. Estimated genetic diversity within populations accounted for 81.3% of the total genetic diversity. For cultivars and advanced selections, genetic diversity estimated by 134 dominant markers was similar to that of wild pawpaw populations (He = 0.071). Thirty-one cultivars and advanced selections were delineated by as few as nine polymorphic AFLP dominant loci. Genetic relationships among wild populations, cultivars and advanced selections were further examined by unweighted pair group method with arithmetic mean (UPGMA) of Nei's unbiased genetic distance. The genetic diversity estimated for wild populations using the clustered polymorphic markers was lower than the result estimated using the nonclustered polymorphic markers. Therefore, this study indicates that the number of sampled genomic regions, instead of the number of markers, plays an important role for the genetic diversity estimates.

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Ying Wang, Laura L. George, Gregory L. Reighard, Ralph Scorza, and Albert G. Abbott

Evergreen genotypes of peach [Prunus persica (L.) Batsch] have been identified in Mexico, where terminal growth on evergreen trees is continuous under favorable environmental conditions. This evergreen trait in peach is controlled by one single gene (evg), and this evergreen condition is homozygous recessive. Four dominant AFLP markers, EAT/MCAC, ETT/MCCA2, EAT/MCTA, and ETT/MACC, were found to be tightly linked to the evg locus at 1 cM, 4.6 cM, 5.8 cM, and 11 cM, respectively. All four markers were sequenced and identified. A peach BAC library was constructed by using the pBeloBAC11 vector for building the physical map for the evg gene. This library represents four times the coverage of the peach genome with the average insert size of 50 to 70 kb. The EAT/MCAC AFLP marker fragment was used for screening the peach BAC library. A single BAC clone, 18F12, was confirmed to contain this fragment. The final BAC contig for this evg gene region and the potential homology between peach and Arabidopsis thaliana will be presented and discussed.

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Douglas G. Bielenberg, Ying Wang, Gregory L. Reighard, and Albert G. Abbott

`Evergrowing' (evg) peach is a naturally occurring mutant unable to enter winter dormancy in response to dormancy inducing conditions. The evg mutant is one of only two described mutants affecting winter dormancy in woody perennial trees. The evg mutation segregates as a single recessive gene and previous work by our group has fine mapped the trait between flanking markers separated by 3.3 centiMorgans. This region was physically mapped using a bacterial artificial chromosome (BAC) library from and a contig of overlapping genomic fragments identified. We have utilized several approaches to complete the sequencing of a 132 kilobase region of the peach genome derived from three overlapping BACs that encompass the complete EVG gene containing region. We present here our analysis and annotation of the genomic region, including putative and experimentally verified gene coding sequences. A primary feature of the region is a large tandem duplication of a region containing a MADS-box type DNA binding transcription factor resulting in six similar copies of the gene, all of which appear to be expressed at the mRNA level in wild-type germplasm. Hybridization analysis revealed the presence of a large deletion in the mutant genome. Five of the identified genes fall within the evg mutation and represent new candidates for the control of entrance into winter dormancy.