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.
Sriyani Rajapakse, Mark Hubbard, Albert Abbott, Robert Ballard and John Kelly
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.
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.
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.
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.
Sriyani Rajapakse, Janice Ryan-Bohac, Sasanda Nilmalgoda, Robert Ballard and Daniel F. Austin
The sweet potato Ipomoea batatas (L.) Lam. is classified in series Batatas (Choisy) in Convolvulaceae, with 12 other species and an interspecific true hybrid. The phylogenetic relationships of a sweetpotato cultivar and 13 accessions of Ipomoeas in the series Batatas were investigated using the nucleotide sequence variation of the nuclear-encoded β-amylase gene. First, flowers were examined to identify the species, and DNA flow cytometry used to determine their ploidy. The sweetpotato accession was confirmed as a hexaploid, I. tabascana a tetraploid, and all other species were diploids. A 1.1–1.3 kb fragment of the β-amylase gene spanning two exons separated by a long intron was PCR-amplified, cloned, and sequenced. Exon sequences were highly conserved, while the intron yielded large sequence differences. Intron analysis grouped species currently recognized as A and B genome types into separate clades. This grouping supported the prior classification of all the species, with one exception. The species I. tiliacea was previously classified as a B genome species, but this DNA study classifies it as an A genome species. From the intron alignment, sequences specific to both A and B genome species were identified. Exon sequences indicated that I. ramosissima and I. umbraticola were quite different from other A genome species. Placement of I. littoralis was questionable: its introns were similar to other B genome species, but exons were quite different. Exon evolution indicated the B genome species evolved faster than A genome species. Both intron and exon results indicated the B genome species most closely related to sweetpotato (I. batatas) were I. trifida and I. tabascana.
Sonja L. Maki, Sriyani Rajapakse, Robert E. Ballard and Nihal C. Rajapakse
Plants grown in far red (FR) light deficient environments are typically shorter because of short internodes, resembling plants treated with GA biosynthesis inhibitors. The role of GAs in the reduction of stem elongation of `Bright Golden Anne' chrysanthemum [Dendranthem ×grandiflora (Ramat.) Kitam. (syn. Chrysanthemum ×morifolium Ramat.)] grown in FR light deficient (-FR) environment was investigated by following the response of chrysanthemums grown in - FR environment to exogenous application of GA1, GA19, or GA20, and the metabolism of GA12 and GA19 in -FR or +FR environment. FR light deficient environment resulted in 25% to 30% shorter plants than in +FR environment. Final height of GA1- and GA20-treated plants followed a quadratic pattern while that of GA19 treated plants followed a linear pattern as the dosage increased from 0 to 50 μg/apex. The response to GA1 was the greatest followed by GA20 and GA19, regardless of the light environment. Application of GA1 (50 μg/apex) increased final height by 65% compared with no GA (0 μg/apex) application under either +FR or -FR light environment, suggesting the response to GA1, which is the active form, remained the same. Responses to GA19 and GA20 declined under -FR light. [14 C]GA12 and [14C]GA19 metabolized slowly in the -FR environment suggesting that the turnover of GAs may have caused in part the lower response to GA19. Although metabolism of GA1 under -FR environments was not investigated, observations with GA1 application experiments support that -FR environment may have enhanced inactivation of GA1. Chemical name used: gibberellic acid (GA).