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  • Author or Editor: Mack Nelson x
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Randomly amplified polymorphic DNA (RAPD) technique is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. It has been widely used for genetic mapping, plant and animal breeding programs, and DNA fingerprinting. However, there is no single set of RAPD-PCR conditions that can be applied to all situations. In order to adjust reaction component concentrations within suggested ranges for efficient amplification during the use of RAPD in detection of genetic variation of genus Camellia, crucial factors, such as concentrations of MgCl2 and DNA, annealing temperature (37 to 44 °C), and the use of an AmpliTaq® DNA polymerase and Stoffel fragment were examined. Five camellia cultivars, `Winter's Beauty', `Pink Icicle', `Polar Ice', `Winter's Hope', and `Snow Flurry', were under investigation. Clear and reproducible amplification products were produced with 3.0 μM MgCl2 and 30 ng template DNA/25 μL reaction mixer at annealing temperature 37 °C and 40 °C, compared with MgCl2 at 1.5, 2.0, and 2.5 μM. When annealing temperature increased, the RAPD-PCR stringency was increased, as expected. Stoffel fragment was found to provide highly reproducible results.

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In this study research was conducted to evaluate the feasibility of characterizing genetic variation within camellia species using random amplified polymorphic DNAs (RAPD) markers. Eight varieties of species Camellia japonica and four varieties of species Camellia reticulata, provided by the America Camellia Society, Fort Valley, Ga., were investigated. RAPD profiles generated by five selected 10-based random primers (out of 20 primers) exhibited distinct patterns of amplified bands for all 12 tested varieties. A total of 344 bands were produced among the eight varieties of species C. japonica, with an average of 8.6 bands, ranging from 220 to 2072 bp in size, scored per primer. Among the 344 amplified bands, 74.4% of the bands presented polymorphic. The four varieties of species C. reticulata produced a total of 180 markers, with an average percentage of 57.8% polymorphisms. The amplified bands were in the range of 236–1760 bp. An average of nine amplified bands was generated per primer. The large percentages of polymorphisms displayed among 12 varieties within the two different species indicate that the expected genetic diversity among varieties within camellia species existed. It was concluded that the RAPD molecular markers are capable of revealing appreciable levels of genetic variation within camellia species.

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The reliability of the random amplified polymorphic DNA (RAPD) technique in amplifying polymorphisim among the hybrids and their parents' genomes of the genus Camellia was evaluated. Three hybrids (`Londontowne Blush', `Ashton's Snow', and `Ashton's Cameo') and one of the parents, C. oleifrea`Plain Jane', provided by the America Camellia Society, Fort Valley, Ga., were investigated. Twenty 10-based random primers were tested in this study. Five out of 20 primers were selected for RAPD analysis based on the ability to produce unambiguously scoreable RAPD bands for evaluation and comparison of the genotypes under investigation. The five primers were selected because they produced distinct patterns of amplified bands for each tested genotype. A total of 162 RAPD bands were produced. Among the 162 bands, 86 bands showed polymorphisms. The amplified band sizes ranged from 236 to 1656 bp. These data indicate that in the three hybrids and one of the parents exist unique genomic regions. Our investigation results showed that the RAPD molecular approach can be used to discriminate genetic variation among hybrids and their parents.

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