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Kirk W. Pomper, Anita N. Azarenko, Joel W. Davis, and Shawn A. Mehlenbacher

Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles and assist in cloning the locus responsible for the sporophytic SI displayed in hazelnut (Corylus avellana L.). DNA was extracted from young leaves collected from field-planted parents and 27 progeny of the cross OSU 23.017 (S1 S12) × VR6-28 (S2 S26). Screening of 10-base oligonucleotide RAPD primers was performed using bulked segregant analysis. DNA samples from six trees each were pooled into four “bulks,” one for each of the following: S1 S2, S1 S26, S2 S12, and S12 S26. “Super bulks” of twelve trees each for S1, S2, S12, and S26 then were created for each allele by combining the appropriate bulks. The DNA from these four super bulks and also the parents was used as a template in the PCR assays. Amplification products were electrophoresed on 2% agarose gels and photographed under UV light after ethidium bromide staining. 200 primers were screened and one RAPD marker each was identified for alleles S2 (OPI-07700) and S1 (OPJ-141700).

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Lianghong Chen, Shizhou Wang, and Mack Nelson

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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Lianghong Chen, Shizhou Wang, and Mack Nelson

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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Raymond J. Schnell and Robert J. Knight

Genetic relationships between commercial mango cultivars are often speculative and only the maternal parent is generally known. RAPD™ primers were used with the polymerase chain reaction (PCR) to provide markers useful in determining individual identity, family relationships, and linkage mapping analysis. In mango, 53 RAPD primers were screened for markers and 27 proved useful. Genomic DNA was isolated from 70 clones of mango maintained in the USDA germplasm collection. DNA from these clones was amplified with each of the 27 primers. Data were scored as the presence or absence of bands. Groupings of the clones using UPGMA based on Nei's genetic distance gave distinct clusters. RAPD clusters vs. clusters based on isozyme analysis are compared.

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Paul. G. Thompson, Liang L. Hong, Kittipat Ukoskit, and Zhiqiang Zhu

RAPD marker analyses were completed on parents and progeny of two sweetpotato [Ipomoea batatas (L.) Lam.] crosses to determine the feasibility of genetic linkage map construction. A total of 100 primers was tested and 96 produced amplified genomic DNA fragments. The average number of polymorphisms per primer was 0.69. A total of 134 polyphorphic markers was observed and 74 (60%) segregated 1 band present : 1 band absent as needed for use in genetic linkage mapping of polyploids. The 60% of RAPD markers that segregated 1:1 shows that genetic linkage mapping of the hexaploid sweetpotato by RAPD marker analysis is feasible. Linkage was determined for all markers that segregated 1:1 and five pairs of linked markers were found. These were the first linked molecular markers found in sweetpotato and they show that construction of a genetic linkage map is feasible. A genetic linkage map will be a valuable tool to assist in genetic improvements.

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Lin Wu and Hong Lin

The polymerase chain reaction (PCR) and RAPD fragments are potentially useful methods for identifying turfgrass cultivar breeding lines. RAPD markers were studied in 25 vegetatively propagated buffalograss lines using oligonucleotide random primers and agarose-gel electrophoresis to determine their potential for identifying cultivar breeding lines. The variation of RAPD markers was extensive. The RAPD markers produced by one random primer were sufficient to separate the 25 buffalograss lines. Cluster analysis baaed on' the RAPD markers produced by two random primers revealed that the 25 buffalograss lines generally fell into two groups: diploid and hexaploid. Three DNA extraction methods—sarcosyl lysis-chloroform extraction-isopropanol precipitation, sodium dodecyl sulfate (SDS) lysine-isopropanol precipitation, and boiling in the presence of Chelex-100 resin—and fresh or oven-dried tissues were tested for reproducibility of RAPD markers. The three DNA extraction methods, using dry or fresh plant tissues, produced highly comparable RAPD marker profiles. More than 80%1 of the RAPD markers was consistently detected in six replicate analyses. The above studies demonstrate that small quantities (5 mg) of oven-dried leaf tissue and several DNA extraction methods can be used for buffalograss fingerprint studies.

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Claudia Cunha, Tana Hintz, and Phillip Griffiths

DNA extractions from 77 snap bean and 2 dry bean cultivars were evaluated for molecular polymorphisms. In total, 100 10-mer oligonuceotide primers were evaluated, and 31 primers that amplified clear and repeatable polymorphisms among bean cultivars were selected. These primers amplified a total of 49 polymorphisms between the cultivars and were used to differentiate the cultivars and evaluate the genetic diversity between them. All cultivars were clustered according to genetic similarities using GenStat 5.0 software, and groupings of pod types were observed when cultivars were separated based on a dissimilarity index. The RAPD polymorphisms will be useful for cultivar determination, seed purity testing and estimation of genetic distances.

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Amnon Levi, Lisa J. Rowland, and John S. Hartung

A procedure for identifying reproducible RAPD markers from woody plant DNA is presented. The procedure relies on using a PCR buffer that contains 1% Triton-X-100 and 0.1 % gelatin [previously described for successful polymerase chain reaction (PCR) amplification of 16S/23S rRNA intergenic spacer regions from eubacteria], and amplification conditions of 50 cycles: 30 sec at 94C, 70 sec at 48C, and 120 sec at 72C. The combination of this buffer and these conditions amplified consistent fragments in higher amounts, as compared to other standard PCR buffers and conditions generally used for RAPD analysis. This procedure resulted in reliable RAPD patterns for all organisms tested. Chemical name used: α-[4-(1,1,3,3,-tetramethylbutyl)phenyl]-cohydroxypoly(oxy-l,2-ethanediyl) (Triton-X-l00).

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C.M. Ronning, R.J. Schnell, and S. Gazit

The native American genus Annona contains many species that are cultivated for their edible fruit, including the custard apple (A. reticuluta L.), soursop (A. muricata L.), cherimoya (A. cherimola L.), sugar apple (A. squamosa L.), and interspecific hybrids, the atemoyas. RAPD analysis of A. cherimola. `Campa' and `Jete,' A. squamosa `Lessard,' and the atemoyas `Ubranitzki,' `Malali,' and `Kaspi' resulted in very distinctive patterns, indicating that RAPD markers, may be an efficient method of fingerprinting genotypes within and between Annona species. All 15 primers used generated repeatable, polymorphic patterns. An F1 population of `Jete' × `Lessard' was analyzed to determine the inheritance of the RAPD banding patterns. Fifty-two polymorphic loci were identified, which segregated in an expected Mendelian fashion.

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C.L. Boehm, H.C. Harrison, G. Jung, and J. Nienhuis

Genetic differences among eleven cultivated and eight wild-type populations of North American ginseng (Panax quinquefolium L.) and four cultivated populations of South Korean ginseng (P. ginseng C.A. Meyer) were estimated using RAPD markers. Cultivated P. ginseng population samples were collected from four regions of S. Korea. Cultivated P. quinquefolium population samples were collected from three regions in North America: Wisconsin, the Southeastern Appalachian region of the United States, and Canada. Wild-type P. quinquefolium was collected from three states in the United States: Pennsylvania, Tennessee, and Wisconsin. Evaluation of germplasm with 10 decamer primers resulted in 100 polymorphic bands. Genetic differences among populations indicate heterogeneity. The genetic distance among individuals was estimated using the ratio of discordant bands to total bands scored. Multidimensional scaling of the relationship matrix showed independent clusters corresponding to the distinction of species, geographical region, and wild versus cultivated types. The integrity of the clusters was confirmed using pooled chi-square tests for fragment homogeneity.