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.
Claudia Cunha, Tana Hintz, and Phillip Griffiths
Virginia P. Roxas and Ellen B. Peffley
Nineteen random primers yielded 36 PCR-amplified products of Allium cepa profiles of each of 15 short-day grano-type onionsgrown commercially in Texas and Southern United States were compared. Several PCR productswere unique among the cultivars and can be used to differentiate among the onion cultivars investigated. A phenogram of the cultivars based on the co-occurrences of the PCR products was derived.
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.
C.M. Ronning, R.J. Schnell, and D.N. Kuhn
RAPD markers have been used successfully in genetic analysis of several crop plants. This method poses difficulties with a highly heterozygous species such as Theobroma cacao because of the dominant phenotypic expression of bands. A backcross family derived from ctultivars Catongo and Pound 12 was analyzed to determine the efficacy of RAPD markers in analyzing cacao populations. A preliminary screen of the parents and the F1 plant used as the backcross parent was conducted with 180 RAPD primers; of these, 26% were polymorphic and reproducible and produced 104 storable loci. Genomic DNA from 54 individuals of the backcross population was then amplified with these primers; 68.3 % of the loci segregated as expected in a Mendelian fashion. Separation of RAPD fragments on acrylamide revealed an additional polymorphic locus from one primer that was indistinguishable on agarose. The results demonstrated that RAPD markers can be used to study the cacao genome.
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).
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.
Mehmet Nuri Nas, Nedim Mutlu, and Paul E. Read
RAPD and phenotypic analysis were conducted to assess clonal stability of hazelnuts generated from axillary buds cultured in vitro for long-term. The nuts produced on in vitro-propagated plants were indistinguishable from those of donor plants. With the exception of rare horizontal (plagiotropic) growth, all in vitro-propagated plants exhibited phenotypes similar to those of donor plants. RAPD analysis did not reveal any somaclonal variation between donor plants from which in vitro cultures were initiated and micropropagated plants (6-year cultures), and no somaclonal variation was detected among in vitro-propagated plants. However, polymorphism (15.6%) was detected between the parent plant and its in vitro-propagated progenies (from seedlings). These results show a good discriminatory power of RAPD to detect polymorphism between samples where it is expected, and it can be effectively used for genetic assessment of micropropagated hazelnut. No evidence of genetic or epigenetic changes was observed in long-term cultured hazelnut, and thus long-term in vitro culture of hazelnut does not seem to limit its clonal propagation.
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.
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.
M.M. Jenderek, K.A. Schierenbeck, and R.M. Hannan
Maintenance of garlic (A. sativum L.) germplasm collections is based on year-to-year vegetative propagation of individual accessions. Several accessions are phenotypically similar, often originating from the same region of the world, but have been collected by different people at different times. These accessions are currently maintained as separate and unique samples, but may represent genetic duplication in the collection. In order to identify genetic duplication in the USDA collection, 45 garlic Plant Introduction accessions from the garlic USDA germplasm collection were analyzed for RAPD marker polymorphism. The samples originated from 20 countries worldwide. RAPD bands were generated by 20 decamer primers, using 100-ng DNA template, and 38 PCR amplification cycles. Polymorphism between accessions was defined as presence or absence of particular bands at given loci. However, a few distinguishing RAPD markers were established for selected accessions, identifying additional molecular markers to wholly assess the similarities or polymorphism of the garlic collection units is necessary.