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A. Belaj, Z. Satovic, L. Rallo and I. Trujillo

The aim of this work was to study in depth the resolving power of RAPD markers for rapid and reliable identification of olive cultivars in germplasm collections. The D parameter (the probability that two randomly chosen cultivars have different banding patterns), used for that purpose, showed high values for most of the 21 primers tested and its values ranged from 0.6114 (OPI-13) to 0.9762 (OPK-16) with a mean value of 0.8566. This parameter was used to select the five most discriminating primers: OPK-16, OPA-19, OPX-09, OPF-06 and OPZ-11. The joint confusion probability and the statistical number of indistinguishable pairs of cultivars were estimated for these primers (under independence hypothesis). The combination of three primers (OPK-16, OPA-19 and OPX-09) was found optimal for rapid discrimination of 103 cultivars with a very low value of cumulative confusion probability (1.72 × 10-5), leaving 0.09 pairs of cultivars indistinguishable. This fact, together with the efficiency of the most discriminating primers combination on an increasing number of cultivars, evidenced the utility of RAPD markers for discrimination of olive cultivars in collections and in nurseries.

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Hongwen Huang, Desmond R. Layne and Thomas L. Kubisiak

Thirty-four extant pawpaw [Asimina triloba (L.) Dunal] cultivars and advanced selections representing a large portion of the gene pool of cultivated pawpaws were investigated using 71 randomly amplified polymorphic DNA (RAPD) markers to establish genetic identities and evaluate genetic relatedness. All 34 cultivated pawpaws were uniquely identified by as few as 14 loci of eight primers. Genetic diversity of the existing gene pool of cultivated pawpaws, as estimated by Nei's gene diversity (He), was similar to that of wild pawpaw populations. The genetic relatedness among the cultivated pawpaws examined by UPGMA cluster analysis separated 34 cultivars and selections into two distinct clusters, a cluster of PPF (The PawPaw Foundation) selections and a cluster including a majority of the extant cultivars selected from the wild and their derived selections. The results are in general agreement with the known selection history and pedigree information available. The consensus fingerprint profile using the genetically defined RAPD markers is a useful and reliable method for establishing the genetic identities of the pawpaw cultivars and advanced selections. This also proved to be an improved discriminating tool over isozyme markers for the assessment of genetic diversity and relatedness. RAPD profiling of data presented in this study provides a useful reference for germplasm curators engaged in making decisions of sampling strategies, germplasm management and for breeders deciding which parents to select for future breeding efforts.

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Riaz Ahmad, Darush Struss and Stephen M. Southwick

We evaluated the potential of microsatellite markers for use in Citrus genome analysis. Microsatellite loci were identified by screening enriched and nonenriched libraries developed from `Washington Navel' Citrus. Microsatellite-containing clones were sequenced and 26 specific PCR primers were selected for cross-species amplification and identification of cultivars/clones in Citrus. After an enrichment procedure, on average 69.9% of clones contained dinucleotide repeats (CA)n and (CT)n, in contrast to <25% of the clones that were identified as positive in hybridization screening of a nonenriched library. A library enriched for trinucleotide (CTT)n contained <15% of the clones with (CTT)n repeats. Repeat length for most of the dinucleotide microsatellites was in the range of 10 to 30 units. We observed that enrichment procedure pulled out more of the (CA)n repeats than (CT)n repeats from the Citrus genome. All microsatellites were polymorphic except one. No correlation was observed between the number of alleles and the number of microsatellite repeats. In total, 118 putative alleles were detected using 26 primer pairs. The number of putative alleles per primer pair ranged from one to nine with an average of 4.5. Microsatellite markers discriminated sweet oranges [Citrus sinensis (L.) osb], mandarin (Citrus reticulata Blanco), grapefruit (Citrus paradisi Macf.), lemon [Citrus limon (L.) Burm.f.], and citrange (hybrids of trifoliate orange and sweet orange), at the species level, but individual cultivars/clones within sweet oranges, mandarins and grapefruit known to have evolved by somatic mutation remained undistinguishable. Since these microsatellite markers were conserved within different Citrus species, they could be used for linkage mapping, evolutionary and taxonomic study in Citrus.

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F. Sanz-Cortés, M.L. Badenes, S. Paz, A. Íñiguez and G. Llácer

Forty olive (Olea europaea L.) cultivars from Valencia, Spain, were screened using random amplified-polymorphic DNA (RAPD) markers. Eighteen selected decamer primers produced 34 reproducible amplification fragments that were then used as polymorphic markers. The resulting combinations of these RAPD markers were used to discriminate 40 cultivars. Results were analyzed for similarity among cultivars and the relatedness of polymorphisms obtained between cultivars agreed with previous results using isozymes. Unweighted pair group method cluster analysis of their similarity values revealed two main groups divided according to geographic origin within Valencia. A third group, which included two Spanish cultivars from regions outside of Valencia, was clustered separately from the Valencian cultivars. RAPD technology proved useful in discriminating closely related cultivars. There was no apparent clustering of cultivars by fruit size or other morphological traits.

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Luís Goulão, Luisa Monte-Corvo and Cristina M. Oliveira

Variability of commercial plum (Prunus L. sp.) cultivars is unknown since breeding often involves intercrossing hybrids with several species but has been based on a low number of parents. Molecular markers like amplified fragment length polymorphisms (AFLP) and inter-simple sequence repeats (ISSR), which sample multiple loci simultaneously, have become increasingly popular, and were used to characterize 24 diploid and four hexaploid cultivars of plum. Seven AFLP and six ISSR primers were used, and resulted in amplification of 379 and 270 products, respectively. Unweighted pair-group method with arithmetic averages (UPGMA) dendrograms, based on similarity coefficients, reflected a clear separation between diploid and hexaploid plums. Among diploid plums, two pairs of cultivars were relatively distinct from the rest, namely `Golden Japan' and `Methley' and `Ozark Premier' and `Songold'. Furthermore, several cultivars were grouped together both with AFLP and ISSR analysis: 1) `Ambra', `Red Beaut', and `Black Beaut', 2) `Black Diamond' and `Royal Diamond', 3) `June Rose', `Santa Rosa', and `Royal Red', and iv) `Freedom', `Larry Ann', and `Queen Rosa'. Although the phenetic classification obtained by the two methods were similar (r = 0.73, for the diploid group), ISSR had a higher reproducibility and percentage of polymorphisms (87.4% vs. 62.8%) than AFLP. Methodological aspects of both markers systems are discussed. Results obtained suggest that the AFLP and ISSR approaches are valuable tools for identification of specific genotypes and analysis of phenetic relationships in plum.

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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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J.F. Hancock, P.A. Callow and Douglas V. Shaw

Eight strawberry cultivars or advanced selections from the Univ. of California, Davis, breeding program were screened for polymorphisms using the polymerase chain reaction (PCR) and 43 random 10-base DNA primers. Over 60% of the primers screened resulted in replicable polymorphic banding patterns (amplification profiles), and a subset of ten primers that exhibited high levels of amplification profile polymorphism was used to identify each of the eight genotypes uniquely. There was also a significant product-moment correlation (r = 0.64, P < 0.01) between number of shared amplification profile phenotypes and pairwise coefficient of coancestry. This technology shows high promise as a means of verifying the identity of cultivars and developing a genetic map of the octoploid cultivated strawberry.

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C.E. Greer, R.E. Schutzki, A. Fernandez and J.F. Hancock

Starch gel electrophoresis was used to fingerprint 55 Taxus plants, listed as 21 species and/or cultivars. Plants were analyzed for six enzymes, representing eight putative loci. Within many of the cultivars, different fingerprints were observed, indicating nomenclatural errors in Taxus.

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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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John H. Culpepper, Luis A. Sayavedra-Soto, Brant J. Bassam and Peter M. Gresshoff

Several horticulturally important members of the genus Cornus were characterized at the DNA level to identify genotypes. Random genomic DNA fragments from Cornus florida L. `Barton' were cloned into pBR322 and λ Gem-11 and used to search for restriction fragment length polymorphisms (RFLPs) among C. sericea L., C. kousa Hance., and four cultivars of C. florida: `Barton', `Cherokee Princess', `Cloud 9', and `Mary Ellen'. Total DNA from these genotypes was restricted with several endonucleases (of which BamHI, EcoRI, and HindIII were used to search for RFLPs), vacuum-blotted onto nylon membranes, and probed with the C. florida `Barton' DNA clones. RFLPs were common among the Cornus species sericea, kousa, and florida, suggesting considerable DNA sequence divergence at the species level. RFLPs were less common among the cultivars of C. florida. These cultivars were selected from a narrow geographical area in North America from nursery-grown trees and exhibit much less DNA sequence divergence.