contain cankers (except basal sprouts) = 100% of stems diseased. Plants scored 0 or 1 were considered resistant to infection by A. anomala . The 0 to 5 scale was converted to percent disease, as described previously, for the QTL analyses. DNA extraction
Josh A. Honig, Megan F. Muehlbauer, John M. Capik, Christine Kubik, Jennifer N. Vaiciunas, Shawn A. Mehlenbacher, and Thomas J. Molnar
Peter H. Velguth and Harold Pellett
We evaluated flow cytometric measurement of nuclear DNA content to determine ploidy level in azalea. If ploidy level correlates with DNA content, ploidy level could be determined more readily than by direct chromosome counts and assist in planning crosses and evaluating progeny. Tested plants included azalea cultivars, materials from the azalea breeding project at the Univ. of Minnesota, and species from the Rhododendron Species Botanic Garden and the North Carolina Arboretum. Data compiled from DNA assays of practically all material analyzed fell into distinct groups consistent with their being either diploid, triploid, or tetraploid. Additionally, a known diploid plant of each of four diploid species, together with a natural or derived tetraploid plant of each of these species was obtained. Results showed that the four diploids had a similar DNA content compared to one another. DNA content of the tetraploids was also similar, and the tetraploid's DNA content was approximately twice that of the diploids, as expected. Unfortunately, success with direct chromosome counts in other material has proven elusive, currently precluding direct correlation of DNA amount with ploidy level across other species and cultivars. Although many cases exist in the literature where DNA content has a direct relationship to ploidy level, this does not always hold. Although the majority of plants tested fell into a diploid, triploid, or tetraploid grouping based on DNA content, further study is required to determine the exact relationship between ploidy level and DNA content in azalea.
U. Lavi, J. Hillel, A. Vainstein, E. Lahav, and D. Sharon
Application of four DNA fingerprint probes to avocado (Persea americana Mill.) resulted in identification of various cultivars, characterization of the three avocado races, and a genetic analysis of family structure. Genomic DNA from 14 cultivars was probed with four DNA fingerprint probes. Three of the probes gave well-resolved bands. The individual-specific patterns obtained for each cultivar validate the use of this technique for definitive cultivar characterization, with the probability of obtaining a similar pattern for two different cultivars being 2 × 10-9. DNA mixes representing either Mexican, Guatemalan, or West-Indian avocado races were hybridized with the DNA fingerprint probes, and a band pattern characteristic for each race was obtained. Progeny of a cross between the cultivars Ettinger and Pinkerton were analyzed. Their DNA fingerprints revealed one pair of linked bands and another band allelic to one of them. The application of these observations to identification, evolutionary studies, and breeding is discussed.
R.N. Trigiano and G. Caetano-Anollés
The primary objectives of these laboratory exercises are to familiarize advanced undergraduate and graduate students (and instructors) with the general concepts, techniques, and uses of DNA fingerprinting and to remove some of the perceived mystique underlying molecular genetics. The technique of DNA amplification fingerprinting (DAF) is partitioned into four independent laboratory exercises that include DNA isolation, DNA amplification, gel electrophoresis and silver staining, and data collection and analysis. Although the DNA amplification and gel electrophoresis exercises are emphasized, very detailed and easy-to-follow instructions and protocols are provided for all aspects of the DNA fingerprinting process. These exercises, or similar ones, have been successfully completed on the first attempt by several classes of novice graduate students and other researchers.
S.M. Scheiber, R. Jarret, and C.D. Robacker
Deciduous azaleas have been gaining popularity because of their showy floral displays and adaptability to adverse environmental conditions. However, an absence of distinguishing morphological characteristics, combined with the wide variability present in most species, has created difficulties in efforts to unambiguously identify the different species. Various DNA isolation protocols were tested in order to determine the most effective methods for isolation of DNA from 22 taxa of Rhododendron for subsequent PCR amplification. DNA yields from the various isolation methods varied widely. A minimum of 50 ng/μL of template DNA was necessary for PCR amplification under standard amplification conditions. Results indicated that the effect of tissue age on the efficiency of DNA isolation was taxa-dependent. For most species, extraction of DNA from freshly harvested young leaf tissue resulted in the highest DNA yields. However, DNA yields from R. serrulatum, R. atlanticum, and R. viscosum `Lemon Drop' were highest when mature leaf tissue was used. Primers designed to amplify the internal transcribed spacer (ITS) region of the nuclear ribosomal genes and the psbD, trnK, and 16S chloroplast genes were tested in various PCR reaction mixes in order to optimize reaction conditions for amplification. Primers to both the ITS and the psbD gene resulted in satisfactory amplification in the presence of 1.5 mM MgCl2 and 50 ng template DNA.
A. Adato, D. Sharon, U. Lavi, J. Hillel, and S. Gazit
DNA fingerprint information was used for identification of mango (Mangifera indica L.) cultivars for genetic relatedness analysis of20 mango cultivars and for genetic analysis of a family structure. Genomic DNA was extracted from young leaves, digested with Hind III or Dra I, and hybridized with 10 different DNA probes. Jeffreys' minisatellite probe 33.6 was the most useful, resulting in well-resolved bands representing highly polymorphic loci. Specific patterns were obtained for each cultivar. The probability of obtaining a similar pattern for two different cultivars was 9.4 × 10-6. Based on DNA fingerprint information, genetic distances between 20 mango cultivars were evaluated and an evolutionary tree was established. Analysis of DNA fingerprint band patterns of 12 progeny resulting from a cross between `Tommy Atkins' and `Keitt' mango revealed neither linked nor allelic bands. Application of the reported results for identification, genetic analyses, and mango breeding is discussed.
Deric D. Picton and Harrison G. Hughes
In this study, 11 species, hybrids, and color variants were characterized using randomly amplified polymorphic DNA (RAPD) analysis. Total genomic DNA was extracted using a 2% CTAB extraction buffer using fresh or frozen leaf material. The DNA was amplified using standard RAPD-PCR protocols utilizing 10-mer primers. All primers utilized exhibited a high degree of polymorphism in their banding patterns among the species and hybrids studied. The primers used produced ≈40 reproducible bands. It was possible to identify and uniquely distinguish all species and hybrids investigated using these bands.
Myneni Aruna, Max E. Austin, and Peggy Ozias-Akins
Cultivars of the economically important rabbiteye blueberry (Vaccinium ashei Reade) were differentiated at the DNA level using the technique of randomly amplified polymorphic DNA. Single decanucleotide primers of arbitrary sequence were used to amplify genomic DNA by the polymerase chain reaction. All cultivars tested exhibited a unique set of collective amplified fragments of distinct molecular weight. A blind fingerprinting experiment resulted in identification of unknown samples without ambiguity. We also clarified the genetic identity of two wild selections of rabbiteye blueberry, `Ethel' and `Satilla', which have been maintained as two different selections, hut are considered by some blueberry breeders to be of the same genetic constitution. The technique also verified the probable identity of two cultivars in a commercial blueberry field by comparing their amplified DNA patterns with those of standard cultivars. No variation was observed between the amplification profiles of `Brightwell' and its presumed sport. A cultivar key based on 11 markers amplified by four primers is presented.
Jack Staub, Jeffery Bacher, and Karl Poetter
The influence of tissue age, pathogen infestation, intrapopulation contamination, and polymerase chain reaction (PCR) conditions were assessed as sources of error in random amplified polymorphic DNA (RAPD) analysis. DNA from young, uninfected tissue provided the most consistent results. Plants infected with Sphaerotheca fuliginea Schl. (ex Fr.) Poll. showed variation in RAPD banding patterns compared to those of uninfected plants. Differences in banding patterns were detectable when DNA from two inbred lines were mixed at dilution ratios of ≤20:1 but not ≥50:1. Differing lots of commercially available 10× reaction buffer, MgCl2 stock solutions, and Taq DNA polymerase affected RAPD banding patterns and overall yield. For reproducibility of RAPD assays, it may be necessary to optimize reactions for specific lots of PCR reagents from either commercial or in-house sources.
Rengong Meng and Chad Finn
Nuclear DNA flow cytometry was used to differentiate ploidy level and determine nuclear DNA content in Rubus. Nuclei suspensions were prepared from leaf discs of young leaves following published protocols with modifications. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose published ploidy ranged from diploid to dodecaploid, indicated that fluorescence increased with an increase in chromosome number. Ploidy level accounted for 99% of the variation in fluorescence intensity (r 2 = 0.99) and variation among ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus Cham. and Schldl., a native blackberry species in the Pacific Northwest, which has been reported to have 6x, 8x, 9x, 10x, 11x, and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x, 11x, and 13x forms were found as well. Attempts to confirm the 13x estimates with manual counts were unsuccessful. Ploidy level of 103 genotypes in the USDA-ARS breeding program was determined by flow cytometry. Flow cytometry confirmed that genotypes from crosses among 7x and 4x parents had chromosome numbers that must be the result of nonreduced gametes. This technique was effective in differentiating chromosome numbers differing by 1x, but was not able to differentiate aneuploids. Nuclear DNA contents of 21 diploid Rubus species from five subgenera were determined by flow cytometry. Idaeobatus, Chamaebatus, and Anaplobatus were significantly lower in DNA content than those of Rubus and Cylactis. In the Rubus subgenus, R. hispidus and R. canadensis had the lowest DNA content and R. sanctus had the highest DNA content, 0.59 and 0.75 pg, respectively. Idaeobatus had greater variation in DNA content among diploid species than the Rubus subgenus, with the highest being from R. ellipticus (0.69 pg) and lowest from R. illecebrosus (0.47 pg).