Steven H. Rogstad

Saturated NaCl-CTAB solution as a means of field preservation of leaves for DNA analyses

Details of a method for preserving leaves for subsequent DNA analyses using a solution that is saturated (obviating precise field weighing of chemicals) with both NaCl and CTAB are presented. Since the dry ingredients are easy to obtain and transport, the method can be utilized even at very remote sites. Results from fresh versus preserved leaves of Podophyllum, Polyalthia (3 spp.), and Taraxacum demonstrate that DNA suitable for restriction fragment analysis can be obtained from leaves that have been stored in the solution for up to one month at ambient temperatures and then frozen for over one year at -20?C (ultracold storage at -70?C is not necessary). Degradation of DNA with this new method appears to be less for some taxa than with previously proposed DNA extraction methods using dried leaves. Analyses of DNA from leaves collected at remote rain forest sites are demonstrated.

Genetic variation detected by use of the M13 'DNA fingerprint' probe in Malus, Prunus, and Rubus (Rosaceae).

SummaryRecently, “DNA fingerprints” have been reported in a wide array of organisms. We used the M13 repeat probe on several genera and species in the angiosperm family Rosaceae. Four apple cultivars could be differentiated when any one of five restriction enzymes was used to analyze minisatellite DNA. Similarly, four individual trees of Prunus serotina (black cherry) exhibited different “fingerprints” with each of four enyzmes. A total of 14 Rubus (blackberries and raspberries) plants representing four species were investigated with two enzymes. Extensive inter-and intraspecific variation was found. However, some closely growing plants had identical “fingerprints”, probably due to their being derived through vegetative propagation.

The tetrapod "DNA fingerprinting" M13 repeat probe reveals genetic diversity and clonal growth in quaking aspen (Populus tremuloides, Salicaceae)

A recently developed class of DNA endonuclease fragment probes (variously termed “minisatellite,” “DNA fingerprinting,” or “variable number tandem repeat loci” probes) has detected extensive intraspecific genetic variation in tetrapods. Here we use one probe from this class, the M 13 repeat probe (shown previously to yield ”DNA fingerprints” in humans) to examine genetic diversity in the quaking aspen. Comparisons of endonuclease fragment profiles of individuals separated by at least 6km reveals that diversification of alleles in this species has occurred to such an extent that the likelihood of two randomly chosen individuals having indistinguishableHaeIII fragment profiles is c. 3.17 × 10−4. Based on this finding, members of interdigitating clones can be assigned to one or another clone with high statistical confidence. Interdigitating, morphologically cryptic clones were also identifiable. These results demonstrate that some minisatellite probes can be applied to very distant taxa to obtain useful information about genetic variation.

Reassignment of Six Polyalthia Species to the New Genus Maasia (Annonaceae): Molecular and Morphological Congruence

Abstract Recently published molecular phylogenies of the Annonaceae have confirmed the long-held hypothesis that the large paleotropical genus Polyalthia is polyphyletic. Species previously assigned to Polyalthia are now known to belong to up to six distinct, generally well-supported clades. Three members of a group of six species previously referred to as the Polyalthia hypoleuca complex form a monophyletic group (with 99% bootstrap support) that is only distantly related to the other species of Polyalthia sampled. Putative morphological synapomorphies are assessed, and justification provided for validating a new generic name, Maasia . Six species names in the Polyalthia hypoleuca complex are accordingly transferred to Maasia: M. discolor, M. glauca, M. hypoleuca, M. multinervis, M. ovalifolia, and M. sumatrana .

Population and species variation of minisatellite DNA in Plantago

Three Plantago species were surveyed for within- and between-population variation at DNA sequences detected with the M13 minisatellite probe. The levels and patterns of variation detected by this probe correspond to those expected from the mating systems of the species. The highly-selfing species P. major has a relatively low variability of minisatellite sequences within populations and considerable differentiation between populations. The outcrossing species P. lanceolata exhibits higher minisatellite variability within populations and moderate differentiation between populations. P. coronopus, with a mixed mating system, has levels of variation intermediate between P. major and P. lanceolata. The levels of variation within and between populations corresponds, in general, to the levels of allozyme variation determined in an earlier study. Mating system and population structure appear to have a major influence on M13-detected fragment variation.

Surveying plant genomes for variable number of tandem repeat loci.

Publisher Summary Different minisatellite DNA systems are taxonomically widely distributed and variation in such systems, where it exists, can be used to investigate various biological questions. Numerous examples of repetitive sequences revealing intraspecific genetic variation are described in the chapter. Randomly chosen repeats often reveal variation in a variety of organisms and intraspecific genetic variation of repetitive sequences can occur whether the repeat sequences are base pairs (bp) or relatively large sequences. Thus, it seems likely that the variation of repetitive sequences is a general characteristic of such sequences regardless of size or composition, rather than a special characteristic of particular repetitive. For any particular organism, it is necessary to survey a variety of minisatellite probes to select those most appropriate for the specific biological issues to be investigated.

Genetic variation across VNTR loci in central North American Taraxacum surveyed at different spatial scales

Dandelions (Taraxacum officinale Weber(sensu lato); Asteraceae) have been introduced to NorthandSouth America with human migration from Europe. While potential sourcepopulations have both sexually and obligate asexually (agamospermous)reproducing lineages, apparently only the latter have successfully colonizedtheAmericas. The consequences of obligate agamospermy on dandelion populationgenetic diversity in North America remain little explored. Here we use fourdifferent synthetic DNA probes that reveal genetic markers at multiplevariable-number-tandem-repeat (VNTR) loci to examine patterns of geneticvariation among plants collected along three different central North Americantransects with plants (21 to 22 individuals per transect) separated by: 1) >2 m and < 60 m (short transect); 2) > 5km and < 30 km (medium transect); and 3) > 30km and < 340 km (long transect). The mean numberofVNTR markers revealed per plant was 59.3. Co-clonal individuals (proportion ofbands shared exceeding 90%) were found in each transect, with the indexof clonality (the percent of co-clonal individuals detected in a transect)ranging from 34.12% for the short transect to 18.65% for the longtransect. Co-clonal individuals were separated by up to 200 km.With redundant examples of co-clonal individuals removed, mean similarity(proportion of band sharing) of distinct genotypes within transects was 0.426,and no statistical differences in level of similarity between transects, norindication of genetic differentiation between transects, was detected (meanFst between transect levels with all individuals included =0.05). These results indicate: 1) that dandelion genetic diversity ofcolonizinglineages in central North America is moderately high and does not reflectextreme bottleneck effects shown by some colonizing species; and 2) thatdandelion seed dispersal can be very effective in maintaining similar levels ofgenetic diversity at the different scales of sampling in this study, withcertain clones maintaining numerous, widespread individuals. Evidence that VNTRmutation is detectable within dandelion clonal lineages is presented,demonstrating that “clonal families” with lines increasinglydifferentiated from one another will continually evolve, and that Mullersrachet is, in all likelihood, turning for asexual lines.

DNA "fingerprints" detect genetic variation in Acer negundo (Aceraceae).

Genomic DNA samples from 21 box elder plants collected in Missouri (U.S.A.) were digested with restriction enzyme and southern blot hybridized with the M13 minisatellite probe. Each plant was found to have a unique DNA fragment pattern. Moreover, levels of genetic variation estimated from a similarity index appear to be related to sampling distances. However, size of the fragments utilized in the analysis affects the estimates of genetic variation to a considerable degree.

Plant DNA extraction using silica

Described here is a method that uses silicon dioxide (silica) to extract whole genomic plant DNA of high molecular weight. The protocol is presented in a microcentrifuge format, and yields were approximately 2–4 μg per 200 mg of plant leaf tissue. The method involves fewer steps than many previous extraction protocols and, as shown here for 4 taxonomically distant angiosperms, produces DNA suitable for digestion with restriction endonucleases. The use of commercial kits is not required; the silica costs are comparatively inexpensive (<

DNA extraction from plants: The use of pectinase

0.03 per tube); and CTAB, rather than the more expensive guanidine thiocyanate salt, is used.