Phantom hitch-hikers mislead estimates of genetic variation in Antarctic mosses

Previous studies of Antarctic mosses employing RAPDs have reported extraordinarily high levels of genetic variation, even within a single clump of moss. This is unexpected given their extreme isolation in Antarctica and lack of sexual reproduction. We offer an alternative explanation: that unusually elevated levels of genetic variability are artefacts from contamination of a number of biota known to be naturally associated with Antarctic mosses. We utilized sequence variation of nrITS and RAPDs to further investigate the effect of naturally occurring contaminants on estimates of genetic variation of mosses. Our results indicate that these ``phantom hitch-hiker' contaminants hinder attempts to accurately and reliably estimate levels of genetic variation by non-specific PCR-based approaches. Furthermore, screening samples via amplification of nrITS failed to identify all contaminated samples, hence we caution against relying solely on ``quick' screening methods and suggest that suspect samples be carefully examined for contamination.     

Fingerprinting cyanobionts and hosts of theAzolla symbiosis by DNA amplification

Cyanobionts of six species of the aquatic fernAzolla were evaluated by specific and random DNA profiles amplified by the DNA polymerase chain reaction. Simultaneous examination of the prokaryoticAnabaena azollae and the host was achieved using primers for the chloroplast-encoded intron of the tRNA-Leucine (UAA) gene. These amplifiedtrnL intron sizes, restriction fragment length polymorphisms of the amplified 16s rRNA gene, and random amplified polymorphic DNAs demonstrated the capacity of this method for the rapid assessment of similarities amongAnabaena azollae and minorAnabaena isolates fromAzolla.     

Population structure of red-cockaded woodpeckers in south Florida: RAPDs revisited

Six south Florida populations of the endangered red-cockaded woodpecker (Picoides borealis) were sampled to examine genetic diversity and population structure in the southernmost portion of the species' range relative to 14 previously sampled populations from throughout the species range. Random amplified polymorphic DNA (RAPD) analyses were used to evaluate the populations (n= 161 individuals, 13 primers, one band/primer). Results suggested that south Florida populations have significant among-population genetic differentiation (F_ST= 0.17, P < 0.000), although gene flow may be adequate to offset drift (N_m= 1.26). Comparison of Florida populations with others sampled indicated differentiation was less in Florida (F_ST for all populations = 0.21). Cluster analyses of all 20 populations did not reflect complete geographical predictions, although clustering of distant populations resulted in a significant correlation between genetic distance and geographical distance. Overall, results suggest populations in south Florida, similar to the remainder of the species, have low genetic diversity and high population fragmentation. Exact clustering of distant populations supports the ability of RAPDs to differentiate populations accurately. Our results further support past management recommendations that translocations of birds among geographically proximate populations is preferable to movement of birds between distant populations.     

Inhibition of random amplified polymorphic DNAs (RAPDs) by plant polysaccharides

A survey of the inhibition of the amplification of spinach DNA by various plant polysaccharides revealed that neutral polysaccharides (arabinogalactan, dextran, gum guar, gum locust bean, inulin, mannan, and starch) were not inhibitory. In contrast, the acidic polysaccharides (carrageenan, dextran sulfate, gum ghatti, gum karaya, pectin, and xylan)were inhibitory. In the process of preparing random amplified polymorphic DNAs (RAPDs), the loss of large DNA bands appears to be an indicator that the fingerprint pattern has been affected by polysaccharides. The addition of various concentrations of Tween 20, DMSO, or PEG 400 to the PCR reaction mixture resulted in partial restoration of amplification of RAPDs for the acidic polysaccharides. The most effective way to eliminate the effects of polysaccharide inhibition was by diluting the DNA extracts, and thereby diluting the polysaccharide inhibitors.     

Hybridization between introduced smooth cordgrass (Spartina alterniflora; Poaceae) and native California cordgrass (S. foliosa) in San Francisco Bay, California, USA

Introduced Spartina alterniflora (smooth cordgrass) is rapidly invading intertidal mudflats in San Francisco Bay, California. At several sites, S. alterniflora co-occurs with native S. foliosa (California cordgrass), a species endemic to California salt marshes. In this study, random amplified polymorphic DNA markers (RAPDs) specific to each Spartina species were identified and used to test for hybridization between the native and introduced Spartina species in the greenhouse and in the field. Greenhouse crosses were made using S. alterniflora as the pollen donor and S. foliosa as the maternal plant, and these crosses produced viable seeds. The hybrid status of the crossed offspring was confirmed with the RAPD markers. Hybrids had low self-fertility but high fertility when back-crossed with S. foliosa pollen. Hybrids were also found established at two field sites in San Francisco Bay; these hybrids appeared vigorous and morphologically intermediate between the parental species. Field observations suggested that hybrids were recruiting more rapidly than the native S. foliosa. Previous work identified competition from introduced S. alterniflora as a threat to native S. foliosa. In this study, we identify introgression and the spread of hybrids as an additional, perhaps even more serious threat to conservation of S. foliosa in San Francisco Bay.     

Phylogenetic study of bisexual Artemia using random amplified polymorphic DNA

Study of polymorphisms in the eukaryotic genome is an important way to discover the evolutionary relationships between species. Artemia (Crustacea, Anostraca) offers a very interesting model for evolutionary studies. In fact the genus, distributed all over the world in hundreds of known biotopes, comprises both bisexual sibling species and parthenogenetic populations easily available from the Artemia Reference Center of Ghent. In spite of great interest in it and its extensive use in aquaculture, little is known about relationships between the different species and intraspecific populations. Recently it has been demonstrated that polymorphisms in genomic fingerprints generated by arbitrarily primed polymerase chain reaction (PCR) can distinguish between strains in many organisms. We have used this technique to estimate the phylogenetic relationships existing between 14 populations living in the American continent, in the Mediterranean area, and in China. The principal coordinate analysis (PCO) obtained from 86 random amplified polymorphic DNA (RAPD) markers indicates that the populations analyzed can be divided into homogeneous clusters representing the four known bisexual species—the American A. franciscana and A. persimilis, the Mediterranean A. salina, and the A. species from China.     

RAPD characterisation of two neotropical hybrid legumes

An investigation of randomly amplified polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker distribution was made for two well-characterised hybrids and their parents,Leucaena leucocephala andL. esculenta andParkinsonia aculeata andCercidium praecox. Three chloroplast DNA (cpDNA) markers identified the maternal parent of eachL. leucocephala ×L. esculenta hybrid. Fifteen species-diagnostic RAPD markers (invariant in one taxon and absent from the other) were always present in theLeucaena hybrid and assumed to be of nuclear origin, whilst three RAPD markers showed expression patterns identical to the cpDNA markers and were assumed to be of organellar origin. No RAPD or PCR-RFLP taxon-diagnostic markers were discovered for eitherP. aculeata orC. praecox. However, 21 RAPD markers were species-specific (polymorphic within one taxon but absent from the other) and Southern analysis indicated that none of the markers were of organellar origin. Only 67% additivity of markers specific toP. aculeata andC. praecox was demonstrated in the hybrids between these two species, whilst inLeucaena 97% additivity was demonstrated. Differences between the two hybridising situations were related to the behaviour of the molecular markers and the biology of the species.     

Isozyme studies in the genusCheirolophus (Asteraceae:Cardueae-Centaureinae) in the Iberian Peninsula, North Africa and the Canary Islands

The phylogenetic and phenetic analysis of 109 RAPD polymorphisms inS. nebrodensis, a perennial and self-incompatible endemic of four mountain ranges in Spain, andS. viscosus, a self-compatible annual widespread in Europe, as well asS. lividus, S. sylvaticus andS. vulgaris revealed a sister group relationship between the first two species. This result contrasts sharply with the earlier hypothesis based on isozyme variation thatS. viscosus originated from within a paraphyleticS. nebrodensis and that the two species represent a progenitor-derivative pair. After considering possible reasons for the sister group relationship found, including the possibility of rooting artefacts, it is concluded that neither the RAPD data nor the isozyme data allow to draw safe conclusions about the mode of speciation and therefore the relative age of the two species. As a consequence, the limited genetic variation ofS. viscosus in comparison toS. nebrodensis as revealed by both the RAPD and the isozyme data may reflect its population history, geographical distribution, reproductive ecology, or mode of dispersal just as well as its recent origin from a paraphyleticS. nebrodensis. The result of this study calls for a critical reexamination of other taxon pairs postulated to have a progenitor-derivative relationship on the basis of isozyme evidence.     

In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plants

A stable salt-tolerant potato cell line, able to grow on media containing 60–450 mM NaCl (i.e. low to high salinity) was selected. Callus grown on 120 or 150 mM NaCl showed higher fresh weights than the rest of the treatments. Replacing NaCl by KCl or Na2SO4 showed that reductions in fresh weight were mainly due to the presence of Na+ ions. When PEG 6000 was added to the medium instead of salt, the salt tolerant cell lines were unable to overcome the PEG-induced water stress. Whole plants, regenerated from salt tolerant callus, exhibited salt stress tolerance as evidenced by their higher fresh and dry weights when watered with 90 mM NaCl, and they also produced more tubers per plant under salt stress. Salt-tolerant plants differed phenotypically from control plants both in terms of leaf shape, tuber flesh and skin colour, which was reddish. In addition, DNA fingerprinting by RAPDs, with 70 different primers, confirmed that the salt tolerant regenerants also differed genotypically from the control, salt sensitive Kennebec potato plants from which they had been selected. This revised version was published online in June 2006 with corrections to the Cover Date.