Regulation of cabbage (Brassica oleracea capitata) hypocotyl elongation, and infection by Peronospora parasitica, with B-indolylacetonitrile and chloramphenicol

Untreated excised segments of the hypocotyls of dark grown cabbage seedlings are always systemically infected when inoculated with the conidia of the obligate parasite Peronospora parasitica (Downy mildew). Cabbage hypocotyl elongation is promoted by 10^–4 M indolylacetonitrile (IAN) and this elongation is inhibited by 100 g mL^–1 chloramphenicol (CAM). The fungus remains localized in 5–8 day old hypocotyl segments exposed to CAM, but this inhibition is reversed by IAN. Indol-3-acetic acid (IAA) has the same effect as IAN. Both gibberellic acid and kinetin inhibit systemic infection. Conidial spore germination is not reduced by the CAM concentration used in these experiments. The success of the pathogen in the host is not correlated with host elongation, but is probably related to a common metabolic site in either host or pathogen affected by both CAM and IAN.Abbreviations IAN indolylacetonitrile - CAM chloramphenicol - IAA indol-3-acetic acid - G gibberellic acid - K kinetin     

Individualization and estimation of relatedness in crocodilians by DNA fingerprinting with a Bkm-derived probe

Individual-specific DNA fingerprints of crocodilians were obtained by the use of Bkm-2(8) probe. Pedigree analyses of Crocodylus palustris, C. porosus and Caiman crocodilus revealed that the multiple bands (22–23 bands with Aludigest) thus obtained were inherited stably in a Mendelian fashion. Unique fingerprints permitted us to identify individuals, assign parentage, and reconstruct the DNA profile of a missing parent. Average band sharing between unrelated crocodiles was found to be 0.37. Band sharing between animals of known pedigrees increased predictably with relatedness and provided a basis for distinguishing relatives from non-relatives. Similar results obtained in other species/genera, using the same probe, suggest that this approach may be applicable to all species of crocodilians, and could facilitate genetic studies of wild and captive populations.     

DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.     

Individual identification of non-human primates using DNA fingerprinting

DNA fingerprints were studied in non-human primates including three species of Old World monkeys and one species of hominoid, using tandem repeats of a 28-base-pair sequence downstream of the human c-Ha-ras-1 oncogene as a probe. We observed Southern hybridization patterns consisting of multiple hypervariable DNA fragments, which were specific to each of the individuals examined. These results indicate that DNA fingerprinting is a powerful tool for identification of individuals among non-human primates, as is the case in man. On leave from the Department of Legal Medicine, Institute of Community Medicine, University of Tsukuba.     

Genetic variation detected by DNA fingerprinting with a rice minisatellite probe in Oryza sativa L.

A rice minisatellite probe detecting DNA fingerprints was used to assess genetic variation in cultivated rice (Oryza sativa L.). Fifty-seven cultivars of rice, including 40 closely related cultivars released in the US, were studied. Rice DNA fingerprinting revealed high levels of polymorphism among distantly related cultivars. The variability of fingerprinting pattern was reduced in the closely related cultivars. A genetic similarity index (S) was computed based on shared fragments between each pair of cultivars, and genetic distance (D) was used to construct the dendrograms depicting genetic relationships among rice cultivars. Cluster analysis of genetic distance tended to group rice cultivars into different units corresponding with their varietal types and breeding pedigrees. However, by comparison with the coefficients of parentage, the criterion of relatedness based on DNA fingerprints appeared to overestimate the genetic relationships between some of the closely related US cultivars. Although this may reduce the power of fingerprints for genetic analysis, we were able to demonstrate that DNA fingerprinting with minisatellite sequences is simpler and more sensitive than most other types of marker systems in detecting genetic variation in rice.This paper reports the results of research only. Mention of a proprietary product does not consititute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the US Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, and the University of Missouri Agricultural Experiment Station Journal Series No. 12178.     

Molecular phylogeny and DNA amplification fingerprinting of Petunia taxa

The relationship of five species of Petunia and ten cultivars of the cultivated petunia, Petunia x hybrida, were investigated using DNA-amplification fingerprinting (DAF). Reproducible banding profiles were obtained from P. parodii and P. axillaris DNA from different seed sources. In contrast, other petunias such as P. inflata, P. violacea and P. integrifolia produced variable fingerprints when different plants were examined. However, representative profiles of the variable Petunia taxa were obtained by bulking the leaf tissue from ten different individual plants. Each of ten octamer primers revealed polymorphic loci between taxa. Among a total of 201 bands produced, 146 (73%) loci were polymorphic and distinguished all species and cultivars. Phenetic and cluster analysis using DAF markers separated P. axillaris from P. parodii and distinguished between the violet-flowered species, P. inflata, P. violacea, and P. integrifolia. P. parodii grouped together with the monophyletic set of the ten cultivars of P. x hybrida examined, indicating that it had made a major contribution to the development of these cultivars. Cultivars were distributed within the dendograms by flower color. The results demonstrated the utility of DAF in establishing relationships among closely related species and cultivars of Petunia.     

Sources of resistance to downy mildew (<Emphasis Type="Italic">Peronospora parasitica</Emphasis> (Pers. (ex Fr.) Fr.) in Sicilian germplasm of cauliflower and broccoli

Downy mildew (Peronospora parasitica (Pers. ex Fr.) Fr.) is a serious disease of brassicas in several countries. Seedlings are very susceptible to this pathogen and crops require frequent fungicide treatments to reach a good marketable yield. The use of resistant cultivars can be the most economical, reliable and environmental friendly method for managing this disease. In this work 32 Sicilian landraces and 16 commercial cultivars of cauliflower and broccoli (B. oleracea) were screened for downy mildew resistance at the cotyledon stage using one P. parasitica strain from Portugal and one from Sicily (Italy). Seven-day old seedlings were inoculated by deposing a droplet of a spore suspension on the cotyledons, incubated under controlled environment and scored 7 days later using a seven-class scale of interaction phenotype (IP), which took into consideration host response and pathogen sporulation. There were no differences in virulence between the two P. parasitica isolates. Accessions ranged from very susceptible to highly resistant to downy mildew showing a variable number of resistant individuals per accession. Forty accessions were very susceptible to downy mildew and are of no interest as sources of resistance, since most of the seedlings were scored in the most susceptible IP classes. Seven accessions had intermediate resistance and included individuals that expressed some degree of resistance. Accession Cv 90 (Cavolfiore Torino) and Br 63 (Sparaceddu) showed the majority of seedlings in the resistant IP classes and may constitute valuable sources of resistance to downy mildew to be used in breeding programs.     

DNA fingerprinting: a tool for determining genetic distances between strains of poultry

Summary DNA fingerprinting, a technique based on the detection of hypervariable minisatellite regions in DNA restriction fragments, was tested for its applicability to conduct population genetics in poultry. Using MspI digestion and phage M13 DNA as a probe, between 25 and 35 minisatellite-containing DNA fragments were observed per bird. Comparison of the banding pattern of offspring with their parents revealed that the bands were inherited as stable genetic traits. The variability of the DNA fingerprinting pattern was reduced in inbred strains. DNA fingerprints of chickens from five well-defined populations of known genetic relationships were analyzed and indices of genetic distances were computed. They correctly reflected the history of these strains, indicating that DNA fingerprinting may be a powerful tool to characterize genetic relationships between different breeding populations of the same species.     

Genetic diversity and population differentiation of chestnut blight fungus, Cryphonectria parasitica, in China as revealed by RAPD

Seventeen Cryphonectria parasitica populations sampled from six regions in China were investigated using RAPD. Across all 169 isolates from the 17 populations evaluated, 52 of the 71 markers (73%) were polymorphic, total genetic diversity (h) was 0.1463, and Shannon’s index was 0.2312. Diversity within populations accounted for 74% of total genetic diversity, and genetic differentiation among populations was 0.26 (G _ST = 0.26). Gene flow was 1.4 among the populations; higher gene flow was found among populations within regions and among regions [N _m (G _SR) = 2.8 and N _m (G _RT) = 3.5]. The unweighted pair group mean analysis (UPGMA) dendrogram revealed two distinct clusters: the northern China group and the southern China group. The spatial autocorrelation analysis revealed that the variation at most loci was randomly distributed and lacked spatial structure, but several loci and closer distances were spatially structured. Human activity and habitat could also be important factors affecting genetic structure among C. parasitica populations in China. Genetic diversity was highest in Southwest China, descending in an orderly fashion to Northeast China. This pattern indicated that Southwest China might be the center of origin of C. parasitica in China. The present study provides useful information for understanding the origin and spread of chestnut blight fungus in China and valuable data for formulating relevant strategies for controlling the disease in China.     

Interpretation of randomly amplified polymorphic DNA marker data for fingerprinting sweet potato (Ipomoea batatas L.) genotypes

In this paper we present a method for the generation of randomly amplified polymorphic DNA (RAPD) markers for sweet potato. These were applied to produce genetic fingerprints of six clonal cultivars and to estimate genetic distances between these cultivars. The level of polymorphism within the species was extremely high. From the 36-decamer random primers used, 170 fragments were amplified, of which 132 (77.6%) were polymorphic. Ten primers resulted in no detected amplification. Of the remaining 26 primers for which amplification was achieved, only one did not reveal polymorphism. Six primers used alone enabled the discrimination of all six genotypes. Pattern analysis, which employed both a classification and ordination method, enabled the grouping of cultivars and the identification of primers which gave greatest discrimination among the cultivars.     

Genetic variation of the St. Lawrence beluga whale population assessed by DNA fingerprinting

Recent surveys suggest that the endangered St. Lawrence beluga ( Delphinapterus leucas ) population is not recovering significantly despite 20 years of protection. Dead individuals that have been autopsied show high levels of tumours and infections. This situation could be a result of pollution, loss of genetic variation, inbreeding depression or a combination of these factors. Analyses of DNA fingerprints from St. Lawrence belugas with three minisatellite probes (Jeffreys 33.6, 33.15 and M13) indicate a reduced level of genetic variation compared to Beaufort Sea animals. The average band-sharing between individuals of the St. Lawrence beluga population for the three probes (0.534, 0.573 and 0.478, respectively) was significantly higher than that of the Beaufort Sea beluga population (0.343, 0.424, 0.314, respectively). Higher levels of mean allele frequency in the St. Lawrence belugas (0.33 vs. 0.21) suggest that this population is composed of individuals which are related. Inbreeding depression could therefore be a factor in the lack of recovery of the St. Lawrence beluga population.     

Atkinsiella parasitica sp. nov. isolated from a rotifer,Brachionus plicatilis

A new species ofAtkinsiella (Lagenidiales, Oomycetes),Atkinsiella parasitica is described and illustrated. It was isolated from the eggs and bodies of a rotifer,Brachionus plicatilis, with fungal infection in 1992. The rotifer was reared in a hatchery as the first food supply for seed production of crustaceans and fishes. The fungus is characterized by producing monoplanetic, lateral biflagellate zoospores, and infrequently branched discharge tubes. Optimum temperature for the fungus was 25°C. The fungus was considered an obligate marine fungus, because its growth was observed only on PYGS medium including seawater.     

DNA fingerprinting of captive breeding pairs of lesser white-fronted geese (Anser erythropus) with unknown pedigrees

For a number of decades, the lesser white-fronted goose (Anser erythropus) has been almost-absent from the Fennoscandian fauna and has a current population size of only about 60 breeding pairs, with fewer than 10 pairs in Sweden. During the period 1981–1991 more than 200 young have been reintroduced in northern Sweden. However, the origin and possible relatedness of lesser white-fronted individuals were unknown when the breeding program started. We have used DNA fingerprinting to assess the similarity of 18 individuals, i.e., the entire captive population used for breeding in 1991 and about 60% of the captive population used in 1981–1991. Minisatellite probe 33.15 provided an index for an average similarity of 0.39 between the mates of the 12 breeding pairs used for producing offspring for reintroduction. This is a higher similarity than in natural populations of birds in general but lower than in populations that have passed through serious population bottlenecks. Individuals originating from different breeders are more dissimilar than those from the same breeder. However, the close relationships (similarity, 0.5–0.6) found in a group of five individuals from different breeders show that selecting individuals from different breeding groups is not sufficient to prevent mating between closely related individuals.     

Estimation of intracolonial worker relationship in a honey bee colony (Apis mellifera L.) using DNA fingerprinting

Summary The number and frequencies of subfamilies in a honey bee colony were determined by DNA fingerprinting. Queen and brood samples were taken from three colonies with artificially inseminated queens and from one colony with a naturally mated queen. UsingHae III restriction enzyme and (GATA)₄ oligonucleotide, the number of subfamilies in the colonies with artificially inseminated queens corresponded with the number of drones used for insemination. In the colony with the naturally mated queen, 12 subfamilies were found in a random sample of 104 workers. Considering that subfamily frequencies range from 1 to 26%, introcolonial worker relationship was estimated to be 0.328, corresponding to a genetical effective number of 6.4 matings.     

Siberian wild rye (Elymus sibiricus L.): Genetic diversity of germplasm determined using DNA fingerprinting and SCoT markers

Elymus sibiricus is a perennial, self-pollinating, allotetraploid grass native to northern Asia. It is widely used in cultivated pastures and natural grassland due to excellent cold and drought tolerance, good forage quality, and adaptability to a variety of habitats. Information on the genetic diversity and variation among worldwide E. sibiricus germplasm is limited but necessary for germplasm collection, conservation and effective commercial use. In this study we ana lyzed genetic diversity and variation of 69 E. sibiricus accessions from the species range and constructed DNA fingerprinting profiles of 24 accessions using SCoT markers. A total of 173 bands were generated from 16 SCoT primers, 154 of which were polymorphic with 89.0% of polymorphic bands (PPB) occurring at the species level. The PPB within 8 geographical regions ranged from 2.3 to 54.3 %. Genetic variation was greater within geographical regions (57.9%) than between regions (42.1%). The 24 accessions from Qinghai-Tibet Plateau, Mongolia Plateau, Kazakhstan, and Russia were distinguished by their unique fingerprinting. This is the first report using SCoT markers for identifying cultivars and accessions of E. sibiricus. The DNA fingerprinting profiles of E. sibiricus were useful in germplasm collection and identification. The genetic diversity of worldwide E. sibiricus germplasm has been substantially affected by ecogeographical factors. Our results suggest that collecting and evaluating E. sibiricus germplasm from major geographic regions and unique environments broadens the available genetic base and illustrates the range of variation.     

Inheritance of polymorphic markers generated by DNA amplification fingerprinting and their use as genetic markers in soybean

DNA amplification fingerprinting (DAF) using a high primer-to-template ratio and single, very short arbitrary primers, was used to generate amplified fragment length polymorphic markers (AFLP) in soybean (Glycine max (L.) Merr.). The inheritance of AFLPs was studied using a cross between the ancestral Glycine soja PI468.397 and Glycine max (L.) Merr. line nts382, F1 and F2 progeny. The amplification reaction was carried out with soybean genomic DNA and 8 base long oligounucleotide primers. Silver-stained 5% polyacrylamide gels containing 7 M urea detected from 11 to 28 DAF products with primers of varying GC content (ranging from 50 to 100% GC). Depending on their intensity, AFLPs were classified into three classes. DAF profiles were reproducible for different DNA extractions and gels. Forty AFLPs were detected by 26 primers when comparing G. soja and G. max. Most AFLPs were inherited as dominant Mendelian markers in F1 and F2 populations. However, abnormal inheritance occured with about 25% of polymorphisms. One marker was inherited as a maternal marker, presumably originating from organelle DNA while another showed apparent paternal inheritance. To confirm the nuclear origin and utility of dominant Mendelian markers, three DAF polymorphisms were mapped using a F11 mapping population of recombinant inbred lines from soybean cultivars Minsoy × Noir 1. The study showed that DAF-generated polymorphic markers occur frequently and reliably, that they are inherited as Mendelian dominant loci and that they can be used in genome mapping.     

Genotype relationships inMicroseris elegans (Asteraceae,Lactuceae) revealed by DNA amplification from arbitrary primers (RAPDs)

The genetic relationships among 10 inbred lines representing 10 populations of the autogamous annualMicroseris elegans from throughout California has been determined using random amplified polymorphic DNAs (RAPDs). Seventeen arbitrary 10 base pair primers produced 134 amplification products; 81 of these were shared by two or more strains. The 3 genotypes from Northern California are closely related as are 3 genotypes from Middle Californian populations which are not nearest neighbors. DNA fingerprinting with the oligonucleotide (GATA)₄ gave compatible results, but the comparison was limited to samples run on one gel. Isoenzyme patterns are compatible with the DNA results, but limited by the very low number of informative polymorphisms. The clustered relationship among genotypes within a species and their geographic distribution suggests very restricted genetic recombination and an origin of new populations from randomly dispersed achenes within the range of the species.     

RAPD fingerprinting of blackcurrant (Ribes nigrum L.) cultivars

Ribes nigrum germplasm was screened for random amplified polymorphic DNA (RAPD) markers. Fiftyfour markers were identified which generated individual fingerprints for each of 21 cultivars. Genetic variation within R. nigrum germplasm, as detected by RAPDs, demonstrated that the genetic basis for improvement of blackcurrant is narrower than would be expected by the analysis of parentage.     

AFLP fingerprinting and genotypic characterization of some serovars of Bacillus thuringiensis

Amplified fragment length polymorphism (AFLP)-based fingerprinting of 24 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 13 EcoR1 (+2) and Mse1 (+3) primer combinations for genotypic characterization. A high degree of polymorphism was established among the Bt serovars. A total of 1107 fragments ranging from 30–850 bp were generated out of which 1106 were polymorphic. Discrimination rates of different primer combinations at various band levels (1–5) among different Bt serovars were more than 90%. Cluster analysis revealed very low similarity values, ranging from 7–50%, among the Bt serovars indicating their remarkable genetic diversity. AFLP analysis establishes the molecular relatedness between the serovars and serotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modelling fungal sink competitiveness with grains for assimilates in wheat infected by a biotrophic pathogen

Background and Aims

Experiments have shown that biotrophic fungi divert assimilates for their growth. However, no attempt has been made either to account for this additional sink or to predict to what extent it competes with both grain filling and plant reserve metabolism for carbon. Fungal sink competitiveness with grains was quantified by a mixed experimental–modelling approach based on winter wheat infected by Puccinia triticina.

Methods

One week after anthesis, plants grown under controlled conditions were inoculated with varying loads. Sporulation was recorded while plants underwent varying degrees of shading, ensuring a range of both fungal sink and host source levels. Inoculation load significantly increased both sporulating area and rate. Shading significantly affected net assimilation, reserve mobilization and sporulating area, but not grain filling or sporulation rates. An existing carbon partitioning (source–sink) model for wheat during the grain filling period was then enhanced, in which two parameters characterize every sink: carriage capacity and substrate affinity. Fungal sink competitiveness with host sources and sinks was modelled by representing spore production as another sink in diseased wheat during grain filling.

Key Results

Data from the experiment were fitted to the model to provide the fungal sink parameters. Fungal carriage capacity was 0·56 ± 0·01 µg dry matter °Cd⁻¹ per lesion, much less than grain filling capacity, even in highly infected plants; however, fungal sporulation had a competitive priority for assimilates over grain filling. Simulation with virtual crops accounted for the importance of the relative contribution of photosynthesis loss, anticipated reserve depletion and spore production when light level and disease severity vary. The grain filling rate was less reduced than photosynthesis; however, over the long term, yield loss could double because the earlier reserve depletion observed here would shorten the duration of grain filling.

Conclusions

Source–sink modelling holds the promise of accounting for plant–pathogen interactions over time under fluctuating climatic/lighting conditions in a robust way.