DNA fingerprinting and analysis of population structure in the chestnut blight fungus, Cryphonectria parasitica.

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).

Spatial analysis of nuclear and mitochondrial RFLP genotypes in populations of the chestnut blight fungus, Cryphonectria parasitica.

Spatial structure of both nuclear and mitochondrial RFLPs were studied in several populations of the chestnut blight fungus, Cryphonectria parasitica, using a variety of spatial autocorrelation tests designed to detect nonrandom patterns. Fungal individuals were sampled from cankers on infected chestnut trees, and the location of each tree was mapped. Single-locus nuclear RFLPs, nuclear fingerprints, and mitochondrial DNA haplotypes were determined for each individual. Individuals with the same DNA fingerprint genotypes occurred closer together than would be expected at random in four of the five plots, while mitochondrial DNA haplotypes were aggregated in all five plots. Genetic distances between individuals, expressed as one minus the proportion of shared restriction fragment size classes for fingerprints and mitochondrial haplotypes, were significantly correlated with Euclidean distances between individuals in four of the five populations, but these correlations were very weak (r < 0.18). The same DNA fingerprint and single-copy nuclear RFLP alleles occurred on the same trees or immediately neighbouring trees more often than would be expected at random. Most of the aggregation for all three genetic markers occurred among individuals within the same cluster of chestnut stems or on neighbouring trees. Lack of spatial autocorrelation in one population was probably due to sampling on a larger scale that was too coarse to detect any patterns. Significant aggregation of genotypes in C. parasitica is most likely caused by some degree of restricted dispersal within populations. The implications of restricted dispersal are discussed in relation to the breeding system and isolation by distance in populations of C. parasitica.

Amplified fragment length polymorphism (AFLP) fingerprinting of symbiotic fungi cultured by the fungus-growing ant Cyphomyrmex minutus.

A PCR-based fingerprinting technique based on amplified fragment length polymorphisms (AFLP) is used to screen symbiotic fungi of the fungus-growing ant Cyphomyrmex minutus for genetic differences. AFLP fingerprints reveal several fungal 'types' that (a) represent distinct clones propagated vegetatively by the ant, or (b) correspond to free-living fungi that may be acquired by the ant. Fungal types identified by AFLP fingerprints correspond to vegetative-compatibility groups established previously, suggesting that vegetative compatibility can be used as a crude indicator of genetic differences between fungi of C. minutus.

Molecular cloning, germ-line transformation, and transcriptional analysis of the zeste locus of Drosophila melanogaster.

Approximately 170 kilobase pairs (kb) of contiguous DNA sequences derived from bands 3A3,4 of the Drosophila melanogaster X chromosome have been isolated by molecular cloning. Sequences required for the wild-type expression of the zeste locus are located within a 6-kb fragment of this chromosomal region, as shown by phenotypic rescue of zeste mutants in P element-mediated germ-line transformation. Expression of zeste is correlated with a 2.2-kb poly(A)+ RNA species transcribed at all postzygotic stages of Drosophila development. Many zeste alleles, including several producing neomorphic phenotypes, are not associated with detectable rearrangements of DNA.