Use of inter-simple sequence repeats and amplified fragment length polymorphisms to analyze genetic relationships among small grain-infecting species of ustilago.

ABSTRACT In the smut fungi, few features are available for use as taxonomic criteria (spore size, shape, morphology, germination type, and host range). DNA-based molecular techniques are useful in expanding the traits considered in determining relationships among these fungi. We examined the phylogenetic relationships among seven species of Ustilago (U. avenae, U. bullata, U. hordei, U. kolleri, U. nigra, U. nuda, and U. tritici) using inter-simple sequence repeats (ISSRs) and amplified fragment length polymorphisms (AFLPs) to compare their DNA profiles. Fifty-four isolates of different Ustilago spp. were analyzed using ISSR primers, and 16 isolates of Ustilago were studied using AFLP primers. The variability among isolates within species was low for all species except U. bullata. The isolates of U. bullata, U. nuda, and U. tritici were well separated and our data supports their speciation. U. avenae and U. kolleri isolates did not separate from each other and there was little variability between these species. U. hordei and U. nigra isolates also showed little variability between species, but the isolates from each species grouped together. Our data suggest that U. avenae and U. kolleri are monophyletic and should be considered one species, as should U. hordei and U. nigra.

DNA markers linked to a T10 loose smut resistance gene in wheat (Triticum aestivum L.).

Screening for loose smut resistance in wheat is difficult. Selecting lines with DNA markers linked to loose smut resistance would be more reliable and less costly. Molecular markers linked to a race T10 loose smut resistance gene were identified using a F6 single seed descent segregating population. A RAPD marker and a RFLP marker were located on opposite flanks of the resistance gene and were shown to be loosely linked. The RAPD marker was converted to a user friendly polymorphic SCAR marker that represented a single genetically defined locus in hexaploid wheat. Using these two bracketing markers simultaneously, the error rate for T10 resistance selection due to crossing-over was reduced to 4%. These markers can be used for a faster and more reliable selection of T10 resistant plants than previous conventional loose smut ratings.

Genomic fingerprinting of "Haemophilus somnus" isolates by using a random-amplified polymorphic DNA assay.

The random-amplified polymorphic DNA (RAPD) assay was used to generate DNA fingerprints for 16 isolates of "Haemophilus somnus," and one isolate each of "Haemophilus agni," "Histophilus ovis," "Actinobacillus seminis," Pasteurella haemolytica, and Escherichia coli. The RAPD assay differentiated among "H. somnus" isolates, which shared similarity coefficients of 0.46 to 1.00 on the basis of pairwise comparisons of RAPD markers produced with nine random decamer primers. Three virulent encephalitic "H. somnus" isolates exhibited identical banding patterns, suggesting a common clonal ancestry. The RAPD assay clearly distinguished between the "H. somnus"-"H. agni"-"H. ovis" group and the other bacterial species tested. The results of the present study suggest that DNA fingerprinting of "H. somnus" isolates by the RAPD assay could be valuable in revealing subspecific divisions within this largely unexplored species.

Species and strain differentiation of Eimeria spp. of the domestic fowl using DNA polymorphisms amplified by arbitrary primers.

Eimeria spp. from the domestic fowl were examined for genetic relatedness by the random amplified polymorphic DNA (RAPD) assay. Nine different oligonucleotide decamers with arbitrary DNA sequences were tested as primers to amplify DNA from six Eimeria species infecting chickens. Two strains each of E. acervulina and E. tenella were used. Depending on the species/strain-primer combination, between 1 and 12 DNA segments ranging in size from 0.16 to 4.95 kb were amplified. The two strains of E. acervulina showed minor and major differences in their amplified DNA patterns, giving a similarity coefficient of 61%. The two strains of E. tenella seemed to be more closely related, yielding a similarity coefficient of 98%. The differences observed between species were greater than those found between strains with every primer used, indicating that the RADP assay could be a useful tool for the study of relationships among these coccidia. The results obtained in this study also indicate the presence of unique, species-specific, amplified DNA segments that could be exploited to identify Eimeria species of the chicken.

Relative abundance of various rDNA repeat types in polytene nuclei of Drosophila melanogaster.

The relative abundance of the five predominant X chromosome nucleolus organizer repeat types was determined in polytene nuclei. Previous studies showed that the major type I insert containing class of repeats was disproportionately underreplicated when compared with the insert-free class of repeats in these nuclei. Our results demonstrate that this disproportionate underreplication also occurs for both the minor type I and type II insert containing repeats. There was not an exclusive polytenization of the insert-free repeats for this wild-type X nucleolus organizer region. A repeat type lacking either insert type and having an unusually long nonstrancribed spacer was identified and found to be preferentially replicated in polytene nuclei. Preliminary mapping studies indicate that this class of repeats has a unique location, in the distal portion of either the X nucleolus organizer region or the centric heterochromatin. Their similarity to the common insert-free repeats includes preferential polytenization and generalized DNase I sensitivity, which suggests that they are transcriptionally active.

Preferential DNase I sensitivity of insert-free ribosomal RNA repeats of Drosophila melanogaster.

The five predominant types of rDNA repeats in D. melanogaster were analyzed with respect to their DNase I sensitivity. Only the insert-free repeats showed a generalized DNase I sensitivity pattern whereas the major type I, both minor type I and type II repeats were not as extensively degraded by the nuclease. For XX and XY embryonic nuclei, where there is rapid cell division, the majority of the In- repeats were DNase I sensitive. This indicated that these In- repeats have the potential to be transcribed during this developmental stage. When compared to the In- repeats, the chromatin configuration of the In+ repeats is indicative of a higher order of chromatin folding. The paucity of In+ primary gene transcripts observed in vivo could result from In+ repeats being packaged into a more condensed form of chromatin.

Nonselective amplification of ribosomal DNA repeat units in compensating genotypes of Drosophila melanogaster.

Compensation is a mechanism by which the X-chromosome nucleolus organizer region of Drosophila melanogaster can increase its ribosomal DNA content up to twofold. It occurs in somatic cells under specific genetic conditions and is mediated by a defined genetic site, the compensatory response locus. The In- and various type I ribosomal DNA repeat units were separated by restriction endonuclease digestion. Comparison of the percentages of these repeat unit types between compensating and noncompensating genotypes showed the same distribution. Therefore no selective amplification of these repeat unit types occurs during ribosomal DNA compensation. These results demonstrate that two processes of rDNA amplification in somatic cells, compensation and independent rDNA polytenization, are exclusive events.

X chromosome nucleolus organizer mutants which alter major type I repeat multiplicity in Drosophila melanogaster.

The nucleolus organizer (NO) of the D. melanogaster X chromosome is composed of ribosomal repeat units which contain two types (I and II) of non-rDNA insertions (In+) and repeats with no insertions (In-). Evidence from other laboratories indicate random interspersion of all types of repeat units within the X NO. An EcoRI and BamHI examination of rDNA from two bobbed mutants, bb2rI and mal12 demonstrates segregation of the major type I repeat units. The 46 rDNA repeats of the bb2rI NO contain no detectable major type I repeats whereas the majority of the 68 rDNA mal12 repeats are major type I and tandemly linked. This observation suggests that gross deletions of rDNA can result in nucleolus organizer regions with predominantly one type of repeat unit. Additivity tests demonstrate that the 46 ribosomal repeats of the bb2rI chromosome revert the phenotype of other bobbed NOs, but the 68 mal12 ribosomal repeats show no or slight additivity. This is in agreement with the observation that In+ repeats do not significantly contribute to functional rRNA. A Southern blot analysis using BamHI which cuts only in type I insertions demonstrates that the majority of major type I In+ repeating units exist in tandem linkage group(s) within the X NO.

Genetic and molecular organization of the 5S locus and mutants in D. melanogaster.

The topography of an entire redundant locus was analyzed by both genetic and molecular means. Three mutants (min0, min1, min2) allelic to the 5S rRNA genetic locus on chromosome 2 of D. melanogaster were isolated. Flies exhibit a mutant phenotype when hemizygous for a min allele, but flies having two doses are wild-type. Saturation hybridization experiments show that the alleles are gross defieciencies each deleting an equal amount of 5S DNA. Each of the three mutant min alleles produces a distinct temperature-sensitive viability phene, and thus they are suggested to be pseudoalleles within the same redundant locus. Using the segmental aneuploid method (Lindsley et al., 1972), the 5S gene cluster was subdivided into proximal and distal halves. Both saturation hybridization experiments and genetic tests show that each half contains about eighty 5S genes. The complementation of the min alleles with the proximal and distal halves of the cluster indicates that both halves function independently. We present evidence which supports the model that all of the 160 5S genes are arranged as a single continuous cluster of tandem repeats with no large interdispersive DNA segments not complementary to 5S rRNA.

A genetic locus having trans and contiguous cis functions that control the disproportionate replication of ribosomal RNA genes in Drosophila melanogaster.

The results of deficiency mapping experiments reveal the presence of a compensatory response (cr+) locus that is located distal to the cluster of ribosomal RNA (rRNA) genes and is responsible for disproportionately replicating these genes when cr+ locus is present in a single dose, as in X/O males or X/sc4-sc8 females. The cr+ locus is novel in that it exhibits both trans and contiguous cis acting properties in somatic cells. It acts in trans to detect the presence of its partner locus in the opposite homolog, and if that partner locus is absent, it acts in cis to drive the disproportionate replication of those rRNA genes (rDNA) that are contiguous with it. The ability of cr+ to function is independent of the number of ribosomal RNA genes present. Furthermore, it can be shown that the cr+ locus is not required for the magnification or reduction of germ line rDNA. Finally, the implication of cr+ for position-effect variegation and the apparent reversion of the abnormal oocyte (abo) phenotype are discussed.

Genetic analysis of the 5S RNA genes in Drosophila melanogaster.

The 5S RNA genes of Drosophila melanogaster in either an isogenic wildtype or a multiply inverted (SM1) chromosome 2 increase their multiplicity when opposite a deficiency for the 5S gene site. This is analogous to the compensation phenomenon previously described for the 18S and 28S ribosomal RNA genes of the X chromosome nucleolus organizer region. Molecular hybridization of 5S RNA to DNA containing various doses of the 56F1-9 region of chromosome 2 demonstrates that most, if not all, of the 5S genes reside in or near this region. Also, a deficiency missing approximately one-half of the wild-type number of 5S genes was isolated and genetically localized. This mutant has a phenotype like that of bobbed, a mutant known to be partially deficient in 18S and 28S ribosomal RAN genes. Finally, we report the existence of a chromosomal rearrangement which splits the second chromosome into two segments, each containing 5S DNA.

Disproportionately-replicated, nonfunctional rDNA in compound chromosomes of Drosophila melanogaster.

The multiplicity of the genes for ribosomal RNA varies in several ways. The series of bb mutants are an example of mutant phenotypes associated with deficiencies of the rDNA. In certain genotypes the amount of rDNA present can disproportionately replicate and in some cases become incorporated into the chromosome and thus inherited. In other cases there is no stabilization of the increased multiplicity of rRna genes. In still other cases the multiplicity of rDNA does not change. We describe cases of increased, but non-functional, multiplicity of rDNA in compound chromosomes routinely used in analyses of these phenomena and discuss the associated problems.