Evidence for outcrossing in Phytophthora sojae and linkage of a DNA marker to two avirulence genes

Two genetically different isolates of the homothallic Oomycete, Phytophthora sojae, were demonstrated to outcross and form hybrid oospores after co-culturing in vitro. Random amplified polymorphic DNA (RAPD) markers revealed ten hybrids among 354 oospores analysed. One F₁ hybrid was allowed to self fertilise and produce an F₂ population of 247 individuals. Among 53 F₂ individuals, selected at random, 18 polymorphic RAPD markers were observed to segregate at near 3:1 Mendelian ratios, consistent with segregation for dominant alleles at single loci. Segregation of virulence against soybean resistance genes Rps1a, 3a, and 5 revealed that the avirulence genes Avr1a, 3a and 5 were dominant to virulence. Avirulence against these three resistance genes appeared to be conditioned by one locus for Avr1a and two independent, complementary dominant loci for both Avr3a and Avr5. Segregation of virulence against Rps6 was in the ratio of 1:2:1 (avirulent:mixed reaction:virulent), suggesting a semi-dominant allele at a single locus. Two avirulence genes and one RAPD marker formed one linkage group, in the order Avr3a, OPH4-1, Avr5, each separated by approximately 5 cM. Our results confirm that outcrossing occurred between the parental isolates, and that sexual recombination under field conditions may play an important role in generating and maintaining genetic diversity in populations of P. sojae.     

Outcrossing in the homothallic oomycete,Pythium ultimum,detected with molecular markers

The oomycete Pythium ultimum is homothallic, thus a single isolate completes the sexual stage in pure culture. It has been generally assumed that homothallic oomycetes are predominantly inbreeding. In P. ultimum, antheridia occasionally develop from hyphae not directly connected to the oogonium and appear to participate in fertilization, suggesting a possible mechanism for outcrossing. We have used molecular markers to confirm that outcrossing can occur between isolates of P. ultimum. Genetic markers based on randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) were used to distinguish isolates in a collection of P. ultimum. Two isolates displaying a high level of polymorphism were mixed and placed on media which allows the development of the sexual stage. RAPD markers were used to screen single oospore progeny to identify potential hybrids between the two parental isolates. Subsequent self-fertilization of one putative F₁ yielded a F₂ population which demonstrated segregation and independent assortment of RAPD and RFLP markers. A similar strategy was used to show that an isolate which is incapable of producing oospores in pure culture can outcross when mixed with a homothallic isolate. These results suggest that other homothallic oomycetes may be capable of outcrossing, and sexual reproduction may, therefore, play an important role in the generation of variation in homothallic oomycetes.     

Introgression mapping in the grasses

The unique properties of Lolium/Festuca hybrids and their derivatives provide an ideal system for intergeneric introgression. At IGER a focus on the Lolium perenne/Festuca pratensis system is being exploited to elucidate genome organization in the grasses, determination of the genetic control of target traits and the isolation of markers for marker-assisted selection in breeding programmes.     

High-GC primers are useful in RAPD analysis of fungi

For genetic analysis of fungal DNAs, we have modified the RAPD method to use primers with G+C contents of 80–100%. In RAPD analysis of Puccinia graminis f. sp. tritici DNAs, these primers generated twice the number of both amplification products per primer and polymorphisms among isolates as compared to the standard 60–70% G+C primers. With respect to segregation and genetic similarity, RAPD markers generated by the high-GC primers behaved as do RAPD markers produced by the standard primers. These high-GC primers also yielded increased numbers of amplification products in RAPDs on the DNAs of a broad range of other fungi. Present address: Department of Biology, Hiram College, Hiram, OH 44234, USA     

Inheritance of mitochondrial and chloroplast genome markers in backcrosses of Chlamydomonas eugametos x Chlamydomonas moewusii hybrids

Summary Southern blot analysis of AvaI-digested total cellular DNA from the interfertile species Chlamydomonas eugametos and Chlamydomonas moewusii with a coxI mitochondrial gene probe from Chlamydomonas reinhardtii revealed single hybridizing fragments of 5.0 and 3.5 kb, respectively. The transmission of these mitochondrial DNA physical markers along with that of chloroplast genetic markers for resistance to streptomycin and resistance to erythromycin was studied in the fourth backcrosses of F₁ hybrids to one or the other parent. Viability in these backcrosses is high in contrast to the cross C. eugametos x C. moewusii and its reciprocal which are associated with considerable meiotic product lethality. The resulting zygospores were found to transmit the mitochondrial and chloroplast genome markers uniparentally or preferentially from the mating-type-plus parent. Thus the species pair C. eugametos and C. moewusii differs from the pair Chlamydomonas reinhardtii and Chlamydomonas smithii in which mitochondrial genome markers are transmitted uniparentally by the mating-type minus parent, while the chloroplast genome markers are transmitted uniparentally by the opposite parental mating-type (Boynton et al. 1987).     

Genes for ribosomal proteins S3, L16, L5 and S14 are clustered in the mitochondrial genome of Brassica napus L.

A PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), was used for assessing genomic variability among a wide range of culture collection strains of black Aspergilli and related species. The performance of this technique is compared with that of the two other genetic techniques most commonly used, namely restriction fragment length polymorphisms on rDNA and isozyme analysis. The eight main groups as assigned by RFLP were also distinguished by RAPD patterns. On the basis of 122 polymorphic RAPD products using six random primers, the 17 collection strains examined could be subdivided into 15 distinct sub-groups. We suggest that the RAPD method is a quick and reliable tool for establishing the amount of genetic variability in closely related species. Our study indicates that the complex group of black Aspergilli is characterized by a high degree of genetic differentiation. This is also apparent from the considerable karyotype variation present in the group.     

Genetic analysis of intergeneric hybrids obtained by protoplast fusion in yeasts

Summary Prototrophic hybrids have been obtained by the fusion of various auxotrophic haploid strains of Saccharomyces cerepisiae and Schwanniomyces castellii. The fusion hybrids showed starch fermentability which derived from one of the fusion parents, S. castellii. Surprisingly, these fusion hybrids were found to exhibit excellent sporulation and spore germination. The progenies of these fusion hybrids showed a few aberrant segregations, but mostly normal segregation for auxotrophic genetic markers. They also showed many tetrads with an apparently digenic segregation (2:2, 3:1 and 4:0) for starch fermentation. On the other hand, mating types of segregants of the fusion hybrids were determined by the prototrophic recovery method. Consequently, tetrad types for mating type were mostly 2a:1:1 non-mater and several asci showed tetrad types of 2a:2 non-mater and 2a:2. The 60 prototrophic fusion hybrids and its segregants did not secrete -amylase on the starch agar plate. However, all of the data suggested that fusion hybrid could carry two dominant genes (STAB and STAC) to ferment starch, and that the two genes STAB and STA2 may be identical or allelic as may be the genes STAC and STA3.     

Assignment of RFLP,RAPD and isoenzyme markers to Aspergillus nidulans chromosomes,using chromosome-substituted segregants of a hybrid of A. nidulans and A. quadrilineatus

Chromosome-substituted haploid segregants were selected from among the benomyl-induced progeny of an interspecific hybrid produced by polyethylene-glycol-induced fusion of protoplasts of an Aspergillus nidulans master strain and an A. quadrilineatus auxotrophic mutant. These segregants were examined by RFLP, RAPD, and isoenzyme analysis. The A. nidulans ribosomal repeat unit was assigned to chromosome V, while the benA and the pyrG genes were assigned to linkage groups VIII and I, respectively, of A. nidulans. None of the other cloned genes tested (gdhA, amdS and 25s rRNA) showed polymorphism between the two parents. The method was also used to assign RAPD markers and isoenzyme bands of -arylesterase, phosphatases, NAD-dependent malate dehydrogenase, and cellulase, to A. nidulans chromosomes and/or to their A. quadrilineatus equivalents. The isoenzyme and DNA sequences assigned to chromosomes could be used to saturate the genetic map of A. nidulans, or could serve as starting points for the construction of a genetic map of A. quadrilineatus. No method affording the same possibilities has been described so far in Aspergilli. This chromosome-assay method may be a useful alternative to pulsed-field-gel electrophoretic procedures for the assignment of molecular markers to chromosomes.     

Nucleotide divergence between populations of trembling aspen (Populus tremuloides) estimated with RAPDs

In the present study, a total of 142 trees sampled from five populations of trembling aspen (Populus tremuloides Michx.) in Alberta was analyzed by the polymerase chain reaction (PCR) with five random oligonucleotide primers. Null-allele frequencies of 28 putative RAPD loci were estimated using the given departure from Hardy-Weinberg equilibrium (F _IS) previously estimated with isozyme markers for the same population. Nucleotide divergence between populations was then estimated in a fashion similar to restriction-fragment data, but considering the dominance of the RAPDs. The average of nucleotide divergence between populations was in the order of 0.0005 and nucleotide divergence were found to be highly correlated with geographic distance. The results suggest that isolation by distance may be an important factor in the genetic differentiation of trembling aspen.     

Genetic variation in <Emphasis Type="Italic">Opisthorchis viverrini</Emphasis> (Trematoda: Opisthorchiidae) from northeast Thailand and Laos PDR based on random amplified polymorphic DNA analyses

Genetic variation in Opisthorchis viverrini adults originating from different locations in northeast Thailand and Laos, People’s Democratic Republic (PDR), was examined using random amplified polymorphic DNA (RAPD) analyses. In an initial analysis, the genomic DNA of one fluke from each of ten localities was amplified using 15 random primers (10-mers); however, genetic variation among O. viverrini specimens was detected reliably for only four primers. A more detailed RAPD analysis using these four primers was conducted on ten individuals from nine localities. Considerable genetic variation was detected among O. viverrini from different geographical areas and among some individuals from the same collecting locality. Comparison of the RAPD profiles revealed that O. viverrini adults from Laos PDR were genetically distinct from those from northeast Thailand. The taxonomic significance of this finding needs to be explored in more detail. The RAPD markers established in the present study provide opportunities to examine the biology and epidemiology of O. viverrini and fish-borne trematodes within the region. Additionally, application of these markers in such studies could have important implications in relation to the prevalence of cholangiocarcinoma in different regions of Asia.     

Intra-specific variation withinEimeria tenella detected by the random amplification of polymorphic DNA

The random amplification of polymorphic DNA (RAPD method), was used to derive fragments of DNA of the coccidial parasiteEimeria tenella for use both as genetic markers and as an aid for the discrimination of different wild-type and attenuated populations. Of 41 arbitrarily chosen 9-to 27-mer primers, 24 yielded useful arrays of fragments following low-stringency annealing conditions and the resultant profiles were generally very reproducible. One non-variant fragment of 2 kb hybridised to a single chromosome (number 12) and four variant fragments were identified. These results strongly suggest that the RAPD method may be an extremely useful tool for studies on various aspects of the genetic organisation of coccidial parasites.     

Diversity within natural progenies of the grapevine dieback fungus Eutypa lata

The diversity within 16 natural progenies of the grapevine dieback fungus, Eutypa lata, was investigated by sampling single-ascospore isolates mainly in France and using random amplified polymorphic DNA (RAPD) markers, vegetative compatibility (VC), and pathogenicity testing. The combination of RAPD and VC data identified each isolate as a unique genotype within each progeny. Only three RAPD haplotypes did not cluster within the expected groups, i.e. the ascospore families. Within each set of clustering haplotypes, Mendelian 1:1 ratios for absence and presence were observed for RAPD markers, indicating that each progeny was the result of a biparental cross. Only one mycelium was obtained when isolation was performed from the discolored wood sustaining the perithecial stroma. This mycelium was identified as a likely parent of the corresponding progeny by RAPD analysis. The level of diversity measured by the average distance between haplotypes calculated from RAPD data, the percentage of vegetatively compatible pairs and the range of pathogenicity appeared similar between all but one progeny, indicating that crosses occurred within a random-mating population. All the results were consistent with the hypothesis that E. lata is a random-mating species having a high degree of genetic diversity. Received: 18 December 1998 / 7 July 1999     

Detection of RAPD Markers Correlated with Chloroquine Resistance in Plasmodium falciparum

We described the use of the random amplified polymorphic DNA (RAPD) technique on Plasmodium falciparum DNA to detect genetic markers for chloroquine-resistant strains. Fourteen RAPD primers were tested, three of which generated banding patterns correlated with chloroquine resistance. To measure this correlation, the RAPD profiles were analyzed using the Nei and Li similarity coefficient. Detection of distinctive RAPD bands allowed us to synthesize specific PCR primers to be used on whole-blood samples. Two primer sets were synthesized and tested on sensitive and resistant strains for their ability to amplify the DNA fragment corresponding to the RAPD marker. These results suggest that RAPD and PCR techniques can be used as powerful tools for the detection of genetic markers associated with drug resistance.     

Transmission and modification of transformation markers during an induced parasexual cycle in Penicillium roqueforti

Summary Protoplasts of two wild-type strains of Penicillium roqueforti were transformed to hygromycin B and phleomycin resistance using resistance genes. DNAs were stably integrated into the fungal chromosomes. Protoplasts from transformed strains have been fused to produce diploid hybrids. After induced haploidization, segregants characterized by a reassortment of the parental genetic markers were isolated indicating that recombination had occurred during the parasexual cycle. Southern blot hydridization experiments revealed that transmission of the transformed phenotype was accompanied by extensive rearrangement and/or deletions dependent on the number of integrated plasmid copies. Methylation of cytosine residues of integrated DNA was also detected. These observations suggest that foreign DNA sequences transmitted during the parasexual cycle undergo modifications similar to the rearrangement of transformed characters induced premeiotically (RIP) through sexual crosses.     

Orthogonal field alternation gelelectrophoresis (OFAGE) as a means for the analysis of somatic hybrids obtained by protoplast fusion of different Saccharomyces strains

Summary PEG-induced fusion of two haploid Saccharomyces strains resulted in three morphologically different types of fusion products. In order to estimate the respective ploidy, one representative of each type — F ₁, F ₂ and F ₃ — was subjected to a measurement of the cellular DNA content and to a meiotic segregation analysis. The data obtained in these analyses suggested the strain F ₁ to be a haploid cybrid resulting from mere plasmogamy, whereas the hybrids F ₂ and F ₃ were likely to be the products of a successful nuclear fusion of two, respectively three, protoplasts of the parental strains. However, the complete genetic composition of the hybrids could only be revealed by OFAGE experiments, as the genetic data solely referred to a few chromosomes with marker genes. All the results of the OFAGE were in full accordance with the assumptions made in the conventional analysis, thus indicating the OFAGE to become a very promising means for the investigation of hybrids inaccessible to genetic analyses.     

Molecular arguments for splitting of Schistosoma intercalatum, into two distinct species

The taxonomic status of the two known strains of Schistosoma intercalatum, the Lower Guinea strain (originating from Edea, Cameroon) and the Zaire strain (originating from Kinshasa, Democratic Republic of Congo, formerly Zaire) was examined using random amplified polymorphic DNA (RAPD) markers. Two additional species within the S. haematobium group, S. haematobium and S. mattheei, were included in the study. DNA was extracted from four male and four female worms of each species and strain under investigation. In all, 13 primers gave reproducible and informative marker patterns; the monomorphic bands in all the males and females of each sample were scored, and 138 bands were included in the final analysis. Overall, 14 RAPD fragments were shared by all the schistosomes studied, and 19 RAPD fragments were considered to be sex markers. Only 22% (20/91) of the RAPD fragments were shared between S. intercalatum Zaire and S. intercalatum Cameroon. The mean values recorded for the Nei and Li's genetic distances between S. haematobium and S. mattheei and between S. intercalatum Zaire and S. intercalatum Cameroon were 0.546 and 0.596, respectively. A principal component analysis and one-way analysis of variance (ANOVA/MANOVA) showed a significant separation between S. intercalatum Zaire and S. intercalatum Cameroon. The data support the hypothesis that S. intercalatum Zaire and S. intercalatum Cameroon are distinct species. Additional molecular-biology studies are in progress that involve the use of nuclear and mitochondrial markers to confirm the extent of the genetic divergence prior to the establishment of final decision on the taxonomic status of the two strains of S. intercalatum. Received: 6 June 2000 / Accepted: 11 July 2000     

Synteny between the Pro+ marker and human glutamate oxaloacetate transaminase

Chinese hamster ovary cells with a specific auxotrophy for proline were fused with human cells from a variety of sources and the resulting hybrids analyzed for human genetic markers. Of 63 hybrid clones examined, 27 possessed both proline and cytoplasmic glutamate oxaloacetate transaminase markers;36 had neither; and no clones were found possessing one and not the other. These results constitute evidence that the proline and glutamate oxaloacetate transaminase markers are syntenic. Evidence for absence of synteny between these and a variety of other human genes is presented. Biochemical tracer experiments established that the proline biosynthetic pathway through glutamate has been restored in the Pro ⁺ hybrids.     

Physical evidence for recombination of chloroplast dna in hybrid progeny of Chlamydomonas eugametos and C. moewusii

Summary Differences in the restriction endonuclease fragmentation patterns of chloroplast DNA (cpDNA) from C. eugametos and C. moewusii have been used to study the inheritance of these DNAs in interspecific hybrids. Analysis of the cpDNAs from ten randomly selected F₁ hybrids, in each case revealed cpDNA to be recombinant for AvaI and BstEII restriction sites, although fragments characteristic of C. eugametos, the mt+ parent, were typically found in excess of those for C. moewusii, the mt– parent. In backcrosses between an F ₁ mt+ hybrid and C. moewusii mt–, seven randomly selected B₁ hybrids showed cpDNA restriction patterns either identical to or highly similar to that of the mt+ parent. We propose that cpDNA molecules are predominantly transmitted by the mt+ parent in both F₁ and B₁ generations but that selection favors survival of F₁ progeny with recombinant chloroplast genomes which avoid interspecific incompatibilities. On the surface, the inheritance of recombinant cpDNA contrasts with the simultaneous uniparental inheritance of two putative chloroplast markers (sr-2 and er-nM1 ⁺). However, it may be that these two markers are by chance associated with cpDNA sequences of the mt+ parent which were selected in all F₁ hybrids.     

Genetic differentiation of cercariae infrapopulations of the avian schistosome <Emphasis Type="Italic">Trichobilharzia szidati</Emphasis> based on RAPD markers and mitochondrial <Emphasis Type="Italic">cox1</Emphasis> gene

Avian schistosome Trichobilharzia szidati is a member of the largest genus within the family Schistosomatidae (Trematoda). Population genetic structure of Trichobilharzia spp. schistosomes, causative agents of cercarial dermatitis in humans, has not been studied yet. The knowledge of the genetic structure of trichobilharzian populations is essential for understanding the host–parasite coevolutionary dynamics and epidemiology strategies. Here we examined genetic diversity in three geographically isolated local populations of T. szidati cercariae inhabiting Russia based on nuclear (randomly amplified polymorphic DNA, RAPD) and mt (cox1) markers. We analyzed T. szidati cercariae shed from seven naturally infected snails of Lymnaea stagnalis. Using three random primers, we demonstrated genetic variation among populations, thus posing genetic structure across geographic sites. Moreover, T. szidati cercariae have been genetically structured among hosts (infrapopulations). Molecular variance analysis was performed to test the significance of genetic differentiation within and between local populations. Of total parasitic diversity, 18.8% was partitioned between populations, whereas the higher contribution (48.9%) corresponds to the differences among individual cercariae within infrapopulations. In contrast to RAPD markers, a 1,125-bp fragment of cox1 mt gene failed to provide any significant within-species structure. The lack of geographic structuring was detected using unique haplotypes which were determined in the current work for Moscow and Western Siberian local populations as well as obtained previously for European isolates (Czech Republic and Germany). All T. szidati/Trichobilharzia ocellata haplotypes were found to be mixed across their geographical origin.     

The use of RAPD markers in the genetic analysis of the plant pathogenic fungus Cladosporium fulvum

RAPD markers have been obtained in Cladosporium fulvum, in order to establish a genetic map based on mitotic recombination in this imperfect fungus. Segregation analysis has provided molecular evidence of a high degree of recombination during the parasexual cycle, and of the ploidy level of the parasexual progeny. A molecular study of 49 RAPDs obtained showed that, with only one exception, all RAPD markers studied represent repetitive DNA. This situation precludes the direct use of these markers to either initiate chromosome walking to a gene of interest or for the assignment of linkage groups to electrophoretically-separated chromosomes. A simple reamplification test has been applied which permitted the quick discrimination between single-copy and repetitive DNA species, without the need for Southern analysis. Additionally, evidence is shown for the presence of single-stranded DNA species in the amplification products, and that these may be present in addition to their double-stranded counterparts. The reamplification test can also identify these DNA species, avoiding misinterpretation of polymorphic bands.