Genetic Mapping and Non-Mendelian Segregation of Mating Type Loci in the Oomycete, Phytophthora Infestans

DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in this normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses as RAPD markers, restriction fragment length polymorphisms (RFLPs), single-strand conformational polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balanced lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude from progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.     

Dominance at a Fungus Mating Type Locus

THE action of the two alleles which control mating type in fungi with bipolar heterothallism is uncertain. A diffusible oligopeptide, produced by the α mating type of Saccharomyces cerevisiae, blocks division and induces copulatory processes in cells of a mating type^2,3 and in some yeasts the mating type locus may be induced to mutate by other genes in the same cell^4,5. These observations in yeast, however, may not be representative of the other fungi with bipolar heterothallism².     

AFLP Linkage Map of the Oomycete Phytophthora infestans

Here we present the first comprehensive genetic linkage map of the heterothallic oomycetous plant pathogen Phytophthora infestans. The map is based on polymorphic DNA markers generated by the DNA fingerprinting technique AFLP (Vos et al., 1995, Nucleic Acids Res. 23: 4407-4414). AFLP fingerprints were made from single zoospore progeny and 73 F1 progeny from two field isolates of P. infestans. The parental isolates appeared to be homokaryotic and diploid, their AFLP patterns were mitotically stable, and segregation ratios in the F1 progeny were largely Mendelian. In addition to 183 AFLP markers, 7 RFLP markers and the mating type locus were mapped. The linkage map comprises 10 major and 7 minor linkage groups covering a total of 827 cM. The major linkage groups are composed of markers derived from both parents, whereas the minor linkage groups contain markers from either the A1 or the A2 mating type parent. Non-Mendelian segregation ratios were found for the mating type locus and for 13 AFLP markers, all of which are located on the same linkage group as the mating type locus. Copyright 1997 Academic Press     

Genetic Map of Randomly Amplified DNA Polymorphisms Closely Linked to the Mating Type Locus of Tetrahymena Thermophila

We have used the PCR-based randomly amplified polymorphic DNA (RAPD) method to efficiently identify and map DNA polymorphisms in the ciliated protozoan Tetrahymena thermophila. The polymorphisms segregate as Mendelian genetic markers. A targeted screen, using DNA from pooled meiotic segregants, yielded the polymorphisms most closely linked to the mat locus. A total of 10 polymorphisms linked to the mat-Pmr segment of the left arm of micronuclear chromosome 2 have been identified. This constitutes the largest linkage group described in T. thermophila. We also provide here the first crude estimate of the frequency of meiotic recombination in the mat region, 20 kb/cM. This frequency is much higher than that observed in most other eukaryotes. Special features of Tetrahymena genetics enhanced the power of the RAPD method: the ability to obtain in a single step meiotic segregants that are whole-genome homozygotes and the availability of nullisomic strains permitting quick deletion mapping of polymorphisms to micronuclear chromosomes or chromosome segments. The RAPD method appears to provide a practical and relatively inexpensive approach to the construction of a high-resolution map of the Tetrahymena genome.     

Genetic and Physical Mapping of the Mouse Ulnaless Locus

The mouse Ulnaless locus is a semidominant mutation which displays defects in patterning along the proximal-distal and anterior-posterior axes of all four limbs. The first Ulnaless homozygotes have been generated, and they display a similar, though slightly more severe, limb phenotype than the heterozygotes. To create a refined genetic map of the Ulnaless region using molecular markers, four backcrosses segregating Ulnaless were established. A 0.4-cM interval containing the Ulnaless locus has been defined on mouse chromosome 2, which has identified Ulnaless as a possible allele of a Hoxd cluster gene(s). With this genetic map as a framework, a physical map of the Ulnaless region has been completed. Yeast artificial chromosomes covering this region have been isolated and ordered into a 2 Mb contig. Therefore, the region that must contain the Ulnaless locus has been defined and cloned, which will be invaluable for the identification of the molecular nature of the Ulnaless mutation.     

Genetic Variability and Distribution of Mating Type Alleles in Field Populations of Leptosphaeria maculans from France

Leptosphaeria maculans is the most ubiquitous fungal pathogen of Brassica crops and causes the devastating stem canker disease of oilseed rape worldwide. We used minisatellite markers to determine the genetic structure of L. maculans in four field populations from France. Isolates were collected at three different spatial scales (leaf, 2-m² field plot, and field) enabling the evaluation of spatial distribution of the mating type alleles and of genetic variability within and among field populations. Within each field population, no gametic disequilibrium between the minisatellite loci was detected and the mating type alleles were present at equal frequencies. Both sexual and asexual reproduction occur in the field, but the genetic structure of these populations is consistent with annual cycles of randomly mating sexual reproduction. All L. maculans field populations had a high level of gene diversity (H = 0.68 to 0.75) and genotypic diversity. Within each field population, the number of genotypes often was very close to the number of isolates. Analysis of molecular variance indicated that >99.5% of the total genetic variability was distributed at a small spatial scale, i.e., within 2-m² field plots. Population differentiation among the four field populations was low (G_ST < 0.02), suggesting a high degree of gene exchange between these populations. The high gene flow evidenced here in French populations of L. maculans suggests a rapid countrywide diffusion of novel virulence alleles whenever novel resistance sources are used.     

Genetic Analysis of Mating Locus Linked Mutations in CHLAMYDOMONAS REINHARDII

The mating-type (mt) locus of Chlamydomonas reinhardii has been analyzed using four mutant strains (imp-1, imp-10, imp-11 and imp-12). All have been shown, or are shown here, to carry mutations linked to either the plus (mt⁺) or the minus (mt^-) locus, and their behavior in complementation tests has allowed us to define several distinct functions for each locus. Specifically, we propose that the mt⁺ locus contains the following genes or regulatory elements: a locus designated sfu, which is necessary for sexual fusion between gametes; a locus designated upp (uniparental plus), which controls aspects of chloroplast gene inheritance and perhaps also zygote maturation; and a locus designated sad, which functions in sexual adhesion. The mt^- locus also contains a sad locus as well as a gene or regulatory element designated mid, which is necessary for the minus dominance in mt⁺/mt^- diploids.     

High Genetic Variability of the agr Locus in Staphylococcus Species

The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.     

Distribution and Expression of Elicitin Genes in the Interspecific Hybrid Oomycete Phytophthora alni

Phytophthora alni subsp. alni, P. alni subsp. multiformis, and P. alni subsp. uniformis are responsible for alder disease in Europe. Class I and II elicitin gene patterns of P. alni subsp. alni, P. alni subsp. multiformis, P. alni subsp. uniformis, and the phylogenetically close species P. cambivora and P. fragariae were studied through mRNA sequencing and 3′ untranslated region (3′UTR)-specific PCRs and sequencing. The occurrence of multiple 3′UTR sequences in association with identical elicitin-encoding sequences in P. alni subsp. alni indicated duplication/recombination events. The mRNA pattern displayed by P. alni subsp. alni demonstrated that elicitin genes from all the parental genomes are actually expressed in this allopolyploid taxon. The complementary elicitin patterns resolved confirmed the possible involvement of P. alni subsp. multiformis and P. alni subsp. uniformis in the genesis of the hybrid species P. alni subsp. alni. The occurrence of multiple and common elicitin gene sequences throughout P. cambivora, P. fragariae, and P. alni sensu lato, not observed in other Phytophthora species, suggests that duplication of these genes occurred before the radiation of these species.     

Genetic Variability at the Esterase-6 Locus in Natural Populations of DROSOPHILA SIMULANS in Relation to Environmental Heterogeneity

The allozymic variation at the esterase-6 locus was examined in fourteen samples of natural populations of Drosophila simulans collected in two different localities. The samples were collected from different species of fruits in the two localities and from banana baits located some meters apart from the fruits. The analysis was done by sequential electrophoresis, using varied gel concentrations and buffers. By this technique, we detected 27 alleles at the esterase-6 locus, where only five alleles were detected by the usual method. The results obtained suggest that habitat choice alone may not be a selective factor, but may be selective in combination with other environmental factors.     

Refinement of the Spinal Muscular Atrophy Locus by Genetic and Physical Mapping

We report the mapping and characterization of 12 microsatellite markers including 11 novel markers. All markers were generated from overlapping YAC clones that span the spinal muscular atrophy (SMA) locus. PCR amplification of 32 overlapping YAC clones shows that 9 of the new markers (those set in italics) map to the interval between the two previous closest flanking markers (D5S629 and D5S557): cen - D5S6 - D5S125 - D5S435 - D5S1407-D5S629-D5S1410-D5S1411/D5S1412-D5S1413-D5S1414-D5Z8-D5Z9-CATT1-D5Z10/D5Z6-D5S557-D5S1408-D5S1409-D5S637-D5S351-MAP1B-tel. Four of these new markers detect multiple loci in and out of the SMA gene region. Genetic analysis of recombinant SMA families indicates that D5S1413 is a new proximal flanking locus for the SMA gene. Interestingly, among the 40 physically mapped loci, the 14 multilocus markers map contiguously to a genomic region that overlaps, and perhaps helps define, the minimum genetic region encompassing the SMA gene(s).     

Genetic and Physical Mapping of the Dreher Locus on Mouse Chromosome 1

Mutations in the mouse dreher (dr) gene cause skeletal defects, hyperactivity, abnormal gait, deafness, white belly spotting, and hypoplasia of Müllerian duct derivatives. To map dr to high resolution, we utilized two crosses. Initially, we analyzed an intersubspecific intercross to construct a detailed genetic map of simple sequence length polymorphism markers within a 6.3-cM region surrounding the dr locus. Subsequently, we analyzed a second intersubspecific intercross segregating for the dr(6J) allele, which positioned dr within a 0.13-cM region between Rxrg and D1Mit370. A physical contig of BAC clones spanning the dr critical region was constructed, and eight potential dr candidate genes were excluded by genetic or physical mapping. Together these results lay the foundation for positional cloning of the dr gene.     

The Role of Autophagy during Development of the Oomycete Pathogen Phytophthora infestans

Phytophthora infestans is a highly destructive plant path- ogen that causes late blight disease in plants such as potato and tomato （Fry and Goodwin, 1997）. As a typical oomycete, the primary infective propagule of P. infestans is the sporangium, which can either germinate directly or release biflagellate zoospores that encyst and germinate on the host subsequently （Grenville-Briggs et al., 2005）. Due to abundant formation of the sporangia and prolific sporulation, P. infestans can spread quickly in the host tissues and cause extensive necrosis of the infected plants （Kamoun and Smart, 2005）.     

Genetic Instability in the Mating Type System of Tetrahymena pigmentosa

Selfing clones of Tetrahymena pigmentosa show several interesting genetic features, and provide some insight into the mechanisms of mating type (mt) determination. They differ significantly from those of Tetrahymena thermophila. They are distributed nonrandomly in crosses. Their rates of stabilization are highly variable, but most are much lower than those reported for T. thermophila. A number of subclones derived from nearly all the selfers have maintained stable mts in culture for several years. However, some subclones manifest persistent selfing, long after the calculated completion of allelic assortment for heterozygous loci. This phenomenon along with the perpetual maintenance of dominant mts in heterozygotes shows that phenotypic assortment is not involved in mt expression. In crosses, many selfers exhibit quantitative and qualitative aberrations in the transmission of alleles to the gametes; some of the micronuclear changes underlying these aberrations occur during vegetative growth. There are rare illegitimate appearances of dominant alleles in sexual progeny, and more common illegitimate appearances of the most recessive phenotype.--Various models to explain mt determination in this species are considered. One which might account for the troubling phenomena of the system consists of an active mat expression site, with "cassettes" at other sites specific for the different dominant alleles and capable of transposition to the expression site.     

Genetic Diversity and Mosaicism at the por Locus of Neisseria gonorrhoeae

The por genes of the predominant serovars of Neisseria gonorrhoeae circulating in a high-frequency transmitter core group located in Nairobi, Kenya, were examined for nucleotide sequence polymorphism. The level of por gene diversity did not differ significantly between core group-derived gonococcal strains and gonococcal strains originating elsewhere. However, por mosaicism appeared to be more frequent among core group-derived strains, suggesting that recombination of different por sequences may be a important strategy by which N. gonorrhoeae generates por gene diversity within core group populations. Despite extensive sequence variability, por expressed by gonococcal isolates of different geographic origin exhibited conserved patterns of nucleotide change, suggesting that diversity among por alleles may also be finite.