A multiplex PCR test for determination of mating type applied to the plant pathogens Tapesia yallundae and Tapesia acuformis

A multiplex PCR test for determining mating type of the pathogens Tapesia yallundae and Tapesia acuformis is described. The test involves three primers: a "common" primer annealing to DNA sequence conserved in the flanking region of both mating-type idiomorphs and two specific primers annealing to sequence in either the MAT-1 or the MAT-2 idiomorphs. Locating the specific primers in different positions relative to the common primer yielded PCR products of 812 or 418 bp from MAT-1 and MAT-2 isolates, respectively. The test was used successfully to determine the mating type of 118 isolates of T. yallundae and T. acuformis from Europe, North America, and New Zealand. Isolates of both mating types were found on all continents for both species despite the rarely observed occurrence of sexual reproduction of T. acuformis. The multiplex test design should be applicable to other ascomycete species, of use in studies of MAT distribution and facilitating sexual crossing by identifying compatible isolates.     

Genomic and experimental evidence for a potential sexual cycle in the pathogenic thermal dimorphic fungus Penicillium marneffei

All meiotic genes (except HOP1) and genes encoding putative pheromone processing enzymes, pheromone receptors and pheromone response pathways proteins in Aspergillus fumigatus and Aspergillus nidulans and a putative MAT-1 alpha box mating-type gene were present in the Penicillium marneffei genome. A putative MAT-2 high-mobility group mating-type gene was amplified from a MAT-1 alpha box mating-type gene-negative P. marneffei strain. Among 37 P. marneffei patient strains, MAT-1 alpha box and MAT-2 high-mobility group mating-type genes were present in 23 and 14 isolates, respectively. We speculate that P. marneffei can potentially be a heterothallic fungus that does not switch mating type.     

Mating factor linkage and genome evolution in basidiomycetous pathogens of cereals

Sex in basidiomycete fungi is controlled by tetrapolar mating systems in which two unlinked gene complexes determine up to thousands of mating specificities, or by bipolar systems in which a single locus (MAT) specifies different sexes. The genus Ustilago contains bipolar (Ustilago hordei) and tetrapolar (Ustilago maydis) species and sexual development is associated with infection of cereal hosts. The U. hordei MAT-1 locus is unusually large (approximately 500 kb) and recombination is suppressed in this region. We mapped the genome of U. hordei and sequenced the MAT-1 region to allow a comparison with mating-type regions in U. maydis. Additionally the rDNA cluster in the U. hordei genome was identified and characterized. At MAT-1, we found 47 genes along with a striking accumulation of retrotransposons and repetitive DNA; the latter features were notably absent from the corresponding U. maydis regions. The tetrapolar mating system may be ancestral and differences in pathogenic life style and potential for inbreeding may have contributed to genome evolution.     

Molecular analysis of the pheromone and pheromone receptor genes of Ustilago hordei

Cell-cell signaling is an integral part of the sexual and disease cycles of the smut fungi, which must mate to be pathogenic. This study reports the cloning and characterization of the pheromone genes Uhmfa1 and Uhmfa2 from MAT-1 and MAT-2 mating types of U. hordei, respectively, and the pheromone receptor gene Uhpra2 from MAT-2 cells. Similar to other fungal pheromone genes, Uhmfa1 and Uhmfa2 encode precursor peptides. Uhpra2 encodes a protein with sequence similarity to the 7-transmembrane class of G-protein coupled receptors. Deletion of Uhmfa1 and Uhpra1, and their subsequent replacement, confirmed the role of these genes in initiation of the sexual cycle. Uhmfa1 and Uhmfa2 were differentially expressed in various cell types and when opposite mating-type cells were grown together. The predicted mature pheromones of each mating type were synthesized, and each specifically induced conjugation tube formation in cells of the opposite mating type.     

Mating-type heterokaryosis and selfing in Cryphonectria parasitica

Selfing in the chestnut blight fungus, Cryphonectria parasitica, occurs by two different genetic mechanisms. Most self-fertile isolates of C. parasitica are heterokaryotic for mating type, and the progeny from selfing segregate for mating type. Further, we resolved mating-type (MAT) heterokaryons into homokaryons of both mating types by isolating uninucleate asexual spores (conidia). However, because ascospore progeny, with rare exceptions, are not MAT heterokaryons, C. parasitica must lack a regular mechanism to maintain heterokaryosis by selfing. We hypothesize that heterokaryon formation may occur either because of recurrent biparental inbreeding, or by mating-type switching, possibly one involving some kind of parasexual process. The second mechanism found for selfing in C. parasitica occurred less frequently. Three single-conidial isolates (MAT-1 and MAT-2) selfed and produced progeny that did not segregate for mating type. It is currently not known if meiosis occurs during ascospore formation by this mechanism.     

Evolution of the gene encoding mitochondrial intermediate peptidase and its cosegregation with the A mating-type locus of mushroom fungi

The high level of DNA polymorphism at the mating-type loci of mushroom fungi has made the cloning of mating-type genes difficult. As an alternative to strategies that employ sequence conservation, an approach utilizing conserved gene order could facilitate the cloning of A mating-type genes from mushroom fungi. It has been shown that a gene encoding a mitochondrial intermediate peptidase (MIP) is very close ( < 1 kbp) to the A mating-type locus of two model mushroom species. In this study, the cosegregation of MIP and the A mating-type locus was studied by genotyping progeny of seven additional mushroom species using PCR and genetic crosses. No evidence of any recombination between MIP and the A mating-type locus was detected among all seven species. Phylogenetic analysis of MIP sequences from diverse mushroom species agrees with the current organismal phylogeny, suggesting the sequences are generally orthologous.     

Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus is a medically important opportunistic pathogen and a major cause of respiratory allergy. The species has long been considered an asexual organism. However, genome analysis has revealed the presence of genes associated with sexual reproduction, including a MAT-2 high-mobility group mating-type gene and genes for pheromone production and detection (Galagan et al., personal communication; Nierman et al., personal communication). We now demonstrate that A. fumigatus has other key characteristics of a sexual species. We reveal the existence of isolates containing a complementary MAT-1 alpha box mating-type gene and show that the MAT locus has an idiomorph structure characteristic of heterothallic (obligate sexual outbreeding) fungi. Analysis of 290 worldwide clinical and environmental isolates with a multiplex-PCR assay revealed the presence of MAT1-1 and MAT1-2 genotypes in similar proportions (43% and 57%, respectively). Further population genetic analyses provided evidence of recombination across a global sampling and within North American and European subpopulations. We also show that mating-type, pheromone-precursor, and pheromone-receptor genes are expressed during mycelial growth. These results indicate that A. fumigatus has a recent evolutionary history of sexual recombination and might have the potential for sexual reproduction. The possible presence of a sexual cycle is highly significant for the population biology and disease management of the species.     

Cloning and analysis of the mating-type idiomorphs from the barley pathogen <Emphasis Type="Italic"> Septoria passerinii</Emphasis>

The genus Septoria contains more than 1000 species of plant pathogenic fungi, most of which have no known sexual stage. Species of Septoria without a known sexual stage could be recent derivatives of sexual species that have lost the ability to mate. To test this hypothesis, the mating-type region of S. passerinii, a species with no known sexual stage, was cloned, sequenced, and compared to that of its close relative S. tritici (sexual stage: Mycosphaerella graminicola). Both of the S. passerinii mating-type idiomorphs were approximately 3 kb in size and contained a single reading frame interrupted by one (MAT-2) or two (MAT-1) putative introns. The putative products of MAT-1 and MAT-2 are characterized by alpha-box and high-mobility-group sequences, respectively, similar to those in the mating-type genes of M. graminicolaand other fungi. The mating-type genes of S. passerinii and M. graminicolaare evolving rapidly, approximately ten times faster than the internal transcribed spacer region of the ribosomal DNA, and are not closely related to those from Cochliobolusor other loculoascomycetes in the order Pleosporales. Therefore, the class Loculoascomycetes may be polyphyletic. Furthermore, differences between the phylogenetic trees may indicate separate evolutionary histories for the MAT-1 and MAT-2 idiomorphs. A three-primer multiplex-PCR technique was developed that allowed rapid identification of the mating types of isolates of S. passerinii. Both mating types were present in approximately equal frequencies and often on the same leaf in fields in Minnesota and North Dakota. Analyses with isozyme and random amplified polymorphic DNA markers revealed that each isolate had a unique genotype. The common occurrence of both mating types on the same leaf and the high levels of genotypic diversity indicate that S. passerinii is almost certainly not an asexual derivative of a sexual fungus. Instead, sexual reproduction probably plays an integral role in the life cycle of S. passerinii and may be much more important than previously believed in this (and possibly other) "asexual" species of Septoria.     

Evolution of uni- and bifactorial sexual compatibility systems in fungi

Mating systems, that is, whether organisms give rise to progeny by selfing, inbreeding or outcrossing, strongly affect important ecological and evolutionary processes. Large variations in mating systems exist in fungi, allowing the study of their origin and consequences. In fungi, sexual incompatibility is determined by molecular recognition mechanisms, controlled by a single mating-type locus in most unifactorial fungi. In Basidiomycete fungi, however, which include rusts, smuts and mushrooms, a system has evolved in which incompatibility is controlled by two unlinked loci. This bifactorial system probably evolved from a unifactorial system. Multiple independent transitions back to a unifactorial system occurred. It is still unclear what force drove evolution and maintenance of these contrasting inheritance patterns that determine mating compatibility. Here, we give an overview of the evolutionary factors that might have driven the evolution of bifactoriality from a unifactorial system and the transitions back to unifactoriality. Bifactoriality most likely evolved for selfing avoidance. Subsequently, multiallelism at mating-type loci evolved through negative frequency-dependent selection by increasing the chance to find a compatible mate. Unifactoriality then evolved back in some species, possibly because either selfing was favoured or for increasing the chance to find a compatible mate in species with few alleles. Owing to the existence of closely related unifactorial and bifactorial species and the increasing knowledge of the genetic systems of the different mechanisms, Basidiomycetes provide an excellent model for studying the different forces that shape breeding systems.     

Genetic Basis of Self-Incompatibility in the Lichen-Forming Fungus Lobaria pulmonaria and Skewed Frequency Distribution of Mating-Type Idiomorphs: Implications for Conservation

Fungal populations that reproduce sexually are likely to be genetically more diverse and have a higher adaptive potential than asexually reproducing populations. Mating systems of fungal species can be self-incompatible, requiring the presence of isolates of different mating-type genes for sexual reproduction to occur, or self-compatible, requiring only one. Understanding the distribution of mating-type genes in populations can help to assess the potential of self-incompatible species to reproduce sexually. In the locally threatened epiphytic lichen-forming fungus Lobaria pulmonaria (L.) Hoffm., low frequency of sexual reproduction is likely to limit the potential of populations to adapt to changing environmental conditions. Our study provides direct evidence of self-incompatibility (heterothallism) in L. pulmonaria. It can thus be hypothesized that sexual reproduction in small populations might be limited by an unbalanced distribution of mating-type genes. We therefore assessed neutral genetic diversity (using microsatellites) and mating-type ratio in 27 lichen populations (933 individuals). We found significant differences in the frequency of the two mating types in 13 populations, indicating a lower likelihood of sexual reproduction in these populations. This suggests that conservation translocation activities aiming at maximizing genetic heterogeneity in threatened and declining populations should take into account not only presence of fruiting bodies in transplanted individuals, but also the identity and balanced representation of mating-type genes.     

Multilocus population structure of Tapesia yallundae in Washington State

Population genetic structure of the fungal wheat pathogen Tapesia yallundae in Washington State was determined using genetically characterized amplified fragment length polymorphic (AFLP) markers and mating-type (MAT1-1 or MAT1-2). Segregation and linkage relationships among 164 AFLP markers and MAT were analysed using 59 progeny derived from an in vitro cross. Alleles at 158 AFLP loci and the mating-type locus segregated in a 1:1 ratio. Ten unlinked markers were chosen to determine genetic and genotypic diversity and to test the hypothesis of random mating and population differentiation among five subpopulations of T. yallundae representative of the geographical distribution of wheat production in eastern Washington. Among 228 isolates collected, overall gene diversity was high (h = 0.425) and a total of 91 unique multilocus genotypes (MLG) were identified, with 32 MLG occurring at least twice. The overall population genetic structure was consistent with random mating based on the segregation of mating-type, index of association (IA), parsimony tree length permutation test (PTLPT) and genotypic diversity analyses. However, clonal genotypes were found within each subpopulation and were also distributed among the five subpopulations. No significant differences in allele frequencies were found among the five subpopulations for all 10 loci based on contingency table analysis (G2) and Wier & Cockerham's population differentiation statistic theta (theta = -0.008, P = 0.722). T. yallundae appears to consist of a large homogeneous population throughout eastern Washington with both sexual and asexual reproduction contributing to the observed population genetic structure despite no report of sexual fruiting bodies of T. yallundae occurring under natural field conditions.     

Isolation and characterization of the mating-type locus of the barley pathogen Pyrenophora teres and frequencies of mating-type idiomorphs within and among fungal populations collected from barley landraces.

Pyrenophora teres f. sp. teres mating-type genes (MAT-1: 1190 bp; MAT-2: 1055 bp) have been identified. Their predicted proteins, measuring 379 and 333 amino acids, respectively, are similar to those of other Pleosporales, such as Pleospora sp., Cochliobolus sp., Alternaria alternata, Leptosphaeria maculans, and Phaeosphaeria nodorum. The structure of the MAT locus is discussed in comparison with those of other fungi. A mating-type PCR assay has also been developed; with this assay we have analyzed 150 isolates that were collected from 6 Sardinian barley landrace populations. Of these, 68 were P. teres f. sp. teres (net form; NF) and 82 were P. teres f. sp. maculata (spot form; SF). Within each mating type, the NF and SF amplification products were of the same length and were highly similar in sequence. The 2 mating types were present in both the NF and the SF populations at the field level, indicating that they have all maintained the potential for sexual reproduction. Despite the 2 forms being sympatric in 5 fields, no intermediate isolates were detected with amplified fragment length polymorphism (AFLP) analysis. These results suggest that the 2 forms are genetically isolated under the field conditions. In all of the samples of P. teres, the ratio of the 2 mating types was consistently in accord with the 1:1 null hypothesis. This ratio is expected when segregation distortion and clonal selection among mating types are absent or asexual reproduction is rare. Overall, sexual reproduction appears to be the major process that equalizes the frequencies of the 2 mating types within populations.     

A species-specific real-time PCR assay for identification of three lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica

We present a multiplex real-time PCR assay for the simultaneous identification of three morphologically similar species of lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica. Based on TaqMan MGB (minor groove binding) probes targeting the fungal internal transcribed spacer (ITS nrDNA) region, our assay unambiguously identifies known samples from all the three species, thus providing a powerful alternative to the more expensive DNA-sequencing techniques.     

Morphological and phylogenetic study of algal partners associated with the lichen-forming fungus <Emphasis Type="Italic">Tephromela atra</Emphasis> from the Mediterranean region

Recent DNA sequence analyses have revealed the diversity of algal partners in lichen symbioses. Although morphologically similar, different genetic lineages of photobionts are detected in wide geographic ranges of the same lichen fungal species. We studied the photobiont of the genus Trebouxia, which are known as partners of diverse lichen-forming fungal species in the Mediterranean region. We studied the phylogeny of these algae with a multilocus dataset including three loci: ITS, rbcL, and actin type I gene. The two lineages found, informally named Trebouxia sp. 1 and Trebouxia sp. 2, are related to Trebouxia arboricola/decolorans. The cultivation under axenic conditions succeeded only for one of them so far. We used light microscopy, confocal laser scanning microscopy and transmission electron microscopy for phenotypic characterisation. The ultrastructural characters currently used to describe species in the genus do not support the segregation of Trebouxia sp.1 from Trebouxia arboricola. The preferential presence in Mediterranean climates of these strains suggests eco-physiological adaptation. Despite their asexuality in long living lichen symbioses, coccoid algal lichen partners have apparently diversified genetically and physiologically.     

毛乌素沙地中间锦鸡儿群体繁育系统的变化

中间锦鸡儿在毛乌素沙地这个介于典型草原和典型荒漠之间的生态过渡带广泛分布 ,是当地灌丛生态系统的一个优势种或建群种[1,2 ] 。我们曾研究表明 ,中间锦鸡儿群体内高度的遗传多样性以及不同景观生态类型群体之间有强大的基因流存在[3 ,4 ] ;特别是同一植株不同种子显示的不同的种子蛋白组成进一步证明该植物的异交性[4 ,5] 。上述群体遗传研究还表明 ,中间锦鸡儿和柠条锦鸡儿共享 1个基因库 ,与徐朗然等 ① 报道的种间杂交结果一致。ＲＡＰＤ研究发现 ,少数多态ＤＮＡ在群体里的频率与所在地区的水分状况有关 :滩地为零或很低 ,生境越干…     

Variation of Breeding Systems in Populations of Caragana intermedia (Leguminosae ) in Maowusu Sandy Grassland

Maowusu sandy grassland locating at an ecotone between typical desert and typical grassland contained several landscape ecotypes or elements where Caragana intermedia Kuang et H.C.Fu naturally distributed as a dominant species in shrub ecosystems. Based on a phenomenon of gene segregation of open-pollinated seeds within each plants similar to Mendel's segregation in F2, a study on testing the breeding systems of populations under 4 landscape ecotypes was conducted. Statistical data showed its availability in estimation of breeding system parameters when isozymes were used as genetic markers. Nei's genetic differentiation GST among 4 ecotypes in Maowusu was estimated at 0.07 from lap loci close to a GST =0.076 reported in the authors' laboratory. The results indicated that breeding systems of populations gradually changed from total outbreeding to partial inbreeding when water conditions worsened. Therefore, the former RAPD data, especially the cline of frequencies for a few polymorphic DNAs in different landscapes can be partially explained by gene fixation caused by selfing or inbreeding probably induced by drought. It was difficult to assess breeding system parameters by using one dimensional SDS polyacrylamide gel electrophoresis of seed proteins of C. intermedia simply due to a difficulty of genetic analysis of seed protein subunits.     

The a and b loci of Ustilago maydis hybridize with DNA sequences from other smut fungi.

The smut fungi are obligately parasitic during the sexual phase of their life cycle, and the mating-type genes of these fungi play key roles in both sexual development and pathogenicity. Among species of smut fungi it is common to find a bipolar mating system in which one locus with two alternate alleles is believed to control cell fusion and establishment of the infectious cell type. Alternatively, several species have a tetrapolar mating system in which two different genetic loci, one of which has multiple alleles, control fusion and subsequent development of the infection hyphae. Cloned sequences from the a and b mating-type loci of the tetrapolar smut fungus Ustilago maydis were used as hybridization probes to DNAs from 23 different fungal strains, including smut fungi with both tetrapolar and bipolar mating systems. In general, all of the smut fungi hybridized with the mating-type genes from U. maydis, suggesting conservation of the sequences involved in mating interactions. A selection of DNAs from other ascomycete and basidiomycete fungi failed to hybridize with the U. maydis mating-type sequences. Exceptions to this finding include hybridization of DNA from the a1 idiomorph of U. maydis to DNA from one strain of U. violacea and hybridization of both a idiomorphs to DNA from Saccharomyces cerevisiae.