Sexual Reproduction in Aspergillus flavus Sclerotia: Acquisition of Novel Alleles from Soil Populations and Uniparental Mitochondrial Inheritance

Aspergillus flavus colonizes agricultural commodities worldwide and contaminates them with carcinogenic aflatoxins. The high genetic diversity of A. flavus populations is largely due to sexual reproduction characterized by the formation of ascospore-bearing ascocarps embedded within sclerotia. A. flavus is heterothallic and laboratory crosses between strains of the opposite mating type produce progeny showing genetic recombination. Sclerotia formed in crops are dispersed onto the soil surface at harvest and are predominantly produced by single strains of one mating type. Less commonly, sclerotia may be fertilized during co-infection of crops with sexually compatible strains. In this study, laboratory and field experiments were performed to examine sexual reproduction in single-strain and fertilized sclerotia following exposure of sclerotia to natural fungal populations in soil. Female and male roles and mitochondrial inheritance in A. flavus were also examined through reciprocal crosses between sclerotia and conidia. Single-strain sclerotia produced ascospores on soil and progeny showed biparental inheritance that included novel alleles originating from fertilization by native soil strains. Sclerotia fertilized in the laboratory and applied to soil before ascocarp formation also produced ascospores with evidence of recombination in progeny, but only known parental alleles were detected. In reciprocal crosses, sclerotia and conidia from both strains functioned as female and male, respectively, indicating A. flavus is hermaphroditic, although the degree of fertility depended upon the parental sources of sclerotia and conidia. All progeny showed maternal inheritance of mitochondria from the sclerotia. Compared to A. flavus populations in crops, soil populations would provide a higher likelihood of exposure of sclerotia to sexually compatible strains and a more diverse source of genetic material for outcrossing.     

Heterothallic life cycle in the white root rot fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

To prepare homologous DNA fragments as restriction fragment length polymorphism (RFLP) markers, the genes encoding phenol oxidase, chitinase, and xylanase were amplified from genomic DNA of Rosellinia necatrix strains. RFLP analysis using the amplified DNA fragments as probe was carried out, with segregation of the markers among two sets of F₁ progenies isolated from an independent perithecium. RFLP was frequently found using rpo1 as the RFLP marker among strains of R. necatrix, which was isolated from single ascospores and the circumference of the perithecium. In each set, RFLPs of some F₁ progenies were different from that of the parent strain. Random amplified polymorphic DNA (RAPD) also revealed that several strains, which were of different genotypes from the parent strain, were contained in the single ascospore culture isolated from the same perithecium. From these results, it is suggested that another strain, which was genetically different, was required for mating and development of the ascus in R. necatrix. Therefore, the life cycle in R. necatrix was presumed to be heterothallism. This is the first report about a heterothallic life cycle in R. necatrix.     

Suppressed recombination and a pairing anomaly on the mating-type chromosome of Neurospora tetrasperma

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, Merino et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.     

A high-resolution map of Arabidopsis recombinant inbred lines by whole-genome exon array hybridization

Recombinant populations were the basis for Mendel's first genetic experiments and continue to be key to the study of genes, heredity, and genetic variation today. Genotyping several hundred thousand loci in a single assay by hybridizing genomic DNA to oligonucleotide arrays provides a powerful technique to improve precision linkage mapping. The genotypes of two accessions of Arabidopsis were compared by using a 400,000 feature exon-specific oligonucleotide array. Around 16,000 single feature polymorphisms (SFPs) were detected in ~8,000 of the ~26,000 genes represented on the array. Allelic variation at these loci was measured in a recombinant inbred line population, which defined the location of 815 recombination breakpoints. The genetic linkage map had a total length of 422.5 cM, with 676 informative SFP markers representing intervals of ~0.6 cM. One hundred fifteen single gene intervals were identified. Recombination rate, SFP distribution, and segregation in this population are not uniform. Many genomic regions show a clustering of recombination events including significant hot spots. The precise haplotype structure of the recombinant population was defined with unprecedented accuracy and resolution. The resulting linkage map allows further refinement of the hundreds of quantitative trait loci identified in this well-studied population. Highly variable recombination rates along each chromosome and extensive segregation distortion were observed in the population.     

Sexual Reproduction as the Cause of Heat Resistance in the Food Spoilage Fungus Byssochlamys spectabilis (Anamorph Paecilomyces variotii)

Paecilomyces variotii is a common cosmopolitan species that is able to spoil various food- and feedstuffs and is frequently encountered in heat-treated products. However, isolates from heat-treated products rarely form ascospores. In this study we examined by using molecular techniques and mating tests whether this species can undergo a sexual cycle and form ascospores. The population structure of this species was examined by analyzing the nuclear ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and the 5.8S rRNA gene, as well as partial β-tubulin, actin, and calmodulin gene sequences. Phylogenetic analyses revealed that P. variotii is a highly variable species. Partition homogeneity tests revealed that P. variotii has a recombining population structure. In addition to sequence analyses, mating experiments indicated that P. variotii is able to form ascomata and ascospores in culture in a heterothallic manner. The distribution of MAT1-1 and MAT1-2 genes showed a 1:1 ratio in the progeny of the mating experiments. From the sequence analyses and mating data we conclude that P. variotii is the anamorph of Talaromyces spectabilis and that it has a biallelic heterothallic mating system. Since Paecilomyces sensu stricto anamorphs group within Byssochlamys, a new combination Byssochlamys spectabilis is proposed.     

RFLP markers show genetic recombination in Botryotinia fuckeliana (Botrytis cinerea) and transposable elements reveal two sympatric species

Molecular markers revealed that Botryotinia fuckeliana (the teleomorph of Botrytis cinerea), a haploid, filamentous, heterothallic ascomycete, contained a large amount of intrapopulation genetic variation. The markers were used to determine the mode of reproduction and the population structure of this fungus. We did not detect any differentiation between isolates from different organs, collection dates, varieties of grape, or locations in the Champagne region of France, but two unexpected sympatric populations were identified. One group of isolates (transposa) contained the transposable elements Boty and Flipper; the other (vacuma) did not. These groups differed from one another for all the other markers. RFLP markers showed that there was genetic recombination in both groups of isolates. We conclude that there are two sympatric populations of B. fuckeliana in Champagne. One species (transposa) seems to be local and well adapted, while the other one (vacuma) is presumably a heterogeneous migrant population.      

Complementation and Genetic Recombination in Candida lipolytica

Nutritional requirements were introduced into wild-type, heterothallic strains of Candida lipolytica by exposing the cells to X rays. Complementing hybrids were recovered from mixtures of the auxotrophic strains, and genetic recombination was observed in individually isolated ascospores from the hybrid strains.     

Mating behavior of Phytophthora parasitica: Evidence for sexual recombination in oospores using DNA restriction fragment length polymorphisms as genetic markers

Cloned nuclear DNA fragments that detected restriction fragment length polymorphisms (RFLPs) in homozygous loci of isolates of Phytophthora parasitica were used as genetic markers to investigate sexual recombination during oospore formation. It was found that the majority of the 23 oospore progeny studied in each of the two crosses carried both of the parental markers. However, aberrant recombination patterns were observed; some of the progeny were homozygous at one RFLP locus, whereas at another locus both of the parental markers were present. Only two of the progeny of each cross did not show sexual recombination with any of the four or five RFLP markers used. Mitochondrial DNA (mtDNA) was uniparentally inherited. In both crosses the majority of the progeny carried the mtDNA type of one of the common parental strains.     

Discovery of a Sexual Cycle in Aspergillus lentulus,a Close Relative of A. fumigatus

Aspergillus lentulus was described in 2005 as a new species within the A. fumigatus sensu lato complex. It is an opportunistic human pathogen causing invasive aspergillosis with high mortality rates, and it has been isolated from clinical and environmental sources. The species is morphologically nearly identical to A. fumigatus sensu stricto, and this similarity has resulted in their frequent misidentification. Comparative studies show that A. lentulus has some distinguishing growth features and decreased in vitro susceptibility to several antifungal agents, including amphotericin B and caspofungin. Similar to the once-presumed-asexual A. fumigatus, it has only been known to reproduce mitotically. However, we now show that A. lentulus has a heterothallic sexual breeding system. A PCR-based mating-type diagnostic detected isolates of either the MAT1-1 or MAT1-2 genotype, and examination of 26 worldwide clinical and environmental isolates revealed similar ratios of the two mating types (38% versus 62%, respectively). MAT1-1 and MAT1-2 idiomorph regions were analyzed, revealing the presence of characteristic alpha and high-mobility-group (HMG) domain genes, together with other more unusual features such as a MAT1-2-4 gene. We then demonstrated that A. lentulus possesses a functional sexual cycle with mature cleistothecia, containing heat-resistant ascospores, being produced after 3 weeks of incubation. Recombination was confirmed using molecular markers. However, isolates of A. lentulus failed to cross with highly fertile strains of A. fumigatus, demonstrating reproductive isolation between these sibling species. The discovery of the A. lentulus sexual stage has significant implications for the management of drug resistance and control of invasive aspergillosis associated with this emerging fungal pathogen.     

Linkage Mapping Identifies the Sex Determining Region as a Single Locus in the Pennate Diatom Seminavis robusta

The pennate diatom Seminavis robusta, characterized by an archetypical diatom life cycle including a heterothallic mating system, is emerging as a model system for studying the molecular regulation of the diatom cell and life cycle. One of its main advantages compared with other diatom model systems is that sexual crosses can be made routinely, offering unprecedented possibilities for forward genetics. To date, nothing is known about the genetic basis of sex determination in diatoms. Here, we report on the construction of mating type-specific linkage maps for S. robusta, and use them to identify a single locus sex determination system in this diatom. We identified 13 mating type plus and 15 mating type minus linkage groups obtained from the analysis of 463 AFLP markers segregating in a full-sib family, covering 963.7 and 972.2 cM, respectively. Five linkage group pairs could be identified as putative homologues. The mating type phenotype mapped as a monogenic trait, disclosing the mating type plus as the heterogametic sex. This study provides the first evidence for a genetic sex determining mechanism in a diatom.     

Population structure of two beetle-associated yeasts: comparison of a New World asexual and an endemic Nearctic sexual species in the <Emphasis Type="Italic">Metschnikowia</Emphasis> clade

The genetic structure of two related yeast species, one sexual and one asexual, was compared using polymorphic DNA markers. Although both yeasts propagate by asexual budding of haploid cells, Metschnikowia borealis reproduces sexually when compatible strains come in contact. To what extent this has occurred in nature was not known. As Candida ipomoeae is a closely related, asexual species, the two yeasts provide an excellent model system to assess the role of sexual reproduction in a biogeographic context. Natural isolates of the two species were characterized using several polymorphic DNA markers. As predicted for an organism whose reproduction is strictly clonal, C. ipomoeae exhibited low haplotype diversity, high linkage disequilibrium, and high population differentiation. In contrast, M. borealis had unique haplotypes in most isolates, lower population differentiation, and little linkage disequilibrium, demonstrating that sexual recombination is prevalent. Geographic gradients were identified in both species, indicating that historical and climatic factors both play a role in shaping the populations. The spatial structure is also thought to be influenced by the ecology of the small floricolous beetles (family Nitidulidae) that vector the yeasts. For example, Hawaiian strains of C. ipomoeae show evidence of having undergone a genetic bottleneck, most likely when the vector was introduced to the islands. The two haplotypes found in Hawaii were nearly identical and were also found in North and Central America. M. borealis had a more continuous distribution where the genetic markers follow latitudinal and longitudinal gradients.     

Ascospores of large-spored Metschnikowia species are genuine meiotic products of these yeasts

The asci of Metschnikowia species normally contain two ascospores (never more), raising the question of whether these spores are true meiotic products. We investigated this problem by crossing genetically-marked strains of the haploid, heterothallic taxa Metschnikowia hawaiiensis, Metschnikowia continentalis var. continentalis, and M. continentalis var. borealis. Asci were dissected and the segregation patterns for various phenotypes analyzed. In all cases (n = 47) both mating types (h+ and h-) were recovered in pairs of sister spores, casting further uncertainty as to whether normal meiosis takes place. However, the segregation patterns for cycloheximide resistance and several auxotrophic markers were random, suggesting that normal meiosis indeed occurs. To explain the lack of second-division segregation of mating types, we concluded that crossing-over does not occur between the mating-type locus and the centromere, and that meiosis I is tied to spore formation, which explains why the number of spores is limited to two. The latter assumption was also supported by fluorescence microscopy. The second meiotic division takes place inside the spores and is followed by the resorption of two nuclei, one in each spore.     

AFLP Linkage Map of the OomycetePhytophthora infestans

Here we present the first comprehensive genetic linkage map of the heterothallic oomycetous plant pathogenPhytophthora infestans.The map is based on polymorphic DNA markers generated by the DNA fingerprinting technique AFLP (Voset al.,1995,Nucleic Acids Res.23:4407–4414). AFLP fingerprints were made from single zoospore progeny and 73 F1 progeny from two field isolates ofP. 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.     

mei-2, a mutagen-sensitive mutant of Neurospora defective in chromosome pairing and meiotic recombination

Summary A Neurospora crassa mutation, mei-2, affecting meiosis and mutagen sensitivity, was characterized for its effect on meiotic recombination and chromosome pairing. Results from homozygous mei-2 crosses involving distant markers on the same chromosome demonstrated a drastic reduction in meiotic recombination. However, mitotic recombination continued to occur. Cytological observations indicated that pairing of homologous chromosomes in zygotene was greatly reduced or absent, resulting in aberrant segregation at anaphase I and often at subsequent divisions as well. The few mature ascospores produced were frequently disomic for one or more chromosomes.     

Clonality Despite Sex: The Evolution of Host-Associated Sexual Neighborhoods in the Pathogenic Fungus Penicillium marneffei

Molecular genetic approaches typically detect recombination in microbes regardless of assumed asexuality. However, genetic data have shown the AIDS-associated pathogen Penicillium marneffei to have extensive spatial genetic structure at local and regional scales, and although there has been some genetic evidence that a sexual cycle is possible, this haploid fungus is thought to be genetically, as well as morphologically, asexual in nature because of its highly clonal population structure. Here we use comparative genomics, experimental mixed-genotype infections, and population genetic data to elucidate the role of recombination in natural populations of P. marneffei. Genome wide comparisons reveal that all the genes required for meiosis are present in P. marneffei, mating type genes are arranged in a similar manner to that found in other heterothallic fungi, and there is evidence of a putatively meiosis-specific mutational process. Experiments suggest that recombination between isolates of compatible mating types may occur during mammal infection. Population genetic data from 34 isolates from bamboo rats in India, Thailand and Vietnam, and 273 isolates from humans in China, India, Thailand, and Vietnam show that recombination is most likely to occur across spatially and genetically limited distances in natural populations resulting in highly clonal population structure yet sexually reproducing populations. Predicted distributions of three different spatial genetic clusters within P. marneffei overlap with three different bamboo rat host distributions suggesting that recombination within hosts may act to maintain population barriers within P. marneffei.     

Relationship between Monokaryotic Growth Rate and Mating Type in the Edible Basidiomycete Pleurotus ostreatus

The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBα and matBβ) and to correlate the presence of specific matBα and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.     

Identification of Glomerella cingulata f. sp phaseoli recombinants by RAPD markers

We examined the capacity of strains of Glomerella cingulata f. sp phaseoli fungus (Colletotrichum lindemuthianum sexual stage) to form recombinants, using random amplified polymorphic DNA (RAPD). Crosses of all possible combinations between strains 40, 42, 20, 21, 22, 23, 24, 25, and 26 were made on Petri dishes using M3 culture medium. The 42 x 21 cross produced the largest number of perithecia and five asci; the respective ascospores were isolated. RAPD analysis was performed on the parents and descendants. The 62 polymorphic RAPD bands obtained were used to assess the genetic similarity using the method of Sorence and Dice and clustering analysis in the form of a dendrogram by the UPGMA method. The RAPD markers allowed identification of recombinants from the cross between strains 42 and 21 of G. cingulata f. sp phaseoli and 40 ascospores presented 63 and 49% genetic similarity with parents 2 (strain 42) and 1 (strain 21), respectively.     

Description of Gibberella sacchari and neotypification of its anamorph Fusarium sacchari

We described the teleomorph of Fusarium sacchari as Gibberella sacchari, sp. nov. This species can be separated from other species of Gibberella on the basis of the longer, narrower ascospores found in G. sacchari and by sexual cross fertility. Female-fertile mating type tester strains were developed that can be used for making sexual crosses with this heterothallic fungus under laboratory conditions. The anamorph, Fusarium sacchari, was neotypified.     

RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS (RAPDs) AS TOOLS FOR GENE MAPPING IN CHLAMYDOMONAS EUGAMETOS (CHLOROPHYTA)1

Studying the sexual behavior of the heterothallic green alga Chlamydomonas eugametos Moewus has revealed genetic differences among the few available strains: different O-methylated sugar patterns on plasma membrane glycoproteins (oms A/B locus) and light-(in)dependent sexual agglutinin activation (lsa locus). However, due to the lack of a reliable linkage map, genes controlling these traits are not accessible by map-based cloning. Here we present a partial linkage map for Chlamydomonas eugametos based on random amplified polymorphic DNA (RAPD) markers and one restriction fragment-length polymorphism (RFLP) marker. Most of these RAPDs (80%) represent unique DNA sequences, which implies that they can be used as starting points for chromosome walking. RAPDs linked to the lsa locus, the mating type locus (mt), and the oms A/B locus were identified by bulk segregant analysis.     

Effect of sexual recombination on population diversity in aflatoxin production by Aspergillus flavus and evidence for cryptic heterokaryosis

Aspergillus flavus is the major producer of carcinogenic aflatoxins (AFs) in crops worldwide. Natural populations of A. flavus show tremendous variation in AF production, some of which can be attributed to environmental conditions, differential regulation of the AF biosynthetic pathway and deletions or loss‐of‐function mutations in the AF gene cluster. Understanding the evolutionary processes that generate genetic diversity in A. flavus may also explain quantitative differences in aflatoxigenicity. Several population studies using multilocus genealogical approaches provide indirect evidence of recombination in the genome and specifically in the AF gene cluster. More recently, A. flavus has been shown to be functionally heterothallic and capable of sexual reproduction in laboratory crosses. In the present study, we characterize the progeny from nine A. flavus crosses using toxin phenotype assays, DNA sequence‐based markers and array comparative genome hybridization. We show high AF heritability linked to genetic variation in the AF gene cluster, as well as recombination through the independent assortment of chromosomes and through crossing over within the AF cluster that coincides with inferred recombination blocks and hotspots in natural populations. Moreover, the vertical transmission of cryptic alleles indicates that while an A. flavus deletion strain is predominantly homokaryotic, it may harbour AF cluster genes at a low copy number. Results from experimental matings indicate that sexual recombination is driving genetic and functional hyperdiversity in A. flavus. The results of this study have significant implications for managing AF contamination of crops and for improving biocontrol strategies using nonaflatoxigenic strains of A. flavus.