Association Mapping in Outbred Populations: Power and Efficiency When Genotyping Parents and Phenotyping Progeny

We develop expressions for the power to detect associations between parental genotypes and offspring phenotypes for quantitative traits. Three different “indirect” experimental designs are considered: full-sib, half-sib, and full-sib–half-sib families. We compare the power of these designs to detect genotype–phenotype associations relative to the common, “direct,” approach of genotyping and phenotyping the same individuals. When heritability is low, the indirect designs can outperform the direct method. However, the extra power comes at a cost due to an increased phenotyping effort. By developing expressions for optimal experimental designs given the cost of phenotyping relative to genotyping, we show how the extra costs associated with phenotyping a large number of individuals will influence experimental design decisions. Our results suggest that indirect association studies can be a powerful means of detecting allelic associations in outbred populations of species for which genotyping and phenotyping the same individuals is impractical and for life history and behavioral traits that are heavily influenced by environmental variance and therefore best measured on groups of individuals. Indirect association studies are likely to be favored only on purely economical grounds, however, when phenotyping is substantially less expensive than genotyping. A web-based application implementing our expressions has been developed to aid in the design of indirect association studies.     

Efficiency of New Dose Escalation Designs in Dose-Finding Phase I Trials of Molecularly Targeted Agents

Background

Statistical simulations have consistently demonstrated that new dose-escalation designs such as accelerated titration design (ATD) and continual reassessment method (CRM)-type designs outperform the standard “3+3” design in phase I cancer clinical trials.

Methods

We evaluated the actual efficiency of different dose escalation methods employed in first-in-human phase I clinical trials of targeted agents administered as single agents published over the last decade.

Results

Forty-nine per cent of the 84 retrieved trials used the standard “3+3” design. Newer designs used included ATD in 42%, modified CRM [mCRM] in 7%, and pharmacologically guided dose escalation in 1%. The median numbers of dose levels explored in trials using “3+3”, ATD and mCRM designs were 6, 8 and 10, respectively. More strikingly, the mean MTD to starting dose ratio appeared to be at least twice as high for trials using mCRM or ATD designs as for trials using a standard “3+3” design. Despite this, the mean number of patients exposed to a dose below the MTD was similar in trials using “3+3”, ATD and mCRM designs.

Conclusion

Our results support a more extensive implementation of innovative dose escalation designs such as mCRM and ATD in phase I cancer clinical trials of molecularly targeted agents.     

Anatomic Characteristics Associated with Head Splitting in Cabbage (Brassica oleracea var. capitata L.)

Cabbage belonging to Brassicaceae family is one of the most important vegetables cultivated worldwide. The economically important part of cabbage crop is head, formed by leaves which may be of splitting and non-splitting types. Cabbage varieties showing head splitting causes huge loss to the farmers and therefore finding the molecular and structural basis of splitting types would be helpful to breeders. To determine which anatomical characteristics were related to head-splitting in cabbage, we analyzed two contrasting cabbage lines and their offspring using a field emission scanning electron microscope. The inbred line “747” is an early head-splitting type, while the inbred line “748” is a head-splitting-resistant type. The petiole cells of “747” seems to be larger than those of “748” at maturity; however, there was no significant difference in petiole cell size at both pre-heading and maturity stages. The lower epidermis cells of “747” were larger than those of “748” at the pre-heading and maturity stages. “747” had thinner epidermis cell wall than “748” at maturity stage, however, there was no difference of the epidermis cell wall thickness in the two lines at the pre-heading stage. The head-splitting plants in the F₁ and F₂ population inherited the larger cell size and thinner cell walls of epidermis cells in the petiole. In the petiole cell walls of “747” and the F₁ and F₂ plants that formed splitting heads, the cellulose microfibrils were loose and had separated from each other. These findings verified that anomalous cellulose microfibrils, larger cell size and thinner-walled epidermis cells are important genetic factors that make cabbage heads prone to splitting.     

Genome-Wide Microsatellite Identification in the Fungus Anisogramma anomala Using Illumina Sequencing and Genome Assembly

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N₅₀ of 10,384 bp and scaffold N₅₀ of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.     

BAC-HAPPY Mapping (BAP Mapping): A New and Efficient Protocol for Physical Mapping

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical “contig” maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning ∼10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual “BAC-HAPPY-mapping” to convert BAC landing data into BAC linkage contigs is possible.     

Discriminating between the Effects of Founding Events and Reproductive Mode on the Genetic Structure of Triops Populations (Branchiopoda: Notostraca)

Crustaceans that initially colonize a freshwater temporary pond can strongly bias the subsequent genetic composition of the population, causing nearby populations to be genetically distinct. In addition, these crustaceans have various reproductive modes that can influence genetic differentiation and diversity within and between populations. We report on two species of tadpole shrimp, Triops newberryi and Triops longicaudatus “short”, with different reproductive modes. Reproduction in the tadpole shrimp can occur clonally (parthenogenesis), with self fertilization (hermaphroditism), or through outcrossing of hermaphrodites with males (androdioecy). For all these reproductive modes, population genetic theory predicts decreased genetic diversity and increased population differentiation. Here we use mitochondrial control region (mtCR) sequences and nuclear microsatellite loci to determine if the difference in reproductive mode affects the high genetic structure typical of persistent founder effects. Previous authors indicated that T. newberryi is androdioecious because populations are composed of hermaphrodites and males, and T. longicaudatus “short” is hermaphroditic or parthenogenetic because males are absent. In our data, T. newberryi and T. longicaudatus “short” populations were highly structured genetically over short geographic distances for mtCR sequences and microsatellite loci (T. newberryi: Φ_ST = 0.644, F _ST = 0.252, respectively; T. l. “short”: invariant mtCR sequences, F _ST = 0.600). Differences between the two Triops species in a number of diversity measures were generally consistent with expectations from population genetic theory regarding reproductive mode; however, three of four comparisons were not statistically significant. We conclude the high genetic differentiation between populations is likely due to founder effects and results suggest both species are composed of selfing hermaphrodites with some level of outcrossing; the presence of males in T. newberryi does not appreciably reduce inbreeding. We cannot exclude the possibility that males in T. newberryi are non-reproductive individuals and the two species have the same mating system.     

Haldane’s Rule Is Linked to Extraordinary Sex Ratios and Sperm Length in Stalk-Eyed Flies

We use three allopatric populations of the stalk-eyed fly Teleopsis dalmanni from Southeast Asia to test two predictions made by the sex chromosome drive hypothesis for Haldane’s rule. The first is that modifiers that suppress or enhance drive should evolve rapidly and independently in isolated populations. The second is that drive loci or modifiers should also cause sterility in hybrid males. We tested these predictions by assaying the fertility of 2066 males derived from backcross experiments involving two pairs of populations and found that the proportion of mated males that fail to produce any offspring ranged from 38 to 60% among crosses with some males producing strongly female-biased or male-biased sex ratios. After genotyping each male at 25–28 genetic markers we found quantitative trait loci (QTL) that jointly influence male sterility, sperm length, and biased progeny sex ratios in each pair of populations, but almost no shared QTL between population crosses. We also discovered that the extant X^SR chromosome has no effect on sex ratio or sterility in these backcross males. Whether shared QTL are caused by linkage or pleiotropy requires additional study. Nevertheless, these results indicate the presence of a “cryptic” drive system that is currently masked by suppressing elements that are associated with sterility and sperm length within but not between populations and, therefore, must have evolved since the populations became isolated, i.e., in <100,000 years. We discuss how genes that influence sperm length may contribute to hybrid sterility.     

A general mixture model approach for mapping quantitative trait loci from diverse cross designs involving multiple inbred lines

Most current statistical methods developed for mapping quantitative trait loci (QTL) based on inbred line designs apply to crosses from two inbred lines. Analysis of QTL in these crosses is restricted by the parental genetic differences between lines. Crosses from multiple inbred lines or multiple families are common in plant and animal breeding programmes, and can be used to increase the efficiency of a QTL mapping study. A general statistical method using mixture model procedures and the EM algorithm is developed for mapping QTL from various cross designs of multiple inbred lines. The general procedure features three cross design matrices, W, that define the contribution of parental lines to a particular cross and a genetic design matrix, D, that specifies the genetic model used in multiple line crosses. By appropriately specifying W matrices, the statistical method can be applied to various cross designs, such as diallel, factorial, cyclic, parallel or arbitrary-pattern cross designs with two or multiple parental lines. Also, with appropriate specification for the D matrix, the method can be used to analyse different kinds of cross populations, such as F2 backcross, four-way cross and mixed crosses (e.g. combining backcross and F2). Simulation studies were conducted to explore the properties of the method, and confirmed its applicability to diverse experimental designs.     

Genetic Distinctions among Clinical and Environmental Strains of Vibrio vulnificus

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n = 35) or clinical (n = 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical “clinical” or “environmental” polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the “environmental” profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical “clinical” profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the “clinical” polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.     

Subtle Population Genetic Structure in Yelloweye Rockfish (Sebastes ruberrimus) Is Consistent with a Major Oceanographic Division in British Columbia,Canada

The boundaries between oceanographic domains often function as dispersal barriers for many temperate marine species with a dispersive pelagic larval phase. Yelloweye rockfish (Sebastes ruberrimus, YR) are widely distributed across the northeastern Pacific Ocean, inhabiting coastal rocky reefs from the Aleutian Islands in Alaska through southern California. This species exhibits an extended pelagic larval duration and has the capacity for long distance larval transport. We assayed 2,862 YR individuals from 13 general areas in the northeast Pacific Ocean for allelic variation at nine microsatellite loci. Bayesian model-based clustering analyses grouped individuals from the Strait of Georgia (SG) into a distinct genetic cluster, while individuals from outer coastal water locations (OCLs) were partitioned equally across two genetic clusters, including the cluster associated with the SG fish. Pairwise F_ST values were consistently an order of magnitude higher for comparisons between the SG and OCLs than they were for all OCL-OCL comparisons (∼0.016 vs. ∼0.001). This same pattern was observed across two time points when individuals were binned into an “old” and “young” group according to birth year (old: ∼0.020 vs. 0.0003; young: ∼0.020 vs. ∼0.004). Additionally, mean allelic richness was markedly lower within the SG compared to the OCLs (8.00 vs. 10.54–11.77). These results indicate that the Strait of Georgia “deep-basin” estuary oceanographic domain acts as a dispersal barrier from the outer coastal waters via the Juan de Fuca Strait. Alternatively, selection against maladapted dispersers across this oceanographic transition may underlie the observed genetic differentiation between the Georgia basin and the outer coastal waters, and further work is needed to confirm the SG-OCL divide acts as a barrier to larval dispersal.     

Breeding designs for recombinant inbred advanced intercross lines

Recombinant inbred lines derived from an advanced intercross, in which multiple generations of mating have increased the density of recombination breakpoints, are powerful tools for mapping the loci underlying complex traits. We investigated the effects of intercross breeding designs on the utility of such lines for mapping. The simplest design, random pair mating with each pair contributing exactly two offspring to the next generation, performed as well as the most extreme inbreeding avoidance scheme at expanding the genetic map, increasing fine-mapping resolution, and controlling genetic drift. Circular mating designs offer negligible advantages for controlling drift and exhibit greatly reduced map expansion. Random-mating designs with variance in offspring number are also poor at increasing mapping resolution. Given equal contributions of each parent to the next generation, the constraint of monogamy has no impact on the qualities of the final population of inbred lines. We find that the easiest crosses to perform are well suited to the task of generating populations of highly recombinant inbred lines.     

Genetic Composition of Laboratory Stocks of the Self-Fertilizing Fish Kryptolebias marmoratus: A Valuable Resource for Experimental Research

The hermaphroditic Mangrove Killifish, Kryptolebias marmoratus, is the world''s only vertebrate that routinely self-fertilizes. As such, highly inbred and presumably isogenic “clonal” lineages of this androdioecious species have long been maintained in several laboratories and used in a wide variety of experiments that require genetically uniform vertebrate specimens. Here we conduct a genetic inventory of essentially all laboratory stocks of the Mangrove Killifish held worldwide. At 32 microsatellite loci, these stocks proved to show extensive interline differentiation as well as some intraline variation, much of which can be attributed to post-origin de novo mutations and/or to the segregation of polymorphisms from wild progenitors. Our genetic findings also document that many of the surveyed laboratory strains are not what they have been labeled, apparently due to the rather frequent mishandling or unintended mixing of various laboratory stocks over the years. Our genetic inventory should help to clarify much of this confusion about the clonal identities and genetic relationships of laboratory lines, and thereby help to rejuvenate interest in K. marmoratus as a reliable vertebrate model for experimental research that requires or can capitalize upon “clonal” replicate specimens.     

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.     

Genetic Architecture Underlying Nascent Speciation—The Evolution of Eurasian Pigs under Domestication

Speciation is a process whereby the evolution of reproductive barriers leads to isolated species. Although many studies have addressed large-effect genetic footprints in the advanced stages of speciation, the genetics of reproductive isolation in nascent stage of speciation remains unclear. Here, we show that pig domestication offers an interesting model for studying the early stages of speciation in great details. Pig breeds have not evolved the large X-effect of hybrid incompatibility commonly observed between “good species.” Instead, deleterious epistatic interactions among multiple autosomal loci are common. These weak Dobzhansky–Muller incompatibilities confer partial hybrid inviability with sex biases in crosses between European and East Asian domestic pigs. The genomic incompatibility is enriched in pathways for angiogenesis, androgen receptor signaling and immunity, with an observation of many highly differentiated cis-regulatory variants. Our study suggests that partial hybrid inviability caused by pervasive but weak interactions among autosomal loci may be a hallmark of nascent speciation in mammals.     

Mapping quantitative trait loci in noninbred mosquito crosses

The identification of genes that affect quantitative traits has been of great interest to geneticists for many decades, and many statistical methods have been developed to map quantitative trait loci (QTL). Most QTL mapping studies in experimental organisms use purely inbred lines, where the two homologous chromosomes in each individual are identical. As a result, many existing QTL mapping methods developed for experimental organisms are applicable only to genetic crosses between inbred lines. However, it may be difficult to obtain inbred lines for certain organisms, e.g., mosquitoes. Although statistical methods for QTL mapping in outbred populations, e.g., humans, can be applied for such crosses, these methods may not fully take advantage of the uniqueness of these crosses. For example, we can generally assume that the two grandparental lines are homozygous at the QTL of interest, but such information is not be utilized through methods developed for outbred populations. In addition, mating types and phases can be relatively easy to establish through the analysis of adjacent markers due to the large number of offspring that can be collected, substantially simplifying the computational need. In this article, motivated by a mosquito intercross experiment involving two selected lines that are not genetically homozygous across the genome, we develop statistical methods for QTL mapping for genetic crosses involving noninbred lines. In our procedure, we first infer parental mating types and use likelihood-based methods to infer phases in each parent on the basis of genotypes of offspring and one parent. A hidden Markov model is then employed to estimate the number of high-risk alleles at marker positions and putative QTL positions between markers in each offspring, and QTL mapping is finally conducted through the inferred QTL configuration across all offspring in all crosses. The performance of the proposed methods is assessed through simulation studies, and the usefulness of this method is demonstrated through its application to a mosquito data set.     

Analysis of ancestry heterozygosity suggests that hybrid incompatibilities in threespine stickleback are environment dependent

Hybrid incompatibilities occur when interactions between opposite ancestry alleles at different loci reduce the fitness of hybrids. Most work on incompatibilities has focused on those that are “intrinsic,” meaning they affect viability and sterility in the laboratory. Theory predicts that ecological selection can also underlie hybrid incompatibilities, but tests of this hypothesis using sequence data are scarce. In this article, we compiled genetic data for F₂ hybrid crosses between divergent populations of threespine stickleback fish (Gasterosteus aculeatus L.) that were born and raised in either the field (seminatural experimental ponds) or the laboratory (aquaria). Because selection against incompatibilities results in elevated ancestry heterozygosity, we tested the prediction that ancestry heterozygosity will be higher in pond-raised fish compared to those raised in aquaria. We found that ancestry heterozygosity was elevated by approximately 3% in crosses raised in ponds compared to those raised in aquaria. Additional analyses support a phenotypic basis for incompatibility and suggest that environment-specific single-locus heterozygote advantage is not the cause of selection on ancestry heterozygosity. Our study provides evidence that, in stickleback, a coarse—albeit indirect—signal of environment-dependent hybrid incompatibility is reliably detectable and suggests that extrinsic incompatibilities can evolve before intrinsic incompatibilities.

This study shows that hybrid incompatibilities between two independent pairs of hybridizing stickleback populations only appear under relevant ecological circumstances, implying that incompatibilities evolve before they can be detected in laboratory studies of speciation.     

Evaluation of soybean RFLP marker diversity in adapted germ plasm

Summary Soybean RFLP markers have been primarily developed and genetically mapped using wide crosses between exotic and adapted genotypes. We have screened 38 soybean lines at 128 RFLP marker loci primarily to characterize germ plasm structure but also to evaluate the utility of RFLP markers identified in unadapted populations. Of these DNA probes 70% detected RFLPs in this set of soybean lines with an average polymorphism index of 0.30. This means that only 1 out of 5 marker loci was informative between any particular pair of adapted soybean lines. The variance associated with the estimation of RFLP genetic distance (GD_R) was determined, and the value obtained suggested that the use of more than 65–90 marker loci for germ plasm surveys will add little precision. Cluster analysis and principal coordinate analysis of the GD_R matrix revealed the relative lack of diversity in adapted germ plasm. Within the cultivated lines, several lines adapted to Southern US maturity zones also appeared as a separate group. GD_R data was compared to the genetic distance estimates obtained from pedigree analysis (GD_P). These two measures were correlated with r = 0.54 for all 38 lines, but the correlation increased to r = 0.73 when only adapted lines were analyzed.     

Rare Branched Fatty Acids Characterize the Lipid Composition of the Intra-Aerobic Methane Oxidizer “Candidatus Methylomirabilis oxyfera”

The recently described bacterium “Candidatus Methylomirabilis oxyfera” couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of “Ca. Methylomirabilis oxyfera” is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of “Ca. Methylomirabilis oxyfera” to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple “Ca. Methylomirabilis oxyfera” enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC_16:0). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the Δ7 position (10MeC_16:1Δ7), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC_16:0 and 10MeC_16:1Δ7 are key and characteristic components of the lipid profile of “Ca. Methylomirabilis oxyfera.” The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.     

Molecular Standardization of Mating Type Terminology in the Gibberella fujikuroi Species Complex

Mating type in the Gibberella fujikuroi species complex is controlled by a single locus with two alleles and is usually identified following sexual crosses with standard, female-fertile tester isolates. The mating type alleles have been arbitrarily designated “+” and “−” within each biological species, and the nomenclature is tied to the standard tester strains. We developed a pair of PCR primers that can be used to amplify a unique fragment of one of the mating type alleles (MAT-2) from at least seven of the biological species in this species complex. Based on the amplification pattern, we propose a replacement for the existing, arbitrary +/− terminology that is presently in use. The new terminology is based on DNA sequence similarities between the mating type allele fragments from the biological species of the G. fujikuroi species complex and the corresponding fragments from other filamentous ascomycetes.