Discrete Phenotypes Research Articles

Embryonic Erythropoiesis and Definitive Hematopoiesis from Human Embryonic Stem Cells Is Regulated by Cytokines Controlling HSC Growth.

Human embryonic stem cells (hESC) provide an invaluable tool for studying the earliest events in human hematopoietic stem cell (HSC) development. We describe novel protocols for the efficient, step-wise differentiation of hESC to embryonic (primitive) erythroid cells and definitive erythro-myeloid cells from embryoid bodies (EB) in semi-solid and liquid cultures. EB cells were re-cultured in semisolid cultures with a cocktail of hematopoietic growth factors at different time points using a modified EB differentiation protocol, and hematopoietic differentiation was analyzed in vitro.The initiation of hematopoiesis, in this model, begins during the first week of EB differentiation. with the formation of primitive macrophages and CD31+/VE-cadherin+ hemato-endothelial clusters that “bud off” primitive embryonic hemoglobin-expressing erythroblasts and multi-potential blast colonies. These clusters ultimately form organized yolk-sac-like structures which produce a loosely adherent primitive hematopoietic cells. After 7–9 days of EB differentiation (prior to CD45 expression), primitive nucleated erythroblast colonies arise and are characterized by a “brilliant red” hemoglobinization under phase microscopy, positivity for embryonic/fetal hemoglobins by Kleihauer-Betke, qRT-PCR assays for epsilon/zeta/gamma chain expression, and a CD71+/glycophorin A+ phenotype. Simultaneously, discrete blast colonies are also shown to develop into mixed multipotential colonies containing secondary erythro-myeloid blast cells, primitive erythroblasts, and macrophages; suggesting a common progenitor for the discrete embryonic phenotypes observed at this stage. Following this first wave of primitive hematopoiesis, definitive CD45+-expressing colony-forming cells (CFC) can be generated from EB cells differentiated for 10–15 days with the sequential appearance of BFU-E, CFU-E, GM-CFC, and multi-lineage CFC. A kinetic expression analysis using qRT-PCR methods, revealed that the first wave of embryonic hematopoiesis at 6–9 days of EB development directly coincides with expression of SCL/TAL1, AML1, GATA1, and GATA2, while the onset of definitive hematopoiesis at 9–15 days directly correlates with increased EB expression of CD34, CD31, CD41, c-myb, and cdx4.In this model, primitive hematopoiesis in EB cells proceeds in the absence of exogenously added growth factors. However, supplementing EB differentiation cultures with FLT3-ligand, KIT-ligand, and THROMBOPOIETIN (FTK), dramatically enhances the number of primitive erythroblast, and multi-lineage blast CFC, as well as the definitive BFU-E, CFU-E, and multi-potent mixed CFC. The kinetics of colony formation for both primitive and definitive CFC is unaffected by FTK supplementation. Moreover, blast cell colonies from EB cells differentiated in the presence of FTK were more potent than those generated without FTK. These blast colonies differentiate into mixed, multi-lineage CD45+/CD13+/CD41+/CD71+/glycophorin A+-expressing colonies that contain both primitive nucleated embryonic hemoglobin-expressing erythroblasts, and definitive mature beta-globin-expressing erythroid cells, neutrophils, monocytes/macrophages, and megakaryocytic precursors. This hESC model reveals the putative existence of a common human progenitor for both embryonic-type and definitive hematopoietic cells, and that cytokines known to expand/self-renew definitive HSC may potentially regulate this differentiation process.

CELLULAR PHENOTYPES AFFECTED BY INDUCTION THERAPY WITH CAMPATH-1H VS THYMOGLOBULIN VS ZENAPAX IN KIDNEY ALLOGRAFT RECIPIENTS

P387 Introduction: Several independent studies have shown the safety and efficacy of antibody induction in renal transplantation using several different maintenance immunosuppressive protocols. This describes an 18 month follow-up in a randomized study, comparing the cytoablative effect of Campath-1H (C) 0.3 mg/kg-2 dosages, Thymoglobulin (T) 1mg/kg-5 dosages and Zenapax (Z) 1mg/kg- 5 dosages on peripheral blood and iliac crest bone marrow of kidney allograft recipients. Methods: In C half maintenance dosages of tacrolimus (tacro) and mycophenolate (MMF) but no corticosteroid were given. In T and Z higher maintenance doses of tacro, MMF as well as steroids were given. Depletion of discrete mononuclear cell phenotypes in peripheral blood and iliac crest bone marrow samples taken sequentially post-transplant at 10, 60, and 200 days were compared. The absolute number of mononuclear cells in peripheral blood or iliac crest bone marrow aspirates per mm3 and the CD3, 19, 56/16 (NK cells), 25, 14 (Monocytes) and 34 (Stem cells) phenotypes in flow cytometry were calculated. Results: In peripheral blood, when compared to normal values, in the C group, there was depletion of virtually all lymphocytes, T cells by 100%, B cells by 98%, NK cells by 95%, monocytes by 83% and Stem cells by 40%, with an earlier recovery (in descending order) of stem cells, B cells, monocytes, and NK cells, and later (at >6 months) with T cells. In contrast, T depleted T cells (99%) and NK cells (98%), but B cells, and monocytes were only moderately affected (20% and 45% respectively). Z depleted all CD25 positive cells with early modest depletion of T cells, B cells and NK cells by 50%, 72% and 62% respectively, and with no effect seen on monocytes. In iliac crest marrow, at 10 days C also strongly depleted all lymphocyte, CD3+, CD19+, CD56/16+, and CD14+ counts by 99%, 98%, 96%, and 68% respectively. However, virtually normal numbers of CD34+ cells were present. At 200 days post-transplant, an increased number of B cells (double the normal values) were seen. The early depletion of affected subsets by T in this compartment were 50% that of C with an earlier return to normal ranges in all phenotypes, while with Z there was only a minimal effect. Conclusion: C is the more potent lymphoablative induction agent (followed by T) allowing in most cases for early and total avoidance of corticosteroids and lower doses of maintenance immunosuppressive agents.

6q subtelomeric deletion: is there a recognizable syndrome?

We report on a girl with an abnormal hybridization pattern for the subtelomeric fluorescence in-situ hybridization (FISH) probe panel showing deletion of the long arm telomeric region of chromosome 6. All other subtelomere DNA probes showed normal hybridization patterns. Metaphase cells analysed from cultures of peripheral blood revealed a normal female chromosome complement at the 650-band level. The deletion was further characterized using genomic microarray analysis. Clinical findings include: developmental delay, seizures, hypoplasia of the corpus callosum, dextrocardia, unusual dimpling of knees and elbows, and minor anomalies. We are aware of only two other reports of isolated cryptic 6q subtelomeric deletions not associated with other chromosomal abnormalities. The absence of retinal abnormalities in our case supports the theory that genes responsible for the retinal abnormalities in other terminal 6q deletions are proximal to 6q27. Subtelomeric FISH probes were useful in establishing a diagnosis in our patient. As more cases are reported, we may be able to establish discrete phenotypes and natural histories that can aid in counselling families.

Is insulin resistance an essential component of PCOS?: The endometriosis syndromes: a clinical classification in the presence of aetiological confusion and therapeutic anarchy.

Clinical confusion and inappropriate management continues to surround endometriosis. It is poorly recognized that the disorder can exist in two different morphological forms that have different symptoms, signs and prognosis. Earlier classification systems have been useful for research but are of limited value in aiding day-to-day management. In the clinic, two discrete phenotypes can be defined by the presence or absence of palpable nodules in the deep pelvis. Patients with such nodules with or without associated ovarian endometrioma usually have severe symptoms with significant risks of bowel and urinary tract involvement. The predominant histological feature of these lesions is extensive fibromuscular hyperplasia (adenomyoma). These patients will often need extensive surgical intervention. Patients without such palpable lesions usually have the classic superficial subperitoneal lesions with endometrial-like glands and stroma on histological examination. This group often has less severe symptoms and has little risk of developing serious associated problems. These lesions may be helped by medications and/or simple ablative surgery. It is suggested that these collections of symptoms and signs or syndromes be named after the pioneers who first described the lesions. Cullen's syndrome can be used to describe those patients with severe symptoms of endometriosis associated with palpable pelvic nodules. Sampson's syndrome can describe those with similar symptoms associated with a structurally normal pelvis.

An experimental investigation into status-dependent male dimorphism in the European earwig, Forficula auricularia

Discrete alternative reproductive phenotypes are probably often due to individuals adopting alternative tactics with unequal fitnesses in conditional strategies with status-dependent selection. One tactic is believed to be favoured below a status switch point and another tactic above it, owing to different fitness functions of the tactics. Males of the European earwig are dimorphic. Macrolabic males are large (high status) and have long forceps, and brachylabic males are small (low status) with short forceps. I tested whether fitness functions, measured as mating success, of the two morphs differed with regard to forceps length and body weight. Macrolabic males with longer forceps than a competing male had higher mating success but brachylabic males benefited by being heavier than their competitor. Thus, different selection regimes were acting on the two morphs, suggesting that their fitness functions differed in relation to status. These observations and those of previous studies, showing that morph expression is environmentally determined and is associated with body size and that the morphs have unequal fitness, provide support for the hypothesis that male dimorphism in this species is a conditional strategy that has evolved under status-dependent selection. The differential investment in forceps growth that characterizes the morphs was manifested in a steeper allometric relation between forceps length and body size in macrolabic than brachylabic males. Behavioural observations showed that males of both morphs engaged in direct contests for females. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.

Hyperactivation of protein kinase B and ERK have discrete effects on survival, proliferation, and cytokine expression in Nf1-deficient myeloid cells

The Nf1 tumor suppressor encodes a GTPase-activating protein for Ras. Previous work has implicated hyperactive Ras in the aberrant growth of Nf1-deficient cells; however, there are limited data on which effectors modulate specific phenotypes. To address this, we generated myeloid cell lines by infecting fetal liver cells with a retrovirus encoding a truncated allele of c-Myb. Granulocyte-macrophage colony stimulating factor (GM-CSF) promoted the survival of wild-type Myb cells in a dose-dependent manner. By contrast, Nf1-deficient myeloid cells deprived of growth factors, were resistant to apoptosis due to hyperactivation of the phosphoinositide-3-OH kinase/protein kinase B cascade. Nf1 −/− cells also demonstrated growth factor-independent proliferation and upregulation of GM-CSF mRNA production that were dependent upon Raf/MEK/ERK signaling. These data link specific Ras effectors with discrete cellular phenotypes in Nf1-deficient cells.

Development and evolution of inner ear sensory epithelia and their innervation.

The development and evolution of the inner ear sensory patches and their innervation is reviewed. Recent molecular developmental data suggest that development of these sensory patches is a developmental recapitulation of the evolutionary history. These data suggest that the ear generates multiple, functionally diverse sensory epithelia by dividing a single sensory primordium. Those epithelia will establish distinct identities through the overlapping expression of genes of which only a few are currently known. One of these distinctions is the unique pattern of hair cell polarity. A hypothesis is presented on how the hair cell polarity may relate to the progressive segregation of the six sensory epithelia. Besides being markers for sensory epithelia development, neurotrophins are also expressed in delaminating cells that migrate toward the developing vestibular and cochlear ganglia. These delaminating cells originate from multiple sites at or near the developing sensory epithelia and some also express neuronal markers such as NeuroD. The differential origin of precursors raises the possibility that some sensory neurons acquire positional information before they delaminate the ear. Such an identity of these delaminating sensory neurons may be used both to navigate their dendrites to the area they delaminated from, as well as to help them navigate to their central target. The navigational properties of sensory neurons as well as the acquisition of discrete sensory patch phenotypes implies a much more sophisticated subdivision of the developing otocyst than the few available gene expression studies suggest.

Different genes, different diabetes: lessons from maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a genetic subgroup of diabetes characterised by an autosomal dominant inheritance and early onset, non-insulin dependent diabetes. This results from a monogenic defect causing beta-cell dysfunction. The defining of five genes in which mutations cause MODY has allowed us to understand the clinical heterogeneity seen in this condition and can guide clinical management. Mutations in the glucokinase gene lead to stable hyperglycaemia, complications are unusual and treatment is rarely needed. Glucokinase patients are often detected during screening in pregnancy. While maternal mutations increase birth weight by increasing maternal glycaemia, fetal mutations reduce birth weight by reducing fetal insulin secretion. Patients with mutation in genes encoding the transcription factors, hepatocyte nuclear factor (HNF)-1 &#102, HNF-4 &#102, HNF-1 &#103 and insulin promoter factor 1 (IPF-1) have a common progressive beta-cell failure resulting in increasing hyperglycaemia and treatment requirements. These patients are at risk of developing microvascular complications. They show a pharmacogenetic effect with a specific sensitivity to sulphonylureas. Patients with transcription factor mutations have a range of discrete extrapancreatic phenotypes including a low renal threshold for glucose with HNF-1 &#102 mutations, altered lipids and lipoproteins with HNF-4 &#102 mutations and a variety of cystic renal diseases and uterine and genital developmental disorders with HNF-1 &#103 mutations. Molecular genetic testing is now available in routine clinical practice. This allows confirmation of a diagnosis of MODY and defines the subgroup. Differences in prognosis and treatment strongly support the increased use of molecular genetic testing in diabetes.

What does Stat3 do?

The Stat protein family was discovered in the course of studies of signaling specificity from IFN receptors (1). The initial finding of a family of related proteins, each activated by a different cytokine receptor, suggested that these proteins would fulfill the requirements predicted for carriers of intracellular signaling information capable of retaining the specificity inherent in cytokine-receptor interactions (2). While studies in cell culture systems belied some of this early promise, the initial findings with genetically manipulated mice, especially using gene-targeting approaches, generally suggested a high degree of specificity for the various Stat proteins in individual signaling pathways. As reviewed elsewhere in this Perspective series, the analysis of mice lacking one or more Stat genes has shown relatively discrete phenotypes, assigning each Stat protein to a relatively specific pathway. Not so with Stat3. Unlike all other members of the Stat gene family, ablation of Stat3 leads to embryonic lethality (3). This finding, along with evidence of its activation by a wide variety of cytokines, growth factors, and other stimuli (4, 5), implied that Stat3 might be more generally deployed than its relatives and has led to the suggestion that it might represent a primordial Stat protein. Recent data, especially from the analysis of conditional loss of Stat3 protein in adult tissues, confirm that Stat3 participates in a wide variety of physiological processes and even directs seemingly contradictory responses.

Different genes, different diabetes: lessons from maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a genetic subgroup of diabetes characterised by an autosomal dominant inheritance and early onset, non-insulin dependent diabetes. This results from a monogenic defect causing beta-cell dysfunction. The defining of five genes in which mutations cause MODY has allowed us to understand the clinical heterogeneity seen in this condition and can guide clinical management. Mutations in the glucokinase gene lead to stable hyperglycaemia, complications are unusual and treatment is rarely needed. Glucokinase patients are often detected during screening in pregnancy. While maternal mutations increase birth weight by increasing maternal glycaemia, fetal mutations reduce birth weight by reducing fetal insulin secretion. Patients with mutation in genes encoding the transcription factors, hepatocyte nuclear factor (HNF)-1 f, HNF-4 f, HNF-1 g and insulin promoter factor 1 (IPF-1) have a common progressive beta-cell failure resulting in increasing hyperglycaemia and treatment requirements. These patients are at risk of developing microvascular complications. They show a pharmacogenetic effect with a specific sensitivity to sulphonylureas. Patients with transcription factor mutations have a range of discrete extrapancreatic phenotypes including a low renal threshold for glucose with HNF-1 f mutations, altered lipids and lipoproteins with HNF-4 f mutations and a variety of cystic renal diseases and uterine and genital developmental disorders with HNF-1 g mutations. Molecular genetic testing is now available in routine clinical practice. This allows confirmation of a diagnosis of MODY and defines the subgroup. Differences in prognosis and treatment strongly support the increased use of molecular genetic testing in diabetes.

Differential Expression of IRS‐1 and IRS‐2 in UterineLeiomyosarcomas with Distinct Oncogenic Phenotypes: Lack ofCorrelation with Downstream Signaling Events

Purpose: Insulin receptor substrates (IRSs) are essential for insulin-induced mitogenic effects on several cell types but they also are involved in cell transformation.We investigated whether the differential constitutive expression and potential distinct downstream signaling events of IRS-1 and IRS-2 might be related to discrete tumourigenic phenotypes of three human uterine leiomyosarcoma cell lines, one of which was specifically isolated for the present study. Methods and results: SK-UT-1B egressed effectively from a gellyfied Matrigel matrix and grew as did DMR cells in an anchorage-independent manner in agar and induced rapidly growing tumours in nude mice. On the contrary, SK-LMS-1 cells did not emigrate from Matrigel, neither grew in agar nor were they tumourigenic. IRS-2 was highly expressed in the more malignant cell lines, whereas IRS-1 was present only in SK-LMS-1 cells. However, upon insulin stimulation both IRS- 1 and IRS-2 were tyrosine phosphorylated with a similar kinetic in the respective cell lines; furthermore, after 1 min of insulin stimulation PI3-kinase associated with IRSs and after 2 min Shc was phosphorylated and associated with Grb2 with minor differences detectable among the various cell lines in the duration of phosphorylation and/or in their association irrespective of whether IRS-1 or IRS-2 were expressed. Discussion: Our findings tend to exclude that the malignancy displayed by uterine leiomyosarcomas might be directly linked to the activation of distinct IRS-1- or IRS-2-dependent pathways.

Correlated response of autogeny to selection for adult starvation resistance in the blowfly, Lucilia cuprina

We withheld sucrose from adults in three lineages of Lucilia cuprina, producing a four-fold greater mortality than in control lineages, in order to impose direct selection for carbohydrate starvation resistance. The frequency of autogeny (maturation of eggs in the absence of adult protein feeding) increased as a correlated response by an average of 4.9 to 9.2% per generation in three lineages subjected to starvation over five generations. The frequency of autogeny fluctuated but did not display a significant net change in three control lineages. Autogeny in L. cuprina seems to behave as a threshold trait, with a continuous, genetically-based underlying disposition producing discrete phenotypes. The heritability of autogeny in our laboratory lineages was estimated to be 0.09 to 0.39, 0.04 to 0.36, and -0.08 to 0.30 (95% confidence intervals). Despite the potential for autogeny to evolve and despite protein limitation of female fecundity in Australian populations of L. cuprina, the trait is rare or absent in the field. Genetic variation for autogeny may be maintained, but at sub-threshold levels, by nutrient availability in the field, while trade-offs associated with autogeny probably limit the net fitness benefit of the trait and prevent the evolution of a noticeable frequency of autogenous females.

Correlated response of autogeny to selection for adult starvation resistance in the blowfly, Lucilia cuprina

We withheld sucrose from adults in three lineages of Lucilia cuprina, producing a four-fold greater mortality than in control lineages, in order to impose direct selection for carbohydrate starvation resistance. The frequency of autogeny (maturation of eggs in the absence of adult protein feeding) increased as a correlated response by an average of 4.9 to 9.2% per generation in three lineages subjected to starvation over five generations. The frequency of autogeny fluctuated but did not display a significant net change in three control lineages. Autogeny in L. cuprina seems to behave as a threshold trait, with a continuous, genetically-based underlying disposition producing discrete phenotypes. The heritability of autogeny in our laboratory lineages was estimated to be 0.09 to 0.39, 0.04 to 0.36, and −0.08 to 0.30 (95% confidence intervals). Despite the potential for autogeny to evolve and despite protein limitation of female fecundity in Australian populations of L. cuprina, the trait is rare or absent in the field. Genetic variation for autogeny may be maintained, but at sub-threshold levels, by nutrient availability in the field, while trade-offs associated with autogeny probably limit the net fitness benefit of the trait and prevent the evolution of a noticeable frequency of autogenous females.

Map-based cloning of quantitative trait loci: progress and prospects.

Map-based cloning has been considered problematic for isolating quantitative trait loci (QTLs) due to the confounding phenotypic effects of environment and other QTLs. However, five recent studies, all in plants, have succeeded in cloning QTLs using map-based methods. We review the important features of these studies and evaluate the prospects for broader application of the techniques. Successful map-based cloning requires that QTLs represent single genes that can be isolated in near-isogenic lines, and that genotypes can be unambiguously inferred by progeny testing. In plants or animals for which map-based cloning of genes with discrete phenotypes is feasible, the modified procedures required for QTLs should not be limiting in most cases. The choice between map-based cloning and alternative methods will depend on details of the species and traits being studied.

Identification of candidate genes in ulcerative colitis and Crohn's disease using cDNA array technology

Inflammatory bowel disease (IBD) follows a multigenic mode of inheritance, encompassing the clinically discrete phenotypes of ulcerative colitis (UC) and Crohn's disease (CD). The risk of malignant transformation of the colon increases with the duration and extent of IBD and is particularly high for patients with a longstanding history of UC. We wished to identify candidate genes that might be involved in disease pathogenesis based on functional plausibility and their putative role in IBD carcinogenesis. Polyadenylated mRNA (PolyA+ mRNA) preparation from inflamed intestinal mucosa of patients with a longstanding history of UC and CD was performed with subsequent hybridization of alpha phosphorus [alpha-32P]-deoxyadenotriphosphate-labeled complementary deoxyribonucleic acid (DNA) populations to nucleic acid arrays. Of 588 different human gene transcripts arrayed, secreted apoptosis-related protein 1 (Sarp1), frizzled (fz) homologues, and disheveled (dvl) were differentially expressed, being elevated in UC as compared to CD. These genes encode proteins involved in the Wingless-type (Wnt)/beta-catenin signaling pathway. The autonomous expression of Sarp1 and Sarp1-compatible fz receptor genes suggests that the Wnt pathway may be involved in UC carcinogenesis.

Genetic control of pathogenic mechanisms in autoimmune demyelinating disease

Multiple sclerosis is a disease of discrete phenotypes in different individuals. Animal models have been useful in identifying self-antigens that become the focus of autoimmune attack and genetic loci that control susceptibility to disease. We have previously demonstrated a role for Fas-dependent pathogenesis in the induction of EAE in B10.PL mice immunized with MBP. Others have indicated a Fas-independent mechanism predominates in SJL mice immunized with PLP. Here we compare the response of (B10.PL×SJL)F1 and parental mice under similar conditions for induction of EAE. The results indicate that immunodominance and dominant pathogenic mechanisms are both under genetic control, but can be inherited independently. The data also indicate that the dominant pathogenic mechanism can change during the course of disease in an individual. Elucidation of the genetic elements controlling pathogenesis during the course of disease would provide important information in designing therapeutic strategies for individuals in a heterogeneous patient population.

Phenotypic identification of the subgroups of murine T-cell receptor alphabeta+ CD4+ CD8- thymocytes and its implication in the late stage of thymocyte development.

Phenotypic analysis of the medullary-type CD4+ CD8- [CD4 single-positive (SP)] thymocytes has revealed phenotypic heterogeneity within this cell population. The characteristic phenotype of mature peripheral T cells can be uniquely marked as Qa-2+ HSA- CD69-, whereas in the medullary-type CD4 SP thymocytes, the expression patterns of many markers were quite different. This suggests that there are many subgroups in the population, which reflects that medullary-type CD4 SP thymocytes may undergo phenotypic maturation. According to the results of two-colour flow cytometry, seven discrete phenotypes were identified by the expression capacity of Qa-2, HSA, CD69, 3G11 and 6C10 molecules. Consequently, the phenotypic precursor-progeny relationship can be envisaged as: 3G11- 6C10+ CD69+ HSAhi -->3G11+ 6C10+ CD69+ HSAhi --> 3G11+ 6C10- CD69+ HSAint -->3G11+ 6C10- CD69- HSAint Qa-2- -->3G11+ HSA-/lo Qa-2lo. At the stage of 3G11+ 6C10- CD69- HSAint Qa-2-, a branch pathway could be initiated, which gave rise to 3G11- HSAl degrees Qa-2- cells, which then, in turn, developed into 3G11- HSA-/loQa-2hi cells, a minor subgroup of the most mature CD4 SP cells. Consistent with this predicted pathway, experiments indicated that the first two subgroups were still cortisone sensitive, whereas the others were cortisone resistant. The cells in the last two Qa-2-positive subgroups are probably ready for emigration into the periphery.

Spatial distribution of discrete RAPD phenotypes of a root endophytic fungus, Phialocephala fortinii, at a primary successional site on a glacier forefront.

Phialocephala fortinii is among the few identified hyphomycetes belonging to the Mycelium radicis atrovirens complex. This 'dark-septate endophyte' has a global distribution and colonizes a wide variety of host plants. In this study, the spatial distribution of discrete genets of P. fortinii on the forefront of a receding glacier was analysed using the randomly amplified polymorphic DNA (RAPD) technique to determine plants colonized and patterns of colonization. In two consecutive years of sampling, a total of 74 isolates of P. fortinii were obtained from nine plant species, typically ectomycorrhizal, ericoid mycorrhizal or non-mycorrhizal. The isolates showed substantial variation, sharing on average approx. half the RAPD markers. In the first year, three isolates belonging to a single genet were obtained from two plants separated by a distance of nearly 1.5 m. The continuity of this genet was assessed by a sampling the following year. No isolates similar to that, or any of the genets collected the year before were observed. Consequently, the identical isolates from the previous year were concluded to represent discontinuous ramets. Two additional large genets were observed during the second year of sampling, which inhabited roots of several plants representing three different species. These data suggest that the sharing of P. fortinii genets among plant species might play a fundamental role in adaptation and interaction within the whole plant community in a system undergoing primary succession.

Markov chain Monte Carlo linkage analysis of a complex qualitative phenotype.

We tested a new computer program, LOKI, that implements a reversible jump Markov chain Monte Carlo (MCMC) technique for segregation and linkage analysis. Our objective was to determine whether this software, designed for use with continuously distributed phenotypes, has any efficacy when applied to the discrete disease states of the simulated data from the Mordor data from GAW Problem 1. Although we were able to identify the genomic location for two of the three quantitative trait loci by repeated application of the software, the MCMC sampler experienced significant mixing problems indicating that the method, as currently formulated in LOKI, was not suitable for the discrete phenotypes in this data set.

Evolution of threshold traits: the balance between directional selection, drift and mutation

Threshold traits are characterized by showing discrete phenotypes (typically two) but by being controlled by many loci of small additive effect, the expression of the phenotype being a consequence of a threshold of sensitivity. In the case of a dimorphic threshold trait, individuals above the threshold display one morph and individuals below the threshold display the alternate. Many threshold traits, such as sex ratio, cyclomorphosis, paedomorphosis and wing dimorphism, are closely connected to fitness but have high heritabilities. The present study investigates the hypothesis that these large heritabilities can be maintained even in the face of directional selection by the countervailing force of mutation. This hypothesis is based on the observation that as selection proceeds to shift the frequency of one morph towards fixation, the selection intensity necessarily declines permitting mutation to restore genetic variation. The hypothesis is tested using a simulation model and a theoretical analysis, the latter assuming no genetic drift. It is shown that over 80 per cent of the original genetic variance can be maintained at equilibrium provided the population (N) and number of loci (n) are reasonably large (N>5000, n=50). However, unless the selection coefficient is very small (<0.001) the equilibrium frequency of the phenotypes (<2 per cent) is considerably below that generally observed. I conclude that mutation could play a significant role in the maintenance of genetic variation in threshold traits but that some form of selection, such as frequency-dependent selection, is required to maintain the phenotypic variation.