Association mapping and meta-analysis: two complementary approaches for the detection of reliable Septoria tritici blotch quantitative resistance in bread wheat (Triticum aestivum L.)

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola, is one of the most ubiquitous and important diseases of bread wheat worldwide. The aim of this study was to identify markers linked to loci conferring resistance to STB from seven biparental populations. Linkage analysis, meta-analysis and association mapping were combined to identify robust quantitative trait loci (QTLs) for resistance. Linkage analysis led to the detection of 115 QTLs for resistance to STB and 66 QTLs linked to plant height and/or earliness. Meta-analysis clustered these 115 QTLs into 27 Meta-QTLs (MQTLs) of pathogen resistance, of which 14 were found to be linked to plant height and/or earliness. Both the relationship between dwarfing and susceptibility to STB and the significant negative correlation between earliness and STB symptoms were confirmed. Eleven loci were linked to STB resistance by association mapping using a general linear model and/or a mixed linear model, of which eight co-located with STB MQTLs and two co-located with individual QTLs. Associated markers located in MQTL regions enhanced the relevance of the results and validated the potential of an association mapping approach. With several biparental populations, meta-analysis is the most relevant form of genetic analysis study, but association mapping can be used as a validation method. Regions linked to resistance in both methods should be relevant for use in breeding programs for improving resistance to STB in wheat varieties. The main interest in comparing both approaches is to detect robust loci that will be functional in many genetic backgrounds rather than just in one or a few specific backgrounds.     

Survival of Male Patients with Incontinentia Pigmenti Carrying a Lethal Mutation Can Be Explained by Somatic Mosaicism or Klinefelter Syndrome

Incontinentia pigmenti (IP), or "Bloch-Sulzberger syndrome," is an X-linked dominant disorder characterized by abnormalities of skin, teeth, hair, and eyes; skewed X-inactivation; and recurrent miscarriages of male fetuses. IP results from mutations in the gene for NF-kappaB essential modulator (NEMO), with deletion of exons 4-10 of NEMO accounting for >80% of new mutations. Male fetuses inheriting this mutation and other "null" mutations of NEMO usually die in utero. Less deleterious mutations can result in survival of males subjects, but with ectodermal dysplasia and immunodeficiency. Male patients with skin, dental, and ocular abnormalities typical of those seen in female patients with IP (without immunodeficiency) are rare. We investigated four male patients with clinical hallmarks of IP. All four were found to carry the deletion normally associated with male lethality in utero. Survival in one patient is explained by a 47,XXY karyotype and skewed X inactivation. Three other patients possess a normal 46,XY karyotype. We demonstrate that these patients have both wild-type and deleted copies of the NEMO gene and are therefore mosaic for the common mutation. Therefore, the repeat-mediated rearrangement leading to the common deletion does not require meiotic division. Hypomorphic alleles, a 47,XXY karyotype, and somatic mosaicism therefore represent three mechanisms for survival of males carrying a NEMO mutation.     

Distribution of mating type alleles in the wheat pathogen Mycosphaerella graminicola over spatial scales from lesions to continents

A total of 2035 Mycosphaerella graminicola strains collected from 16 geographic locations on four continents were assayed for the mating type locus. RFLP fingerprints were used to identify clones in each population. At the smallest spatial scale analyzed, both mating types were found among fungal strains sampled from different lesions of the same leaf as well as from different pycnidia in the same lesion. At larger spatial scales, the two mating types were found at equal frequencies across spatial scales ranging from several square meters to several thousand square kilometers. Though the absolute frequencies of the two mating types sometimes varied for different sampling units within the same spatial scale in the hierarchy (plots within a field, fields within a country, or different continents of the world), none of the differences were statistically significant from the null hypothesis of equal frequencies for the two mating types. The evolutionary forces likely to maintain the even distribution of the two mating types in this pathogen were discussed.     

Hemophilia A and B are associated with abnormal spatial dynamics of clot growth

To gain greater insight into the nature of the bleeding tendency in hemophilia, we compared the spatial dynamics of clotting in platelet-free plasma from healthy donors and from patients with severe hemophilia A or B (factor VIII:C or IX:C<1%). Clotting was initiated via the intrinsic or extrinsic pathway in a thin layer of nonstirred plasma by bringing it in contact with the glass or fibroblast monolayer surface. The results suggest that clot growth is a process consisting of two distinct phases, initiation and elongation. The clotting events on the activator surface and the preceding period free of visible signs of clotting are the initiation phase. In experiments with and without stirring alike, this phase is prolonged in hemophilic plasma activated by the intrinsic, but not the extrinsic pathway. Strikingly, both hemophilia A and B are associated with a significant deterioration in the elongation phase (clot thickening), irrespective of the activation pathway. The rate of clot growth in hemophilic plasma is significantly lower than normal and declines quickly. The resulting clots are thin, which may account for the bleeding disorder.     

Avirulence in the wheat septoria tritici leaf blotch fungus Mycosphaerella graminicola is controlled by a single locus

Segregation of avirulence in Mycosphaerella graminicola, a heterothallic ascomycete that causes wheat septoria tritici leaf blotch, was studied in F1, BC1, and F2 populations by inoculation assays on five wheat cultivars in the seedling stage and by amplified fragment length polymorphism and random amplified polymorphic DNA analyses. F1 was generated by crossing isolates IPO323 (avirulent) and IPO94269 (virulent). All F1, BC1, and F2 progeny isolates were virulent on the susceptible check cultivar Taichung 29 and were avirulent on the resistant check cultivar Kavkav-K4500. Avirulence segregation was observed in F1 and in several BC1 and F2 generations on the differential cultivars Shafir, Kavkaz, and Veranopolis at a 1:1 ratio. Avirulence for the three differential cultivars always cosegregated. We conclude that avirulence in isolate IPO323 is controlled by a single, seemingly complex locus.     

A novel mode of chromosomal evolution peculiar to filamentous Ascomycete fungi

Background  

Gene loss, inversions, translocations, and other chromosomal rearrangements vary among species, resulting in different rates of structural genome evolution. Major chromosomal rearrangements are rare in most eukaryotes, giving large regions with the same genes in the same order and orientation across species. These regions of macrosynteny have been very useful for locating homologous genes in different species and to guide the assembly of genome sequences. Previous analyses in the fungi have indicated that macrosynteny is rare; instead, comparisons across species show no synteny or only microsyntenic regions encompassing usually five or fewer genes. To test the hypothesis that chromosomal evolution is different in the fungi compared to other eukaryotes, synteny was compared between species of the major fungal taxa.     

MgSlt2, a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth

Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1242-bp open reading frame, and encodes a 414-amino-acid protein. We designated this homolog MgSlt2, generated MgSlt2 knockout strains in M. graminicola isolate IPO323, and found several altered phenotypes in vitro as well as in planta. In yeast glucose broth, MgSlt2 disruptants showed a defective polarized growth in the tip cells upon aging, causing substantial local enlargements culminating in large swollen cells containing two to four nuclei. The MgSlt2 disruptants showed a significantly increased sensitivity to several fungicides, including miconazole (2x), bifonazole (>4x), imazalil (5x), and cyproconazole (10x), and were hypersensitive to glucanase. Unlike the wild type, MgSlt2 disruptants did not produce aerial mycelia and did not melanize on potato dextrose agar. Although cytological analysis in planta showed normal penetration of wheat stomata by the germ tubes of the MgSlt2 disruptants, subsequently formed hyphal filaments frequently were unable to branch out and establish invasive growth resulting in highly reduced virulence, and prevented pycnidia formation. Therefore, we conclude that MgSlt2 is a new pathogenicity factor in M. graminicola.     

Antiproteinuric treatment reduces urinary loss of vitamin D-binding protein but does not affect vitamin D status in patients with chronic kidney disease

Vitamin D deficiency is common in chronic kidney disease (CKD). Increased urinary loss of vitamin D binding protein (VDBP), the main transporter of 25-hydroxyvitamin D(3) in the circulation, has been postulated to contribute to vitamin D deficiency in proteinuria. To test this hypothesis we analyzed urinary and plasma levels of VDBP, 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) from proteinuric patients, before and after antiproteinuric interventions. We performed a post-hoc analysis of a clinical trial in CKD patients (n=13, creatinine clearance median 60 (range 25-177) ml/min) subjected to the following study periods: washout (no antiproteinuric treatment, 4 weeks), lisinopril 40mg QD (ACEi, 6 weeks), or indomethacin 75mg BID (NSAID, 4 weeks) in randomized sequence. Healthy subjects screened for donation (n=10) served as controls. Plasma and urine VDBP levels were measured by ELISA, 25-hydroxyvitamin D(3) levels by LC-MS and 1,25-dihydroxyvitamin D(3) levels by radioimmunoassay. In CKD patients urinary VDBP excretion was strongly increased (median (range) 5413 (155-211,027) μg/24h) as compared to healthy controls (64 (23-111) μg/24h, p<0.001). Both NSAID and ACEi significantly decreased urinary VDBP excretion, in proportion to proteinuria reduction. Plasma VDBP, 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) levels, however, were similar between patients and controls and not affected by antiproteinuric intervention. Urinary VDBP excretion is markedly increased in proteinuria and responds to antiproteinuric treatment. Urinary VDBP loss is not associated with plasma VDBP or vitamin D(3) levels, suggesting that urinary loss of VDBP does not affect vitamin D status.     

Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.     

The European carbon balance. Part 3: forests

We present a new synthesis, based on a suite of complementary approaches, of the primary production and carbon sink in forests of the 25 member states of the European Union (EU‐25) during 1990–2005. Upscaled terrestrial observations and model‐based approaches agree within 25% on the mean net primary production (NPP) of forests, i.e. 520±75 g C m^?2 yr^?1 over a forest area of 1.32 × 10⁶ km² to 1.55 × 10⁶ km² (EU‐25). New estimates of the mean long‐term carbon forest sink (net biome production, NBP) of EU‐25 forests amounts 75±20 g C m^?2 yr^?1. The ratio of NBP to NPP is 0.15±0.05. Estimates of the fate of the carbon inputs via NPP in wood harvests, forest fires, losses to lakes and rivers and heterotrophic respiration remain uncertain, which explains the considerable uncertainty of NBP. Inventory‐based assessments and assumptions suggest that 29±15% of the NBP (i.e., 22 g C m^?2 yr^?1) is sequestered in the forest soil, but large uncertainty remains concerning the drivers and future of the soil organic carbon. The remaining 71±15% of the NBP (i.e., 53 g C m^?2 yr^?1) is realized as woody biomass increments. In the EU‐25, the relatively large forest NBP is thought to be the result of a sustained difference between NPP, which increased during the past decades, and carbon losses primarily by harvest and heterotrophic respiration, which increased less over the same period.