Primers for the amplification of sequence‐characterized loci in Cryphonectria cubensis populations

We describe the development of DNA markers for the fungal pathogen of Eucalyptus, Cryphonectria cubensis. These markers originated from cloned intershort sequence repeat polymerase chain reactions, which enrich for medium to highly repetitive DNA sequences. In total, 10 markers were isolated, eight of which were polymorphic, and these can subsequently be applied to study populations of C. cubensis.     

Unequivocal determination of sire allele origin for multiallelic microsatellites when only the sire and progeny are genotyped

The effect of a segregating economic trait locus (ETL) can be detected with the aid of a linked genetic marker, if specific alleles of each locus are in association among the individuals genotyped for the genetic marker. For dairy cattle this can be achieved by application of the ‘granddaughter design’. If only the sires and their sons are genotyped for the genetic markers, then the allele origin of sons having the same genotypes as their sires cannot be determined. Seven sires and 101 sons were genotyped for five microsatellites. The mean frequency of heterozygous sires was 77%. The mean number of alleles per locus was 8.2. Frequency of informative sons per locus ranged from 60% to 80% with a mean of 72%. With highly polymorphic microsatellites, at least 60% more grandsire families can be included in the analysis, and the number of sons assayed can be reduced by 40%, as compared to diallelic markers.     

AFLP characterization of natural populations of Berberis (Berberidaceae) in Patagonia, Argentina

 AFLP markers were used to analyse the intra- and interspecific relationships among 22 natural populations of 13 Patagonian species of Berberis and the relationships among the taxa belonging to homoploid and polyploid complexes. Seven primer combinations gave rise to 231 AFLP bands, of which 199 were polymorphic. Correspondence between AFLP data, morphological traits and seed protein bands was also assessed. The dendrogram inferred from AFLP fingerprints showed that, in general, populations of the same species formed closely related groups with high coefficients of similarity. Principal co-ordinates analysis showed two separate subgroups: (i) B. bidentata and their putative ancestors –B. darwinii and B. linearifolia– which form a homogamic group, and (ii) B. buxifolia, B. heterophylla and B. parodii– which could form a polyploid hybrid complex. Received March 21, 2001 Accepted September 11, 2001     

Chronobiology of Coronary Risk Markers in Greenland Eskimos: A Comparative Study with Caucasians Residing in the Same Arctic Area

We report a comparison of fibrinolytic variables between 10 Caucasians on a predominantly European diet and 10 Greenland Eskimos on a traditional Inuit diet containing a substantial amount of fish and sea animals. We studied the diurnal variation in tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) antigens and activities during a 24-h period. Blood samples were taken every 4 h. The variations of the sinusoidal curves were evaluated by the Friedman χ² test. t-PA and PAI-1 antigen in plasma fluctuated significantly during the 24 h (Eskimos p < 0.000007 and p < 0.0007; Caucasians p < 0.00003 and p < 0.02), with a peak in the early morning and a nadir in the afternoon. This also held true for PA1 activity (Eskimos p < 0.0008; Caucasians p < O.Ol), whereas t-PA activity showed an inverse but still significant pattern (Eskimos p < 0.006; Caucasians p < 0.0008). Amplitudes, areas underneath, and overall medians of the sinusoidal curves did not deviate between the two groups with respect to t-PA and PAL In contrast to the significant variation of t-PA and PAI, the plasma concentrations of fibrin degradation products (D-Dimer), a measure of effective fibrinolysis, remained constant during the 24 h, and the absolute differences between groups did not reach statistical significance. These findings suggest that circadian variation of fibrinolytic activators and inhibitors is a basic biologic phenomenon, which is not affected by life-style, dietary habits, or ethnic differences. Furthermore, the lack of diurnal variation in D-Dimer raises the question of whether there is a causal relationship between low morning activities of t-PA and the frequent onset of myocardial infarction at that time of day, as suggested by several authors.     

An improvement on the maximum likelihood reconstruction of pedigrees from marker data

Many methods have been proposed to reconstruct the pedigree of a sample of individuals from their multilocus marker genotypes. These methods, like those in other fields of statistical inferences, may suffer from both type I (falsely related) and type II (falsely unrelated) errors. In sibship reconstruction, type I errors come from the spurious fusion of two or more small sibships into a single sibship, and type II errors originate from the spurious splitting of a large sibship into two or more small sibships. In this study I investigate the tendencies of both types of errors made by the likelihood methods in sibship reconstruction, using both analytical and simulation approaches. I propose an improvement on the likelihood methods to reduce sibship splitting, and thus type II errors by downscaling the number of inferred siblings sharing the same genotype at a locus. Simulations are then conducted to compare the accuracy of the original and improved likelihood methods in sibship reconstruction of a large sample of individuals in full-sib families of the same small size, the same large size and highly variable sizes, using a variable number of loci with a variable number of alleles per locus. The methods were also applied to the analysis of a salmon data set. I show that my scaling scheme prevents effectively the splitting of large sibships, and reduces type II errors greatly with little increase in type I errors. As a result, it improves the overall accuracy of sibship assignments, except when sibships are expected to be uniformly small or marker information is unrealistically scarce.     

Issues in using whole slide imaging for diagnostic pathology: “routine” stains,immunohistochemistry and predictive markers

The traditional microscope, together with the “routine” hematoxylin and eosin (H & E) stain, remains the “gold standard” for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of “stains” available. Because of the need for specific identification and even measurement of “biomarkers,” immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.     

Genetic and allelopathic differences between populations of daisy fleabane Erigeron annuus (L.) Pers. (Asteraceae) from disturbed and stable habitats

The interactions between invasive plants and their habitats may vary at different phases of the invasion process and depend on the phenotypic plasticity or local adaptations of each species. In this study, we investigated whether habitat changes during the invasion process are related to variations in the physiological traits (allelopathic properties) and genetic differentiation of daisy fleabane (Erigeron annuus (L.) Pers.). E. annuus is a winter annual invasive species that originated in North America and is now distributed throughout Europe. Genetic and genotypic diversity analyses were performed for 37 populations of E. annuus based on inter simple sequence repeat (ISSR) polymorphisms. In total, 684 plants were analyzed; 342 were from stable habitats and 342 were from disturbed habitats. The genetic differences among the populations from the different habitats were studied using a Bayesian cluster analysis and an analysis of molecular variance (AMOVA) and by calculating the genetic and genotypic diversity parameters. A germination test using the juglone index was employed to examine the potential allelopathic properties of the plants from the different habitats. Bayesian cluster analysis, AMOVA and allelopathic effects evaluation revealed differences in the allelopathic potential and genetic structure of the E. annuus populations from the disturbed and stable habitats. This differentiation of populations could be associated with founder effects or with different selection pressures among habitats.     

Twenty-one new microsatellite markers for the ecologically important midge Heterotrissocladius marcidus,and their use in studies of alpine lakes

Heterotrissocladius marcidus (Walker, 1856) (Diptera: Chironomidae) inhabits aquatic habitats across the Holarctic and Oriental regions, including remote alpine glacial lakes. It is thus a promising species for studies of population structure and genetic diversity in these highly vulnerable habitats. Here, 21 polymorphic microsatellites for H. marcidus are described, obtained using 454 GS-FLX pyrosequencing. The number of alleles per locus varied from 2 to 9 and no significant linkage disequilibrium was observed. To test applicability, 40 individuals were used from two lakes in the Tatra Mts (Western Carpathians). Observed and expected heterozygosity varied from 0 to 0.9 and 0.102 to 0.888, respectively. In both populations, three loci deviated significantly from Hardy–Weinberg equilibrium after Bonferroni correction, probably due to presence of null alleles or undetected biological processes. Application of microsatellites was tested on six alpine lake populations. Bayesian cluster analysis assigned individuals to lakes of their origin and revealed limited gene-flow between them. Five loci were successfully cross-amplified in the related midge Macropelopia sp. (Tanypodinae) and two in Pseudodiamesa branickii (Nowicki, 1873) (Diamesinae). The microsatellites described herein proved to be useful for genetic studies of alpine populations, and can provide important data for management and conservation of these threatened habitats.     

Characterization of rabbit DNA micros extracted from the EMBL nucleotide sequence database

Microsatellite polymorphisms are invaluable for mapping vertebrate genomes. In order to estimate the occurrence of microsatellites in the rabbit genome and to assess their feasibility as markers in rabbit genetics, a survey on the presence of all types of mononucleotide, dinucleotide, trinucleotide and tetranucleotide repeats, with a length of about 20 bp or more, was conducted by searching the published rabbit DNA sequences in the EMBL nucleotide database (version 32). A total of 181 rabbit microsatellites could be extracted from the present database. The estimated frequency of microsatellites in the rabbit genome was one microsatellite for every 2–3 kb of DNA. Dinucleotide repeats constituted the prevailing class of microsatellites, followed by trinucleotide, mononucleotide and tetranucleotide repeats, respectively. The average length of the microsatellites, as found in the database, was 26, 23, 23 and 22 bp for mono-, di-, tri- and tetranucleotide repeats, respectively. The most common repeat motif was AG, followed by A, AC, AGG and CCG. This group comprised about 70% of all extracted rabbit microsatellites. About 61% of the microsatellites were found in non-coding regions of genes, whereas 15% resided in (protein) coding regions. A significant fraction of rabbit microsatellites (about 22%) was found within interspersed repetitive DNA sequences.