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Fine-scale genetic structure and gene dispersal inferences in 10 neotropical tree species.

The extent of gene dispersal is a fundamental factor of the population and evolutionary dynamics of tropical tree species, but directly monitoring seed and pollen movement is a difficult task. However, indirect estimates of historical gene dispersal can be obtained from the fine-scale spatial genetic structure of populations at drift-dispersal equilibrium. Using an approach that is based on the slope of the regression of pairwise kinship coefficients on spatial distance and estimates of the effective population density, we compare indirect gene dispersal estimates of sympatric populations of 10 tropical tree species. We re-analysed 26 data sets consisting of mapped allozyme, SSR (simple sequence repeat), RAPD (random amplified polymorphic DNA) or AFLP (amplified fragment length polymorphism) genotypes from two rainforest sites in French Guiana. Gene dispersal estimates were obtained for at least one marker in each species, although the estimation procedure failed under insufficient marker polymorphism, limited sample size, or inappropriate sampling area. Estimates generally suffered low precision and were affected by assumptions regarding the effective population density. Averaging estimates over data sets, the extent of gene dispersal ranged from 150 m to 1200 m according to species. Smaller gene dispersal estimates were obtained in species with heavy diaspores, which are presumably not well dispersed, and in populations with high local adult density. We suggest that limited seed dispersal could indirectly limit effective pollen dispersal by creating higher local tree densities, thereby increasing the positive correlation between pollen and seed dispersal distances. We discuss the potential and limitations of our indirect estimation procedure and suggest guidelines for future studies.

French Guiana↗

Microsatellite markers identify three lineages of Phytophthora ramorum in US nurseries, yet single lineages in US forest and European nursery populations.

Analysis of 12 polymorphic simple sequence repeats identified in the genome sequence of Phytophthora ramorum, causal agent of 'sudden oak death', revealed genotypic diversity to be significantly higher in nurseries (91% of total) than in forests (18% of total). Our analysis identified only two closely related genotypes in US forests, while the genetic structure of populations from European nurseries was of intermediate complexity, including multiple, closely related genotypes. Multilocus analysis determined populations in US forests reproduce clonally and are likely descendants of a single introduced individual. The 151 isolates analysed clustered in three clades. US forest and European nursery isolates clustered into two distinct clades, while one isolate from a US nursery belonged to a third novel clade. The combined microsatellite, sequencing and morphological analyses suggest the three clades represent distinct evolutionary lineages. All three clades were identified in some US nurseries, emphasizing the role of commercial plant trade in the movement of this pathogen.

Europe↗

High genetic diversity in a rare and endangered sunflower as compared to a common congener.

Determining the genetic structure of isolated or fragmented species is of critical importance when planning a suitable conservation strategy. In this study, we use nuclear and chloroplast SSRs (simple sequence repeats) to investigate the population genetics of an extremely rare sunflower, Helianthus verticillatus Small, which is known from only three locations in North America. We investigated levels of genetic diversity and population structure compared to a more common congener, Helianthus angustifolius L., using both nuclear and chloroplast SSRs. We also investigated its proposed hybrid origin from Helianthus grosseserratus Martens and H. angustifolius. Twenty-two nuclear SSRs originating from the cultivated sunflower (Helianthus annuus L.) expressed sequence tag (EST) database, and known to be transferable to H. verticillatus and its putative parental taxa, were used in this study thereby allowing for statistical control of locus-specific effects in population genetic analyses. Despite its rarity, H. verticillatus possessed significantly higher levels of genetic diversity than H. angustifolius at nuclear loci and equivalent levels of chloroplast diversity. Significant levels of population subdivision were observed in H. verticillatus but of a magnitude comparable to that of H. angustifolius. Inspection of multilocus genotypes also revealed that clonal spread is highly localized. Finally, we conclude that H. verticillatus is not of hybrid origin as it does not exhibit a mixture of parental alleles at nuclear loci, and it does not share a chloroplast DNA haplotype with either of its putative parents.

Base Sequence↗

Evolutionary mode, tempo, and phylogenetic association of continuous morphological traits in the aquatic moss genus Amblystegium.

Evolutionary significance of morphological characters that have traditionally been used for species delineation in the aquatic moss genus Amblystegium was tested by partitioning the environmentally and genetically induced morphological variation and focusing on morphological evolution using comparative methods. Cultivation experiments under controlled condition showed that most of the morphological variation in nature resulted from plasticity. Information regarding genetically fixed morphological variation and genetic similarity derived from polymorphic inter-simple sequence repeat markers was combined into an explicit model of morphological evolution. Maximum likelihood estimates of the model parameters indicated that evolution of most characters tended to accelerate in the most recent taxa and was often independent from the phylogeny. Constraining the different characters to be independent from each other most often produced a less likely result than when the characters were free to evolve in a correlated fashion. Thus, the morphological characters that have traditionally been used to circumscribe different Amblystegium species lack the independence, diagnostic value for specific lineages, and stability that would be required for distinguishing different species.

Analysis of Variance↗

Population dynamics of the ectomycorrhizal fungal species Tricholoma populinum and Tricholoma scalpturatum associated with black poplar under differing environmental conditions.

Fungi combine sexual reproduction and clonal propagation. The balance between these two reproductive modes affects establishment dynamics, and ultimately the evolutionary potential of populations. The pattern of colonization was studied in two species of ectomycorrhizal fungi: Tricholoma populinum and Tricholoma scalpturatum. The former is considered to be a host specialist whereas T. scalpturatum is a generalist taxon. Fruit bodies of both basidiomycete species were mapped and collected over several years from a black poplar (Populus nigra) stand, at two different sites. Multilocus genotypes (= genets) were identified based on the analysis of random amplified polymorphic DNA (RAPD) patterns, inter-simple sequence repeat (ISSR) patterns and restriction fragment length polymorphisms (RFLPs) in the ribosomal DNA intergenic spacer (rDNA IGS). The genetic analyses revealed differences in local population dynamics between the two species. Tricholoma scalpturatum tended to capture new space through sexual spores whereas T. populinum did this by clonal growth, suggesting trade-offs in allocation of resources at the genet level. Genet numbers and sizes strongly differ between the two study sites, perhaps as a result of abiotic disturbance on mycelial establishment and genet behaviour.

Agaricales↗

PCR-based DNA fingerprinting of Staphylococcus haemolyticus to investigate nosocomial infections.

OBJECTIVE: To apply PCR-based DNA fingerprinting in a clinical microbiology laboratory to investigate nosocomial infections with Staphylococcus haemolyticus. METHODS: DNA fingerprints were generated by PCR on 99 S. haemolyticus isolates using different primer combinations based on ERIC, REP or arbitrarily chosen simple repeat sequences. RESULTS: Primer combinations REP1+(GTC)6 and ERIC1+ERIC2 had sufficient discrimatory power and were chosen to analyze the clinical isolates. DNA fingerprint patterns from strains isolated from the patients nursed in the same hospital ward in the period 1991--94 were approximately 90% similar to each other. One staff member, sampled in 1991, carried a strain with a similar fingerprint. CONCLUSIONS: PCR based DNA fingerprinting is a suitable method to perform in a clinical laboratory. An S. haemolyticus strain appeared to be endemic in the hospital ward and had most probably been transmitted from patient to patient. S. haemolyticus may carry glycopeptide resistance and needs attention as a causative agent of nosocomial infections.

Journal Article↗

Genetic structure of Cenococcum geophilum populations in primary successional volcanic deserts on Mount Fuji as revealed by microsatellite markers.

Polymorphic simple sequence repeat (SSR) markers were used to investigate the genetic structure in a Cenococcum geophilum population associated with Salix reinii in an early successional volcanic desert at Gotenba, on the south-eastern slope of Mount Fuji in Japan, and in three other populations associated with the same host at more developed sites on the mountain, one at Fujinomiya and two at Subashiri. The genotype richness of C. geophilum tended to be higher in more developed vegetation patches as well as in more developed sites, suggesting that genotype richness increased with advanced succession because new genotypes might have been introduced into these sites over time. High genotypic similarity was observed between the Gotenba and Fujinomiya populations but not between the Gotenba and Subashiri populations, suggesting that C. geophilum genotypes in Gotenba were introduced from the direction of Fujinomiya. Genotypes in the Gotenba population were clearly distinguishable into two groups. The absence of any intermediate genotype suggests the absence of frequent recombination in this C. geophilum population associated with early successional vegetation.

Ascomycota↗

The role of hybridization, polyploidization and glaciation in the origin and evolution of the apomictic Ranunculus cassubicus complex.

The Ranunculus cassubicus complex, comprising diploids and polyploids, is a good model for studying the role of hybridization and polyploidy in the origin of apomixis. Results from amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses performed on 448 individuals were combined with evidence from morphology, isozymes, karyology and distribution. Our results indicated a unique hybrid origin for the apomictic hexaploid R. carpaticola from north-western Slovakia, involving two sexual parents: autotetraploid R. cassubicifolius from the northern pre-Alps, and diploid R. carpaticola from central Slovakia. The hybrids were intermediate to the parents, but unique alleles have resulted from genomic reorganisation in the allopolyploids, which might also have triggered apomixis. Their distribution patterns and estimated ages suggest that hybridization may be correlated with the last glacial period. Hybridization seems to be the major origination for apomicts in the R. cassubicus complex. Polyploidy creates novel sexual genotypes and acts as a springboard for the production of new hybrids, but it only results in a combination with hybridization in apomixis. In turn, asexuality has permitted the perpetuation and establishment of ecologically divergent hybrid genotypes.

Alleles↗

Population genetic structure of Carchesium polypinum (Ciliophora: Peritrichia) in four Chinese lakes inferred from ISSR fingerprinting: high diversity but low differentiation.

Although the peritrichous ciliate Carchesium polypinum is common in freshwater, its population genetic structure is largely unknown. We used inter-simple sequence repeat (ISSR) fingerprinting to analyze the genetic structure of 48 different isolates of the species from four lakes in Wuhan, central China. Using eight polymorphic primers, 81 discernible DNA fragments were detected, among which 76 (93.83%) were polymorphic, indicating high genetic diversity at the isolate level. Further, Nei's gene diversity (h) and Shannon's Information index (I) between the different isolates both revealed a remarkable genetic diversity, higher than previously indicated by their morphology. At the same time, substantial gene flow was found. So the main factors responsible for the high level of diversity within populations are probably due to conjugation (sexual reproduction) and wide distribution of swarmers. Analysis of molecular variance (AMOVA) showed that there was low genetic differentiation among the four populations probably due to common ancestry and flooding events. The cluster analysis and principal component analysis (PCA) suggested that genotypes isolated from the same lake displayed a higher genetic similarity than those from different lakes. Both analyses separated C. polypinum isolates into subgroups according to the geographical locations. However, there is only a weak positive correlation between the genetic distance and geographical distance, suggesting a minor effect of geographical distance on the distribution of genetic diversity between populations of C. polypinum at the local level. In conclusion, our studies clearly demonstrated that a single morphospecies may harbor high levels of genetic diversity, and that the degree of resolution offered by morphology as a marker for measuring distribution patterns of genetically distinct entities is too low.

Animals↗

Development of a yeast-based assay system for monitoring microsatellite instability.

Simple sequence repeats (microsatellites) are found in all eukaryotic genomes. Instabilities within these sequences have been associated with several human disorders including Huntington's chorea and myotonic dystrophy. Further studies have identified links between microsatellite instability, faulty mismatch repair and certain human cancers, in particular a form of hereditary colorectal cancer. The assay system described here consists of a congenic set of yeast strains mutated in DNA replication and mismatch repair genes and assay plasmids with which it is possible to measure differences in microsatellite stability in the range of 5-850-fold. The development of this technology will allow monitoring of environmental and dietary influences on the genomic stability in the context of human disease.

Base Pair Mismatch↗

Evaluation of molecular techniques for the genetic characterization of Saccharomyces cerevisiae strains.

The applicability of different PCR-based techniques, random amplified polymorphic DNA, cleaved amplified polymorphic sequence and SSRs (simple sequence repeats) to genetically identify, typify and discriminate among Saccharomyces cerevisiae strains, was investigated. The analysis and comparison of the genetic profiles obtained for each of the techniques allowed determination of the degree of polymorphism, genetic diversity and relationships among the strains. Consequently, the discriminatory power and usefulness of the molecular techniques assayed was established. SSR analysis was the most effective method due to its high level of polymorphism, the ease of interpretation of the genetic profiles obtained, and the speed of use.

Genetic Techniques↗

Comparison of RAMP and SSR markers for the study of wild barley genetic diversity.

Two molecular marker technologies, random amplified microsatellite polymorphism (RAMP) and simple sequence repeats (SSR), were used to determine genetic diversity of 27 accessions of the wild barley Hordeum vulgare ssp. spontaneum. 19 primer combinations were used to generate RAMP fragments and 16 SSR loci were analysed. A high level of polymorphism was found with both kind of markers as revealed by the mean polymorphism information content (PIC) values obtained: 0.838 and 0.855 for RAMP and SSR, respectively. Genetic dissimilarities between genotypes were estimated from RAMP and SSR data. A lack of correlation was found between both sets of data. This was reflected in the two dendrograms obtained which presented accessions clustered differently. The results suggest that both sets of markers reveal genetic variation induced by different mechanisms. The dendrogram produced from the RAMP dissimilarity estimates showed most of the groups related to the geographic origin of the accessions.

Alleles↗

Genetic analysis of Indian mulberry varieties through molecular markers.

India is one of the countries where sericulture is being practiced traditionally. Due to the higher economic return and the greater employment potential, attempts are being made to increase the productivity by developing high yielding mulberry varieties. At the present, Mysore local, Bomaypiasbari, Kanva-2, Bilidevalaya, Kajli, S1, BC(2)59, C776, RFS-175, S-36 and Victory-1 are being cultivated extensively in different parts of India for rearing the silkworm Bombyx mori L. Using 17 random amplified polymorphic DNA (RAPD) and 11 inter-simple sequence repeat (ISSR) primers the genetic relationships among these varieties were analyzed. The RAPD and ISSR primers revealed more than 75% polymorphism among the varieties. The genetic similarity estimated from RAPD markers varied from 0.645, between Kajli and Victory-1 to 0.887, between Kanva-2 and Bilidevalaya. Similarly, the genetic similarity estimated from the ISSR markers ranged from 0.600, between Kajli and Victory-1, to 0.873 between Kanva-2 and BC(2)59. The dendrogram constructed from these markers grouped the varieties into three major groups comprising the low yielding, medium yielding and high yielding. The low genetic similarity between the group of varieties originating from the eastern regions with that of the southern region encourages formation of extensive breeding programs between these groups as to transfer the high yield potential of the southern varieties to the low yielding but highly adaptive eastern varieties.

DNA, Plant↗

Sequencing and analysis of a genomic fragment provide an insight into the Dunaliella viridis genomic sequence.

Dunaliella is a genus of wall-less unicellular eukaryotic green alga. Its exceptional resistances to salt and various other stresses have made it an ideal model for stress tolerance study. However, very little is known about its genome and genomic sequences. In this study, we sequenced and analyzed a 29,268 bp genomic fragment from Dunaliella viridis. The fragment showed low sequence homology to the GenBank database. At the nucleotide level, only a segment with significant sequence homology to 18S rRNA was found. The fragment contained six putative genes, but only one gene showed significant homology at the protein level to GenBank database. The average GC content of this sequence was 51.1%, which was much lower than that of close related green algae Chlamydomonas (65.7%). Significant segmental duplications were found within this fragment. The duplicated sequences accounted for about 35.7% of the entire region. Large amounts of simple sequence repeats (microsatellites) were found, with strong bias towards (AC)(n) type (76%). Analysis of other Dunaliella genomic sequences in the GenBank database (total 25,749 bp) was in agreement with these findings. These sequence features made it difficult to sequence Dunaliella genomic sequences. Further investigation should be made to reveal the biological significance of these unique sequence features.

Animals↗

Random amplified polymorphic DNA analysis of Ustilago violacea.

Isolates of the two mating type strains of the basidiomycete phytopathogen Ustilago violacea (Pers.) Roussel [a.k.a Microbotryum violaceum (Pers.:Pers.) Deml and Oberw] are restricted in their host range to one or a few species of Caryophyllaceae (Pinks). Molecular genetics maps in this species are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross and more recently through electrophoretic karyotypes and chromosomal polymorphism using CHEF gel analysis. However, currently, polymorphisms in genomic fingerprints generated by arbitrarily primed polymerase chain reaction (PCR) can distinguish between strains of almost any organism, which is useful in genetic mapping. The objective of this project was to use PCR technology, 40 Operon 10-mer primers, and 5 simple sequence repeat (SSR) primers, designed on microsatellite sequences to determine their efficiency in detecting intraspecific differences between the genomic DNA of the two mating type strains of U. violacea (a1 and a2). Polymorphism in the banding patterns of the DNA samples was detected after PCR and electrophoresis in 1.4% agarose gels. This polymorphic intraspecific variation will be utilized to detect cryptic and trans-active transposable elements in U. violacea.

DNA Primers↗

Alternative splice acceptor site in MSH4 gene is responsible for male sterility conferred by ms5 in soybean.

In soybean breeding, using the recessive male-sterile ms5 gene, derived from fast neutron mutagenesis, for recurrent selection is advantageous because of the d2 locus, which controls cotyledon color in mature seeds and can be used as a phenotypic selection marker for ms5 male sterility. However, occasional self-fertilization occurs because of the elimination of d2 linkage and instability of male sterility. Elucidating the mechanism and the gene responsible for ms5 male sterility may resolve these problems. Using fine mapping with 15 simple sequence repeat (SSR) markers, we narrowed down the candidate ms5 locus to a 54-kbp region. Bulked-DNA analysis using next-generation sequencing revealed a deletion as a candidate variation in the region. This 15-bp deletion and a nucleotide substitution were identified in intron 1 of MutS homolog (GmMSH4), which modulates chromosomal recombination in meiosis. The ms5 transcript contained a novel exon with a premature termination codon. This exon originated from an alternative splice acceptor site caused by the deletion and nucleotide substitution, disrupting gene function. Co-segregation of male sterility with five independent mutations in GmMSH4 was confirmed using progeny of mutant lines. Mutations in GmMSH4 led to biased DNA partitioning during meiosis, resulting in collapsed or enlarged pollen and suggesting that ms5 male sterility is caused by the failure of pollen formation during meiosis due to the loss of function of GmMSH4. These findings could help explain the mechanism of instability of ms5 male sterility and improve the efficiency of recurrent selection using DNA markers in soybean breeding.

Glycine max↗

Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype.

Cancers of the microsatellite mutator phenotype (MMP) show exaggerated genomic instability at simple repeat sequences. More than 50 percent (21 out of 41) of human MMP+ colon adenocarcinomas examined were found to have frameshift mutations in a tract of eight deoxyguanosines [(G)8] within BAX, a gene that promotes apoptosis. These mutations were absent in MMP- tumors and were significantly less frequent in (G)8 repeats from other genes. Frameshift mutations were present in both BAX alleles in some MMP+ colon tumor cell lines and in primary tumors. These results suggest that inactivating BAX mutations are selected for during the progression of colorectal MMP+ tumors and that the wild-type BAX gene plays a suppressor role in a p53-independent pathway for colorectal carcinogenesis.

Adenocarcinoma↗

Mutations of GTBP in genetically unstable cells.

The molecular defects responsible for tumor cell hypermutability in humans have not yet been fully identified. Here the gene encoding a G/T mismatch-binding protein (GTBP) was localized to within 1 megabase of the related hMSH2 gene on chromosome 2 and was found to be inactivated in three hypermutable cell lines. Unlike cells defective in other mismatch repair genes, which display widespread alterations in mononucleotide, dinucleotide, and other simple repeated sequences, the GTBP-deficient cells showed alterations primarily in mononucleotide tracts. These results suggest that GTBP is important for maintaining the integrity of the human genome and document molecular defects accounting for variation in mutator phenotype.

Base Sequence↗