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PubMed2003

Genetic diversity of forest arabica coffee (Coffea arabica L.) in Ethiopia as revealed by random amplified polymorphic DNA (RAPD) analysis.

Genetic diversity within the forest Coffea arabica L. gene pool in Ethiopia has not been extensively examined with molecular markers. In the present study, a total of 75 polymorphic RAPD bands generated by twelve random primers were used to assess genetic diversity among 144 genotypes representing 16 C. arabica populations. The number of polymorphic bands detected with each primer ranged from 2 to 9 with a mean of 6.25 bands per primer. Banding patterns ranged in percentage polymorphism from 37% to 73% with an overall mean of 56% for the populations analyzed. The amount of genetic variation among populations estimated by Shannon-Weaver diversity index was (H = 0.30). The within population and between populations differentiation values were 0.65 and 0.35, respectively. Genetic differentiations within and between zones of sample collection sites were 0.80 and 0.20, respectively. Within population average similarities estimated by simple matching coefficients ranged from 0.72 to 0.85, with an overall average of 0.78. In the cluster analysis that used individual samples as operational taxonomic units, most of the representatives of the same population failed to cluster before they joined members of other populations. Nevertheless, most of the populations were clustered on the basis of their geographic closeness and an east west differentiation was observed at approximately 75% similarity. The results obtained provide information on how to select sites for in situ conservation of C. arabica germplasm.

Coffea↗
PubMed2004

A metabolomic analysis of medicinal diversity in Huang-qin (Scutellaria baicalensis Georgi) genotypes: discovery of novel compounds.

In vitro manipulation of plant regeneration in the Chinese medicinal species Scutellaria baicalensis Georgi (Huang-qin) resulted in 26 chemically distinct germplasm lines. Antioxidant potential, growth rate and concentration of baicalin, baicalein, melatonin, and wogonin were the selective markers used to identify elite lines. Metabolomic analysis of a subset of the most distinct lines revealed that Huang-qin extracts contained over 2,000 compounds including 781 determined to be of putative medicinal importance as determined by a database search, as well as previously unidentified amino-derivatives of baicalin and wogonin. Huang-qin also contained a metabolite with the same net formula as hyperforin, previously thought to be unique to Hypericum perforatum L. Together these results provide new insights into the biochemical complexity of an important medicinal species and demonstrate the power of in vitro manipulation in combination with untargeted metabolomic screening for the production of new germplasm.

Antioxidants↗
PubMed2006

AFLP fingerprinting for the analysis of genetic diversity within Necator americanus.

In the present study, we utilised the method of AFLP to screen for genetic variation within and among individuals of the blood-feeding human hookworm Necator americanus (Nematoda) from Africa, Asia and South America. A total of 45 adult worms (i.e. 20 from Ghana, 16 from Colombia and 9 from Nepal) were subjected to analysis using the restriction enzyme/primer combination HindIII+AG/BglII+AC. Cluster analysis divided N. americanus into multiple, genetically distinct groups, consistent with previous findings using ribosomal and mitochondrial DNA data sets. The results demonstrated the usefulness of AFLP fingerprinting for establishing genetic variation within N. americanus and reinforce its applicability to other parasitic helminths of human and/or veterinary health importance.

Animals↗
PubMed2001

Amplified fragment length polymorphism analysis of genetic diversity of Haemonchus contortus during selection for drug resistance.

For the first time we used amplified fragment length polymorphism on individual nematode parasites to analyse the genetic diversity between and within isolates during consecutive stages of increased benzimidazole resistance and of increased levamisole resistance of Haemonchus contortus. The genetic diversity of the H. contortus genome turned out to be unusually high, within and between the isolates. The difference between individuals of an isolate could be as high as between individuals of two different mammalian species that do not interbreed. During benzimidazole selection the genetic constitution of the population was changed, but did not lead to a decrease in the genetic diversity. The selection for levamisole resistance resulted in a limited reduction of the genetic diversity only after the first selection step. The extensive genetic diversity apparently has allowed a fast and flexible response of H. contortus to drug selection as shown by the appearance of drug resistant isolates. This selection however has little or no effect on the extent of the genetic diversity of these resistant isolates. Implications for more sustainable control methods are discussed.

Animals↗
PubMed2005

Comparative analysis of morphological diversity: does disparity accumulate at the same rate in two lineages of centrarchid fishes?

Evolutionary lineages differ with regard to the variety of forms they exhibit. We investigated whether comparisons of morphological diversity can be used to identify differences in ecological diversity in two sister clades of centrarchid fishes. Species in the Lepomis clade (sunfishes) feed on a wider range of prey items than species in the Micropterus clade (black basses). We quantified disparity in morphology of the feeding apparatus as within-clade variance on principal components and found that Lepomis exhibits 4.4 and 7.4 times more variance than Micropterus on the first two principal components. However, lineages are expected to diversify morphologically and ecologically given enough time, and this pattern could have arisen due to differences in the amount of time each clade has had to accumulate variance. Despite being sister groups, the age of the most recent common ancestor of Lepomis is approximately 14.6 million years ago and its lineages have a total length of 86.4 million years while the age of the most recent common ancestor of Micropterus is only about 8.4 million years ago, and it has a total branch length of 42.9 million years. We used the Brownian motion model of character evolution to test the hypothesis that time of independent evolution of each clade's lineages accounts for differences in morphological disparity and determined that the rates of evolution of the first two principal components are 4.4 and 7.7 times greater in Lepomis. Thus, time and phylogeny do not account for the differences in morphological disparity observed in Lepomis and Micropterus, and other diversity-promoting mechanisms should be investigated.

Adaptation, Biological↗
PubMed2000

Harnessing the power of forward genetics--analysis of neuronal diversity and patterning in the zebrafish retina.

The seven major cell classes of the vertebrate retina are organized with remarkable precision into distinct layers. The appearance of this architecture during embryogenesis raises two questions of general importance. How do individual cell classes acquire their specialized structures and functions if they all originate from a morphologically uniform cell population? What mechanisms are responsible for the formation of such a complex and exact pattern? Recent advances present an opportunity to apply the tools of forward genetic analysis to identify mutations that affect these mechanisms in zebrafish. Molecular characterization will follow, providing insight into the basis of neuronal patterning in the vertebrate CNS.

Animals↗
PubMed2001

Analysis of the diversity of African streak mastreviruses using PCR-generated RFLPs and partial sequence data.

Maize streak virus (MSV) is the most economically significant member of a diverse group of African grass-infecting Mastrevirus species in the family Geminiviridae. We designed a single set of degenerate primers which enables the PCR amplification of an approximately 1300 bp DNA fragment spanning both conserved (the RepA gene) and variable (the long intergenic region and MP gene) portions of these viruses' genomes. Using restriction fragment length polymorphism (RFLP) analysis of PCR products obtained from 39 MSV, one SSV, and two PanSV isolates, it was possible to both identify the different virus species, which differ in nucleotide sequence by up to 40%, and to differentiate between MSV isolates sharing up to 99% sequence identity. The reliability of the RFLP data for typing the MSV isolates was verified by the phylogenetic analysis of the partial genomic nucleotide sequences of a representative subset of the MSV isolates. Based on both the RFLP and sequence data, the MSV isolates could be clearly differentiated into the four groups: these were a group of predominantly maize-infecting isolates, and three groups containing grass/wheat-infecting isolates. RFLP analysis also revealed a number of mixed virus infections in which, in certain instances, it was possible to identify individual population members.

Africa↗
PubMed2003

[Microsatellite analysis of genetic diversity and phylogenetic relationship of eight sheep breeds in Xinjiang].

To reveal the genetic diversity and syseighttemic relationship of main sheep breeds in north Xingjiang, the genetic polymorphisms of 10 microsatellites in 8 sheep breeds and one first filial generation (F1) in north Xinjiang were studied by means of PCR, polyacrylamide gel electrophoresis and silver staining. Number of alleles, average effective number of alleles (E) and average rates of homozygote of each breeds were counted. According to allele frequencies of ten microsatellites, polymorphism information content (PIC), mean heterozygosity (h) and genetic distances were calculated for each breeds. By using the Neighbor-joining method of Molecular Evolutionary Genetics Analysis software, a dendrogram was obtained based on genetic distances. Another dendrogram was obtained by Maximum Likelihood method in PHYLIP (3.6) software. The bootstrap values were evaluated for each crunode of the dendrogram by means of bootstrap test. The systemic relationship was analyzed as well. The results showed that 8 of 10 microsatellite loci were highly polymorphic, but BM1824 and MAF65 were low and medium polymorphic respectively, so the 8 microsatellite loci were effective markers for analysis of genetic relationship among sheep breeds. The average PIC (0.5631), h (0.5721) and E(2.9) of the whole population was all lower than those of other sheep breeds reported in the documents, which showed the gene polymorphisms and genetic diversity in these sheep breeds are relative rare. The genetic distances of native Aletai, Kazak and Bashibai sheeps in Xingjiang from foreign sheep breeds and cultivated breeds bearing foreign bloodline are relatively large. Consequently, they clustered in two groups. The phylogenetic relationship between different sheep breeds was in accordance with their resource, breeding history, differentiation and localities.

Algorithms↗
PubMed2006

Quantitative structure analysis of genetic diversity among spring bread wheats (Triticum aestivum L.) from different geographical regions.

Genetic diversity in spring bread wheat (T. aestivum L.) was studied in a total of 69 accessions. For this purpose, 52 microsatellite (SSR) markers were used and a total of 406 alleles were detected, of which 182 (44.8%) occurred at a frequency of <5% (rare alleles). The number of alleles per locus ranged from 2 to 14 with an average of 7.81. The largest number of alleles per locus occurred in the B genome (8.65) as compared to the A (8.43) and D (5.93) genomes, respectively. The polymorphism index content (PIC) value varied from 0.24 to 0.89 with an average of 0.68. The highest PIC for all accessions was found in the B genome (0.71) as compared to the A (0.68) and D genomes (0.63). Genetic distance-based method (standard UPGMA clustering) and a model-based method (structure analysis) were used for cluster analysis. The two methods led to analogical results. Analysis of molecular variance (AMOVA) showed that 80.6% of the total variation could be explained by the variance within the geographical groups. In comparison to the diversity detected for all accessions (H ( e ) = 0.68), genetic diversity among European spring bread wheats was H ( e ) = 0.65. A comparatively higher diversity was observed between wheat varieties from Southern European countries (Austria/Switzerland, Portugal/Spain) corresponding to those from other regions.

Alleles↗
PubMed1991

Clonal analysis of astrocyte diversity in neonatal rat spinal cord cultures.

Within the mammalian CNS, astrocytes appear to be a heterogeneous class of cells. To assay the number of distinct types of astrocytes in the rat spinal cord, cell lineage and phenotypic analyses were carried out on cultures from newborn rat spinal cord and five distinct types of astrocytes were observed. Proliferating precursors for each class of astrocyte were isolated by low density culture and shown to give rise to 5 distinct and morphologically homogeneous clusters of GFAP + astrocytes. Immunocytochemical analysis with antibodies A2B5 and Ran-2, which identify different glial lineages in optic nerve cultures, demonstrated that many clusters included both A2B5+ and A2B5- cells. Similarly, many clusters also possessed a mixture of Ran-2+ and Ran-2-cells, suggesting that in spinal cord cultures, in contrast to optic nerve cultures, expression of these antigens is regulated by individual cells rather than by cell lineage. Single-cell cloning studies, revealed that the abundance and proliferative capacity of individual astrocyte precursors differed depending on the type of astrocyte. To assay the effects of a complex cellular environment on the composition of astrocyte clones, lineage analysis was performed in complete spinal cord cultures using a replication deficient retrovirus. Although similar morphologically homogeneous clones of cells to those seen with single-cell clones were observed, the proliferative capacity and relative abundance of the distinct astrocyte precursors differed from that seen in single-cell cloning studies. Together these observations suggest that in spinal cord, gliogenesis is considerably more complex than in the optic nerve and that cultures of newborn rat spinal cord contain multiple, distinct populations of astrocytes.

Animals↗
PubMed2002

16S ribosomal DNA-based analysis of bacterial diversity in purified water used in pharmaceutical manufacturing processes by PCR and denaturing gradient gel electrophoresis.

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.

Bacteria↗
PubMed2005

[RAPD analysis on genetic diversity of the preconcentrated core germplasms of Camellia Sinensis in China].

The study was to evaluate the genetic diversity of 69 tea cultivars of the preconcentrated core germplasms of Camellia Sinensis in China by the random amplified polymorphic DNA (RAPD). Among 50 arbitrary primers, 32 primers could generate enough amplified bands for all the strains in this study. Among a total of 348 bands observed, 328 (94.3%)bands were polymorphic in the 69 cultivars tested except additional 20 cultivars. Genetic distances between the cultivars varied from 0.223 to 0.723. The study indicated that the pre-concentrated core germplasms of Camellia Sinensis in China could well represent the whole collection in respect of genetic structure and genetic diversity and genetic distance. At the same time, it was the best option to establish core collection of Camellia Sinensis in China by combining morphological markers with DNA molecular markers.

Base Sequence↗
PubMed2006

[Analysis of nucleotide diversity at the cytochrome b and cytochrome oxidase 1 genes at the population, species, and genus levels].

Algorithms of nucleotide diversity measures and other measures of genetic divergence at the molecular level are analyzed. Based on a database of p-distances, we have compared genetic divergence of populations (1) and taxa of different rank, such as sibling species (2), species within a genus (3), and species from different genera within a family (4). Based on the theory and algorithms of distance calculation from the primary DNA sequences, as well as the actual distances estimated from literature, it is recommended to use in analysis of experimental data a specific model selected from the eight available ones. The empirical data for more than 24,000 vertebrate and invertebrate species demonstrate that the data series are realistic and interpretable when p-distance or its various estimates are used. This testifies to the applicability of p-distance for most interspecies and intraspecies comparisons of genetic divergence up to the family level by two genes compared. Data on p-distances revealed various and increasing levels of genetic divergence of the sequences of genes Cyt-b and Co-1 in four groups compared. Mean unweighted scores of distances for the four groups were as follows: Cyt-b (1) 1.55 +/- 0.56, (2) 5.52 +/- 1.34, (3) 10.69 +/- 1.34, (4) 18.51 +/- 2.09 and Co-1 (1) 0.55 +/- 0.19, (2) 4.91 +/- 0.83, (3) 9.66 +/- 0.72, (4) 14.69 +/- 1.02. Differences in divergence between the genes themselves at the four levels were also found, although the total mean distances for the two genes did not show statistically significant differences. This conforms to the ample evidence showing different and nonuniform evolution rates of these and other genes and their various regions. The results of the analysis of the nucleotide and allozyme divergence within species and higher taxa of animals, first, are in a good agreement with these results, including data on protein gene markers, and, second, this evidence suggests that in animals, phyletic evolution is likely to prevail at the molecular level, while speciation mainly corresponds to the type D1 geographic model). The prevalence of the D1 speciation mode does not mean that the other modes are absent. There are at least seven various modes of speciation. Recognition of speciation modes is a task that seems to require construction of a quantitative genetic model (theory) of speciation. Although, in view of a vast diversity of the possible causes of reproductive isolating barriers (RIBs) and speciation initiation, as well as the "empirical nature" of the formalized approach, proposed in the present work, some newly arising questions may be left without an answer. Their solution probably lied in increasing the number of descriptors and members of equations, proposed in this study, on the basis of DNA markers and other genomic characteristics.

Animals↗
PubMed2000

Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora.

N(2) fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the gamma subdivision of the division Proteobacteria (gamma-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the alpha-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.

Bacteria, Anaerobic↗
PubMed2002

Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis.

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

Cheese↗
PubMed2001

Helicobacter pylori CagA: analysis of sequence diversity in relation to phosphorylation motifs and implications for the role of CagA as a virulence factor.

CagA is transported into host target cells and subsequently phosphorylated. Clearly this is a mechanism by which Helicobacter pylori could take control of one or more host cell signal transduction pathways. Presumably the end result of this interaction favors survival of H. pylori, irrespective of eventual damage to the host cell. CagA is noted for its amino acid (AA) sequence diversity, both within and outside the variable region of the molecule. The primary purpose of this review is to examine how variation in the type and number of CagA phosphorylation sites might determine the outcome of infection by different strains of H. pylori. The answer to this question could help to explain the widely disparate results obtained when H. pylori CagA status has been compared to type and severity of disease outcome in different populations, that is in different countries. Analysis of all available CagA sequences revealed that CagA contains both tyrosine phosphorylation motifs (TPMs) and cyclic-AMP-dependent phosphorylation motifs (CPMs). There are two potential CPMs near the N-terminus of CagA and at least two in the repeat region; these are not all equally well conserved. We also defined a 48-residue AA sequence, which includes the N-terminal TPM at tyrosine (Y)-122, which distinguishes between Eastern (Hong Kong-Taiwan-Japan-Thailand) H. pylori isolates and those from the West (Europe-Africa-the Americas-Australia). All 28 of the Eastern type CagA proteins have a functional N-terminal TPM whereas 11 of 47 (23.4%) of the Western type contain an inactive motif, with threonine (T) replacing the critical aspartic acid (D) residue. Only 13 of 24 (54%) known CagA sequences have an active TPM in the repeat region and only one has two TPMs in this region. The potential TPM near the C-terminus of CagA is not likely to be important since only 3 of 24 (12.5%) sequences were found to be intact. Protein database searches revealed that the AA sequence immediately following the TPM at Y-122 in CagA is homologous with a pair of PDZ domains which are common in signal transducing proteins, particularly tyrosine phosphatases. This provides a theoretical link between CagA and many of the observed responses of host cells to H. pylori. In summary, not all CagA proteins are equal in their potential for initiating host cell responses via signal transduction pathways. The degree of functional diversity of this protein depends upon which phosphorylation motifs are critical to the biological activity of CagA.

Amino Acid Motifs↗
PubMed1998

Genetic analysis of the diversity and origin of hantaviruses in Peromyscus leucopus mice in North America.

Nucleotide sequences were determined for the complete M genome segments of two distinct hantavirus genetic lineages which were detected in hantavirus antibody- and PCR-positive white-footed mice (Peromyscus leucopus) from Indiana and Oklahoma. Phylogenetic analyses indicated that although divergent from each other, the virus lineages in Indiana and Oklahoma were monophyletic and formed a newly identified unique ancestral branch within the clade of Sin Nombre-like viruses found in Peromyscus mice. Interestingly, P. leucopus-borne New York virus was found to be most closely related to the P. maniculatus-borne viruses, Sin Nombre and Monongahela, and monophyletic with Monongahela virus. In parallel, intraspecific phylogenetic relationships of P. leucopus were also determined, based on the amplification, sequencing, and analysis of the DNA fragment representing the replication control region of the rodent mitochondrial genome. P. leucopus mitochondrial DNA haplotypes were found to form four separate genetic clades, referred to here as Eastern, Central, Northwestern, and Southwestern groups. The distinct Indiana and Oklahoma virus lineages were detected in P. leucopus of the Eastern and Southwestern mitochondrial DNA haplotypes, respectively. Taken together, our current data suggests that both cospeciation of Peromyscus-borne hantaviruses with their specific rodent hosts and biogeographic factors (such as allopatric migrations, geographic separation, and isolation) have played important roles in establishment of the current genetic diversity and geographic distribution of Sin Nombre-like hantaviruses. In particular, the unusual position of New York virus on the virus phylogenetic tree is most consistent with an historically recent host-switching event.

Animals↗