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PubMed2000

A microsatellite marker reveals population heterogeneity within human and animal genotypes of cryptosporidium parvum.

Isolates of the protozoan parasite Cryptosporidium parvum have been differentiated into 2 genotypes: genotype 'H', which is associated only with human infections, and genotype 'C', which is associated with both human and animal infections. To date, the analysis of polymorphisms of genes and of the small subunit ribosomal DNA have revealed no heterogeneity within the 2 genotypes. In the present study, a locus containing simple sequence repeats (microsatellites) was PCR amplified and sequenced from 94 C. parvum isolates, which were collected from humans (immunocompetent and immunocompromized individuals, outbreak and single cases) and from several animal hosts in 3 continents. The analysis revealed that genotype 'H' can be further differentiated into 2 subgenotypes, and genotype 'C' can be further differentiated into 4 subgenotypes. The 6 subgenotypes differ in terms of expansions/contractions of the microsatellite repeats and by point mutations. Some subgenotypes showed a wide geographical distribution, whereas others were restricted to specific regions. Therefore, microsatellites are informative markers for more defined studies on the epidemiology, the transmission routes, and the population structure of this parasite.

Animals↗

PubMed2003

Genetic structure of Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota).

The genetic structure of five Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota) was investigated using codominant PCR-RFLP, allele specific amplification (ASA) markers, inter simple sequence repeat (ISSR) markers and mating studies. Sequence analyses of a subset of single spore isolates revealed sequence variation in four target sequences; internal transcribed spacer (ITS) and intergenic spacer (IGS1) of the nuclear ribosomal DNA, the translation elongation factor 1 alpha (efa) gene and the super oxide dismutase (sod) gene. No sequence variation was found in amplified portions of the mitochondrial large and small rRNA genes. Genotype distributions were mostly (90%) in accordance with Hardy-Weinberg expectations, and the nrDNA markers (ITS/IGS1), efa and sod were in most cases (87%) in linkage equilibrium, indicating an outcrossing reproductive mode, panmictic conditions and large population sizes of the fungus. Mating tests confirmed that F. rosea exhibits an outcrossing bipolar heterothallic mating system. Mating allele richness was high in two investigated populations. Phylogenetic analyses of ITS and IGS1 sequences from the five geographic populations revealed some geographic sub-structuring of the ITS sequences, but no sub-structuring of IGS1. The nrDNA (ITS/IGS1), efa and sod markers gave a low overall FST (0.013). The ISSR markers gave no clustering of the populations in UPGMA, and the between-population variance component was very low in AMOVA (0.4%), indicating a high level of gene flow.

Alleles↗

PubMed2003

Mycelial incompatibility operative in pairings between single basidiospore isolates of Helicobasidium mompa.

Helicobasidium mompa is a clampless basidiomycete and binucleate both in secondary and primary hyphae. Single basidiospore isolates were paired in sibling and non-sibling combinations. We did not observe any alterations in colony morphology, such as tuft formation, but mycelial incompatibility, as indicated by the presence of a dark demarcation line between colonies, occurred in a unifactorial manner. Techniques using DNA molecular markers, such as internal transcribed spacer-restriction fragment-length polymorphism (ITS-RFLP), inter-simple-sequence-repeat polymerase chain reaction (PCR), and universally primed PCR, failed to identify nuclear migration to opposite single basidiospore isolates in all but one of the 92 pairings. These results imply that single basidiospore isolates of H. mompa may be incompetent to mate under laboratory conditions and that a single mycelial incompatibility factor operates in single basidiospore isolates.

Basidiomycota↗

PubMed2003

Distribution of Amanita spp. genotypes under eastern Australian sclerophyll vegetation.

Basidiomes of Amanita alboverrucosa, A. ochrophylla, and A. pyramidifera were collected from native mixed sclerophyll forest sites and of A. conicoverrucosa and A. punctata from planted stands of Eucalyptus maculata in New South Wales, Australia. DNA was extracted from stipe tissue and subjected to inter-simple sequence repeat (ISSR) PCR analysis conducted using the primers (GTG)5 and (GACA)4 in order to determine genotype distribution at each site. Two to nine genotypes of one of the species were identified at each field site. Genotypes of A. ochrophylla, A. conicoverrucosa and A. punctata were spread over areas of ca 10-60 m diam, suggesting vegetative spread via large below-ground mycelial genets. In contrast, genotypes of A. alboverrucosa were more spatially restricted, suggesting recent establishment via basidiospores and more limited below-ground vegetative spread. Two groups of A. pyramidifera basidiomes that were separated by ca 600 m were found to be of the same genotype. While this might reflect long distance spread of below-ground mycelium in this taxon, the proximity of the basidiomes to a roadway makes movement of vegetative basidiome tissue via vehicular activity and subsequent establishment equally plausible.

Amanita↗

PubMed2005

SSR-based detection of genetic variability in the charcoal root rot pathogen Macrophomina phaseolina.

Macrophomina phaseolina, the causal agent of charcoal root or collar rot, is an important plant pathogen especially in soybean and cotton. Single primers of simple sequence repeats (SSR) or microsatellite markers have been used for the characterization of genetic variability of different populations of M. phaseolina obtained from soybean and cotton grown in India and the USA. Genetic similarity between isolates was calculated, and cluster analysis was used to generate a dendrogram showing relationships between isolates collected from the two hosts. Forty isolates could be clustered into three major groups corresponding to their hosts and geographical region. The wide distribution of microsatellites in M. phaseolina genome was assessed by agarose gel electrophoresis of the PCR products generated by direct amplification of inter SSR regions DNA. This is the first report of the use of microsatellite markers to characterize the charcoal root rot pathogen. The SSR fingerprints (0.25-3.5 kb) generated using DNA from different populations of M. phaseolina of two hosts indicated that these repeats are interspersed within the genome of this pathogen. The variability found within closely related isolates of M. phaseolina indicated that such microsatellites are useful in population studies and represents a step towards identification of potential isolate diagnostic markers specific to soybean and cotton.

Ascomycota↗

PubMed2004

Geographic isolation of Diplodia scrobiculata and its association with native Pinus radiata.

Diplodia pinea (syn. Sphaeropsis sapinea) is a well-known latent pathogen of Pinus spp. with a worldwide distribution. As such, this fungus is native where pines are endemic in the northern hemisphere and it has been introduced into all countries of the Southern Hemisphere where pines are exotic. The newly described D. scrobiculata (formerly known as the B morphotype of D. pinea) is thought to have a much more limited distribution. D. scrobiculata was first reported as an endophyte and weak pathogen of P. banksiana, where it was found to coexist with D. pinea. Diplodia scrobiculata is now known to have a much broader distribution in Northern America and Europe. In this study, seven Simple Sequence Repeat (SSR) markers were used to evaluate genetic diversity and gene flow between populations of D. scrobiculata. Results indicate a strong geographic isolation between populations of D. scrobiculata from different regions in North America, with unique alleles fixed in the different populations. The data fits the isolation by distance model indicating limited dispersal. Geographic isolation in combination with isolation by distance suggests prolonged reproductive isolation. Intensive collections of endophytes from native P. radiata in California have yielded only D. scrobiculata and not the significantly more pathogenic D. pinea. SSR analysis of three populations of D. scrobiculata from native P. radiata identified many shared alleles among the populations and moderate to high gene flow between them. The three Californian populations are distant and distinct from populations of D. scrobiculata from elsewhere. Under stress conditions, P. radiata is known to be very susceptible to D. pinea in plantations in the Southern Hemisphere. Native P. radiata is currently experiencing severe stress due to pitch canker caused by Fusarium circinatum. Such stress would provide ideal conditions for an associated outbreak of D. pinea. Thus, it is critical to prevent the movement of D. pinea into the last remaining native stands of P. radiata.

Ascomycota↗

PubMed2004

Statistical correlations of primer thermodynamic stability DeltaG degrees for enhanced flax ISSR-PCR cultivar authentication.

We revealed four statistically significant correlations related to inter-simple-sequence repeat (ISSR) patterns: (1) between thermodynamic free energy DeltaG degrees of ISSR primer sequence and PCR reamplification intensity (dA(i)), (2) between free energy DeltaG degrees of ISSR primer sequence and PIC coefficient quantifying the polymorphism of ISSR patterns, (3) and (4) between free energy DeltaG degrees of anchor sequence of primer and the number of total, and polymorphic bands in ISSR patterns, respectively. Methodological recommendations for effective ISSR primer design were inferred based on revealed correlations. In particular, free energy of ISSR primer sequence is recommended to be DeltaG degrees > 160 kJ/mol of interaction and free energy of flanking anchor sequence in primer to be around DeltaG degrees = 28 kJ/mol of interaction to produce ISSR patterns displaying maximum polymorphism of flax germplasm.

DNA Primers↗

PubMed2005

Effects of nucleo-cytoplasmic interactions on leaf volatile compounds from citrus somatic diploid hybrids.

Three diploid citrus somatic hybrids (cybrids) were produced by fusions combining nucellar callus-derived protoplasts of Willow Leaf mandarin (Citrus deliciosa Ten.) and Commune clementine (Citrus clementina Hort. ex Tan.) with, respectively, leaf protoplasts of Eureka lemon [Citrus limon (L.) Burm.] and Marumi kumquat [Fortunella japonica (Thunb.) Swing.] and leaf protoplasts of Marumi kumquat. Ploidy and origins of the nuclear, chloroplastic, and mitochondrial genomes were investigated by flow cytometry and nuclear and cytoplasmic simple sequence repeat analyses. Volatile compounds were extracted from the leaves of the three cybrids by a pentane/ether (1:1) mixture, analyzed by GC-MS, and compared to those of their parents. The cybrids were found to be very close to their nucleus-giving parent, suggesting that the main information for volatile compounds biosynthesis is contained in the nucleus. However, nucleo-cytoplasmic interactions occurred: the (mandarin + lemon) cybrid, possessing nucleus and chloroplasts of lemon and mitochondria from mandarin, synthesizes more monoterpene alcohols and esters than its nucleus-giving parent; the (clementine + kumquat) cybrid, possessing nucleus from kumquat and organelles from mandarin, synthesizes more monoterpene and sesquiterpene hydrocarbons and sesquiterpene alcohols than its nucleus-giving parent.

Cell Nucleus↗

PubMed2006

Non-concordance between genetic profiles of olive oil and fruit: a cautionary note to the use of DNA markers for provenance testing.

To investigate the contribution of paternal alleles to the DNA content of olive oil, genetic analyses of olive DNA samples from fruits, leaves, and oil derived from the same tree (cv. Leccino) were carried out. DNA extracted from maternal tissues--leaves and flesh--from different fruits showed identical genetic profiles using a set of DNA markers. Additional simple sequence repeat (SSR) alleles, not found in the maternal samples, were amplified in the embryos (stone), and they were also detected in DNA extracted from the paste obtained by crushing whole fruits and from the oil pressed from this material. These results demonstrate that the DNA profile obtained from olive oil is likely to represent a composite profile of the maternal alleles juxtaposed with alleles contributed by various pollen donors. Therefore, care needs to be taken in the interpretation of DNA profiles obtained from DNA extracted from oil for resolving provenance and authenticity issues.

Alleles↗

PubMed2000

Use of three different marker systems to estimate genetic diversity of Indian elite rice varieties.

Genetic diversity among 42 Indian elite rice varieties, which is important for selection of parents for conventional breeding and hybrid program, was evaluated using three different types of DNA markers and parentage analysis. Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and sequence tagged microsatellite site (STMS) markers resulted in mean heterozygosity values of 0.429, 0.675 and 0.882 over all loci, respectively, and marker index values of 2.21, 4.05 and 5.49, respectively. The three molecular marker systems together provide wider genome coverage and, therefore, would be a better indicator of the genetic relationships among the 42 elite rice cultivars than those revealed using individual molecular markers. A total of 153 bands (91%) were polymorphic out of 168 bands amplified, considering all the markers together. The average genetic similarity coefficient across all the 861 cultivar pairs was 0.70 while the average coefficient of parentage was 0.10. Cluster analysis revealed that there was a very poor correlation (correlation coefficient <0.1) between dendrograms generated using coefficients of parentage and molecular marker generated genetic similarities, which can be attributed to selection pressure, genetic drift, sampling of loci and unknown relationships among supposedly unrelated ancestors.

DNA, Plant↗

PubMed2001

Functional characterisation of urease accessory protein G (ureG) from potato.

The activation of the nickel metalloenzyme urease is a complex process. In bacteria, several urease accessory proteins are essential for incorporation of nickel into the active centre of urease. Comparatively little is known about the activation process and the proteins involved in plants. We cloned five different cDNAs encoding isoforms of urease accessory protein G (ureG) in potato. The 5'-coding region of these cDNAs is highly polymorphic within Solanum tuberosum ssp. tuberosum, containing mainly a simple sequence repeat encoding histidine and aspartate. Mapping on an ultrahigh-density map of the potato genome and Southern blot analysis showed that the isoforms arise from allelic differences of a single-copy gene which was located on chromosome 2. Expression analysis at the mRNA and protein levels indicated the presence of ureG in almost all tissues examined, consistent with the ubiquitous expression of urease. An attempt to correlate urease activity with ureG expression levels in different tissues was made. Allelic copies of ureG were expressed in a tissue-specific manner. UreG from potato and the Klebsiella aerogenes urease operon defective in bacterial ureG were co-expressed in Escherichia coli. The plant gene complements the K. aerogenes ureG mutation, demonstrating that it encodes a urease accessory protein and indicating a structural conservation between the plant and the bacterial urease activation complexes.

Alleles↗

PubMed1998

Molecular-cytogenetic characterization of the Vicia faba genome--heterochromatin differentiation, replication patterns and sequence localization.

A comprehensive survey of the molecular-cytogenetic features of the Vicia faba chromosome complement (2n = 12) is given. It includes previous as well as new original data. Various Giemsa, restriction endonuclease and fluorochrome banding patterns, azacytidine-mediated segment extension, replication patterns, lateral A/T asymmetry and sequence localization data for tandemly arranged simple sequence repeats, dispersed repeats and coding sequences as well as histone acetylation patterns are considered. This allows not only to distinguish and characterize telomeres, subtelomeres, centromeres and the NOR, but also the structure of the 5S rRNA gene loci and two main types of interstitial heterochromatin. Additionally, it offers physical landmarks within euchromatic areas. Thus, the field bean genome, exemplified by the reconstructed karyotype ACB, belongs to the cytogenetically best investigated plant genomes.

Chromosome Banding↗

PubMed2001

Comparative analysis of genetic diversity among Indian populations of Scirpophaga incertulas by ISSR-PCR and RAPD-PCR.

Genetic variation between 28 Indian populations of the rice pest, Scirpophaga incertulas was evaluated using inter-simple sequence repeats (ISSR)-PCR assay. Nine SSR primers gave rise to 79 amplification products of which 67 were polymorphic. A dendrogram constructed from this data indicates that there is no geographical bias to the clustering and that gene flow between populations appears to be relatively unrestricted, substantiating our earlier conclusion based on the RAPD (random amplified polymorphic DNA) data. The dendrograms obtained using each of these marker systems were poorly correlated with each other as determined by Mantel's test for matrix correlation. Estimates of expected heterozygosity and marker index for each of these marker systems suggests that both these marker systems are equally efficient in determining polymorphisms. Matrix correlation analyses suggest that reliable estimates of genetic variation among the S. incertulas pest populations can be obtained by using RAPDs alone or in combination with ISSRs, but ISSRs alone cannot be used for this purpose.

Animals↗

PubMed2001

Molecular marker analysis of protein content using PCR-based markers in wheat.

Grain protein concentration (GPC) of hexaploid wheat is one of the important factors that determines the end-product quality as well as playing a pivotal role in human nutrition. In an attempt to identify PCR-based DNA markers linked to GPC, 106 recombinant inbred lines (RILs) were developed from a cross between two wheat cultivars PH132 and WL711, which differ significantly in GPC, by the single seed descent method. The RILs were phenotyped for GPC at two diverse agroclimatic locations, namely Pune and Ludhiana, to study the influence of genotype and environment interactions on this trait. The parents were screened with 85 inter simple sequence repeat (ISSR) primers and 350 random primers. The selective genotyping and whole population analysis revealed nine DNA markers associated with the trait. Three markers (UBC8441100, UBC8801000, and OPA4800) were observed to be associated with the trait in both locations, whereas two markers (OPH41400) and UBC873750) werefound to be specific to Pune, and four markers (OPM5870, OPO10870, OPV141200, and UBC8251000) were specific to Ludhiana. Together five markers at the Pune location representing five QTLs and seven markers at Ludhiana representing four QTLs accounted for 13.4 and 13.5% of total phenotypic variation, respectively. This study clearly demonstrates that GPC is highly influenced by the environment, and the applicability of ISSR and RAPD markers in finding regions on chromosomes associated with quantitative characters in wheat such as GPC.

Breeding↗

PubMed2001

A genome-wide scan of 1842 DNA markers for allelic associations with general cognitive ability: a five-stage design using DNA pooling and extreme selected groups.

All measures of cognitive processes correlate moderately at the phenotypic level and correlate substantially at the genetic level. General cognitive ability (g) refers to what diverse cognitive processes have in common. Our goal is to identify quantitative trait loci (QTLs) associated with high g compared with average g. In order to detect QTLs of small effect size, we used extreme selected samples and a five-stage design with nominal alpha levels that permit false positive results in early stages but remove false positives in later stages. As a first step toward a systematic genome scan for allelic association, we used DNA pooling to screen 1842 simple sequence repeat (SSR) markers approximately evenly spaced at 2 cM throughout the genome in a five-stage design: (1) case-control DNA pooling (101 cases with mean IQ of 136 and 101 controls with mean IQ of 100), (2) case-control DNA pooling (96 cases with IQ > 160 and 100 controls with mean IQ of 102), (3) individual genotyping of Stage 1 sample, (4) individual genotyping of Stage 2 sample, (5) transmission disequilibrium test (TDT; 196 parent-child trios for offspring with IQ > 160). The over all Type I error rate is 0.000125, which robustly protects against false positive results. The numbers of markers surviving each stage using a conservative allele-specific directional test were 108, 6, 4, 2, and 0, respectively, for the five stages. A genomic control test using DNA pooling suggested that the failure to replicate the positive case-control results in the TDT analysis was not due to ethnic stratification. Several markers that were close to significance at all stages are being investigated further. Relying on indirect association based on linkage disequilibrium between markers and QTLs means that 100,000 markers may be needed to exclude QTL associations. Because power drops off precipitously for indirect association approaches when a marker is not close to the QTL, we are not planning to genotype additional SSR markers. Instead we are using the same design to screen markers such as cSNPs and SNPs in regulatory regions that are likely to include functional polymorphisms in which the marker can be presumed to be the QTL.

Adolescent↗

PubMedDate not supplied

Development and mapping of SSR markers for maize.

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 x Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.

Chromosome Mapping↗

PubMed2004

Molecular genetic variation in Chinese populations of three subspecies of Hippophae rhamnoides.

To provide a population-level genetic profile for investigation and conservation of genetic diversity of the sea buckthorn (Hippophae rhamnoides L.), 300 individuals of fifteen natural populations of sea buckthorn in China were analyzed by using ISSR (inter-simple sequence repeats) markers. On the basis of Shannon's index and Nei's genetic diversity, the mean genetic diversity detected in the natural populations of Hippophae rhamnoides ssp. yunnanensis, ssp. sinensis, and ssp. gyantsensis was 0.1944, 0.2169, and 0.1372, respectively. The coefficient of gene differentiation (Gst) of seven ssp. yunnanensis populations is 0.2790, and that of seven ssp. sinensis populations is 0.4184. This means that 28% of the total molecular variance of seven ssp. yunnanensis populations existed among populations, and 42% for seven ssp. sinensis populations, suggesting that the subspecies have different genetic structures. No significant correlation between genetic and geographic distances of the populations was found using ISSR markers.

China↗

PubMed1989

Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops.

The telomeric ends of eukaryotic chromosomes are composed of simple repeating sequences in which one DNA strand contains short tracts of guanine residues alternating with short tracts of A/T-rich sequences. The guanine-rich strand is always oriented in a 5'-3' direction towards the end of the chromosome and is extended to produce a 3' overhang of about two repeating units in species where the telomeric terminus is known. This overhang has been implicated in the formation of several unusual intra-and intermolecular DNA structures, although none of these structures has been characterized fully. We now report that oligonucleotides encoding Tetrahymena telomeres dimerize to form stable complexes in solution. This salt-dependent dimerization is mediated entirely by the 3'-terminal telomeric overhang (TT-GGGGTTGGGG) and produces complexes in which the N7 position of every guanine in the overhangs is chemically inaccessible. We therefore propose that telomeric DNA dimerizes by hydrogen bonding between two intramolecular hairpin loops, to form antiparallel quadruplexes containing cyclic guanine base tetrads. These novel hairpin dimers may be important in telomere association and recombination and could also provide a general mechanism for pairing two double helices in other recombinational processes.

Animals↗