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Isolation and characterization of the mating-type idiomorphs from the wheat septoria leaf blotch fungus Mycosphaerella graminicola.

Both mating-type loci from the wheat septoria leaf blotch pathogen Mycosphaerella graminicola have been cloned and sequenced. The MAT1-2 gene was identified by screening a genomic library from the MAT1-2 isolate IPO94269 with a heterologous probe from Tapesia yallundae. The MAT1-2 idiomorph is 2772 bp and contains a single gene encoding a putative high-mobility-group protein of 394 amino acids. The opposite idiomorph was obtained from isolate IPO323, which has the complementary mating type, by long-range PCR using primers derived from sequences flanking the MAT1-2 idiomorph. The MAT1-1 locus is 2839 bp in size and contains a single open reading frame encoding a putative alpha1-domain protein of 297 amino acids. Within the nonidiomorphic sequences, homology was found with palI, encoding a membrane receptor from Aspergillus nidulans, and a gene encoding a putative component of the anaphase-promoting complex from Schizosaccharomyces pombe and a DNA-(apurinic or apyrimidinic) lyase from S. pombe. For each of the MAT genes specific primers were designed and tested on an F1 mapping population that was generated from a cross between IPO323 and IPO94269. An absolute correlation was found between the amplified allele-specific fragments and the mating type as determined by backcrosses of each F1 progeny isolate to the parental isolates. The primers were also used to screen a collection of field isolates in a multiplex PCR. An equal distribution of MAT1-1 and MAT1-2 alleles was found for most geographic origins examined.

Amino Acid Sequence↗

Areas 3a, 3b, and 1 of human primary somatosensory cortex. Part 2. Spatial normalization to standard anatomical space.

Interindividual topographical variability of cytoarchitectonically defined somatosensory areas 3a, 3b, and 1 was analyzed in the standard anatomical format of a computerized brain atlas. T1-weighted magnetic resonance images were obtained from 10 postmortem brains. The brains were serially sectioned at 20 mcm, sections were stained for cell bodies, and areas 3a, 3b, and 1 were defined with an observer-independent cytoarchitectonic technique. After correction of the sections for deformations due to histological processing, the 3-D reconstructed histological volumes of the individual brains and the volume representations of the cytoarchitectonic areas were adapted to the reference brain of a computerized atlas. Corresponding areas were superimposed in the 3-D space of the reference brain. These population maps describe, for each voxel, how many brains have a representation of one particular cytoarchitectonic area. Each area's extent is very variable across different brains, but representations of areas 3a, 3b, and 1 in >/=50% of the brains were found in the fundus of the central sulcus, its caudal bank, and on the crown of the postcentral gyrus, respectively. Volumes of interest (VOIs) were defined for each area in which >/=50% of the brains have a representation of that area. Despite close spatial relationship of areas 3a, 3b, and 1 in the postcentral gyrus, the three VOIs overlap by <1% of their volumes. Functional imaging data can now be brought into the same standard anatomical format, and changes in regional cerebral blood flow can be calculated in VOIs of areas 3a, 3b, and 1, which are derived from genuine cytoarchitectonic data.

Adult↗

Genetics of oxidative phosphorylation: mitochondrial loci determining ossamycin-, venturicidin- and oligomycin-resistance in yeast.

With a view towards identifying new ATPase loci on the mitochondrial genome a large number of oligomycin-, ossamycin- and venturicidin-resistant mutants were isolated after MnCl2 mutagenesis. The mutants were subjected to mass-screens which divided them into different cross-resistance phenotype-classes and also distinguished the common OLI1 mutations from the mutations at all other loci. Allelism tests between examples of the different classes of phenotype indicated that the majority of mutations in the population mapped at the previously known loci OLI1, OLI2, OLI3, and OLI4. Mutations conferring specific ossamycin resistance defined two new loci, namely OSS1 and OSS2 which are linked to the OLI2 and OLI1 loci respectively. A few rare mutations comprise a new locus OLI5 which is linked to the OLI1 locus (12.6% total recombination). In conclusion we can now say that that there are two unlinked segments of the mitochondrial genome, each of which is composed of several distinct, genetically-linked loci. One segment contains the OLI1, OLI3, OLI5 and OSS2 loci and the other the OLI2, OLI4 and OSS1 loci. The phenotypically-distinguishable mutations described herein should facilitate fine-structure mapping of these two segments.

Adenosine Triphosphatases↗

Identification of microsatellite markers linked to Russian wheat aphid resistance genes Dn4 and Dn6.

The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), is a serious economic pest of wheat worldwide. Host plant resistance is the preferred method to control RWA infestations. The identification and mapping of RWA-resistant genes and the development of resistant wheat cultivars can be facilitated through the use of molecular markers. In the present study, microsatellite (SSR) markers linked to the RWA-resistant genes Dn4 and Dn6 were identified using several F(2) mapping populations derived from crosses of susceptible wheat cultivars and resistant sources. Two flanking microsatellite markers Xgwm106 and Xgwm337 are linked in coupling phase with Dn4 on the short arm of wheat chromosome 1D at 7.4 cM and 12.9 cM, respectively. Two other microsatellite markers Xgwm44 and Xgwm111 are linked to Dn6 in coupling phase near the centromere on the short arm of chromosome 7D at 14.6 cM and 3.0 cM, respectively. This is the first report on the chromosomal location of Dn6, which proved to be either allelic or tightly linked to Dn1, Dn2 and Dn5. This result of Dn6 location contradicts previous reports that Dn6 was independent of Dn1, Dn2 and Dn5. The linked markers can be conveniently used for marker-assisted selection in wheat breeding programs for the identification and/or pyramiding of Dn4 and Dn6 genes.

Journal Article↗

Investigation of crossover interference in barley ( Hordeum vulgare L.) using the coefficient of coincidence.

Interference, the interaction between recombination events, was analysed in seven mapping populations of barley ( Hordeum vulgare L.). The coefficient of coincidence was applied to investigate the type and position of interference within the genome. Interference was analysed with respect to dependence on the recombination fraction, and simulations were used to obtain test statistics which consider the small sample size of 71-150 double haploid lines. In addition to positive interference in intermediate intervals, strong negative interference, i.e. encouraged double recombination, was found in short intervals. The relationship between recombination fraction and interference could not be described with a uniform function, neither for the entire genome nor for individual chromosomes. The analysis of the position of interference within the genome revealed that interference does not act in the same way in the whole genome. Intervals spanning the centromere exhibited significantly higher means for the coefficient of coincidence than intervals within the chromosome arms, especially with regard to small intervals. In general, positive interference was found in the chromosome arms and no or negative interference in the genetically small but physically large centromeric region.

Journal Article↗

Identification of QTLs controlling tissue-culture traits in barley ( Hordeum vulgare L.).

Quantitative trait loci (QTLs) controlling callus growth (CG), subsequent shoot differentiation ratio (SD) and green shoot ratio (GS) in immature embryo culture were identified in barley. A base map was developed from 99 recombinant inbred lines (RILs) of 'Azumamugi' x 'Kanto Nakate Gold'. The tissue-culture traits were evaluated at the F(7) and F(10) generations of the RILs. The RILs showed wide and continuous variations in each of the three tissue-culture traits. Three QTLs for CG, three QTLs for SD and two QTLs for GS were detected by using composite interval mapping. A QTL for SD on chromosome 3H had a large effect, and 'Kanto Nakate Gold', which has a high differentiation ability, contributed to this QTL. The location of this QTL is identical to, or very close to, the uzu locus. We discuss the relationships between tissue-culture loci in 'Azumamugi' x 'Kanto Nakate Gold' and those in other mapping populations.

Journal Article↗

Mapping of quantitative trait loci determining agronomic important characters in hexaploid wheat ( Triticum aestivum L.).

A set of 114 recombinant inbred lines of the 'International Triticeae Mapping Initiative' mapping population was grown during the seasons 1997, 1998, 1999 and 2000 under several environments. Twenty morphological (glume colour, awn colour, waxiness, leaf erectness, peduncle length), agronomical (ear emergence time, flowering time, grain filling time, ear length, plant height, lodging, grain number, thousand-grain-weight, grain weight per ear, grain protein content, winter hardiness) and disease resistance (powdery mildew, yellow rust, leaf rust, fusarium) traits were studied. Not all traits were scored in each experiment. In total 210 QTLs with a LOD threshold of >2.0 (minor QTLs) were detected of which 64 reached a LOD score of >3.0 (major QTLs). Often QTLs were detected in comparable positions in different experiments. Homologous and homoeologous relationships of the detected QTLs, and already described major genes or QTLs determining the same traits in wheat or other Triticeae members, are discussed.

Journal Article↗

Development of CAPS markers based on three key genes of the phenylpropanoid pathway in tea, Camellia sinensis (L.) O. Kuntze, and differentiation between assamica and sinensis varieties.

The genetic diversity of tea, Camellia sinensis (L.) O. Kuntze, including the two main cultivated sinensis and assamica varieties, was investigated based on PCR-RFLP analysis of PAL, CHS2 and DFR, three key genes involved in catechin and tannin synthesis and directly responsible for tea taste and quality. Polymorphisms were of two types: amplicon length polymorphism (ALP) due to the presence of indels in two introns of PAL and DFR, and point mutations detected after restriction of amplified fragments with appropriate enzymes. A progeny test showed that all markers segregated in a Mendelian fashion and that polymorphisms were exclusively co-dominant. CHS2, which belongs to a multi-gene family, allowed for greater variation than the single-copy PAL gene. Based on Nei's gene diversity index, var. sinensis was revealed to be more variable than var. assamica, and that a higher proportion of overall diversity resided within varieties as compared to between varieties. Even though no specific DNA profile was found for either tea varieties following any single PCR-RFLP analysis, a factorial correspondence analysis carried out on all genotypes and markers separated the tea samples into two distinct groups according to their varietal status. This reflects the large difference between var. sinensis and var. assamica in their polyphenolic profiles. The STS-based markers developed in this study will be very useful in future mapping, population genetics and fingerprinting studies of this important crop species and other Camellia species, as the primers have also proven successful in the three other subgenera of this genus.

Acyltransferases↗

Inheritance of superoxide dismutase ( Sod-1) in a perennial x annual ryegrass cross and its allelic distribution among cultivars.

Identifying annual ryegrass contamination in perennial ryegrass seed lots has been of major interest in seed-testing laboratories and for seed regulatory agencies in the USA for many years. This study was conducted to characterize a superoxide dismutase locus ( Sod-1) and determine its potential to distinguish cultivated ryegrass species. The inheritance of Sod-1 was evaluated in a three-generation annual x perennial ryegrass mapping population and segregation fitted an expected 1:2:1 ratio for a single locus with two alleles. The molecular form of the Sod-1 locus was determined by H(2)O(2) and KCN inhibitor assays which indicated that the Sod-1, and a second independently segregating Sod-2, locus were both Cu/ZnSod enzymes. The common alleles at the Sod-1 locus were scored in 13 annual and 24 perennial ryegrass cultivars to determine the potential of using this locus for species separation. The Sod-1b allele was homozygous in 98% of perennial ryegrass individuals from 24 cultivars, but those not 100% homozygous for Sod-1b were seed lots with unknown contamination from annual ryegrass. These results indicate that the Sod-1b allele in the homozygous condition is a good indicator of perenniality. All eight annual ryegrass cultivars originating in Europe or Asia had a low frequency of Sod-1b homozygous individuals or none at all. The five cultivars originating in the Western Hemisphere, however, had genotype frequencies for homozygous Sod-1b of up to 56%. The potential of the Sod-1 locus to serve as a test to separate the two growth forms depends on the source of the annual-type contamination.

Journal Article↗

Flowering response of rice to photoperiod and temperature: a QTL analysis using a phenological model.

In this study we have attempted to quantify the thermal and photoperiodical responses of rice (Oryza sativa L.) flowering time QTLs jointly by a 'date-of-planting' field experiment of a mapping population, and a 'phenological model' analysis that separately parameterizes the two responses, based on daily temperature, daily photoperiod and flowering date. For this purpose, the 'three-stage Beta model', which parameterizes the sensitivity to temperature (parameter alpha), the sensitivity to photoperiod (parameter beta), and earliness under optimal conditions (10 h photoperiod at 30 degrees C) (parameter G), was applied to 'Nipponbare' x 'Kasalath' backcross inbred lines that were transplanted on five dates. QTLs for the beta value were detected in the four known flowering time QTL (Hd1, Hd2, Hd6 and Hd8) regions, while QTLs for the G value were detected only in the Hd1 and Hd2 regions. This result was consistent with previous reports on near-isogenic lines (NILs) of Hd1, Hd2 and Hd6, where these loci were involved in photoperiod sensitivity, and where Hd1 and Hd2 conferred altered flowering under both 10 and 14 h photoperiods, while Hd6 action was only affected by the 14 h photoperiod. Hd8 was shown to control photoperiod sensitivity for the first time. Interestingly, Hd1 and Hd2 were associated with a QTL for the alpha value, which might support the previous hypothesis that the process of photoinduction depends on temperature. These results demonstrate that our approach can effectively quantify environmental responses of flowering time QTLs without controlled environments or NILs.

Flowers↗

Methods for predicting superior genotypes under multiple environments based on QTL effects.

Methods were developed for predicting two kinds of superior genotypes (superior line and superior hybrid) based on quantative trait locus (QTL) effects including epistatic and QTL x environment interaction effects. Formulae were derived for predicting the total genetic effect of any individual with known QTLs genotype derived from the mapping population in a specific environment. Two algorithms, enumeration algorithm and stepwise tuning algorithm, were used to select the best multi-locus combination of all the putative QTLs. Grain weight per plant (GW) in rice was analyzed as a working example to demonstrate the proposed methods. Results showed that the predicted superior lines and superior hybrids had great superiorities over the F(1) hybrid, indicating large breeding potential remained for further improvement on GW. Results also showed that epistatic effects and their interaction with environments largely contributed to the superiorities of the predicted superior lines and superior hybrids. User-friendly software, QTLNetwork, version 1.0, was developed based on the methods in the present paper.

Algorithms↗

Microsatellite marker diversity in common bean (Phaseolus vulgaris L.).

A diversity survey was used to estimate allelic diversity and heterozygosity of 129 microsatellite markers in a panel of 44 common bean (Phaseolus vulgaris L.) genotypes that have been used as parents of mapping populations. Two types of microsatellites were evaluated, based respectively on gene coding and genomic sequences. Genetic diversity was evaluated by estimating the polymorphism information content (PIC), as well as the distribution and range of alleles sizes. Gene-based microsatellites proved to be less polymorphic than genomic microsatellites in terms of both number of alleles (6.0 vs. 9.2) and PIC values (0.446 vs. 0.594) while greater size differences between the largest and the smallest allele were observed for the genomic microsatellites than for the gene-based microsatellites (31.4 vs. 19.1 bp). Markers that showed a high number of alleles were identified with a maximum of 28 alleles for the marker BMd1. The microsatellites were useful for distinguishing Andean and Mesoamerican genotypes, for uncovering the races within each genepool and for separating wild accessions from cultivars. Greater polymorphism and race structure was found within the Andean gene pool than within the Mesoamerican gene pool and polymorphism rate between genotypes was consistent with genepool and race identity. Comparisons between Andean genotypes had higher polymorphism (53.0%) on average than comparisons among Mesoamerican genotypes (33.4%). Within the Mesoamerican parental combinations, the intra-racial combinations between Mesoamerica and Durango or Jalisco race genotypes showed higher average rates of polymorphism (37.5%) than the within-race combinations between Mesoamerica race genotypes (31.7%). In multiple correspondance analysis we found two principal clusters of genotypes corresponding to the Mesoamerican and Andean gene pools and subgroups representing specific races especially for the Nueva Granada and Peru races of the Andean gene pool. Intra population diversity was higher within the Andean genepool than within the Mesoamerican genepool and this pattern was observed for both gene-based and genomic microsatellites. Furthermore, intra-population diversity within the Andean races (0.356 on average) was higher than within the Mesoamerican races (0.302). Within the Andean gene pool, race Peru had higher diversity compared to race Nueva Granada, while within the Mesoamerican gene pool, the races Durango, Guatemala and Jalisco had comparable levels of diversity which were below that of race Mesoamerica.

Alleles↗

Identification of c-Fos immunoreactive brainstem neurons activated during fictive mastication in the rabbit.

In the present study we used the expression of the c-Fos-like protein as a "functional marker" to map populations of brainstem neurons involved in the generation of mastication. Experiments were conducted on urethane-anesthetized and paralyzed rabbits. In five animals (experimental group), rhythmical bouts of fictive masticatory-like motoneuron activity (cumulative duration 60-130 min) were induced by electrical stimulation of the left cortical "masticatory area" and recorded from the right digastric motoneuron pool. A control group of five animals (non-masticatory) were treated in the same way as the experimental animals with regard to surgical procedures, anesthesia, paralysis, and survival time. To detect the c-Fos-like protein, the animals were perfused, and the brainstems were cryosectioned and processed immunocytochemically. In the experimental group, the number of c-Fos-like immunoreactive neurons increased significantly in several brainstem areas. In rostral and lateral areas, increments occurred bilaterally in the borderzones surrounding the trigeminal motor nucleus (Regio h); the rostrodorsomedial half of the trigeminal main sensory nucleus; subnucleus oralis-gamma of the spinal trigeminal tract; nuclei reticularis parvocellularis pars alpha and nucleus reticularis pontis caudalis (RPc) pars alpha. Further caudally-enhanced labeling occurred bilaterally in nucleus reticularis parvocellularis and nucleus reticularis gigantocellularis (Rgc) including its pars-alpha. Our results provide a detailed anatomical record of neuronal populations that are correlated with the generation of the masticatory motor behavior.

Anesthesia↗

Marker-assisted introgression of trypanotolerance QTL in mice.

A marker-assisted introgression (MAI) experiment was conducted to use genetic markers to transfer each of the three trypanotolerance QTL from a donor mouse strain, C57BL/6, into a recipient mouse strain, A/J. We used a backcross strategy that consisted of selecting two lines, each carrying two of the donor QTL alleles through the backcross (BC) phase. At the fourth BC generation, single-carrier animals were selected for the production of homozygous animal in the intercross phase. The QTL regions (QTLR) were located on chromosomes MMU1, MMU5, and MMU17. Groups of mice with different genotypes and the parental lines were subjected to a challenge with Trypanosoma congolense. The results show that trypanotolerance QTL was successfully moved into the recipient background genotype, yielding a longer survival time. The mean estimated survival time was 57.9, 49.5, and 46.8 days for groups of mice carrying the donor QTL on MMU1, MMU5, and MMU17 on A/J background. The mean estimated survival time was 29.7 days for the susceptible A/J line and 68.8 days for the resistant C57BL/6 line. The estimated QTLR effects are close to 30% smaller than those in the original mapping population which was likely caused by the difference in the background on which the effects of QTLR are tested. This is the first report of successful marker-assisted introgression of QTL in animals. It is experimental proof of the use of genetic markers for marker-assisted introgression in animal breeding.

Animals↗

Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat.

Nearly 900 SSRs (simple sequence repeats) were identified among 15,000 ESTs (expressed sequence tags) belonging to bread wheat ( Triticum aestivumL.). The SSRs were defined by their minimum length, which ranged from 14 to 21 bp. The maximum length ranged from 24 to 87 bp depending upon the length of the repeat unit itself (1-7 bp). The average density of SSRs was one SSR per 9.2 kb of EST sequence screened. The trinucleotide repeats were the most abundant SSRs detected. As a representative sample, 78 primer pairs were designed, which were also used to screen the dbEST entries for Hordeum vulgare and Triticum tauschii (donor of the D-genome of cultivated wheat) using a cut-off E (expectation) value of 0.01. On the basis of in silico analysis, up to 55.12% of the primer pairs exhibited transferability from Triticum to Hordeum, indicating that the sequences flanking the SSRs are not only conserved within a single genus but also between related genera in Poaceae. Primer pairs for the 78 SSRs were synthesized and used successfully for the study of (1) their transferability to 18 related wild species and five cereal species (barley, oat, rye, rice and maize); and (2) polymorphism between the parents of four mapping populations available with us. A subset of 20 EST-SSR primers was also used to assess genetic diversity in a collection of 52 elite exotic wheat genotypes. This was done with a view to compare their utility relative to other molecular markers (gSSRs, AFLPs, and SAMPL) previously used by us for the same purpose with the same set of 52 bread wheat genotypes. Although only a low level of polymorphism was detected, relative to that observed with genomic SSRs, the study suggested that EST-SSRs can be successfully used for a variety of purposes, and may actually prove superior to SSR markers extracted from genomic libraries for diversity estimation and transferability.

Bread↗

Retrotransposon-based molecular markers for linkage and genetic diversity analysis in wheat.

Retrotransposon-based molecular markers have been developed to study bread wheat ( Triticum aestivum) and its wild relatives. SSAP (Sequence-Specific Amplification Polymorphism) markers based on the BARE-1/ Wis-2-1A retrotransposons were assigned to T. aestivum chromosomes by scoring nullisomic-tetrasomic chromosome substitution lines. The markers are distributed among all wheat chromosomes, with the lowest proportion being assigned the D wheat genome. SSAP markers for BARE-1/ Wis-2-1A and three other wheat retrotransposons, Thv19, Tagermina and Tar1, are broadly distributed on a wheat linkage map. Polymorphism levels associated with these four retrotransposons vary, with BARE-1/ Wis-2-1A and Thv19 both showing approximately 13% of bands polymorphic in a mapping population, Tagermina showing approximately 17% SSAP band polymorphism and Tar1 roughly 18%. This suggests that Tagermina and Tar1 have been more transpositionally active in the recent evolutionary past, and are potentially the more useful source of molecular markers in wheat. Lastly, BARE-1 / Wis-2-1A markers have also been used to characterise the genetic diversity among a set of 35 diploid and tetraploid wheat species including 26 Aegilops and 9 Triticum accessions. The SSAP-based diversity tree for Aegilops species agrees well with current classifications, though the Triticum tree shows several significant differences, which may be associated with polyploidy in this genus.

DNA, Plant↗

Genome-wide linkage analysis of Arabidopsis genes required for leaf development.

In most crop species, primary productivity depends mainly on the leaf. However, the genes that contribute to the making of plant leaves remain largely unknown. With a view to identifying the genes involved in leaf development in Arabidopsis thaliana, we previously isolated EMS-induced mutants with abnormally shaped leaves and demonstrated that they fall into 94 complementation groups. We present here the map positions of 76 of these genes, which have been obtained using a high-throughput genetic mapping method, based on the simultaneous coamplification by PCR of 21 polymorphic microsatellites and the semiautomated fluorescent detection of the products. The map positions and F2 mapping populations obtained in this work will be instrumental in the positional cloning of these genes, which are essential for leaf development.

Alleles↗

Analysis of genetic heterogeneity within the type strain of satellite tobacco mosaic virus reveals variants and a strong bias for G to A substitution mutations.

Satellite tobacco mosaic virus (STMV) is a small plant virus that is dependent for its replication on the presence of a helper tobamovirus. RNase protection analysis of genomic RNA of the STMV type strain revealed that it was composed of two major genome types which differed at a single detectable site near nucleotide 753. Analyses of 42 full-length STMV clones for sequence heterogeneity resulted in the identification of 16 variants distinguishable by unique RNase protection assay patterns. Characterization of these variants confirmed the presence of a major heterogeneity site at nucleotide 751 and identified several sites of sequence microheterogeneity typical of an RNA quasispecies population. Mapping of the heterogeneity sites revealed an apparently random distribution along the length of the STMV genome, with no significant clustering or preference for noncoding regions. Infectivity experiments in tobacco showed that RNA transcripts of 13 of the 16 variant clones were infectious, indicating that most of the variants represent functional genomes coexisting in the type strain with the two major genome types. Sequence analyses revealed that most of the heterogeneity sites detected, including the major site of heterogeneity, were single base differences. Assessment of all the heterogeneity sites found in the total of 10,545 nt sequenced allowed us to estimate that the RNase protection assays detected approximately 50% of the differences present in the 16 clones studied. The nature of these differences was highly biased in that 18 of the 29 single base differences characterized (62%) were G to A substitutions.

Base Sequence↗