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PubMed2002

Comparison of the waxy locus sequence from a non-waxy strain and two waxy mutants of spontaneous and artificial origins in barley.

Molecular characterization of 3 alleles of the waxy gene from a non-waxy strain "Shikoku hadaka No. 84" (SH84), an indigenous waxy strain "Mochimugi D" (MMD), and an artificial waxy mutant strain "Shikoku hadaka No. 97" (SH97) of barley (Hordeum vulgare ssp. vulgare) was performed via a PCR direct sequencing strategy. The 3 haplotypes were analyzed in terms of single nucleotide polymorphisms, insertion/deletion mutations, and simple sequence repeat polymorphisms. In comparison with the barley non-waxy gene sequence deposited in the public DNA database, 110 polymorphic sites were found in the 5,190-bp sequenced region of the non-waxy strain SH84. A 418-bp deletion in the 5' non-coding sequence was identified in the indigenous waxy strain MMD. Except for the deletion in the promoter region, the spontaneous mutant wax allele and non-waxy allele were identical. Such highly conserved sequences provide evidence for the recent occurrence of a deletion event in the cultivated barley gene pool. Compared to the original variety SH84, induced waxy mutant SH97 had a base substitution of a C to T in the exon 5, which converting Gln-89 of the wild-type gene into a stop codon, suggesting the involvement of a nonsense-mediated mRNA decay. These results will be helpful for understanding the mechanism of the variable amylose content in waxy cultivars of cereal species.

Amylopectin↗

PubMed2004

Chloroplast and nuclear DNA variation in common wheat: insight into the origin and evolution of common wheat.

To understand the origin and evolution of common wheat, chloroplast (ct) and nuclear DNA variations were studied in five hexaploid and three tetraploid wheat subspecies. Based on chloroplast simple sequence repeats at 24 loci, they were classified into two major plastogroups. Plastogroup I consisted of 11 plastotypes, including the major plastotype H10 that occurred at the highest frequency (59%) in common wheat. Plastogroup II consisted of five plastotypes and occurred in eight out of 27 accessions of T. aestivum ssp. spelta and one accession of ssp. aestivum. As for nuclear DNA variations, AFLP data using 10 primer sets revealed two major clades of a phylogenetic tree constructed by UPGMA (unweighted pair-group method with arithmetic mean), one consisting of common wheat and the other of emmer wheat. The clade of common wheat was further divided into two major and six minor subclades. One of the major subclades consisted only of non-free-threshing ssp. spelta accessions, which were grouped into two clusters, one consisting only of accessions with plastogroup I ctDNA and the other with both plastogroups I and II. T. aestivum ssp. macha, another non-free-threshing common wheat, formed the other cluster. Taken together, our data indicate the existence of at least two maternal lineages in common wheat and support the hypothesis that European spela wheat originated in Europe separately from other groups of common wheat.

Biological Evolution↗

PubMedDate not supplied

Development and partial characterization of heliothine cell lines from embryonic and differentiated tissues.

The goal of this study was to generate cell lines from a variety of insect tissues that could be useful for developing in vitro assays with tissue-specific properties. In this article, we describe the establishment of new cell cultures from differentiated (primarily neural) and undifferentiated tissues (primarily embryonic) and their initial characterization. Cell lines were established from the following tissues of the budworm, Heliothis virescens, and the bollworm, Helicoverpa zea: larval ventral nerve cords (4 lines), larval midguts (1 line), adult ovaries (1 line), and embryonic tissues (11 lines). Cell lines were primarily characterized by morphological examination and polymerase chain reaction (PCR) (both deoxyribonucleic acid amplification fingerprinting and inter-simple sequence repeats PCR).

Animals↗

PubMed2005

Sorghum genome sequencing by methylation filtration.

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.

Arabidopsis↗

PubMed2005

Microsatellite instability: an indirect assay to detect defects in the cellular mismatch repair machinery.

The DNA mismatch repair (MMR) pathway plays a prominent role in the correction of errors made during DNA replication and genetic recombination and in the repair of small deletions and loops in DNA. Mismatched nucleotides can occur by replication error, damage to nucleotide precursors, damage to DNA, or during heteroduplex formation between two homologous DNA molecules in the process of genetic recombination. Defects in MMR can precipitate instability in simple sequence repeats (SSRs), also referred to as microsatellite instability (MSI), which appears to be important in certain types of cancers, both spontaneous and hereditary. Variation in the highly polymorphic alleles of specific microsatellite repeats can be identified using PCR with primers derived from the unique flanking sequences. These PCR products are analyzed on denaturing polyacrylamide gels to resolve differences in allele sizes of more than 2 bp. Although (CA)n repeats are the most abundant class among dinucleotide SSRs, trinucleotide and tetranucleotide repeats are also frequent. These polymorphic repeats have the advantage of producing band patterns that are easy to analyze and can be used as an indication of a possible MMR defect in a cell. The presumed association between such allelic variation and an MMR defect should be confirmed by molecular analysis of the structure and/or expression of MMR genes.

Animals↗

PubMed2006

The Genome of Dictyostelium discoideum.

The Dictyostelium discoideum genome has been sequenced, assembled and annotated to a high degree of reliability. The parts-list of proteins and RNA encoded by the six chromosomes can now be accessed and analyzed. One of the initial surprises was the remarkably large number of genes that are shared with plants, animals, and fungi that must have been present in their common progenitor over a billion years ago. The genome encodes a total of about 10,300 proteins including protein families involved in cytoskeletal control, posttranslational protein modification, detoxification, secondary metabolism, cell adhesion, and signal transduction. The genome has a higher proportion of homopolymeric tracts and simple sequence repeats, such as [CAA]n, than most other genomes. Triplet repeats in translated regions produce the highest known proportion of polyglutamine tracts in any known proteome. Phylogenetic analyses based on complete proteomes confirm that the amoebozoa are a sister group to the animals and fungi, distinct from plants and early diverging species such as Leishmania, Plasmodium, or Giardia. The completed Dictyostelium sequence opens the door to large-scale functional exploration of its genome.

Animals↗

PubMed2004

ISSR-based clustering of cultivated flax germplasm is statistically correlated to thousand seed mass.

Inter-simple sequence repeat (ISSR)-polymerase chain reaction (PCR) polymorphism was generated to provide useful markers for assessment of genetic diversity within flax germplasm collections. We used nine previously selected anchored ISSR primers for fingerprinting of 53 flax cultivars or genotypes and obtained 62 scorable bands, from which 45 bands (72.6%) were polymorphic. An efficient separation of 53 flax accessions into four groups and eight subgroups was achieved using unweighted pair group method with arithmetic means (UPGMA) clustering procedure based on genetic similarity expressed by the Jaccard similarity coefficient (JSC). Clustering procedure within both groups and subgroups successfully produced smaller homogenous clusters, whereas clustering between the main four groups of flax accessions displayed only a continuous decrease of similarity with a weak clustering effect. Statistical significance of grouping and subgrouping within a cluster dendrogram was estimated by calculation of the error flag and cophenetic correlation parameter for each branch. Principal coordinates (PCO) analysis mostly confirmed the separation by UPGMA clustering. We observed a statistically significant correlation between the number of total vs polymorphic bands in ISSR patterns. A one-way analysis of variance (ANOVA) test confirmed statistically significant differences in the average thousand seed mass (TSM) between eight subclusters of flax accessions from an ISSR-PCR-based UPGMA dendrogram, which indicate statistical correlation between flax ISSR polymorphism (the structure of ISSR-based clustering) TSM.

Cluster Analysis↗

PubMedDate not supplied

Interpopulation congruence in Chinese Primula ovalifolia revealed by chemical and molecular markers using essential oils and ISSRs.

The chemical composition of the essential oils of five natural populations of P. ovalifolia from central and southwest China and their interpopulation variability were first analyzed by using GC-MS. Twenty-two essential oil compounds were obtained, in which eighteen ones were identified and characterized representing 95%-96% of the oil composition. Three main chemotypes, i.e., the methyl-acetyl-hydroquinone-rich, hydroquinone-rich, and acetyl-hydroquinone-rich chemotypes, were then differentiated, corresponding to the three groups obtained from the cluster analysis based on the essential oil composition percentages. Genetic variations among the five populations were also investigated using the Inter-Simple Sequence Repeats (ISSR) markers. Finally, the Mantel test showed that there was a significant correlation between two distance matrices based on the chemical compounds of essential oils and ISSR markers, confirming the congruence of interpopulation relationships in the P. ovalifolia revealed by the chemical and molecular markers

China↗

PubMedDate not supplied

New source of genetic polymorphisms in Lepidoptera?

The variability level of the ISSR (inter-simple sequences repeat) primer (GACA)4 was examined in the three Lepidoptera families Pyralidae, Sphingidae and Pieridae. Our study shows that the tetra-repeat (GACA)n is evidently present in sufficient numbers in these butterflies to provide informative DNA fingerprints. The variability is mostly rather high, but within a comparable range to other ISSR studies. Although less polymorphisms may be encountered in some butterfly families, this study indicates that high variability of this marker may be a common characteristic of Lepidoptera genomes. An appeal for a minimal level of standardization of ISSR-PCR data analysis is formulated to enable an exact comparison between the groups of organisms studied with this fingerprint technique.

Animals↗

PubMed2004

Genetic influences on ovulation of primary oocytes in LT/Sv strain mice.

A high proportion of LT/Sv strain oocytes arrest in meiotic metaphase I (MI) and are ovulated as diploid primary oocytes rather than haploid secondary oocytes. (Mus musculus castaneus x LT/SvKau)F1 x LT/SvKau backcross females were analysed for the proportion of oocytes that arrested in MI and typed by PCR for a panel of microsatellite DNA sequences (simple sequence repeat polymorphisms) that differed between strain LT/SvKau and M. m. castaneus. This provided a whole genome scan of 86 genetic markers distributed over all 19 autosomes and the X chromosome, and revealed genetic linkage of the MI arrest phenotype to markers on chromosomes 1 and 9. Identification of these two chromosomal regions should facilitate the identification of genes involved in mammalian oocyte maturation and the control of meiosis.

Animals↗

PubMed2005

An analysis of genetic diversity across the maize genome using microsatellites.

How domestication bottlenecks and artificial selection shaped the amount and distribution of genetic variation in the genomes of modern crops is poorly understood. We analyzed diversity at 462 simple sequence repeats (SSRs) or microsatellites spread throughout the maize genome and compared the diversity observed at these SSRs in maize to that observed in its wild progenitor, teosinte. The results reveal a modest genome-wide deficit of diversity in maize relative to teosinte. The relative deficit of diversity is less for SSRs with dinucleotide repeat motifs than for SSRs with repeat motifs of more than two nucleotides, suggesting that the former with their higher mutation rate have partially recovered from the domestication bottleneck. We analyzed the relationship between SSR diversity and proximity to QTL for domestication traits and observed no relationship between these factors. However, we did observe a weak, although significant, spatial correlation for diversity statistics among SSRs within 2 cM of one another, suggesting that SSR diversity is weakly patterned across the genome. Twenty-four of 462 SSRs (5%) show some evidence of positive selection in maize under multiple tests. Overall, the pattern of genetic diversity at maize SSRs can be explained largely by a bottleneck effect with a smaller effect from selection.

DNA, Plant↗

PubMed2005

Genetic consequences of selection during the evolution of cultivated sunflower.

We mapped quantitative trait loci (QTL) controlling differences in seed oil content and composition between cultivated and wild sunflower and used the results, along with those of a previous study of domestication-related QTL, to guide a genome-wide analysis of genetic variation for evidence of past selection. The effects of the seed oil QTL were almost exclusively in the expected direction with respect to the parental phenotypes. A major, oil-related QTL cluster mapped near a cluster of domestication-related QTL on linkage group six (LG06), the majority of which have previously been shown to have effects that are inconsistent with the parental phenotypes. To test the hypothesis that this region was the target of a past selective sweep, perhaps resulting in the fixation of the antagonistic domestication-related QTL, we analyzed simple sequence repeat (SSR) diversity from 102 markers dispersed throughout the sunflower genome. Our results indicate that LG06 was most likely the target of multiple selective sweeps during the postdomestication era. Strong directional selection in concert with genetic hitchhiking therefore offers a possible explanation for the occurrence of numerous domestication-related QTL with apparently maladaptive phenotypic effects.

Biological Evolution↗

PubMed2005

Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map.

The concept of selective (or bin) mapping is used here for the first time, using as an example the Prunus reference map constructed with an almond x peach F2 population. On the basis of this map, a set of six plants that jointly defined 65 possible different genotypes for the codominant markers mapped on it was selected. Sixty-three of these joint genotypes corresponded to a single chromosomal region (a bin) of the Prunus genome, and the two remaining corresponded to two bins each. The 67 bins defined by these six plants had a 7.8-cM average length and a maximum individual length of 24.7 cM. Using a unit of analysis composed of these six plants, their F1 hybrid parent, and one of the parents of the hybrid, we mapped 264 microsatellite (or simple-sequence repeat, SSR) markers from 401 different microsatellite primer pairs. Bin mapping proved to be a fast and economic strategy that could be used for further map saturation, the addition of valuable markers (such as those based on microsatellites or ESTs), and giving a wider scope to, and a more efficient use of, reference mapping populations.

Chromosome Mapping↗

PubMed2005

Association mapping of kernel size and milling quality in wheat (Triticum aestivum L.) cultivars.

Association mapping is a method for detection of gene effects based on linkage disequilibrium (LD) that complements QTL analysis in the development of tools for molecular plant breeding. In this study, association mapping was performed on a selected sample of 95 cultivars of soft winter wheat. Population structure was estimated on the basis of 36 unlinked simple-sequence repeat (SSR) markers. The extent of LD was estimated on chromosomes 2D and part of 5A, relative to the LD observed among unlinked markers. Consistent LD on chromosome 2D was <1 cM, whereas in the centromeric region of 5A, LD extended for approximately 5 cM. Association of 62 SSR loci on chromosomes 2D, 5A, and 5B with kernel morphology and milling quality was analyzed through a mixed-effects model, where subpopulation was considered as a random factor and the marker tested was considered as a fixed factor. Permutations were used to adjust the threshold of significance for multiple testing within chromosomes. In agreement with previous QTL analysis, significant markers for kernel size were detected on the three chromosomes tested, and alleles potentially useful for selection were identified. Our results demonstrated that association mapping could complement and enhance previous QTL information for marker-assisted selection.

Chromosome Mapping↗

PubMed2006

Maize adaptation to temperate climate: relationship between population structure and polymorphism in the Dwarf8 gene.

To investigate the genetic basis of maize adaptation to temperate climate, collections of 375 inbred lines and 275 landraces, representative of American and European diversity, were evaluated for flowering time under short- and long-day conditions. The inbred line collection was genotyped for 55 genomewide simple sequence repeat (SSR) markers. Comparison of inbred line population structure with that of landraces, as determined with 24 SSR loci, underlined strong effects of both historical and modern selection on population structure and a clear relationship with geographical origins. The late tropical groups and the early "Northern Flint" group from the northern United States and northern Europe exhibited different flowering times. Both collections were genotyped for a 6-bp insertion/deletion in the Dwarf8 (D8idp) gene, previously reported to be potentially involved in flowering time variation in a 102 American inbred panel. Among-group D8idp differentiation was much higher than that for any SSR marker, suggesting diversifying selection. Correcting for population structure, D8idp was associated with flowering time under long-day conditions, the deletion allele showing an average earlier flowering of 29 degree days for inbreds and 145 degree days for landraces. Additionally, the deletion allele occurred at a high frequency (>80%) in Northern Flint while being almost absent (<5%) in tropical materials. Altogether, these results indicate that Dwarf8 could be involved in maize climatic adaptation through diversifying selection for flowering time.

Alleles↗

PubMed2006

Species-specific SCAR markers for authentication of Sinocalycanthus chinensis.

Sinocalycanthus chinensis, an endangered species endemic to China, is cultivated as an ornamental landscape tree in China. However, S. chinensis, Chimonanthus species and Calycanthus floridus are difficult to be distinguished in seedling market because of their similar morphological characters. In this study, ISSR (inter-simple sequence repeats) were applied to detect S. chinensis from its closely related species. A unique 748-bp band was found in all accessions of S. chinensis. SCAR (sequence characterized amplified regions) markers were created by cloning and sequencing the specific band, and designing a pair of primers to amplify the band of 748 bp. Diagnostic PCRs were performed using the primer pair with the total DNAs of S. chinensis, Chimonanthus species and C. floridus as templates, with only S. chinensis being able to be amplified. This amplification is not only rapid (results can be obtained in less than 3 h), but is also easy to perform. Hence it is a feasible method for identifying S. chinensis in seedling market.

Calycanthaceae↗

PubMed2000

Randomly amplified polymorphic DNA (RAPD) polymerase chain reaction assay for identification of Schistosoma mansoni strains sensitive or tolerant to anti-schistosomal drugs.

The genetic differences between Schistosoma mansoni strains from different geographic areas that were reportedly resistant or sensitive to anti-schistosomal drugs were studied with randomly amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) polymerase chain reaction (PCR) assays. Of the 20 RAPD primers we chose, 19 showed the capacity to produce a medium to high level of amplification and 6 revealed difference PCR bands between drug-resistant and drug-sensitive strains. One particular primer, 5'-CAGCGACAAG-3', showed 2 major difference bands between praziquantel (PZQ)-resistant and PZQ-sensitive strains from the endemic area of Egypt. These results demonstrate that defined sequence primers could be applied as a useful tool for differentiating drug-resistant and -sensitive schistosome parasites in the field.

Animals↗

PubMed2000

Use of haploid mixtures and heteroduplex analysis enhance polymorphisms revealed by denaturing gradient gel electrophoresis.

PCR-based codominant genetic markers were developed by using primer sequences designed from cDNA clones of loblolly pine (Pinus taeda L.). Such markers offer certain advantages relative to simple-sequence repeat (SSR), also known as short-tandem repeat (STR) markers, and include the ability to quantify and map DNA polymorphisms in expressed genes. However, detecting these DNA polymorphisms is more problematic because many DNA polymorphisms in genes involve base substitutions rather than insertions or deletions. Denaturing gradient gel electrophoresis (DGGE) is a sensitive and efficient method for detecting sequence differences among PCR fragments. This paper demonstrates the application of DGGE to genetically map expressed genes in loblolly pine. Also, heteroduplex DNA fragments, formed during the amplification of DNA from heterozygotes and from mixes of haploid DNAs from megagametophytes, enhanced and strengthened genetic interpretations and genotypic classifications.

Alleles↗