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PubMed2004

DNA methylation polymorphism in a set of elite rice cultivars and its possible contribution to inter-cultivar differential gene expression.

RAPD (randomly amplified polymorphic DNA) and ISSR (inter-simple sequence repeat) fingerprinting on HpaII/MspI-digested genomic DNA of nine elite japonica rice cultivars implies inter-cultivar DNA methylation polymorphism. Using both DNA fragments isolated from RAPD or ISSR gels and selected low-copy sequences as probes, methylation-sensitive Southern blot analysis confirms the existence of extensive DNA methylation polymorphism in both genes and DNA repeats among the rice cultivars. The cultivar-specific methylation patterns are stably maintained, and can be used as reliable molecular markers. Transcriptional analysis of four selected sequences (RdRP, AC9, HSP90 and MMR) on leaves and roots from normal and 5-azacytidine-treated seedlings of three representative cultivars shows an association between the transcriptional activity of one of the genes, the mismatch repair (MMR) gene, and its CG methylation patterns.

Azacitidine↗

PubMed2004

Characterization of the genetic basis for yield and its component traits of rice revealed by doubled haploid population.

Main-effect QTL, epistatic effects and their interactions with environment are important genetic components of quantitative traits. In this study, we analyzed the QTLs, epistatic effects and QTL by environment interactions (QE) underlying nine traits of yield and yield-component, using a doubled-haploid (DH) population consisted of 190 lines from the cross between an indica parent Zhenshan 97 and a japonica parent WYJ 2, and tested in two-year replicated field trials. A genetic linkage map with 179 SSR (simple sequence repeat) marker loci was constructed. A mixed linear model approach was applied to detect QTLs, digenic interactions and QEs for the nine traits. In total, 57 QTLs of main effects, 41 digenic interactions, eight QEs and seven interactions of epistasis by environment were detected. Each of the main-effect QTLs individually explained 1.3 % to 25.8% of the phenotypic variations. And they collectively explained 11.5% to 66.8% of the phenotypic variations for these traits. Most of the traits (except seed setting) had the QTLs simultaneously detected in two years. Many of the traits shared same QTLs with each other, which is consistent with their significant phenotypic correlations. The pleiotropism or tight linkage of QTLs for different traits might be the important genetic base for trait correlations. The environmental influences on the stability of the trait performance were also discussed.

Crops, Agricultural↗

PubMed2004

[Isolation and characterization of nuclear microsatellites from Chamaecypress obtusa Endl].

Using 300 approximately 1 000 bp DNA fragments retrieved from 2% agarose gel electrophoresis on nuclear DNA samples of Chamaecypress obtusa with Nde II restricted digestion, microsatellites were efficiently isolated by using Dynabeads M280 streptavitin beads. These beads could immobilize hybrid products between biotin-labeled (CT) 15 probe, and 300 approximately 1 000 bp DNA fragments previously linkaged to Sau3A I cassette. Two enriched microsatellite libraries with about 2 200 positive white clones were constructed by inserting potential microsatellite fragments into vector pUC118 BamH I /BAP and culturing with host E. coli. After sequence analysis on 480 randomly picked out positive clones, it was revealed that the number of SSR-bearing clones accounts for 65.6%, and 215 clones were identified as non-homology. Furthermore,380 microsatellite locus were found in 215 non-homology clones indicating 1.7 loci per clone. Among the existed microsatellites,the expected type of (GA/CT) is the most popular which accounts for 86.1% and were compatible to the (CT) 15 probe, however some other types including (TG/AC)n and more complicated simple sequence repeats with 3 approximately 4 bases, such as (ATAG)n, (CAGA)n, (TGA)n, (ACG)n, (TCA) n, (TCT)n, could be also detected in these two libraries. Finally, primers for amplifying 128 microsatellite locus from 114 SSR-bearing clones were designed by Oligo 5. 1 (http: // lxg. users4. 50megs. com/ssr. html).

Base Sequence↗

PubMed2004

[Assesment of genetic diversity of allelopathic rice germplasm based on RAPD and ISSR].

Random Amplified Polymorphic DNA (RAPD) and Inter-simple Sequence Repeat (ISSR) methods were used to detect the genetic diversity of 57 allelopathic rice accessions which were introduced from 10 countries or areas. A total of 12 RAPD primers and seven ISSR primers were indentified with polymorphism among the entries. For RAPD markers, 85 polymorphic bands were produced, percentage of polymorphic bands (PPB) were 69.4%. For ISSR marker, 34 polymorphic bands were generated, PPB were 53.0%. The result from the clustering analysis by UPGMA indicated that those accessions from the same geographical location were clustered into one group. It was also found that some rice accessions with higher allelopathic potential were clustered together, implying that the genes conferring allelopathy in those rice accessions might be isolocus. However, some rice accessions with different allelopathic potential clustered into the same group performed lower level of generic polymorphism which was attributed to oriented selection for other traits in breeding program. The estimates of correlation coefficient of RAPD and ISSR based on the genetic similarity matrices were significantly correlated.

Genetic Variation↗

PubMed2004

Analysis of microsatellites derived from bee Ests.

To accelerate the molecular analysis of genetics,evolution and behavior, etc. in the honey bee (Apis), the frequency and density of simple sequence repeats (SSRs) have been analyzed from the bee EST (expressed sequence tag) database comprising 15 869 sequences amounting to 7.9 Mb. Results showed that the frequency of SSRs was 1/0.52 kb in bee ESTs, and hexanucleotide repeats (45.0%) motifs appeared to be the most abundant type in bee,the dinucleotide, mononucleotide, trinucleotide, tetranucleotide and pentanucleotide repeats are 17.9%, 14.1%, 11.6%, 9.2% and 2.2%, respectively. Meanwhile, the A-rich repeats are predominant in each type of SSRs, such as A, AT, AG, AC, AAT, AAG, AAC, AAAT, AAAG, AAAAG, AAAAT, AATAT, AAAAAG and AAAAAT repeats, whereas G-rich repeats are rare in the coding regions. The further analysis suggests that, apart from minor deviations, there is no significant difference in the distribution and density of microsatellites in the redundant and non-redundant set of bee ESTs. Furthermore, the availability of microsatellite markers can be expected to enhance the power and resolution of genome analysis in bee.

Animals↗

PubMed2003

[Progress of telomere and telomerase in higher plant].

Telomere is an important DNA-protein structure. It caps the ends of linear eukaryotic chromosomes. Telomeric DNA consists of tandemly repeated simple sequences. Telomere is synthesized with the action of telomerase, a ribonucleoprotein with reverse transcriptase activity. Telomere plays an important role in maintaining the stability of intact chromosome,genome and cell. This paper is a review of telomere, telomerase, telomere-binding protein and developmental control of telomere change, telomerase activity in higher plant.

English Abstract↗

PubMed2003

[The evolution and application of microsatellites].

Microsatellites,simple sequence repeats (SSR),are abundant and distributed throughout the eukaryote genome. The contents of microsatellites are variant in different creatures. There are also different types of microsatellites, which are dominant in different creatures. One of the most noticeable characters of microsatellites is that they are easy to expand during DNA replication. It is thought to attribute to DNA slippage. This kind of mutation is affected by many factors. It is guessed that microsatellites come from pro-microsatellites, while the pro-microsatellites origin from random point mutations. The length of microsatellites can be maintained under relative conservative ranges during species evolution. As they are abundant, codominatnt,distributed over the euchromatic part of the genome, and have the character of highly polimorphic, microsatellites are useful tools for gene mapping, clinical diagnosis and predicting, paternity or pedigree analysis, evolution study, and marker-assisted breeding.

English Abstract↗

PubMed2004

Verification of STS markers for leaf rust resistance genes of wheat by seven European laboratories.

A set of Thatcher near-isogenic lines and two breeding lines were used to examine sequence tagged site (STS) markers linked to leaf rust resistance genes Lr9, Lr10, Lr19, Lr24, Lr28, Lr29, Lr35, and a simple sequenced repeat (SSR) marker for Lr39. The selected STS markers for resistance genes Lr9, Lr10, Lr19, Lr24 and Lr28 were identified in seven accessions by seven European laboratories. Near-isogenic lines of the spring wheat Thatcher were used as positive controls. Markers for resistance genes Lr9, Lr10, Lr19, Lr24 were identified in all seven laboratories as amplification products of 1100 bp, 310 bp, 130 bp and 310 bp, respectively. The STS markers linked to resistance genes Lr9, Lr10, Lr19, Lr24, Lr29, Lr35 and the SSR marker for Lr39 were robust and highly specific for these genes and will be useful in marker-assisted selection in wheat. However, the amplification product of 378 bp that corresponded with resistance gene Lr28 was detected in all accessions including genotypes lacking this gene in all seven laboratories. This marker needs to be improved.

Basidiomycota↗

PubMed2004

Combined use of linked markers for genotyping the Pm1 locus in common wheat.

Genotyping of 98 wheat cultivars/lines was carried out with molecular markers that are linked to the Pm1 locus: two bi-allelic (dominant) markers: the sequence-tagged site Xsts638-7A and the amplified fragment length polymorphism XE39M58-77-7A; and the multi-allelic simple sequence repeat marker Xgwm344-7A. Employing segregation data recorded in the population Chinese Spring x Virest (Pm1e), genetic mapping revealed that Xgwm344-7A and XE39M58-77-7A were distally linked to Pm1e in the repulsion phase with respective linkage distances of 0.9 cM and 4.8 cM, while Xsts638-7A was found to co-segregate with Pm1e in the coupling phase. The genotyping results of Xsts638-7A and XE39M58-77-7A confirmed disease scoring, except for the accessions of cultivars Omega, Remus and Weihenstephan Stamm M1N. The SSR marker Xgwm344 amplified 15 different fragments ranging from 102 bp to 147 bp, with 15 entries being null-allelic at the 7A and 7B homoeoloci. It was found that wheat lines having resistance alleles at the Pm1 locus mainly show the null allele at the Xgwm344-7A locus. Due to their fast-evolving nature, the use of multi-allelic SSRs for genotype determination may be complicated. However, the combined use of multiple linked marker alleles seems to be a promising approach for genotyping a broad range of plant materials.

Alleles↗

PubMed2004

[Molecular study and C-banding of chromosomes in common wheat alloplasmic lines obtained from the backcross progeny of barley-wheat hybrids Hordeum vulgare L. (2n = 14) x Triticum aestivum L. (2n = 42) and differing in fertility].

We studied common wheat alloplasmic lines differing in fertility traits, which had been obtained from the backcross progeny of barley-wheat hybrids Hordeum vulgare L. (2n = 14) x Triticum aestivum L. (2n = 42), using molecular analysis and chromosome C-banding. It was found that the nuclei of all alloplasmic lines studied, regardless of their fertility traits, contained only the common wheat chromosomes (2n = 42). The formation of line L-79(10)(3)F6, stable for self-fertility, from line L-79(10)(3)F6 was accompanied by changes of the proportions of simple sequence repeats of the parental common wheat varieties in the nuclear genome. The presence of barley genome fragments in line accessions with incomplete self-fertility was shown by RAPD. Heteroplasmy for mitochondrial genome loci was detected in these lines with the use of primers specific to the tMet-18S-5S repeat of mitochondrial ribosomal genes.

Chimera↗

PubMed2004

Dna probes for identification of leptospires and disease diagnosis.

A newly identified 1 kb DNA fragment amplified by PCR using (AG)8T inter-simple sequence repeats (ISSR) primer and a 631 bp segment of 16S rRNA ribosomal gene amplified by PCR using reported primers were labeled with a alpha32P dCTP for use as DNA probes. These probes were hybridized with DNA extracted from 19 standard pathogenic serovars, 3 standard saprophytic serovars, 33 pathogenic isolates (12 from patients, 1 from a tapwater source, and 20 from rodents), and 22 saprophytic isolates from environmental sources. The pathogen-specific 16S rRNA DNA probe specifically hybridized all 33 standard pathogenic serovars, to 13 pathogenic isolates. Similarly, the saprophyte specific 1 kb ISSR DNA probe specifically hybridized the 3 standard saprophytic serovars and the 22 saprophytic Leptospira isolates. The sensitivity of the 1 kb labeled saprophytic Leptospira specific DNA probe was 1.95 ng, and for the 16S rRNA pathogen specific probe 3.90 ng. The 16S rRNA gene segment DNA probe could also identify the leptospiremic stage in mice or guinea pigs infected experimentally with the pathogenic serovars australis, autumnalis or icterohaemorrhagiae. DNA probes therefore, owing to their high specificity and sensitivity, appear useful for easy, rapid, and reliable differentiation of pathogenic Leptospira strains and also hold promise for direct identification of organisms in blood samples to diagnose leptopsirosis.

Animals↗

PubMed2005

[RAPD and ISSR analyses of regenerated pea Pisum sativum L. plants].

Long-term pea callus cultures of different genotypes (mutants R-9 and W-1 and cultivar Viola) were used to regenerate plants (generation R0). The regenerants displayed changes both in qualitative and in quantitative traits. The most dramatic morphological alterations and complete sterility were observed in regenerants of the cultivar Viola. To estimate the genetic differences, regenerants were compared with the original lines with the use of RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeat) analyses. The extent of divergence varied among regenerants and depended mostly on the original genotype. The genetic difference from the original line was no more than 1% in W-1 regenerants, 0.7-5.3% in R-9 regenerants, and 10-15% in sterile regenerants of the cultivar Viola. The genetic variation of plants regenerated from a callus culture maintained for ten years did not exceed that of plants obtained from a culture maintained for two years.

Base Sequence↗

PubMed2005

[Kohonen network study of the results of RAPD and ISSR analyses of genomic polymorphism in the genus Capsicum L].

The results of studies based on multilocus molecular analyses, including random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) analyses, are usually presented in the form of images (electrophoregrams, photographs, etc.). The interpretation of this information is complicated, labor-consuming, and subjective. Artificial neural networks (ANNs), which are ideal "image processors," may be useful when solving such tasks. The possibility of using ANNs for the treatment of the results of RAPD and ISSR analyses has been studied. The RAPD and ISSR spectra have been studied in fragments of DNA of plants from the genus Capsicum L. (peppers). The results of clustering the accessions studied by means of the unweighted pair-group method with arithmetic averages (UPGMA), which is often used for phylogenetic constructions based on RAPD and ISSR data, serve as expert estimates. Fundamentally new methods of genetic polymorphism estimation using ANN technologies, namely, self-organizing feature maps (SOFMs) have been developed. The results show that the clusters obtained with the use of UPGMA and SOFM coincide by more than 90%; taking into account that ANNs can deal with high noise levels and incomplete or contradictory data, the approach proposed may prove to be efficient.

Capsicum↗

PubMed2005

[Studying plant genome variation using molecular markers].

The authors' studies on the organization and variation of plant genome with the use of molecular markers are briefly reviewed with special emphasis on random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), sequence characterized amplified region (SCAR), and cleaved amplified polymorphic sequence (CAPS) markers detected with the use of polymerase chain reaction (PCR). These markers have been demonstrated to be promising for identifying cultivars and determining the purity of genetic strains of pea. Genetic relationships between strains, cultivars, and mutants of pea have been studied. The role of molecular markers in molecular genetic mapping and localizing the genes of commercially important characters of pea has been shown. The possibility of the use of molecular markers for studying somaclonal variation and detecting mutagenic factors in plants during long-term spaceflights is considered. The prospects of using DNA markers for understanding the organization and variability of higher plant genomes are discussed.

Genetic Markers↗

PubMed2002

[ISSR markers and their applications in plant genetics].

Recently, inter-simple sequence repeat (ISSR) markers have emerged as an alternative system with reliability and advantages of microsatellites (SSR). The technique involves amplification of genomic segments flanked by inversely oriented and closely spaced microsatellite sequences by a single primer or a pair of primers based on SSRs anchored 5' or 3' with 1-4 purine or pyramidine residues. The sequences of repeats and anchor nucleates are arbitrarily selected. Coupled with the separation of amplification products on a polyacrylamide or agarose gels,ISSR amplification can reveal a much larger number of fragments per primer than RAPD. It is concluded that ISSR technique provides a quick, reliable and highly informative system for DNA fingerprinting.ISSR markers are inherited in Mendelin mode and segregated as dominant markers. This technique has been widely used in the studies of cultivar identification, genetic mapping, gene tagging,genetic diversity, evolution and molecular ecology.

English Abstract↗

PubMed2005

Towards an expanded linkage map and exploration on co-dominant scoring of AFLPs in maize.

Simple sequence repeats (SSRs) and amplified fragment length polymorphisms (AFLPs) have become the most important markers for molecular mapping. Primarily based on restriction fragment length polymorphism (RFLP) markers, extensive linkage maps of maize had been developed. To construct a near-saturated genetic linkage map, an expanded maize genetic linkage map was constructed using a population of 234 F2 individuals derived from a cross of X178 and B73 base on an essential SSR framework map of maize. The level of polymorphisms and genetic properties of SSR and AFLP markers were characterized. A total of 249 markers consisting of 130 SSRs and 119 AFLPs have been landed on 10 chromosomes of maize. The 249-locus map spanned 1 659.3 centi-morgans (cM) and had a mean density of 6.66 cM. This mapping population and related information should connect further research involving analysis of quantitative trait loci, comparative genomics, and heterosis. Moreover, in many studies, AFLPs were analyzed on the basis of the presence or absence of a band on the electrophoresis gels. A new method based on double polymorphic bands of co-dominant scoring of AFLPs was explored according to the similarity of loci amplified from AFLP enzyme combination.

Amplified Fragment Length Polymorphism Analysis↗

PubMed2005

[Genetic diversity of yam (Dioscorea opposita Thunb) detected by ISSR markers].

Genetic diversity of 28 cultivars of yam (Dioscorea opposita Thunb) was assessed by means of Inter-simple sequence repeat (ISSR) markers. The results showed that seven proper primers, with rich polymorphism, could be selected from a total of forty four ISSR ones; distinct differences appeared among 28 cultivars amplified bands, and the rate of polymorphic bands was 83.01%; Shannon's Information index was 0.3191; a Jaccard's genetic similarity matrix and a dendrogram for these cultivars were formed, in which they could be divided into four groups: Group 1 was composed of D. opposita. cv. Ribenbai, D. opposita. cv. Huashanyao and D. opposita. cv. Ribenyuan; Group2 contained D. opposita. cv. Xiaoye; Group 3 contained D. opposita. cv. No.1 Songye; other 23 cultivars were put into Group4. PCA(Principal component analysis) was employed to evaluate the resolving power of the markers to differentiate among them. This laid the foundation of the identification of yam cultivars and the efficient use of its germplasm resources.

DNA, Plant↗

PubMed2005

[Genetic diversity of flue-cured tobacco varieties based on ISSR markers].

Genetic diversity was assessed among 24 flue-cured tobacco varieties by ISSR (inter simple sequence repeats). A total of 100 ISSR primers were used to amplify the DNA from these varieties, of which 10 primers produced reproducible amplified products. Using polyacrylamide gel electrophoresis 208 bands were identified, of which 141 bands were polymorphic among the flue-cured tobacco varieties analyzed. Each primer produced 7-37 bands, the length of which ranged 200-2,400 bp. The ratio of polymorphic bands (PPB) was 67.79%. By cluster analysis based on ISSR markers using UPGMA, 24 varieties were divided into 5 major groups, in which the biggest group consisted of 12 varieties derived from Coker319. The genetic similarity index was 0.66-0.85 among 24 flue-cured tobacco varieties. Low genetic diversity among flue-cured tobacco varieties suggested that it is necessary to expand the genetic base of the flue-cured tobacco. 24 varieties could be distinguished by using 2 ISSR markers. The result also indicated that ISSR analysis was suitable for varietal identification and the study on genetic diversity of tobacco germplasm.

DNA, Plant↗