Identification of repetitive, genome-specific probes in crucifer oilseed species

Genome ◽  
2002 ◽  
Vol 45 (3) ◽  
pp. 485-492 ◽  
Author(s):  
Daryl J Somers ◽  
Goewin Demmon
Keyword(s):  
Genomic Dna ◽  
Tandem Repeats ◽  
Pcr Primers ◽  
Specific Marker ◽  
Dna Fragments ◽  
Minisatellite Dna ◽  
High Affinity ◽  
B Genome ◽  
A Genome ◽  
Direct Amplification

Direct amplification of minisatellite DNA by PCR (DAMD PCR) was used to amplify and subsequently clone several fragments of DNA from crucifer species. The PCR-derived fragments of DNA were generated using known minisatellite core sequences as PCR primers. Southern hybridization of these putative minisatellite DNA fragments revealed that many were genome-specific; they hybridized with high affinity only to the genomic DNA of the species from which they were cloned. The DNA fragments were believed to be dispersed in the genome, based on smear-like hybridization signals on EcoRI-, BamHI-, and HindIII-digested genomic DNA. Genome-specific probes were specifically isolated from Brassica rapa (A genome), Brassica nigra (B genome), and Sinapis alba in addition to several other crucifer species. The sequence of a B. rapa specific probe (pBr17.1.3A) contained a minisatellite region that could be divided into three tandem repeats; each repeat contained between two and five subrepeats and each subrepeat shared a highly conserved core region of 29 bp. This minisatellite sequence also hybridized with high affinity to the A genome species B. napus and B. juncea. This research showed that dispersed, genome-specific probes can be isolated using DAMD PCR and that these probes could be used to detect and quantify alien DNA present in progeny from intergeneric or interspecific crosses.Key words: Brassica, genome specific, marker-assisted selection, minisatellite.

Characterization of six wheat × Thinopyrum intermedium derivatives by GISH, RFLP, and multicolor GISH

Genome ◽  
2003 ◽  
Vol 46 (3) ◽  
pp. 490-495 ◽  
Author(s):  
F P Han ◽  
G Fedak ◽  
A Benabdelmouna ◽  
K Armstrong ◽  
T Ouellet
Keyword(s):  
Genomic Dna ◽  
Aegilops Tauschii ◽  
Rflp Analysis ◽  
Aegilops Speltoides ◽  
Thinopyrum Intermedium ◽  
B Genome ◽  
A Genome ◽  
Genetic Stocks ◽  
Disomic Addition Lines ◽  
Thinopyrum Elongatum

Restriction fragment length polymorphism (RFLP) analysis and multicolor genomic in situ hybridization (GISH) are useful tools to precisely characterize genetic stocks derived from crosses of wheat (Triticum aestivum) with Thinopyrum intermedium and Thinopyrum elongatum. The wheat × Th. intermedium derived stocks designated Z1, Z2, Z3, Z4, Z5, and Z6 were initially screened by multicolor GISH using Aegilops speltoides genomic DNA for blocking and various combinations of genomic DNA from Th. intermedium, Triticum urartu, and Aegilops tauschii for probes. The probing (GISH) results indicated that lines Z1 and Z3 were alien disomic addition lines with chromosome numbers of 2n = 44. Z2 was a substitution line in which chromosome 2D was substituted by a pair of Th. intermedium chromosomes; this was confirmed by RFLP and muticolour GISH. Z4 (2n = 44) contained two pairs of wheat – Th. intermedium translocated chromosomes; one pair involved A-genome chromosomes, the other involved D- and A-genome chromosomes. Z5 (2n = 44) contained one pair of wheat – Th. intermedium translocated chromosomes involving the D- and A-genome chromosomes of wheat. Z6 (2n = 44) contained one pair of chromosomes derived from Th. intermedium plus another pair of translocated chromosomes involving B-genome chromosomes of wheat. Line Z2 was of special interest because it has some resistance to infection by Fusarium graminearum.Key words: wheat, Thinopyrum intermedium, addition, substitution, and translocation lines, GISH, multicolor GISH, RFLP.

scholarly journals SCAR Marker for the A Genome of Bananas (Musa spp. L.) Supports Lack of Differentiation between the A and B Genomes

2017 ◽  
Vol 9 (6) ◽  
pp. 64
Author(s):  
Lloyd Mabonga ◽  
Michael Pillay
Keyword(s):  
Scar Marker ◽  
Agronomic Traits ◽  
Random Amplified Polymorphic Dna ◽  
Diploid Species ◽  
Specific Marker ◽  
Correct Identification ◽  
Musa Spp ◽  
B Genome ◽  
A Genome ◽  
Putative Marker

  Bananas (Musa spp. L.) are grouped on the basis of their genomic origins in relation to Musa acuminata (A genome) and M. balbisiana (B genome). The two ancestral wild seeded diploid species evolved in vastly different geographical areas and contributed several agronomic traits towards the present genetic composition of cultivated bananas. Most cultivated bananas are triploid (AAA, AAB and ABB), some are diploid (AA, BB and AB) and a few are tetraploids (AAAA, AAAB, AABB and ABBB). Limitations on the correct identification of the A and B genomes in Musa have generated need for the development of new and more reliable techniques. Distinguishing the A and the B genome remains practically and theoretically important for banana breeders. The aim of the research was to develop a DNA based A genome specific marker for the identification of the A genome in bananas. A putative marker (600 bp) specific to the A genome was identified by Random Amplified Polymorphic DNA (RAPD) technique. A sequence characterised amplified region (SCAR) marker was developed from the RAPD amplicon. The SCAR primers annealed a 500 bp fragment specific to the A genome in a sample of 22 randomly selected homo- and heterogenomic A genome containing accessions representing different genome combinations. The 500 bp SCAR marker is useful for the identification of the A genome. However an additional 700 bp fragment annealed in all M. balbisiana genotypes and in five of the eight heterogenomic accessions, suggesting lack of differentiation between the A and B genome. This study has provided a 500 bp A genome SCAR marker and recent evidence that the A and B genomes of banana may not be as differentiated as previously considered.

A direct repeat sequence associated with the centromeric retrotransposons in wheat

Genome ◽  
2004 ◽  
Vol 47 (4) ◽  
pp. 747-756 ◽  
Author(s):  
Hidetaka Ito ◽  
Shuhei Nasuda ◽  
Takashi R Endo
Keyword(s):  
Genomic Dna ◽  
Direct Repeat ◽  
Repeat Sequence ◽  
Triticum Monococcum ◽  
Fish Analysis ◽  
Long Terminal Repeats ◽  
D Genome ◽  
B Genome ◽  
Cereal Species ◽  
A Genome

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.Key words: centromere, Ty3-gypsy retrotransposon, FISH, wheat, repetitive element.

scholarly journals Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xianqiang Li ◽  
Xin Jiang ◽  
Meiying Xu ◽  
Yun Fang ◽  
Yan Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Environmental Stress ◽  
Genomic Dna ◽  
Luciferase Reporter ◽  
Functional Screening ◽  
Dna Fragments ◽  
Control Of Gene Expression ◽  
E Coli ◽  
A Genome ◽  
Stress Changes

Abstract Bacteria promoters along with operators are crucial elements in the control of gene expression in microbes in response to environmental stress changes. A genome-wide promoter DNA regulatory library is in demand to be developed for a microbe reporter method to monitor the existence of any given environmental stress substance. In this study, we utilized Escherichia coli (E. coli) as a model system for the preparation of both cell lysates and genomic DNA fragments. Through enriching protein-bound DNA fragments to construct luciferase reporter libraries, we found that, of 280 clones collected and sequenced, 131 clones contained either the promoter-35 and -10 conservative sequences and/or an operator transcription factor binding sites (TFBS) region. To demonstrate the functionality of the identified clones, five of 131 clones containing LexA binding sequence have been demonstrated to be induced in response to mitomycin C treatment. To evaluate our libraries as a functional screening library, 80 randomly picked up clones were cultured and treated with and without MMC, where two clones were shown to have greater than twofold induction. In addition, two arsenite-responsive clones were identified from 90 clones, one having the well-known ArsR and another having the osmotically inducible lipoprotein (OsmE1). The newly discovered osmE1 has been quantitatively validated to be induced by arsenite treatment with real-time PCR in a dose response and time course manner. This enriching protein-bound DNA luciferase reporter libraries and functional screening facilitate the identification of stress-responsive transcriptional factors in microbes. We developed functional libraries containing E. coli genomic-wide protein-bound DNA as enhancers/operators to regulate downstream luciferase in response to stress.

scholarly journals Physical mapping of repetitive oligonucleotides facilitates the establishment of a genome map-based karyotype to identify chromosomal variations in peanut

2020 ◽  
Author(s):  
Liuyang Fu ◽  
Qian Wang ◽  
Lina Li ◽  
Tao Lang ◽  
Junjia Guo ◽  
...  
Keyword(s):  
Physical Mapping ◽  
Tandem Repeats ◽  
Genetic Research ◽  
Diploid Species ◽  
B Genome ◽  
A Genome ◽  
Genome Map ◽  
Reference Sequences ◽  
Chromosomal Variants

Abstract Background: Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut. Thus, the identification of chromosomal variants in peanut remains a challenge, owing to a lack of efficient chromosomal markers. Results: A total 114 new oligo probes were developed, based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligos were classified into 28 types, based on their positions, and overlapping signals in chromosomes. For each oligo types, a single and representative oligos was selected and modified with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA). Based on these 28 probes, a new multiplex #3 cocktail was developed with FAM-modified TIF-439, TIF-185-1, TIF-134-3, and TIF-165-3, and TAMRA-modified Ipa-1162, Ipa-1137, DP-1, and DP-5. This cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of peanut induced by radiation. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, and intercalary and terminal regions; four were B genome-specific; one was chromosome-specific; and the other 15 were extensively mapped in the pericentric regions of chromosomes. Conclusions: The development of new oligo probes provides effective tools, which can be used to distinguish various chromosomes of peanut. Physical mapping reveals the genomic organization of repetitive oligos in peanut chromosomes by FISH. Following comparisons with their positions in the reference sequences, a genome map-based karyotype was established and used for the identification of chromosome variations in peanut.

scholarly journals Physical mapping of repetitive oligonucleotides facilitates the establishment of a genome map-based karyotype to identify chromosomal variations in peanut

2021 ◽  
Author(s):  
Liuyang Fu ◽  
Qian Wang ◽  
Lina Li ◽  
Tao Lang ◽  
Junjia Guo ◽  
...  
Keyword(s):  
Physical Mapping ◽  
Tandem Repeats ◽  
Genetic Research ◽  
Diploid Species ◽  
Reference Sequence ◽  
B Genome ◽  
A Genome ◽  
Genome Map ◽  
Chromosomal Variants

Abstract Background: Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut due to the lack of efficient chromosomal markers. Until now, the identification of chromosomal variants in peanut has remained a challenge.Results: A total of 114 new oligo probes were developed based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligos were classified into 28 types based on their positions and overlapping signals in chromosomes. For each type, a representative oligo was selected and modified with green fluorescein 6-carboxyfluorescein (FAM) or red fluorescein 6-carboxytetramethylrhodamine (TAMRA). Two cocktails, Multiplex #3 and Multiplex #4, were developed by pooling the fluorophore conjugated probes. Multiplex #3 included FAM-modified oligo TIF-439, oligo TIF-185-1, oligo TIF-134-3, and oligo TIF-165. Multiplex #4 included TAMRA-modified oligo Ipa-1162, oligo Ipa-1137, oligo DP-1, and oligo DP-5. Each cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of peanut induced by radiation exposure. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, intercalary, and terminal regions; four were B genome-specific; one was chromosome-specific; and the remaining 15 were extensively mapped in the pericentric regions of chromosomes.Conclusions: The development of new oligo probes provides an effective set of tools which can be used to distinguish the various chromosomes of the peanut. Physical mapping by FISH reveals the genomic organization of repetitive oligos in peanut chromosomes. A genome map-based karyotype was established and used for the identification of chromosome variations in peanut following comparisons with their reference sequence positions.

Genomic interspersions determine the size and complexity of transgene loci in transgenic plants produced by microprojectile bombardment

Genome ◽  
2001 ◽  
Vol 44 (4) ◽  
pp. 691-697 ◽  
Author(s):  
Sergei K Svitashev ◽  
David A Somers
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Tandem Repeats ◽  
Microprojectile Bombardment ◽  
Culture Conditions ◽  
Plant Genome ◽  
Dna Fragments ◽  
Chromosomal Breakage ◽  
Multiple Copies ◽  
Transgene Locus

The structure of transgene loci in six transgenic allohexaploid oat (Avena sativa L.) lines produced using microprojectile bombardment was characterized using fluorescence in situ hybridization (FISH) on extended DNA fibers (fiber-FISH). The transgene loci in five lines were composed of multiple copies of delivered DNA interspersed with genomic DNA fragments ranging in size from ca. 3 kb to at least several hundred kilobases, and in greater numbers than detected using Southern blot analysis. Although Southern analysis predicted that the transgene locus in one line consisted of long tandem repeats of the delivered DNA, fiber-FISH revealed that the locus actually contained multiple genomic interspersions. These observations indicated that transgene locus size and structure were determined by the number of transgene copies and, possibly to a greater extent, the number and the length of interspersing genomic DNA sequences within the locus. Large genomic interspersions detected in several lines were most likely the products of chromosomal breakage induced either by tissue culture conditions or, more likely, by DNA delivery into the nucleus using microprojectile bombardment. We propose that copies of transgene along with other extrachromosomal DNA fragments are used as patches to repair double-strand breaks (DSBs) in the plant genome resulting in the formation of transgene loci.Key words: genetic transformation, microprojectile bombardment, transgenic oat, FISH, transgene locus structure.

Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1356-1363 ◽  
Author(s):  
Barbara P. Schick ◽  
David Maslow ◽  
Adrianna Moshinski ◽  
James D. San Antonio
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Tandem Repeats ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

scholarly journals A Microsatellite Map of Wheat

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 2007-2023 ◽  
Author(s):  
Marion S Röder ◽  
Victor Korzun ◽  
Katja Wendehake ◽  
Jens Plaschke ◽  
Marie-Hélène Tixier ◽  
...  
Keyword(s):  
Bread Wheat ◽  
Reference Population ◽  
Triticum Aestivum L ◽  
Wheat Genome ◽  
D Genome ◽  
B Genome ◽  
Microsatellite Map ◽  
A Genome ◽  
Polymorphic Microsatellite ◽  
Primer Sets

Abstract Hexaploid bread wheat (Triticum aestivum L. em. Thell) is one of the world's most important crop plants and displays a very low level of intraspecific polymorphism. We report the development of highly polymorphic microsatellite markers using procedures optimized for the large wheat genome. The isolation of microsatellite-containing clones from hypomethylated regions of the wheat genome increased the proportion of useful markers almost twofold. The majority (80%) of primer sets developed are genome-specific and detect only a single locus in one of the three genomes of bread wheat (A, B, or D). Only 20% of the markers detect more than one locus. A total of 279 loci amplified by 230 primer sets were placed onto a genetic framework map composed of RFLPs previously mapped in the reference population of the International Triticeae Mapping Initiative (ITMI) Opata 85 × W7984. Sixty-five microsatellites were mapped at a LOD >2.5, and 214 microsatellites were assigned to the most likely intervals. Ninety-three loci were mapped to the A genome, 115 to the B genome, and 71 to the D genome. The markers are randomly distributed along the linkage map, with clustering in several centromeric regions.

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