Preparation of high molecular weight plant DNA and analysis by pulsed field gel electrophoresis

Daelen, R.A.J.J. van; Zabel, P.

doi:10.1007/978-94-011-0511-8_26

Cited by 5 publications

(5 citation statements)

References 118 publications

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“…BAC filter hybridization was based on the Southern blotting protocol described by Van Daelen & Zabel (1994) with some modification using non-radioactive dig-11-dUTP labelling (Roche). One BAC filter of the tomato Heinz 1706 BAC library (LeHba-A, http://www.genome.arizona.edu) representing 2Â tomato genome equivalents was hybridized with tomato Cot-100 DNA, TGRII, and TGRIII repeats.…”

Section: Bac Filter Hybridizationmentioning

confidence: 99%

FISH mapping and molecular organization of the major repetitive sequences of tomato

et al. 2008

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This paper presents a bird_s-eye view of the major repeats and chromatin types of tomato. Using fluorescence insitu hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA] 4 , a microsatellite that also forms part of the pericentromeres, together with [GA] 8 , [GATA] 4 and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6)

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Section: Bac Filter Hybridizationmentioning

confidence: 99%

FISH mapping and molecular organization of the major repetitive sequences of tomato

et al. 2008

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“…High molecular weigth (Ͼ 2 Mb) genomic tomato DNA was isolated from young leaves according to Van Daelen and Zabel (1994) and digested with BglII or EcoRV . Restriction fragments in the range 50-2000 kb were resolved on a CHEF gel (electrophoresis conditions: 60 sec pulses for 16 h followed by 90 sec pulses for 7 h at 6 V cm -1 ).…”

Section: Pulsed Field Gel Electrophoresismentioning

confidence: 99%

“…Restriction fragments in the range 50-2000 kb were resolved on a CHEF gel (electrophoresis conditions: 60 sec pulses for 16 h followed by 90 sec pulses for 7 h at 6 V cm -1 ). Southern blotting and hybridization were performed as described elsewhere (Van Daelen and Zabel, 1994) using α[ 32 P]dATP-labelled probes.…”

Section: Pulsed Field Gel Electrophoresismentioning

confidence: 99%

FISH studies reveal the molecular and chromosomal organization of individual telomere domains in tomato

Zhong¹,

Fransz²,

Eden³

et al. 1998

The Plant Journal

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The molecular and cytological organization of the telomeric repeat (TR) and the subtelomeric repeat (TGR1) of tomato were investigated by fluorescence in situ hybridization (FISH) techniques. Hybridization signals on extended DNA fibres, visualized as linear fluorescent arrays representing individual telomeres, unequivocally demonstrated the molecular co-linear arrangement of both repeats. The majority of the telomeres consisted of a TR and a TGR1 region separated by a spacer. Microscopic measurements of the TR and TGR1 signals revealed high variation in length of both repeats, with maximum sizes of 223 and 1330 kb, respectively. A total of 27 different combinations of TR and TGR1 was detected, suggesting that all chromosome ends have their own unique telomere organization. The fluorescent tracks on the extended DNA fibres were subdivided into four classes: (i) TR-spacer-TGR1; (ii) TR-TGR1; (iii) only TR; (iv) only TGR1. FISH to pachytene chromosomes enabled some of the TR/TGR1 groups to be assigned to specific chromosome ends and to interstitial regions. These signals also provided evidence for a reversed order of the TR and TGR1 sites at the native chromosome ends, suggesting a backfolding telomere structure with the TGR1 repeats occupying the most terminal position of the chromosomes. The FISH signals on diakinesis chromosomes revealed that distal euchromatin areas and flanking telomeric heterochromatin remained highly decondensed around the chiasmata in the euchromatic chromosome areas. The rationale for the occurrence and distribution of the TR and TGR1 repeats on the tomato chromosomes are discussed.

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“…Isolation and restriction digestion of megabasesized tomato DNA from nematode-resistant (VFNT Cherry) and susceptible (Moneymaker) genotypes was performed as described [36]. For double restriction digestions, the enzyme used in the first reaction was removed by washing the plugs overnight at 4°C in a large volume (50-100ml) of 10mM Tris-HC1 pH 8.0, 10mM EDTA before addition of the second enzyme.…”

Section: Isolation Of High-molecular-weight Dna and Restriction Digesmentioning

confidence: 99%

“…Electrophoresis conditions and markers are specified in the figure legends. Gels were blotted as described [36]. Hybridizations were performed as described [23] using probes radiolabelled according to the random hexamer priming procedure [ 13 ].…”

Section: Pulsed-filed Gel Electrophoresis (Pfge) Southern Blotting mentioning

confidence: 99%

Long-range physical maps of two loci (Aps-1 and GP79) flanking the root-knot nematode resistance gene (Mi) near the centromere of tomato chromosome 6

Daelen¹,

Gerbens²,

Ruissen³

et al. 1993

Plant Mol Biol

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The root knot nematode resistance gene Mi in tomato has been mapped in the pericentromeric region of chromosome 6. With the objective of isolating Mi through a map-based cloning approach, we have previously identified and ordered into a high-resolution genetic linkage map a variety of tightly linked molecular markers. Using pulsed-field gelelectrophoresis and various rarely cutting restriction enzymes in single, double and partial digestions, we now report long-range physical maps of the two closest flanking markers, acid phosphatase-1 (Aps-1) and GP79, which span over 400 and 800 kb, respectively. It is concluded that the physical distance between both markers is larger than predicted on the basis of genetic linkage analysis. Furthermore, two RFLP markers (H3F8 and H4H10) which map genetically to the same locus as Aps-1 do not show physical linkage, indicating severe suppression of recombination in this region of the chromosome. Finally, no evidence was obtained showing the presence of a CpG island near Aps-1.

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Preparation of high molecular weight plant DNA and analysis by pulsed field gel electrophoresis

Cited by 5 publications

References 118 publications

FISH mapping and molecular organization of the major repetitive sequences of tomato

FISH mapping and molecular organization of the major repetitive sequences of tomato

FISH studies reveal the molecular and chromosomal organization of individual telomere domains in tomato

Long-range physical maps of two loci (Aps-1 and GP79) flanking the root-knot nematode resistance gene (Mi) near the centromere of tomato chromosome 6

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