Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescencein situ hybridization (FISH)

Zhong, Xiao‐bo; Jong, J.H. de; Zabel, P.

doi:10.1007/bf02254940

Cited by 140 publications

(87 citation statements)

References 32 publications

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“…The material was squashed with a fine needle, covered with acetic acid (60%) and then stirred and baked on a slide warmer at 45°C to spread chromosomes on slides. Cells were rinsed with ice-cold acetic acid fixative and left to air-dry for at least 2 h. DNA of the 346H10 BAC (1$lg) was labelled with biotin-16-dUTP with a nick translation kit using the manufacturer's protocol (Roche) and FISH was performed according to the methods of Zhong et al (1996). Chromosomes were counterstained in 5 lg/ml DAPI in Vectashield anti-fade (Vector laboratories).…”

Section: Methodsmentioning

confidence: 99%

Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice

Calderini¹,

Chang

Jong

et al. 2006

Theor Appl Genet

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Apomixis in plants is a form of clonal reproduction through seeds. A BAC clone linked to apomictic reproduction in Paspalum simplex was used to locate the apomixis locus on meiotic chromosome preparations. Fluorescent in situ hybridisation revealed the existence of a single locus embedded in a heterochromatin-poor region not adjacent to the centromere. We report here for the first time information regarding the sequencing of a large DNA clone from the apomixis locus. The presence of two genes whose rice homologs were mapped on the telomeric part of the long arm of rice chromosome 12 confirmed the strong synteny between the apomixis locus of P. simplex with the related area of the rice genome at the map level. Comparative analysis of this region with rice as representative of a sexual species revealed large-scale rearrangements due to transposable elements and small-scale rearrangements due to deletions and single point mutations. Both types of rearrangements induced the loss of coding capacity of large portions of the ''apomictic'' genes compared to their rice homologs. Our results are discussed in relation to the use of rice genome data for positional cloning of apomixis genes and to the possible role of rearranged supernumerary genes in the apomictic process of P. simplex.

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Section: Methodsmentioning

confidence: 99%

Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice

Calderini¹,

Chang

Jong

et al. 2006

Theor Appl Genet

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“…Any oil on the cover glass was removed by wiping with 100% ethanol, and the cover glass and mounting medium were removed by washing in 2× salt-sodium citrate (SSC). FISH was performed as described by Zhong et al (1996) except that no RNase or pepsin treatments were used. The hybridization mixture (20μl per slide) included 50% formamide, 2× SSC, 10% sodium dextran sulfate, 0.25% sodium dodecyl sulfate, and 1ng biotin-labeled A. thaliana telomere repeat probes (Richards and Ausubel 1988).…”

Section: Fluorescence In Situ Hybridizationmentioning

confidence: 99%

Cytological analysis of MRE11 protein during early meiotic prophase I in Arabidopsis and tomato

et al. 2008

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Early recombination nodules (ENs) are multiprotein complexes that are thought to be involved in synapsis and recombination, but little is known about their components or how they may be involved in these events. In this study, we describe the cytological behavior of a possible EN component, MRE11, a protein that is important for the repair of the numerous, programmed deoxyribonucleic acid double-strand breaks (DSBs) that occur early in the meiotic prophase. By immunofluorescence, many MRE11 foci were associated with chromosomal axes during early prophase I in both wild-type Arabidopsis and tomato primary microsporocytes. Similar patterns of MRE11 foci were observed in two Arabidopsis mutants (Atspo11-1 and Atprd1) that are defective in DSB formation and synapsis. In tomato chromosomes, MRE11 foci were more common in distal euchromatin than in proximal heterochromatin, consistent with known EN patterns. However, electron microscopic immunogold localization demonstrated that only about 10% of ENs were labeled, and most MRE11 label was associated with synaptonemal complex components. Thus, in plants, MRE11 foci are not dependent on DSB formation, and most MRE11 foci do not correspond to ENs. More generally, our results show that the simple presence of large numbers of fluorescent foci associated with synapsing chromosomes is insufficient evidence to equate these foci with ENs.

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“…Meiotic pachytene chromosomes have been shown to be a better target for high-resolution FISH mapping in plants (Xu and Earle, 1996;Zhong et al, 1996aZhong et al, , 1996bFransz et al, 1998;Chen et al, 2000). First, pachytene chromosomes are generally 10 to 20 times longer than somatic metaphase chromosomes and possess many cytological landmarks Chen et al, 2000;see Supplemental Table 1 online).…”

Section: Introductionmentioning

confidence: 99%

High-Resolution Single-Copy Gene Fluorescence in Situ Hybridization and Its Use in the Construction of a Cytogenetic Map of Maize Chromosome 9

2006

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High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by ;100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contigbased physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.

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Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescencein situ hybridization (FISH)

Cited by 140 publications

References 32 publications

Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice

Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice

Cytological analysis of MRE11 protein during early meiotic prophase I in Arabidopsis and tomato

High-Resolution Single-Copy Gene Fluorescence in Situ Hybridization and Its Use in the Construction of a Cytogenetic Map of Maize Chromosome 9

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