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Fluorescence in Situ Hybridization Techniques for Cytogenetic and Genomic Analyses

  • Jason G. Walling
  • Wenli Zhang
  • Jiming JiangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 956)

Abstract

Fluorescent in situ hybridization (FISH) is a powerful method to visualize DNA sequences in the context of the whole chromosome. Yet despite the value of FISH analysis for cytogenetic studies, there are surprisingly few labs that are able to adapt the technique for their experiments in chromosomal and genome biology. Here we present a comprehensive FISH protocol acquired from over 20 years of collective experience using different plant species. Our description uses rice as a model for performing a complete FISH procedure, but the protocol can be readily adapted for other plant species. We have provided more specialized instruction beyond routine FISH, which includes the preparation of meiotic and mitotic samples suitable for FISH analysis, procedures for direct and indirect labeling of DNA probes, and techniques for increasing signal strength using layers of antibodies.

Key words

Fluorescent in situ hybridization Mitotic metaphase chromosomes Pachytene chromosomes 

Notes

Acknowledgments

Cytogenetic studies of Oryza species in our lab have been supported by grant 2006-35604-16649 from the US Department of Agriculture Cooperative State Research, Education, and Extension Service (CSREES) and grant DBI-0603927 from the National Science Foundation.

References

  1. 1.
    Chen MS, Presting G, Barbazuk WB, Goicoechea JL, Blackmon B, Fang FC, Kim H, Frisch D, Yu YS, Sun SH, Higingbottom S, Phimphilai J, Phimphilai D, Thurmond S, Gaudette B, Li P, Liu JD, Hatfield J, Main D, Farrar K, Henderson C, Barnett L, Costa R, Williams B, Walser S, Atkins M, Hall C, Budiman MA, Tomkins JP, Luo MZ, Bancroft I, Salse J, Regad F, Mohapatra T, Singh NK, Tyagi AK, Soderlund C, Dean RA, Wing RA (2002) An integrated physical and genetic map of the rice genome. Plant Cell 14:537–545PubMedCrossRefGoogle Scholar
  2. 2.
    Jiang JM, Gill BS, Wang GL, Ronald PC, Ward DC (1995) Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes. Proc Natl Acad Sci USA 92:4487–4491PubMedCrossRefGoogle Scholar
  3. 3.
    Cheng Z, Presting GG, Buell CR, Wing RA, Jiang JM (2001) High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice. Genetics 157:1749–1757PubMedGoogle Scholar
  4. 4.
    Kurata N, Omura T, Iwata N (1981) Studies on centromere, chromomere and nucleolus in pachytene nuclei of rice, Oryza sativa, microsporocytes. Cytologia 46:791–800CrossRefGoogle Scholar
  5. 5.
    Khush GS, Singh RJ, Sur SC, Librojo AL (1984) Primary trisomics of rice: origin, morphology, cytology and use in linkage mapping. Genetics 107:141–163PubMedGoogle Scholar
  6. 6.
    Chung MC, Wu HK (1987) Karyotype analysis of ‘IR36’ and two trisomic lines of rice. Bot Bull Acad Sin 28:289–304Google Scholar
  7. 7.
    Cheng Z, Buell CR, Wing RA, Gu M, Jiang JM (2001) Toward a cytological characterization of the rice genome. Genome Res 11:2133–2141PubMedCrossRefGoogle Scholar
  8. 8.
    Kao FI, Cheng YY, Chow TY, Chen HH, Liu SM, Cheng CH, Chung MC (2006) An integrated map of Oryza sativa L. chromosome 5. Theor Appl Genet 112:891–902PubMedCrossRefGoogle Scholar
  9. 9.
    Cheng ZK, Buell CR, Wing RA, Jiang JM (2002) Resolution of fluorescence in-situ hybridization mapping on rice mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers. Chromosome Res 10:379–387PubMedCrossRefGoogle Scholar
  10. 10.
    Dong F, Jiang JM (1998) Non-Rabl patterns of centromere and telomere distribution in the interphase nuclei of plant cells. Chromosome Res 6:551–558PubMedCrossRefGoogle Scholar
  11. 11.
    Fukui K, Ohmido N, Khush GS (1994) Variability in rDNA loci in the genus Oryza detected trough fluorescence in-situ hybridization. Theor Appl Genet 87:893–899CrossRefGoogle Scholar
  12. 12.
    Ohmido N, Fukui K (1997) Visual verification of close disposition between a rice A genome-specific DNA sequence (TrsA) and the telomere sequence. Plant Mol Biol 35:963–968PubMedCrossRefGoogle Scholar
  13. 13.
    Ohmido N, Kijima K, Akiyama Y, de Jong JH, Fukui K (2000) Quantification of total genomic DNA and selected repetitive sequences reveals concurrent changes in different DNA families in indica and japonica rice. Mol Gen Genet 263:388–394PubMedCrossRefGoogle Scholar
  14. 14.
    Cheng Z, Stupar RM, Gu M, Jiang JM (2001) A tandemly repeated DNA sequence is associated with both knob-like heterochromatin and a highly decondensed structure in the meiotic pachytene chromosomes of rice. Chromosoma 110:24–31PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of HorticultureUniversity of Wisconsin-MadisonMadisonUSA

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