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  • 1 Hungarian Academy of Sciences Agricultural Institute, Centre for Agricultural Reseach Martonvásár Hungary
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Fluorescence in situ hybridization (FISH) is the most versatile and accurate molecular cytogenetic technique for determining euchromatic-heterochromatic boundaries and the locations of repetitive and single-copy DNA sequences and of chromosome-specific BAC clones on chromosomes. The combination of cytogenetic and genetic methods yields a highresolution physical map. FISH allows direct mapping of specific DNA sequences inside the cell (interphase nuclei), along meiotic pachytene chromosomes and isolated chromatin (DNA fibres). The increased sensitivity of the technique and its ability to detect gene locations provide a powerful research tool for genetic and pre-breeding studies. FISH-based physical mapping plays an important role and is increasingly used for studies at the cytological level on the chromatin organization that controls gene expression and regulation. The present minireview describes some of the benefits of alternative FISH-based techniques and their application for studying plant chromosomes and genomes.

  • Buongiorno-Nardelli, M., Amaldi, F. (1969): Autoradiographic detection of molecular hybrids between rRNA and DNA in tissue sections. Nature (London), 225, 946–947.

    Amaldi F. , 'Autoradiographic detection of molecular hybrids between rRNA and DNA in tissue sections ' (1969 ) 225 Nature (London) : 946 -947.

    • Search Google Scholar
  • Carlton, P. M. (2008): Three-dimensional structured illumination microscopy and its application to chromosome structure. Chromosome Res., 16, 351–365.

    Carlton P. M. , 'Three-dimensional structured illumination microscopy and its application to chromosome structure ' (2008 ) 16 Chromosome Res. : 351 -365.

    • Search Google Scholar
  • Catalan, P., Muller, J., Hasterok, R., Jenkins, G., Mur, L. A. J., Langdon, T., Betekhtin, A., Siwinska, D., Pimentel, M., Lopez-Alvarez, D. (2012): Evolution and taxonomic split of the model grass Brachypodium distachyon. Ann. Bot., 109, 385–405.

    Lopez-Alvarez D. , 'Evolution and taxonomic split of the model grass Brachypodium distachyon ' (2012 ) 109 Ann. Bot. : 385 -405.

    • Search Google Scholar
  • Cheng, Z., Presting, G. G., Buell, C. R., Wing, R. A., Jiang, J. (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, 1749–1757. PMID:11290728.

    Jiang J. , '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 ' (2001 ) 157 Genetics : 1749 -1757.

    • Search Google Scholar
  • de Jong, J. H. (2003): Visualizing DNA domains and sequences by microscopy: a fifty-year history of molecular cytogenetics. Genome, 46, 943–946.

    Jong J. H. , 'Visualizing DNA domains and sequences by microscopy: a fifty-year history of molecular cytogenetics ' (2003 ) 46 Genome : 943 -946.

    • Search Google Scholar
  • de Jong, J. H., Fransz, P., Zabel, P. (1999): High resolution FISH in plants — techniques and applications. Trends Plant Sci., 4, 258–263.

    Zabel P. , 'High resolution FISH in plants — techniques and applications ' (1999 ) 4 Trends Plant Sci. : 258 -263.

    • Search Google Scholar
  • Devos, K. M. (2010): Grass genome organization and evolution. Curr. Opin. Plant Biol., 13, 139–145.

    Devos K. M. , 'Grass genome organization and evolution ' (2010 ) 13 Curr. Opin. Plant Biol. : 139 -145.

    • Search Google Scholar
  • Doležel, J., Kubaláková, M., Paux, E., Bartos, J., Feuillet, C. (2007): Chromosome-based genomics in the cereals. Chromosome Res, 15, 51–66.

    Feuillet C. , 'Chromosome-based genomics in the cereals ' (2007 ) 15 Chromosome Res : 51 -66.

  • Fransz, P. F., Alonso-Blanco, C., Liharska, T. B., Peeters, A. J. M., Zabel, P., de Jong, J. H. (1996): High-resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres. Plant J., 9, 421–430.

    Jong J. H. , 'High-resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres ' (1996 ) 9 Plant J. : 421 -430.

    • Search Google Scholar
  • Fransz, P. F., Armstrong, S., de Jong, J. H., Parnell, L. D., van Drunen, G., Dean, C, Zabel, P., Bisseling, T., Jones, G. H. (2000): Integrated cytogenetic map of chromosome arm 4S of A. thaliana: structural organization of heterochromatic knob and centromere region. Cell, 100, 367–376.

    Jones G. H. , 'Integrated cytogenetic map of chromosome arm 4S of A. thaliana: structural organization of heterochromatic knob and centromere region ' (2000 ) 100 Cell : 367 -376.

    • Search Google Scholar
  • Fransz, P., Linc, G., Ali, M., Schubert, I., Wennekes, J., de Jong, H., Koorneef, M., Peters, J., Gerats, T. (2005): Genetic and epigenetic consequences of a paracentric inversion in Arabidopsis thaliana. Chromosome Res., 1, 71–72.

    Gerats T. , 'Genetic and epigenetic consequences of a paracentric inversion in Arabidopsis thaliana ' (2005 ) 1 Chromosome Res. : 71 -72.

    • Search Google Scholar
  • Gad, S., Klinger, M., Caux-Moncoutier, V., Pages-Berhouet, S., Gauthier-Villars, M., Coupier, I., Bensimon, A., Aurias, A., Stoppa-Lyonnet, D. (2002): Bar code screening on combed DNA for large rearrangements of the BRCA1 and BRCA2 genes in French breast cancer families. J. Med. Genet., 39, 817–821.

    Stoppa-Lyonnet D. , 'Bar code screening on combed DNA for large rearrangements of the BRCA1 and BRCA2 genes in French breast cancer families ' (2002 ) 39 J. Med. Genet. : 817 -821.

    • Search Google Scholar
  • Gill, B. S., Friebe, B. R. (1998): Plant cytogenetics at the dawn of the 21st century. Curr. Opin. Plant Biol., 1, 109–115.

    Friebe B. R. , 'Plant cytogenetics at the dawn of the 21st century ' (1998 ) 1 Curr. Opin. Plant Biol. : 109 -115.

    • Search Google Scholar
  • Heng, H. H. Q., Squires, J., Tsui, L-C. (1992): High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc. Natl. Acad. Sci. USA., 89, 9505–9513.

    Tsui L.-C. , 'High-resolution mapping of mammalian genes by in situ hybridization to free chromatin ' (1992 ) 89 Proc. Natl. Acad. Sci. USA. : 9505 -9513.

    • Search Google Scholar
  • Huang, B., Wang, W., Bates, M., Zhuang, X. (2008): Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science, 319, 810–813.

    Zhuang X. , 'Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy ' (2008 ) 319 Science : 810 -813.

    • Search Google Scholar
  • Idziak, D., Betekhtin, A., Wolny, E., Lesniewska, K., Wright, J., Febrer, M., Bevan, M. W., Jenkins, G., Hasterok, R. (2011): Painting the chromosomes of Brachypodium — current status and future prospects. Chromosoma, 120, 469–479.

    Hasterok R. , 'Painting the chromosomes of Brachypodium — current status and future prospects ' (2011 ) 120 Chromosoma : 469 -479.

    • Search Google Scholar
  • Islam-Faridi, M. N., Childs, K. L., Klein, P. E., Hodnett, G., Menz, M. A., Klein, R. R., Rooney, W. L., Mullet, J. E., Stelly, D. M., Price, H. J. (2002): A molecular cytogenetic map of sorghum chromosome 1: Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes. Genetics, 161, 345–353. PMID:12019248.

    Price H. J. , 'A molecular cytogenetic map of sorghum chromosome 1: Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes ' (2002 ) 161 Genetics : 345 -353.

    • Search Google Scholar
  • Jackson, S. A., Cheng, Z. K., Wang, M. L., Goodman, H. M., Jiang, J. (2000): Comparative fluorescence in situ hybridization mapping of a 431-kb Arabidopsis thaliana bacterial artificial chromosome contig reveals the role of chromosomal duplications in the expansion of the Brassica rapa genome. Genetics, 156, 833–838. PMID:11014828.

    Jiang J. , 'Comparative fluorescence in situ hybridization mapping of a 431-kb Arabidopsis thaliana bacterial artificial chromosome contig reveals the role of chromosomal duplications in the expansion of the Brassica rapa genome ' (2000 ) 156 Genetics : 833 -838.

    • Search Google Scholar
  • Jiang, J., Gill, B. S. (2006): Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research. Genome, 49, 1057–1068.

    Gill B. S. , 'Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research ' (2006 ) 49 Genome : 1057 -1068.

    • Search Google Scholar
  • Jin, W. W., Melo, J. R., Nagaki, K., Talbert, P. B., Henikoff, S., Dawe, D. K., Jiang, J. (2004): Maize centromeres: organization and functional adaptation in the genetic background of oat. Plant Cell, 16, 571–581.

    Jiang J. , 'Maize centromeres: organization and functional adaptation in the genetic background of oat ' (2004 ) 16 Plant Cell : 571 -581.

    • Search Google Scholar
  • John, H. L., Birnstiel, M. L., Jones, K. W. (1969): RNA-DNA hybrids at the cytological level. Nature (London), 223, 912–913.

    Jones K. W. , 'RNA-DNA hybrids at the cytological level ' (1969 ) 223 Nature (London) : 912 -913.

    • Search Google Scholar
  • Khrustaleva, L. I., Kik, C. (2001): Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplification. Plant J., 25, 699–707.

    Kik C. , 'Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplification ' (2001 ) 25 Plant J. : 699 -707.

    • Search Google Scholar
  • Kim, J. S., Islam-Faridi, M. N., Klein, P. E., Stelly, D. M., Price, H. J., Klein, R. R., Mullet, J. E. (2005): Comprehensive molecular cytogenetic analysis of sorghum genome architecture: distribution of euchromatin, heterochromatin, genes and recombination in comparison to rice. Genetics, 171, 1963–1976. doi:10.1534/genetics.105.048215. PMID:16143604.

    Mullet J. E. , 'Comprehensive molecular cytogenetic analysis of sorghum genome architecture: distribution of euchromatin, heterochromatin, genes and recombination in comparison to rice ' (2005 ) 171 Genetics : 1963 -1976.

    • Search Google Scholar
  • Kolas, N. K., Marcon, E., Crackower, M. A., Höög, C., Penninger, J. M., Spyropoulos, B., Moens, P. B. (2005): Mutant meiotic chromosome core components in mice can cause apparent sexual dimorphic endpoints at prophase or X-Y defective male-specific sterility. Chromosoma, 114, 92–102.

    Moens P. B. , 'Mutant meiotic chromosome core components in mice can cause apparent sexual dimorphic endpoints at prophase or X-Y defective male-specific sterility ' (2005 ) 114 Chromosoma : 92 -102.

    • Search Google Scholar
  • Koumbaris, G. L., Bass, H. W. (2003): A new single-locus cytogenetic mapping system for maize (Zea mays L.): overcoming FISH detection limits with marker-selected sorghum (S. propinquum L.) BAC clones. Plant J., 35, 647–659. doi:10.1046/j.1365-313X.2003.01829.x.PMID:12940957.

    Bass H. W. , 'A new single-locus cytogenetic mapping system for maize (Zea mays L.): overcoming FISH detection limits with marker-selected sorghum (S. propinquum L.) BAC clones ' (2003 ) 35 Plant J. : 647 -659.

    • Search Google Scholar
  • Kulikova, O., Gualtieri, G., Kim, D. J., Cook, D., Huguet, T., Bisseling, T. (2001): Integration of the FISH pachytene and genetic maps of Medicago truncatula. Plant J., 27, 49–58.

    Bisseling T. , 'Integration of the FISH pachytene and genetic maps of Medicago truncatula ' (2001 ) 27 Plant J. : 49 -58.

    • Search Google Scholar
  • Lilly, J. W., Havey, M. J., Jackson, S. A., Jiang, J. (2001): Cytogenomic analyses reveal the structural plasticity of the chloroplast genome in higher plants. Plant Cell, 13, 245–254.

    Jiang J. , 'Cytogenomic analyses reveal the structural plasticity of the chloroplast genome in higher plants ' (2001 ) 13 Plant Cell : 245 -254.

    • Search Google Scholar
  • Linc, G. M., Hoda, M. A., Schubert, I., Fransz, P. F. (2004): Euchromatic arrangements in knob and knobless accessions of Arabidopsis thaliana. Plant and Animal Genome XII, San Diego, CA, USA. p. 93.

  • Lou, Q., Iovene, M., Spooner, D. M., Buell, C. R., Jiang, J. (2010): Evolution of chromosome 6 of Solanum species revealed by comparative fluorescence in situ hybridization mapping. Chromosoma, 119, 435–442.

    Jiang J. , 'Evolution of chromosome 6 of Solanum species revealed by comparative fluorescence in situ hybridization mapping ' (2010 ) 119 Chromosoma : 435 -442.

    • Search Google Scholar
  • Lysak, M. A., Berr, A., Pecinka, A., Schmidt, R., McBreen, K., Schubert, I. (2006): Mechanisms of chromosome number reduction in Arabidopsis thaliana and related Brassicaceae species. Proc. Natl. Acad. Sci. USA, 103, 5224–5229.

    Schubert I. , 'Mechanisms of chromosome number reduction in Arabidopsis thaliana and related Brassicaceae species ' (2006 ) 103 Proc. Natl. Acad. Sci. USA : 5224 -5229.

    • Search Google Scholar
  • Lysak, M. A., Fransz, P. F., Ali, H. B., Schubert, I. (2001): Chromosome painting in Arabidopsis thaliana. Plant J., 28, 689–697.

    Schubert I. , 'Chromosome painting in Arabidopsis thaliana ' (2001 ) 28 Plant J. : 689 -697.

  • Lysak, M. A., Koch, M. A., Pecinka, A., Schubert, I. (2005): Chromosome triplication found across the tribe Brassiceae. Genome Res., 15, 516–525. doi:10.1101/gr.3531105. PMID:15781573.

    Schubert I. , 'Chromosome triplication found across the tribe Brassiceae ' (2005 ) 15 Genome Res. : 516 -525.

    • Search Google Scholar
  • Lysak, M. A., Mandakova, T., Lacombe, E. (2010): Reciprocal and multispecies chromosome BAC painting in crucifers (Brassicaceae). Cytogenet. Genome Res., 129, 184–189.

    Lacombe E. , 'Reciprocal and multispecies chromosome BAC painting in crucifers (Brassicaceae) ' (2010 ) 129 Cytogenet. Genome Res. : 184 -189.

    • Search Google Scholar
  • Ma, L., Vu, G. T., Schubert, V., Watanabe, K., Stein, N., Houben, A., Schubert, I. (2010): Synteny between Brachypodium distachyon and Hordeum vulgare as revealed by FISH. Chromosome Res., 18, 841–850.

    Schubert I. , 'Synteny between Brachypodium distachyon and Hordeum vulgare as revealed by FISH ' (2010 ) 18 Chromosome Res. : 841 -850.

    • Search Google Scholar
  • Molnár, I., Kubaláková, M., Šimková, H., Cseh, A., Molnár-Láng, M., Doležel, J. (2011): Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. Geniculate. PLoS ONE, 6(11), e27708. doi:10.1371/journal.pone.0027708

    Doležel J. , 'Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. Geniculate ' (2011 ) 6 PLoS ONE : e27708 -.

    • Search Google Scholar
  • Nagaki, K., Cheng, Z., Ouyang, S., Talbert, P. B., Kim, M., Jones, K. M., Henikoff, S., Buell, C. R., Jiang, J. (2004): Sequencing of a rice centromere uncovers active genes. Nat. Genet., 36, 138–145.

    Jiang J. , 'Sequencing of a rice centromere uncovers active genes ' (2004 ) 36 Nat. Genet. : 138 -145.

    • Search Google Scholar
  • Pardue, M. L., Gall, J. G. (1969): Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl. Acad. Sci. USA, 64, 600–604.

    Gall J. G. , 'Molecular hybridization of radioactive DNA to the DNA of cytological preparations ' (1969 ) 64 Proc. Natl. Acad. Sci. USA : 600 -604.

    • Search Google Scholar
  • Pecinka, A., Schubert, V., Meister, A., Kreth, G., Klatte, M., Lysak, M., Fuchs, J., Schubert, I. (2004): Chromosome territory arrangement and homologous pairing in nuclei of Arabidopsis thaliana are predominantly random except for NOR-bearing chromosomes. Chromosoma, 113, 258–269.

    Schubert I. , 'Chromosome territory arrangement and homologous pairing in nuclei of Arabidopsis thaliana are predominantly random except for NOR-bearing chromosomes ' (2004 ) 113 Chromosoma : 258 -269.

    • Search Google Scholar
  • Pietro, P., Moore, G., Shaw, P. (2007): Fluorescence in situ hybridization on vibratome sections of plant tissues. Nat. Protoc., 2, 1831–1838.

    Shaw P. , 'Fluorescence in situ hybridization on vibratome sections of plant tissues ' (2007 ) 2 Nat. Protoc. : 1831 -1838.

    • Search Google Scholar
  • Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J, Gray, J. (1988): Fluorescence in situ hybridization with human chromosome specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. USA, 85, 9138–9142.

    Gray J. , 'Fluorescence in situ hybridization with human chromosome specific libraries: detection of trisomy 21 and translocations of chromosome 4 ' (1988 ) 85 Proc. Natl. Acad. Sci. USA : 9138 -9142.

    • Search Google Scholar
  • Schwartz, D. C., Li, X., Hernandez, L. I., Ramnarain, S. P., Huff, E. J., Wang, Y. K. (1993): Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping. Science, 262, 110–114.

    Wang Y. K. , 'Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping ' (1993 ) 262 Science : 110 -114.

    • Search Google Scholar
  • Stephens, J. L., Brown, S. E., Lapitan, N. L. V., Knudson, D. L. (2004): Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique. Genome, 47, 179–189.

    Knudson D. L. , 'Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique ' (2004 ) 47 Genome : 179 -189.

    • Search Google Scholar
  • Szinay, D., Chang, S. B., Khrustaleva, L., Peters, S., Schijlen, E., Bai, Y., Stiekema, W. J., Van Ham, R. C. H. J., de Jong, H., Lankhorst Klein, R. M. (2008): High-resolution chromosome mapping of BACs using multicolour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6. Plant J., 56, 627–637.

    Lankhorst Klein R. M. , 'High-resolution chromosome mapping of BACs using multicolour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6 ' (2008 ) 56 Plant J. : 627 -637.

    • Search Google Scholar
  • Tang, X., de Boer, J. M., van Eck, H. J., Bachem, C., Visser, R. G. F., de Jong, H. (2009): Assignment of genetic linkage maps to diploid Solanum tuberosum pachytene chromosomes by BAC-FISH technology. Chromosome Res., 17, 899–915.

    Jong H. , 'Assignment of genetic linkage maps to diploid Solanum tuberosum pachytene chromosomes by BAC-FISH technology ' (2009 ) 17 Chromosome Res. : 899 -915.

    • Search Google Scholar
  • Tessadori, F., van Driel, R., Fransz, P. (2004): Cytogenetics as a tool to study gene regulation. Trends Plant Sci., 9, 147–153.

    Fransz P. , 'Cytogenetics as a tool to study gene regulation ' (2004 ) 9 Trends Plant Sci. : 147 -153.

    • Search Google Scholar
  • The Arabidopsis Genome Initiative (2000): Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 408, 796–815.

    'Analysis of the genome sequence of the flowering plant Arabidopsis thaliana ' (2000 ) 408 Nature : 796 -815.

    • Search Google Scholar
  • The International Brachypodium Initiative (IBI) (2010): Genome sequence analysis of the model grass Brachypodium distachyon: insights into grass genome evolution. Nature, 463, 763–768.

    'Genome sequence analysis of the model grass Brachypodium distachyon: insights into grass genome evolution ' (2010 ) 463 Nature : 763 -768.

    • Search Google Scholar
  • Valarik, M., Bartos, J., Kovarova, P., Kubalakova, M., de Jong, J. H., Dolezel, J. (2004): Highresolution FISH on super-stretched flow-sorted plant chromosomes. Plant J., 37, 940–950.

    Dolezel J. , 'Highresolution FISH on super-stretched flow-sorted plant chromosomes ' (2004 ) 37 Plant J. : 940 -950.

    • Search Google Scholar
  • Wang, Y., Tang, X., Cheng, Z., Mueller, L., Giovannoni, J., Tanksley, S. D. (2006): Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome. Genetics, 172, 2529–2540.

    Tanksley S. D. , 'Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome ' (2006 ) 172 Genetics : 2529 -2540.

    • Search Google Scholar
  • Watson, J. D., Crick, F. H. C. (1953): Molecular structure of nucleic acids — a structure for deoxyribose nucleic acids. Nature (London), 171, 737–738.

    Crick F. H. C. , 'Molecular structure of nucleic acids — a structure for deoxyribose nucleic acids ' (1953 ) 171 Nature (London) : 737 -738.

    • Search Google Scholar
  • Wiegant, J., Kalle, W., Mullenders, L., Brookes, S., Hoovers, J. M., Dauwerse, J. G., van Ommen, G. J., Raap, A. K. (1992): High-resolution in situ hybridization using DNA halo preparations. Hum. Mol. Genet., 1, 587–591.

    Raap A. K. , 'High-resolution in situ hybridization using DNA halo preparations ' (1992 ) 1 Hum. Mol. Genet. : 587 -591.

    • Search Google Scholar
  • Zhao, Q., Zhang, Y., Cheng, Z. (2002): A fine physical map of the rice chromosome 4. Genome Res., 12, 817–823.

    Cheng Z. , 'A fine physical map of the rice chromosome 4 ' (2002 ) 12 Genome Res. : 817 -823.

  • Zhong, X. B., Lizardi, P. M., Huang, X. H., Bray-Ward, P. L., Ward, D. C. (2001): Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibres using rolling circle DNA amplification. Proc. Natl. Acad. Sci. USA, 98, 3940–3945.

    Ward D. C. , 'Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibres using rolling circle DNA amplification ' (2001 ) 98 Proc. Natl. Acad. Sci. USA : 3940 -3945.

    • Search Google Scholar
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