Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established.
References
[1]
Schwarzacher T, Anamthawat-Jonsson K, Harrison GE (1992) Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat. Theor Appl Genet 84: 778–786. DOI:10.1007/BF00227384.
[2]
Mukai Y, Friebe B, Hatchett JH, Yamamoto M, Gill BS (1993) Molecular cytogenetic analysis of radiation-induced wheat-rye terminal and intercalary chromosomal translocations and the detection of rye chromatin specifying resistance to Hessian fly. Chromosoma 102: 88–95. DOI:10.1007/BF00356025.
[3]
Shishido R, Apistwanich S, Ohmido N, Okinaka Y, Mori K, et al. (1998) Detection of specific chromosome reduction in rice somatic hybrids with the A, B and C genomes by multi-color genomic in situ hybridization. Theor Appl Genet 97: 1013–1018. DOI:10.1007/s001220050985.
[4]
Ma N, Li ZY, Cartagena JA, Fukui K (2006) GISH and AFLP analyses of novel Brassica napus lines derived from one hybrid between B. napus and Orychophragmus violaceus. Plant Cell Rep 25: 1089–1093. DOI:10.1007/s00299-006-0171-0.
[5]
Stephens JL, Brown SE, Lapitan NLV, Knudson DL (2004) Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique. Genome 47: 179–189. DOI:10.1139/g03-084.
[6]
Wang CJR, Harper L, Cande ZW (2006) High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9. Plant Cell 18: 529–544. DOI:10.1105/tpc.105.037838.
[7]
Fransz PF, Alonso-Blanco C, Liharska TB, Peters AJ, Zabel P, et al. (1996) High resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibers. Plant J 9: 421–430. DOI:10.1046/j.1365-313X.1996.09030421.x.
[8]
Ohmido N, Kijima K, Hirose T, de Jong JH, Fukui K (1999) Recent advances in the physical mapping of genes by fluorescence in situ hybridization (FISH) of rice. Am Biotech Lab 17: 56–58. DOI:10.2183/pjab.86.103.
[9]
Kato S, Ohmido N, Fukui K (2003) Development of a quantitative pachytene chromosome map in Oriza sativa by imaging methods. Genes & Genet Syst 78: 155–161. DOI:10.1266/ggs.78.155.
[10]
Wang K, Song XL, Han ZG, Guo WZ, Yu JZ, et al. (2006) Complete assignment of the chromosomes of Gossypium hirsutum L. by translocation and fluorescence in situ hybridization mapping. Theor Appl Genet 113: 73–80. DOI:10.1007/s00122-006-0273-7.
[11]
Wang K, Guo WZ, Zhang TZ (2007) Development of one set of chromosome-specific microsatellite- containing BACs and their physical mapping in Gossypium hirsutum L. Theor Appl Genet 115: 675–682. DOI:10.1007/s00122-007-0598-x.
[12]
Ji Y, Zhao X, Paterson AH, Price HJ, Stelly DM (2007) Integrative mapping of Gossypium hirsutum L. by meiotic fluorescent in situ hybridization of a tandemly repetitive sequence (B77). Genetics 176: 115–123. DOI:10.1534/genetics.107.071738.
[13]
Feng Q, Zhang YJ, Hao P, Wang SY, Fu G, et al. (2002) Sequence and analysis of rice chromosome 4. Nature 420: 316–320. DOI:10.1038/nature01183.
[14]
Sasaki T, Matsumoto T, Yamamoto K, Sakata K, Baba T, et al. (2002) The genome sequence and structure of rice chromosome 1. Nature 420: 312–316. DOI:10.1038/nature01184.
[15]
Yu YS, Rambo T, Currie J, Saski C, Kim HR, et al. (2003) In-depth view of structure, activity, and evolution for rice chromosome 10. Science 300: 1566–1569. DOI:10.1126/science.1083523.
[16]
Cheng Z, Dong F, Langdon T, Ouyang S, Buell CB, et al. (2002) Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon. Plant Cell 14: 1691–1704. DOI:10.1105/tpc.003079.
[17]
Hiroshi M, Wu JZ, Hiroyuki K, Masaki F, Nobukazu N, et al. (2006) Sequencing and characterization of telomere and subtelomere regions on rice chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S. The Plant J 46: 206–217. DOI:10.1111/j.1365-313X.2006.02684.x.
[18]
Zhong XB, Fransz PF, Wennekes-van Eden J, Ramanna MS, vanKammen A, et al. (1998) FISH studies reveal the molecular and chromosomal organization of individual telomere domains in tomato. Plant J 13: 507–517. DOI:10.1046/j.1365-313X.1998.00055.x.
[19]
Heng HHQ, Squire J, Tsui LC (1992) High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc Natl Acad Sci 89: 9509–9513. http://www.pnas.org/content/89/20/9509.
[20]
Weier HU, Wang M, Mullikin JC, Zhu Y, Cheng JF, et al. (1995) Quantitative DNA fiber mapping. Hum Mol Genet 4: 1903–1910. DOI:10.1093/hmg/4.10.1903.
[21]
Zhong XB (1996) Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescence in situ hybridization (FISH). Chromosome Research 4: 24–28. DOI:10.1007/BF02254940.
[22]
Jackson SA, Dong FG, Jiang JM (1999) Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization. Plant J 17: 581–587. DOI:10.1046/j.1365–313X.1999.00398x.
[23]
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: 388–394. DOI:10.1007/s004380051182.
[24]
Cheng Z, Buell CR, Wing RA, Gu M, Jiang J (2001) Toward a cytological characterization of the rice genome. Genome Research 11: 2133–2141. DOI:10.1101/gr.194601.
[25]
Cheng Z, Presting GG, Buell CR, Wing RA, 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.
[26]
de Jong JH, Fransz P, Zabel P (1999) High resolution FISH in plants-techniques and applications. Trends Plant Science 4: 258–263. DOI:10.1016/S1360-1385(99)01436-3.
[27]
Cheng Z, Buell CR, Wing RA, Jiang J (2002) Resolution of fluorescence in situ hybridization mapping on rice mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers. Chromosome Research 10: 379–387. DOI:10.1023/A:1016849618707.
[28]
Ohmido N, Fukui K (2004) Recent advances in FISH analysis of plant chromosomes. Recent Res Devel Biochem 5: 267–269. ISBN:81-271-0049-8.
[29]
Jiang J, Gill BS (2006) Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research. Genome 49: 1057–1068. DOI:10.1139/G06-076.
[30]
Shiels C, Coutelle C, Huxley C (1997) Analysis of ribosomal and alphoid repetitive DNA by fiber-FISH. Cytogenet Cell Genet 76: 20–22. DOI:10.1159/000134504.
[31]
Jackson SA, Cheng Z, Wang LM, Goodman HM, Jiang J (2000) Comparative fluorescence in situ hybridization mapping of a 431-kb Arabidopsis thaliana bacterial chromosomal duplications in expansion of the Brassica rapa genome. Genetics 156: 833–838. http://www.genetics.org/cgi/reprint/156/？2/833.
[32]
Nagaki K, Song J, Stupar SM, Parokonny AS, Yuan Q, et al. (2003) Molecular and cytological analyses of large tracks of centromeric DNA reveal the structure and evolutionary dynamics of maize centromeres. Genetics 163: 759–770. http://www.genetics.org/cgi/reprint/163/？2/759.
[33]
Li ZY, Qin R, Jin WW, Xiong ZY, Song YC (2005) FISH analysis of pachytene chromosome and DNA fiber of telomere sequence in rice (Oryza stativa l. Indica). Acta Genetica Sinica 32: 832–836. DOI:CNKI:ISSN:0379-4172.0.2005-08-010.
[34]
Jeffrey CZ, Scheffler BE, Dennis E, Triplett BA, Zhang T, et al. (2007) Toward sequencing cotton (Gossypium) genomes. Plant Physiol 145: 1304–1310. DOI:10.1104/pp.107.107672.
[35]
Paterson Andrew H (2008) Sequencing the cotton genomes-Gossypium spp. Cotton Science 20(Suppl.): 3. DOI:CNKI:SUN:MHXB.0.2008-S1-003.
[36]
Wilkins TA (2008) Sequencing of a cuitivated dipoid cotton genome-Gossypium arboreum. Cotton Science 20(Suppl.): 5. CNKI:SUN:MHXB.0.2008-S1-005.
[37]
Fryxell PA (1992) A revised taxonomic interpretation of Gossypium L. (Malvaceae). Rheedea 2: 108–165.
[38]
Beasley JO (1942) Meiotic chromosome behavior in species hybrids, haploids, and induced polyploids of Gossypium. Genetics 27: 25–54. PMID:17247031.
[39]
Endrizzi JE, Turcotte EL, Kohel RJ (1985) Genetics, cytology, and evolution of Gossypium. Adv Genet 23: 271–375. http://dx.doi.org/10.1016/S0065-2660(08)？60515-5.
[40]
Percival AE, Wendel JF, Stewart JM (1999) Taxonomy and germplasm resources. In: Smith CW, Cothren JT, editors. Cotton: origin, history, technology, and production. pp. 33–63. ISBN 0-471-18045-9.
[41]
Xiang XL, Shen DZ (1989) Chinese Asian Cotton (Gossypium arboreum). Chinese Agricultural Press, Beijing.
[42]
Wendel JF, Schnabei A, Seelanan T (1995) An unusual ribosomal DNA sequence from Gossypium gossypioides reveals ancient, cryptic, intergenomic introgression. Mol Phylogenet Evol 4: 298–313. http://dx.doi.org/10.1006/mpev.1995.1027.
[43]
Hanson RE, Zwick MS, Choi SD, Islamfaridi MN, Mcknight TD, et al. (1995) Fluorescent in situ hybridization of a bacterial artificial chromosome. Genome 38: 646–651. DOI:10.1139/g95-082.
[44]
Zhao XP, Yang S, Hanson RE, Crane CF, Price HJ, et al. (1998) Dispersed repetitive DNA has spread to new genome since polyploid formation in cotton. Genome Research 8: 479–492. DOI:10.1101/gr.8.5.479.
[45]
Ji YF, Raska DA, Mcknight TD (1997) Use of meiotic FISH for identification of a new monosome in Gossypium hirsutum L. Genome 40: 34–40. DOI:10.1139/g97-005.
[46]
Wang KB, Wang WK, Wang CY, Song GL, Cui RX, et al. (2001) Investigation of Gossypium babardense L. by FISH and karyotype analysis. Acta Genetica Sinica 28: 69–75.DOI: CNKI:ISSN:0379-4172.0.2001-01-011
[47]
Liu SH, Wang KB, Song GL, Wang CY, Liu F, et al. (2005) Primary investigation on GISH-NOR in cotton. Chinese Science Bulletin 50: 425–429. DOI:10.1007/BF02897457.
[48]
Wang K, Yang ZJ, Shu CS, Hu J, Lin QY, et al. (2009) Higher axial-resolution and sensitivity pachytene fluorescence in situ hybridization protocol in tetroploid cotton. Chromosome Research 17(8): 1041–1050. DOI:10.1007/s10577-009-9085-3.
[49]
Wang K, Guo WZ, Yang ZJ, Hu Y, Zhang WP, et al. (2010) Structure and size variations between 12A and 12D homoeologous chromosomes based on high-resolution cytogenetic map in allotetraploid cotton. Chromosoma 119(3): 255–266. DOI:10.1007/s00412-009-0254-0.
[50]
Ijdo JW, Wells RA, Baldini A, Reeders ST (1991) Improved telomere detection using a telomere repeat probe (TTTAGGG)n generated by PCR. Nucleic Acids Research 19: 478. http://www.pubmedcentral.nih.gov/picrend？er.fcgi?artid=328734&blobtype.
[51]
Goel S, Chen Z, Conner JA, Akiyama Y, Hanna WW, et al. (2003) Delineation by fluorescence in situ hybridization of a single hemizygous chromosomal region associated with aposporous embryo sac formation in Pennisetum squamulatum and Cenchrus ciliaris. Genetics 163: 1069–1082. http://www. genetics.org/cgi/reprint/163/3/1069.
[52]
Li LJ, Yang JL, Tong Q, Zhao LJ, Song YC (2005) A novel approach to prepare extended DNA in plant. Cytometry (Part A) 63A: 114–117. DOI:10.1002/cyto.a.20111.
[53]
Liu Y, Whittier RF (1994) Rapid preparation of metabase plant DNA from nuclei in agarose plugs and microbeads. Nucleic Acids Research 22: 2168–2169. DOI:10.1093/nar/22.11.2168.
[54]
Zhang HB (2000) Manual for construction and manipulation of large insert bacterial clone libraries (on-line). http://hbz7.tamu.edu/homelinks/tool/bac_？content.htm.
[55]
Wang XF, Ma J, Wang WS, Zheng YM, Zhang GY, et al. (2006) Construction and characterization of the first bacterial artificial chromosome library for the cotton species Gossypium barbadense L. Genome 49: 1393–1398. DOI:10.1139/G06-113.
[56]
Lavania UC, Yamamoto M, Mukai Y (2003) Extended chromatin and DNA fibers from active plant nuclei for high-resolution FISH. J Histochem Cytochem 51: 1249–1253. DOI:10.1177/002215540305101001.
[57]
Fransz PF, Armstrong S, Alonso-Blanco C, Fischer TC, Torres-Ruiz RA, et al. (1998) Cytogenetics for the model system Arabidopsis thaliana. Plant J 13: 867–876. DOI:10.1046/j.1365-313X.1998.00086.x.
[58]
Kim JS, Islam-Faridi MN, Klein PE, Stelly DM, Price HJ, et al. (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.
[59]
Wiegant J, Kalle W, Mullenders L, Brookes S, Hoovers JMN, Dauwerse JG, et al. (1992) High-resolution in situ hybridization using DNA halo preparations. Hum Mol Genet 1: 587–591. DOI:10.1093/hmg/1.8.587.
[60]
Windle B, Silvas E, Parra I (1995) High resolution microscopic mapping of DNA using multi-color fluorescent hybridization. Electrophoresis 16: 273–278. DOI:10.1002/elps.1150160143.
[61]
Fransz PF, Armstrong S, de Jong JH, Parnell LD, van Druneu C, et al. (2000) Integrated cytogenetic map of chromosome arm 4S of A. thaliana: structural organization of heterochromatic knob and centromere region. Cell 100: 367–376. DOI:10.1016/S0092-8674(00)80672-8.
[62]
van de Rijke FM, Florijn RJ, Tanke HJ, Raap AK (2000) DNA fiber-FISH staining mechanism. The Journal of Histochemistry & Cytochemistry 48: 743–745. http://www.jhc.org/cgi/reprint/48/6/743.
