Please wait a minute...
   Search  |  ADV Search
Cotton Science    2017, Vol. 29 Issue (3) : 213-221     DOI: 10.11963/1002-7807.lylprh.20170427
Construction and Primary Application of Oligos Fluorescence in situ Hybridization Technology in Cotton
Yuling Liu1,2(),Zhen Liu1,Zhaoguo Li1,Yuhong Wang2,Zhongli Zhou2,Xiaoyan Cai2,Xingxing Wang2,Xiaoyan Wang1,Shulin Zhang1,Haiyan Zhao1,Zhenmei Zhang2,Kunbo Wang2,Fang Liu2,*(),Renhai Peng1,2,*()
1. Anyang Institute of Technology, Anyang, Henan 455000, China
2. Institute of Cotton Research of Chinese Academy of Agricultural Sciences / State Key Laboratory of Cotton Biology, Anyang, Henan 455000, China
Download: PDF(1304 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks    

[Objective] Fluorescence in situ hybridization (FISH) can achieve the accurate chromosomal localization of DNA sequences, which is one of the important techniques for genome in-depth study. The acquisition of chromosome-specific probes is the key for FISH application. The aim of this study is to explore a system of cotton oligonucleotides (oligos) fluorescence in situ hybridization (Oligo-FISH) technology. [Method] Based on the published cotton genome sequence, the chromosome-specific oligos library was obtained by bioinformatics method. Then, the obtained oligos were labeled through emulsion polymerase chain reaction, and used for FISH of cotton mitotic chromosome. [Results] A system of cotton Oligo-FISH technology was set up and successfully applied in cotton chromosome identification. [Conclusion] This system can be used for dissection of cotton genomes.

Keywords cotton      oligos      fluorescence in situ hybridization      chromosome     
Corresponding Authors: Fang Liu,Renhai Peng     E-mail:;;
Issue Date: 02 June 2017
E-mail this article
E-mail Alert
Articles by authors
Yuling Liu
Zhen Liu
Zhaoguo Li
Yuhong Wang
Zhongli Zhou
Xiaoyan Cai
Xingxing Wang
Xiaoyan Wang
Shulin Zhang
Haiyan Zhao
Zhenmei Zhang
Kunbo Wang
Fang Liu
Renhai Peng
Cite this article:   
Yuling Liu,Zhen Liu,Zhaoguo Li, et al. Construction and Primary Application of Oligos Fluorescence in situ Hybridization Technology in Cotton[J]. Cotton Science, 2017, 29(3): 213-221.
URL:     OR
Fig. 1  Procedure for design of single chromosome-specific oligos of cotton
水相Aqueous phase油相Oil phase
成分Component用量Usage /μL成分Component用量Usage /μL
Nuclease free water99.5矿物油Mineral oils10 000
5 × HF buffer30.0Triton X-1005
50 mmol L-1MgCl23.0ABIL EM90400
MYtags PCR Primer mix1.5
10 mmol L-1dNTP3.0
10 g L-1BSA (High Q)7.5
Table 1  Composition and quantity of aqueous phase and oil phase of emulsified PCR
Fig. 2  Detection of emulsion PCR product and labeled probe by gel
Fig. 3  Oligo-FISH on mitotic metaphase chromosomes ofGossypium raimondii
[1] Speicher MR,Ballard SG,Ward DC.Karyotyping human chromosomes by combinatorial multi-fluor FISH[J].Nature Genetics,1996,12:368-375.
[2] Lysak MA,PecinkaA,SchubertI.Recent progress in chromosome painting ofArabidopsisand related species[J].Chromosome Research,2003,11:195-204.
doi: 10.1023/A:1022879608152 pmid: 12769287 url:
[3] Ferguson-Smith MA,TrifonovV.Mammalian karyotype evolution[J].Nature Reviews Genetics,2007,8:950-962.
[4] Beliveau BJ,Joyce EF,ApostolopoulosN,et al.Versatile design and synthesis platform for visualizing genomes with oligopaint FISH probes[J].Proceedings of the National Academy of the Sciences of the United States of America,2012,109:21301-21306.
doi: 10.1073/pnas.1213818110 pmid: 23236188 url:
[5] FuchsJ,HoubenA,BrandesA,et al.Chromosome 'painting' in plants — A feasible technique?[J].Chromosoma,1996,104(5):315-320.
doi: 10.1007/BF00337219 pmid: 8575242 url:
[6] Lysak MA,MandákováT.Analysis of plant meiotic chromosomes by chromosome painting[J].Methods in Molecular Biology,2013,990:13-24.
[7] Betekhtin A, Jenkins G, Hasterok R. Reconstructing the evolution of Brachypodium genomes using comparative chromosome painting[J/OL]. PLoS ONE, 2014, e115108 [2016-10-27]. .
url: http://
[8] Lou QF,Zhang YX,He YH,et al.Single-copy gene-based chromosome painting in cucumber and its application for chromosome rearrangement analysis inCucumis[J].The Plant Journal,2014,78:169-179.
doi: 10.1111/tpj.12453 pmid: 24635663 url:
[9] Han YH,ZhangT,ThammapichaiP,et al.Chromosome-specific painting inCucumisspecies using bulked oligonucleotides[J].Genetics,2015,200:771-779.
doi: 10.1534/genetics.115.177642 pmid: 25971668 url:
[10] BoyleS,Rodesch MJ,Halvensleben HA,et al.Fluorescencein situhybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis[J].Chromosome Research,2011,19:901-909.
[11] Yamada NA,Rector LS,TsangP,et al.Visualization of fine-scale genomic structure by oligonucleotide-based high-resolution FISH[J].Cytogenetic and Genome Research,2011,132:248-254.
doi: 10.1159/000322717 pmid: 21178330 url:
[12] Wang KB,Wang ZW,Li FG,et al.The draft genome of a diploid cottonGossypium raimondii[J].Nature Genetics,2012,44:1098-1103.
[13] Paterson AH,Wendel JF,GundlachH,et al.Repeated polyploidization ofGossypiumgenomes and the evolution of spinnable cotton fibres[J].Nature,2012,492:423-427.
doi: 10.1038/nature11798 pmid: 23257886 url:
[14] Li FG,Fan GY,Wang KB,et al.Genome sequence of the cultivated cottonGossypium arboreum[J].Nature Genetics,2014,46:567-572.
[15] Li FG,Fan GY,Lu CR,et al.Genome sequence of cultivated upland cotton (Gossypium hirsutumTM-1) provides insights into genome evolution[J].Nature Biotechnology,2015,33:524-530.
doi: 10.1038/nbt.3208 pmid: 25893780 url:
[16] Zhang TZ,HuY,Jiang WK,et al.Sequencing of allotetraploid cotton (Gossypium hirsutumL. acc. TM-1) provides a resource for fiber improvement[J].Nature Biotechnology,2015,33:531-537.
doi: 10.1038/nbt.3207 pmid: 25893781 url:
[17] Yuan D J, Tang Z H, Wang M J, et al. The genome sequence of Sea-Island cotton (Gossypium barbadense) provides insights into the allopolyploidization and development of superior spinnable fibres[J/OL]. Scientific Reports, 2015, 5(6): 17662 [2016-10-27]. . DOI: 10.1038/srep17662.
[18] Liu X, Zhao B, Zheng H J, et al. Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites[J/OL]. Scientific Reports, 2015, 5: 14139 [2016-10-27]. . DOI: 10.1038/srep14139.
[19] Kent WJ.BLAT—The BLAST-like alignment tool[J].Genome Research,2002,12:656-664.
[20] UntergasserA,CutcutacheI,KoressaarT,et al.Primer3 — new capabilities and interfaces[J].Nucleic Acids Research,2012,40:e115.
[21] Murgha Y E, Rouillard J M, Gulari E. Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries[J/OL]. PLoS ONE, 2014, 9: e94752 [2016- 10-27]. . DOI: 10.1371/journal.pone.0094752.
[22] 王春英,王坤波,王文奎,等.棉花gDNA体细胞染色体FISH技术[J].棉花学报,1999,11(2):79-83.
[22] WangChunying,WangKunbo,WangWenkui,et al.Protocol of cotton FISH of somatic chromosomes with gDNA as probes[J].Cotton Science,1999,11(2):79-83.
[23] Huang SW,Li RQ,Zhang ZH,et al.The genome of the cucumber,Cucumis sativusL.[J].Nature Genetics,2009,41:1275-1281.
[24] Jiang JM,Gill BS.Current status and the future of fluorescencein situhybridization (FISH) in plant genome research[J].Genome,2006,49:1057-1068.
doi: 10.1068/d0505 pmid: 17110986 url:
[25] Cheng ZK,Presting GG,Buell CR,et al.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[J].Genetics,2001,157:1749-1757.
[26] Cui XL,LiuF,LiuY,et al.Construction of cytogenetic map ofGossypium herbaceumchromosome 1 and its integration with genetic maps[J/OL]. Molecular Cytogenetics,2015, 8: 2 [2016-10-27]. DOI:10.1186/s13039-015-0106-y.
[27] MukaiY,FriebeB,Hatchett JH,et al.Molecular cytogenetic analysis of radiation-induced wheat-rye terminal and intercalary chromosomal translocations and the detection of rye chromatin specifying resistance to Hessian fly[J].Chromosoma,1993,10:88-95.
doi: 10.1007/BF00356025 url:
[28] KatoA,Lamb JC,Birchler JA.Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize[J].Proceedings of the National Academy of the Sciences of the United States of America,2004,101:13554-13559.
[29] Yuan WY,MotonoriT.Centromeric distribution of 350-family inDasypyrum villosumand its application to identifyingDasypyrumchromatin in the wheat genome[J].Hereditas,2009,146:58-66.
[30] Xiong ZY,Pires JC.Karyotype and identification of all homoeologous chromosomes of allopolyploidBrassica napusand its diploid progenitors[J].Genetics,2011,187:37-49.
doi: 10.1534/genetics.110.122473 pmid: 21041557 url:
[31] ChesterM,Lipman MJ,Gallagher JP,et al.An assessment of karyotype restructuring in the neoallotetraploidTragopogon miscellus(Asteraceae)[J].Chromosome Research,2013,21:75-85.
[32] ZhangP,Li WL,FellersJ,et al.BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements[J].Chromosoma,2004,112:288-299.
doi: 10.1007/s00412-004-0273-9 pmid: 14986017 url:
[33] JandaJ,SafarJ,KubalakovaM,et al.Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B[J].The Plant Journal,2006,47:977-986.
doi: 10.1111/j.1365-313X.2006.02840.x pmid: 16911585 url:
[34] SuzukiG,OgakiY,HokimotoN,et al.Random BAC FISH of monocot plants reveals differential distribution of repetitive DNA elements in small and large chromosome species[J].Plant Cell Reports,2012,31:621-628.
doi: 10.1007/s00299-011-1178-8 pmid: 22083649 url:
[35] Tang ZX,Yang ZJ,Fu SL.Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis[J].Journal of Applied Genetics,2014,55:313-318.
doi: 10.1007/s13353-014-0215-z pmid: 24782110 url:
[36] Jiang JM,FriebeB,Gill BS.Recent advances in alien gene transfer in wheat[J].Euphytica,1994,73:199-212.
doi: 10.1007/BF00036700 url:
[37] JanskyS.Breeding for disease resistance in potato[J].Plant Breeding Reviews,2000,19:69-155.
doi: 10.1023/A:1022075604669 url:
[38] SybengaJ.Recombination and chiasmata: Few but intriguing discrepancies[J].Genome,1996,39:473-484.
doi: 10.1139/g96-061 pmid: 18469909 url:
[39] Jena,KK,Khush G S. Monosomic alien addition lines of rice - production, morphology, cytology, and breeding behavior[J].Genome,1989,32:449-455.
doi: 10.1139/g89-468 url:
[40] SchawarzaeherT,AnamthawatJ,Harrison GE,et al.Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat[J].Theoretical and Applied Genetics,1992,84:778-786.
doi: 10.1007/BF00227384 pmid: 24201474 url:
[41] 黄滋康,季道藩,潘家驹.中国棉花遗传育种学[M].济南:山东科学技术出版社,2003.
[41] HuangZikang,JiDaofan,PanJiaju.Genetics and breeding of Chinese cotton[M].Jinan:Shandong Science and Technology Press,2003.
[1] Luo Junyu, Zhang Shuai, Ren Xiangliang, Lü Limin, Zhang Lijuan, Ji Jichao, Ma Yan, Cui Jinjie. Research Progress of Cotton Insect Pests in China in Recent Ten Years[J]. Cotton Science, 2017, 29(增刊): 100-112.
[2] Li Pengcheng, Zheng Cangsong, Sun Miao, Pang Chaoyou, Zhang Siping, Liu Shaodong, Chen Jing, Li Yabing, Dong Helin, Zhao Xinhua. Research Progress on Fertilization Technology and Nutrition Mechanism of cotton[J]. Cotton Science, 2017, 29(增刊): 118-130.
[3] Zhao Yunlei, Wang Ning, Ge Xiaoyang, Zhang Chaojun, Yan Gentu, Wang Hongmei, Li Fuguang. Research and Application of Genetic Improvement for Cotton Stress Resistance[J]. Cotton Science, 2017, 29(增刊): 11-19.
[4] Fu Xiaoqiong, Peng Jun. Prospect and Retrospection of National Cotton Regional Test of China in Last Decade[J]. Cotton Science, 2017, 29(增刊): 113-117.
[5] Du Xiongming, Liu Fang, Wang Kunbo, Jia Yinhua, Zhou Zhongli, He Shoupu, Gong Wenfang, Pan Zhao’e, Wang Liru, Geng Xiaoli, Pang Baoyin. Collection, Evaluation and Utilization of Cotton Germplasm[J]. Cotton Science, 2017, 29(增刊): 51-61.
[6] . Advances of Light and Simplified Cultivation Technologies in China[J]. Cotton Science, 2017, 29(增刊): 80-88.
[7] Wei Shoujun, Tang Shurong, Kuang Meng, Wang Yanqin, Ma Lei. Research Progress of Quality & Safety Risk Assessment for Cotton Production[J]. Cotton Science, 2017, 29(增刊): 89-99.
[8] . Research Progress and Application of Early Maturity in Upland Cotton[J]. Cotton Science, 2017, 29(增刊): 1-10.
[9] . The Progress and Perspective of Cotton Functional Genomics Research[J]. Cotton Science, 2017, 29(增刊): 20-27.
[10] Zhu Heqin, Li Zhifang, Feng Zili, Feng Hongjie, Wei Feng, Zhao Lihong, Shi Yongqiang, Liu Shichao, Zhou Jinglong . Overview of Cotton Verticillium Wilt Research over the Past Decade in China and Its Prospect in Future[J]. Cotton Science, 2017, 29(增刊): 37-50.
[11] . Ten-Year Achievements and Future Development of Cotton Heterosis Utilization[J]. Cotton Science, 2017, 29(增刊): 28-36.
[12] Shang Haihong, Yu Jiwen, Shi Yuzhen, Gong Wankui, Li Junwen, Wu Man, Ge Qun, Gong Juwu, Fan Senmiao, Yuan Youlu. Identification of QTL/Genes of Fiber Yield, Quality and Disease Resistance, and Molecular breeding in Cotton[J]. Cotton Science, 2017, 29(增刊): 62-71.
[13] Zhang Xiling, Song Meizhen, Gui Huiping, Pang Nianchang, Wang Xiangru, Zhang Hengheng, Dong Qiang. The Progress and Prospect of the Cotton Planting Technology Standard in China[J]. Cotton Science, 2017, 29(增刊): 72-79.
[14] Zhou Na, Wang Luyao, Zhang Tianzhen, Hu Yan. Genome-Wide Analysis of MIKCC Gene Family in Cotton[J]. Cotton Science, 2017, 29(6): 495-503.
[15] Kong Fanjin, Deng Yongsheng, Shen Guifang, Wang Jinghui, Han Zongfu, Wang Zongwen, Duan Bing, Zhang Lingshan, Li Ruzhong. Analysis on Competitive Heterosis of Hybrid F1 and F2 between Transgenic Bt Varieties of Upland Cotton[J]. Cotton Science, 2017, 29(6): 504-512.
Full text