Article | 12. 2015 Vol. 33, Issue. 6
FISH Karyotype Analysis of Four Wild Cucurbitaceae Species Using 5S and 45S rDNA Probes and the Emergence of New Polyploids in Trichosanthes kirilowii Maxim.

Plant Biotechnology Institute, Department of Life Science, Sahmyook University1
Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University2

2015.12. 869:876


Wild relative species of domesticated crops are useful genetic resources for improving agronomic traits. Cytogenetic investigations based on chromosome composition provide insight into basic genetic and genomic characteristics of a species that can be exploited in a breeding program. Here, we used FISH analysis to characterize the ploidy level, chromosome constitution, and genomic distribution of 5S and 45S ribosomal DNA (rDNA) in four wild Cucurbitaceae species, namely, Citrullus lanatus (Thunb.) Mansf. var. citroides L. H. Bailey (2n = 22), Melothria japonica Maxim. (2n = 22), Sicyos angulatus L. (2n = 24), and Trichosanthes kirilowii Maxim. (2n = 66, 88, 110 cytotypes), collected in different areas of Korea. All species were diploids, except for T. kirilowii, which included hexa-, octa-, and decaploid cytotypes (2n = 6x = 66, 8x = 88, and 10x = 110). All species have small metaphase chromosomes in the range of 2–5 μm. The 45S rDNA signals were localized distally compared to the 5S rDNA. C. lanatus var. citroides and M. japonica showed one and two loci of 45S and 5S rDNA, respectively, with co-localization of rDNA signals in one M. japonica chromosome. S. angulatus showed two co-localized signals of 5S and 45S rDNA loci. The hexaploid T. kirilowii cytotype showed five signals each for 45S and 5S rDNA, with three being co-localized. This is the first report of hexaploid and decaploid cytotypes in T. kirilowii. These results will be useful in future Cucurbitaceae breeding programs.

1. Albert, P. S., Z. Gao, T. V. Danilova, and J. A. Birchler. 2010. Diversity of chromosomal karyotypes in maize and its relatives. Cytogenet. Genome. Res. 129:6-16.  

2. Anamthawat-Jónsson, K. 2001. Molecular cytogenetics of intro-gressive hybridization in plants. Methods Cell Sci. 23:141-150.  

3. Antão, C. M. and F. X. Malcata. 2005. Plant serine proteases: biochemical, physiological and molecular features. Plant Physiol. Biochem. 43:637-650.  

4. Balao, F., J. Herrera, and S. Talavera. 2011. Phenotypic consequences of polyploidy and genome size at the microevolutionary scale: a multivariate morphological approach. New Phytol. 192: 256-265.  

5. Benavente, E., M. Cifuentes, J. C. Dusautoir, and J. David. 2008. The use of cytogenetic tools for studies in the crop-to-wild gene transfer scenario. Cytogenet. Genome Res. 120:384-395.  

6. Bennetzen, J. L. 2005. Transposable elements, gene creation and genome rearrangement in flowering plants. Curr. Opin. Genet. Dev. 15:621-627.  

7. Bennetzen, J. L., J. Ma, and K. M. Devos. 2005. Mechanisms of recent genome size variation in flowering plants. Ann. Bot. 95:127-132.  

8. Bertioli, D.J., M.C. Moretzsohn, L.H. Madsen, N. Sandal, S.C. Leal-bertioli, P.M. Guimaraes, B.K. Hougaard, J. Fredslund, L. Schauser, A.M. Nielsen, S. Sato, S. Tabata, S.B. Cannon, and J. Stougaard. 2009. An analysis of synteny of Arachis with Lotus and Medicago sheds new light on the structure, stability and evolution of legume genomes. BMC Genomics 10:45.  

9. Brar, D. S. and G. S. Khush. 1997. Alien introgression in rice. Plant Mol. Biol. 35:35-47.  

10. Bruggmann, R., A. K. Bharti, H. Gundlach, J. Lai, S. Young, A. C. Pontaroli, F. Wei, G. Haberer, G. Fuks, C. Du, C. Raymond, M. C. Estep, R. Liu, J. L. Bennetzen, A. P. Chan, P. D. Rabinowicz, J. Quackenbush, W. B. Barbazuk, R. A. Wing, B. Birren, C. Nusbaum, S. Rounsley, K. F. Mayer, and J. Messing. 2006. Uneven chromosome contraction and expansion in the maize genome. Genome Res. 16:1241-1251.  

11. Chen, Z. J. and H. H. Yu. 2013. Genetic and epigenetic mechanisms for polyploidy and hybridity, p. 335-354. In: Z. J. Chen and J.A. Birchler. Polyploid and hybrid genomics. John Wiley & Sons, Inc., Ames, Iowa, USA.  

12. Clarkson, J. J., K. Y. Lim, A. Kovarik, M. W. Chase, S. Knapp, and A. R. Leitch. 2005. Long-term genome diploidization in allopolyploid Nicotiana section Repandae (Solanaceae). New Phytol. 168:241-252.  

13. Comai, L. 2005. The advantages and disadvantages of being polyploid. Nat. Rev. Genet. 6:836-846.  

14. Delannay, I. Y., J. E. Staub, and J. F. Chen. 2010. Backcross Introgression of the Cucumis hystrix genome increases genetic diversity in US Processing cucumber. J. Am. Soc. Hortic. Sci. 135:351-361.  

15. Garcia-Mas, J., A. Benjak, W. Sanseverino, M. Bourgeois, G. Mir, V. M. Gonzalez, E. Henaff, F. Camara, L. Cozzuto, E. Lowy, T. Alioto, S. Capella-Gutierrez, J. Blanca, J. Canizares, P. Ziarsolo, D. Gonzalez-Ibeas, L. Rodriguez-Moreno, M. Droege, L. Du, M. Alvarez-Tejado, B. Lorente-Galdos, M. Mele, L. Yang, Y. Weng, A. Navarro, T. Marques-Bonet, M. A. Aranda, F. Nuez, B. Pico, T. Gabaldon, G. Roma, R. Guigo, J. M. Casacuberta, P. Arus, and P. Puigdomenech. 2012. The genome of melon (Cucumis melo L.). Proc. Natl. Acad. Sci. USA 109:11872-11877.  

16. Gerlach, W. and J. Bedbrook. 1979. Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 7:1869-1885.  

17. Guerra, M. 2008. Chromosome numbers in plant cytotaxonomy: concepts and implications. Cytogenet. Genome Res. 120:339-350.  

18. Guo, S., J. Zhang, H. Sun, J. Salse, W. J. Lucas, H. Zhang, Y. Zheng, L. Mao, Y. Ren, Z. Wang, J. Min, X. Guo, F. Murat, B. K. Ham, Z. Zhang, S. Gao, M. Huang, Y. Xu, S. Zhong, A. Bombarely, L. A. Mueller, H. Zhao, H. He, Y. Zhang, Z. Zhang, S. Huang, T. Tan, E. Pang, K. Lin, Q. Hu, H. Kuang, P. Ni, B. Wang, J. Liu, Q. Kou, W. Hou, X. Zou, J. Jiang, G. Gong, K. Klee, H. Schoof, Y. Huang, X. Hu, S. Dong, D. Liang, J. Wang, K. Wu, Y. Xia, X. Zhao, Z. Zheng, M. Xing, X. Liang, B. Huang, T. Lv, J. Wang, Y. Yin, H. Yi, R. Li, M. Wu, A. Levi, X. Zhang, J. J. Giovannoni, J. Wang, Y. Li, Z. Fei, and Y. Xu. 2013. The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nat. Genet. 45:51-58.  

19. Huang, S., R. Li, Z. Zhang, L. Li, X. Gu, W. Fan, W. J. Lucas, X. Wang, B. Xie, P. Ni, Y. Ren, H. Zhu, J. Li, K. Lin, W. Jin, Z. Fei, G. Li, J. Staub, A. Kilian, E. A. van der Vossen, Y. Wu, J. Guo, J. He, Z. Jia, G. Tian, Y. Lu, J. Ruan, W. Qian, M. Wang, Q. Huang, B. Li, Z. Xuan, J. Cao, Asan, Z. Wu, J. Zhang, Q. Cai, Y. Bai, B. Zhao, Y. Han, Y. Li, X. Li, S. Wang, Q. Shi, S. Liu, W. K. Cho, J. Y. Kim, Y. Xu, K. Heller-Uszynska, H. Miao, Z. Cheng, S. Zhang, J. Wu, Y. Yang, H. Kang, M. Li, H. Liang, X. Ren, Z. Shi, M. Wen, M. Jian, H. Yang, G. Zhang, Z. Yang, R. Chen, L. Ma, H. Liu, Y. Zhou, J. Zhao, X. Fang, L. Fang, D. Liu, H. Zheng, Y. Zhang, N. Qin, Z. Li, G. Yang, S. Yang, L. Bolund, K. Kristiansen, S. Li, X. Zhang, J. Wang, R. Sun, B. Zhang, S. Jiang, and Y. Du. 2009. The genome of the cucumber, Cucumis sativus L. Nat. Genet. 41:1275-1281.  

20. Humphreys, M., H.-M. Thomas, J. Harper, G. Morgan, A. James, A. Ghamari-Zare, and H. Thomas. 1997. Dissecting drought-and cold-tolerance traits in the Lolium-Festuca complex by introgression mapping. New Phytol. 137:55-60.  

21. Icso, D., M. Molnar-Lang, and G. Linc. 2014. Constructing an alternative wheat karyotype using barley genomic DNA. J. Appl. Genet. 56:45-48.  

22. Jeffrey, C. S. 2005. A new system of Cucurbitaceae. Bot. Zhurn. 90:332-335.  

23. Jiang, J., B. Friebe, and B. Gill. 1993. Recent advances in alien gene transfer in wheat. Euphytica 73:199-212.  

24. Karmakar, K., R. K. Sinha, and S. Sinha. 2013. Karyological and electrophoretic distinction between sexes of Trichosanthes bracteata. Am. J. Plant Sci. 4:494.  

25. Kellogg, E.A. 2013. Grasses, Synteny, Evolution, and Molecular Systematics. In: S.M. HUGHES (ed.). Brenner’s Encyclopedia of Genetics (Second Edition). San Diego: Academic Press.  

26. Kocyan, A., L. B. Zhang, H. Schaefer, and S. S. Renner. 2007. A multi-locus chloroplast phylogeny for the Cucurbitaceae and its implications for character evolution and classification. Mol. Phylogenet. Evol. 44:553-577.  

27. Koo, D. H., Y. W. Nam, D. Choi, J. W. Bang, H. de Jong, and Y. Hur. 2010. Molecular cytogenetic mapping of Cucumis sativus and C. melo using highly repetitive DNA sequences. Chromosome Res. 18:325-336.  

28. Kovarik, A., S. Renny-Byfield, M.-A. Grandbastien, and A. Leitch 2012. Evolutionary implications of genome and karyotype restructuring in Nicotiana tabacum L., p. 209-224. In: P. S. Soltis and D. E. Soltis. Polyploidy and genome evolution., Springer, Berlin Heidelberg, Germany.  

29. Lashermes, P., S. Andrzejewski, B. Bertrand, M. C. Combes, S. Dussert, G. Graziosi, P. Trouslot, and F. Anthony. 2000. Molecular analysis of introgressive breeding in coffee (Coffea arabica L.). Theor. Appl. Genet. 100:139-146.  

30. Lee-Huang, S., P. L. Huang, H. F. Kung, B. Q. Li, P. L. Huang, P. Huang, H. I. Huang, and H. C. Chen. 1991. TAP 29: an anti-human immunodeficiency virus protein from Trichosanthes kirilowii that is nontoxic to intact cells. Proc. Natl. Acad. Sci. USA 88:6570-6574.  

31. Lee, J.-M. 1994. Cultivation of grafted vegetables I. Current status, grafting methods, and benefits. Hortscience. 29:235-239.  

32. Liao, P.-C., C.-C. Tsai, C.-H. Chou, and Y.-C. Chiang. 2012. Introgression between cultivars and wild populations of Momordica charantia L. (Cucurbitaceae) in Taiwan. Int. J. Mol. Sci. 13: 6469-6491.  

33. Macas, J., P. Neumann and A. Navratilova. 2007. Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive charac-terization using 454 sequencing and comparison to soybean and Medicago truncatula. BMC Genomics 8:427.  

34. Mandáková, T., M. E. Schranz, T. F. Sharbel, H. de Jong, and M. A. Lysak. 2015. Karyotype evolution in apomictic Boechera and the origin of the aberrant chromosomes. Plant J. 82:785-793.  

35. Mayer, K. F. X., M. Martis, P. E. Hedley, H. Šimková, H. Liu, J. A. Morris, B. Steuernagel, S. Taudien, S. Roessner, H. Gundlach, M. Kubaláková, P. Suchánková, F. Murat, M. Felder, T. Nussbaumer, A. Graner, J. Salse, T. Endo, H. Sakai, T. Tanaka, T. Itoh, K. Sato, M. Platzer, T. Matsumoto, U. Scholz, J. Doležel, R. Waugh, and N. Stein. 2011. Unlocking the barley genome by chromosomal and comparative genomics. Plant Cell 23:1249-1263.  

36. Mayer, R. A., P. A. Sergios, K. Coonan, and L. O’Brien. 1992. Trichosanthin treatment of HIV-induced immune dysregulation. Eur. J. Clin. Invest. 22:113-122.  

37. McGrath, C. L. and M. Lynch. 2012. Evolutionary significance of whole-genome duplication, p.1-20. In: P. S. Soltis and D. E. Soltis (eds.). Polyploidy and genome evolution. Springer, Berlin Heidelberg, Germany.  

38. McGrath, M. S., K. M. Hwang, S. E. Caldwell, I. Gaston, K. C. Luk, P. Wu, V. L. Ng, S. Crowe, J. Daniels, J. Marsh, and et al. 1989. GLQ223: an inhibitor of human immunodeficiency virus replication in acutely and chronically infected cells of lymphocyte and mononuclear phagocyte lineage. Proc. Natl. Acad. Sci. USA 86:2844-2848.  

39. Minh, C. V., N. X. Nhiem, H. T. Yen, P. V. Kiem, B. H. Tai, H. Le Tuan Anh, T. T. T. Hien, S. Park, N. Kim, and S. H. Kim. 2015. Chemical constituents of Trichosanthes kirilowii and their cytotoxic activities. Arch. Pharm. Res. 1-6.  

40. Mun, J. H., S. J. Kwon, T. J. Yang, Y. J. Seol, M. Jin, J. A. Kim, M. H. Lim, J. S. Kim, S. Baek, B. S. Choi, H. J. Yu, D. S. Kim, N. Kim, K. B. Lim, S. I. Lee, J. H. Hahn, Y. P. Lim, I. Bancroft, and B. S. Park. 2009. Genome-wide comparative analysis of the Brassica rapa gene space reveals genome shrinkage and differential loss of duplicated genes after whole genome triplication. Genome Biol. 10:R111.  

41. Ni, L., X. Zhu, C. Gong, Y. Luo, L. Wang, W. Zhou, S. Zhu, and Y. Li. 2015. Trichosanthes kirilowii fruits inhibit non-small cell lung cancer cell growth through mitotic cell-cycle arrest. Am. J. Chin. Med. 43:349-364.  

42. Otto, S. P. 2007. The evolutionary consequences of polyploidy. Cell 131:452-462.  

43. Qian, C.-L., X.-H. Qi, J.-H. Yang, and M.-F. Zhang. 2012. Molecular phylogeny of Chinese snakegourd (Trichosanthes kirilowii Maxim.) based on cytological and AFLP analyses. Caryologia 65:216-222.  

44. Ramsey, J. 2011. Polyploidy and ecological adaptation in wild yarrow. Proc. Natl. Acad. Sci. 108:7096-7101.  

45. Robinson, R. W. and D. S. Decker-Walters 1997. Cucurbits. Wallingford, UK, CABI Publishing.  

46. Sain, R. S., P. Joshi and E. V. Divakara Sastry. 2002. Cytogenetic analysis of interspecific hybrids in genus Citrullus (Cucurbitaceae). Euphytica 128:205-210.  

47. Seo, C.-S. S., T.-W. Kim, Y.-J. Kim, S.-R. Park, H. Ha, H.-K. Shin, and J.-Y. Jung. 2015. Trichosanthes kirilowii ameliorates cisplatin-induced nephrotoxicity in both in vitro and in vivo. Nat. Prod. res. 29:554-557.  

48. Shengping, Z., G. Xingfang, and W. Ye. 2006. Effect of bur cucumber (Sicyos angulatus L.) as rootstock on growth physiology and stress resistance of cucumber plants. Acta Hortic. Sin. 33:1231-1236.  

49. Singh, A. K. 1979. Cucurbitaceae and polyploidy. Cytologia 44: 897-905.  

50. Snowdon, R. J., W. Köhler, W. Friedt, and A. Köhler. 1997. Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids. Theor. Appl. Genet. 95:1320-1324.  

51. Soltis, D. E., R. J. A. Buggs, W. B. Barbazuk, S. Chamala, M. Chester, J. P. Gallagher, P. S. Schnable, and P. S. Soltis 2012. The early stages of polyploidy: Rapid and repeated evolution in Tragopogon, p.271-292. In: Polyploidy and genome evolution. P. S. Soltis and D. E. Soltis, Springer, Berlin, Heidelberg, Germany.  

52. Szinay, D., Y. Bai, R. Visser, and H. de Jong. 2010. FISH appli-ca-tions for genomics and plant breeding strategies in tomato and other solanaceous crops. Cytogenet. Genome Res. 129:199-210.  

53. Thies, J. A. and A. Levi. 2007. Characterization of watermelon (Citrullus lanatus var. citroides) germplasm for resistance to root-knot nematodes. Hortscience. 42:1530-1533.  

54. Tzonev, R. 2005. Sicyos angulatus (Cucurbitaceae): a new adventive species for the flora of Bulgaria. Phytologia Balcanica 11:67-68.  

55. Uchikoba, T., S. Hosoyamada, M. Onjyo, K. Arima, H. Yonezawa, and M. Kaneda. 2001. A serine endopeptidase from the fruits of Melothria japonica (Thunb.) Maxim. Phytochemistry 57:1-5.  

56. Vrana, J., H. Simkova, M. Kubalakova, J. Cihalikova and J. Dolezel. 2012. Flow cytometric chromosome sorting in plants: The next generation. Methods 57:331-337.  

57. Waminal, N., H. M. Park, K. B. Ryu, J. H. Kim, T. J. Yang, and H. H. Kim. 2012. Karyotype analysis of Panax ginseng C.A. Meyer, 1843 (Araliaceae) based on rDNA loci and DAPI band distribution. Comp. Cytogenet. 6:425-441.  

58. Waminal, N. E. and H. H. Kim. 2012. Dual-color FISH karyotype and rDNA distribution analyses on four Cucurbitaceae species. Hortic. Environ. Biotechnol. 53:49-56.  

59. Waminal, N. E., N. S. Kim, and H. H. Kim. 2011. Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species. Genes Genom. 33:521-528.  

60. Waminal, N. E., K. B. Ryu, B. R. Park, and H. H. Kim. 2014. Phylogeny of Cucurbitaceae species in Korea based on 5S rDNA non-transcribed spacer. Genes Genom. 36:57-64.  

61. Wang, Q., H. Liu, A. Gao, X. Yang, W. Liu, X. Li, and L. Li. 2012. Intergenomic rearrangements after polyploidization of Kengyilia thoroldiana (Poaceae: Triticeae) affected by environmental factors. PloS one 7.  

62. Weeden, N. F. and R. W. Robinson. 1986. Allozyme segregation ratios in the interspecific cross Cucurbita maxima x C. ecuadorensis suggest that hybrid breakdown is not caused by minor alterations in chromosome structure. Genetics 114:593-609.  

63. Wendel, J. F. 2000. Genome evolution in polyploids. Plant Mol. Biol. 42:225-249.  

64. Weng, Y. and Z. Sun 2012. Major cucurbit crops, p. 1-16. In: Y.-H. Wang, T. K. Behera, and C. Kole. Genetics, genomics and breeding of cucurbits. Science Publishers, New Hampshire, USA.  

65. Xiong, Z. Y. and J. C. Pires. 2011. Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors. Genet. 187:37-49.  

66. Zamir, D. 2001. Improving plant breeding with exotic genetic libraries. Nat. Rev. Genet. 2:983-989.  

67. Zhuang, F. Y., J. F. Chen, J. E. Staub, and C. T. Qian. 2006. Taxonomic relationships of a rare Cucumis species (C. hystrix Chakr.) and its interspecific hybrid with cucumber. Hortscience 41:571-574.