Article | . 2018 Vol. 36, Issue. 1
Triple-color FISH Karyotype Analysis of Four Korean Wild Cucurbitaceae Species

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

2018.. 98:107


Cytogenetic mapping of DNA markers provides insights into a species’ basic genomic structure and facilitates the deduction of phylogenetic relationships between related species. The family Cucurbitaceae has numerous economically and medicinally important crop species. In addition, wild Cucurbitaceae species can provide important genetic resources for crop improvement. However, cytogenetic information for many species is still insufficient. Here, triple-color fluorescence in situ hybridization (FISH) was performed on four Korean wild Cucurbitaceae species of Actinostemma tenerum Grift, Thladiantha dubia Bunge, Sechium edule (Jacq.) Swartz, and Gynostemma pentaphyllum (Thunb.) Makino using 5S rDNA, 45S rDNA, and telomeric-repeat probes. The chromosome numbers of the four species were 2n = 2x = 16, 18, 28, and 2n = 6x = 66, respectively. The four species have relatively small chromosomes, ranging from 2.16 to 5.38 µm. One 5S and three 45S rDNA signals were observed in T. dubia, with one colocalization on a satellite chromosome (1:3:1), while signal patterns were 1:1:0, 1:3:0, and 4:5:0 for A. tenerum, S. edule, and G. pentaphyllum, respectively. Compared with that of 5S rDNA, 45S rDNA localization was more distal, except on chromosome 8 of A. tenerum and chromosomes 2 and 3 of S. edule. The species exhibited telomeric signals on the chromosomal terminal region, while additional signals hybridized on the pericentromeric region in A. tenerum chromosomes 1, 2, 4, and 5. These results will contribute to elucidating phylogenetic relationships among Cucurbitaceae species and improve on-going Cucurbitaceae breeding programs.

1. Abd El-Twab MH, Kondo K (2006) FISH physical mapping of 5S, 45S and Arabidopsis-type telomere sequence repeats in Chrysanthemum zawadskii showing intra-chromosomal variation and complexity in nature. Chromosom Bot 1:1-5. doi:10.3199/iscb.1.1  

2. Agarwal P, Roy R (1976) Natural polyploids in Cucurbitaceae I. Cytogenetical studies in triploid Momordica dioica Roxb. Caryologia 29:7-13. doi:10.1080/00087114.1976.10796644  

3. Albert PS, Gao Z, Danilova TV, Birchler JA (2010) Diversity of chromosomal karyotypes in maize and its relatives. Cytogenet Genome Res 129:6-16. doi:10.1159/000314342  

4. Anamthawat-Jõnsson K (2001) Molecular cytogenetics of introgressive hybridization in plants. Methods Cell Sci 23:141-150. doi:10.1007/978-94-010-0330-8_14  

5. Balao F, Herrera J, Talavera S (2011) Phenotypic consequences of polyploidy and genome size at the microevolutionary scale: a multivariate morphological approach. New Phytol 192:256-265. doi:10.1111/j.1469-8137.2011. 03787.x  

6. Baloglu MC, Ulu F, Altunog YC, Pekol S, Alagoz G, Ese O (2015) Identification, molecular characterization and expression analysis of RPL24 genes in three Cucurbitaceae family members: cucumber, melon and watermelon. Biotechnol Biotechnol Equip 29:1024-1034. doi:10.1080/13102818.2015.1079144  

7. Beevy SS, Kuriachan P (1996) Chromosome numbers of south Indian Cucurbitaceae and a note on the cytological evolution in the family. J Cytol Genet 31:65-71  

8. Bertioli D, Moretzsohn MC, Madsen LH, Sandal N, Leahl-Bertioli SC, Guimaraes PM, Hougaard BK, Freslund J, Schauser L, et al (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. doi:10.1186/1471-2164-10-45  

9. Brar DS, Khush GS (1997) Alien introgression in rice. Plant Mol Biol 35:35-47. doi:10.1023/A:1005825519998  

10. Castilho A, Heslop-Harrison J (1995) Physical mapping of 5S and 18S-25S rDNA and repetitive DNA sequences in Aegilops umbellulata. Genome 38:91-96. doi:10.1139/g95-011  

11. Coluccia E, Pichiri G, Nieddu M, Coni P, Manconi S, Deiana A, Salvadori S, Mezzanotte R (2011) Identification of two new repetitive elements and chromosomal mapping of repetitive DNA sequences in the fish Gymnothorax unicolor (Anguilliformes: Muraenidae). Eur J Histochem 55:12. doi:10.4081/ejh.2011.e12  

12. Cota JH, Philbrick CT (1994) Chromosome number variation and polyploidy in the genus Echinocereus (Cactaceae). Am J Bot 81:1054-1062  

13. Datson PM, Murray BG (2006) Ribosomal DNA locus evolution in Nemesia: transposition rather than structural rearrangement as the key mechanism? Chromosome Res 14:845-57. doi:10.1007/s10577-006-1092-z  

14. De Wilde WJJO, Duyfjes BEE (2007) Gynostemma (Cucurbitaceae) in Thailand and Malaysia. Blumea-Biodivers Evol Biogeogr Plants 52:263-280  

15. Delannay IY, Staub JE, Chen JF (2010) Backcross introgression of the Cucumis hystrix genome increases genetic diversity in US processing cucumber. J Am Soc Hortic Sci 135:351-361  

16. Devi J, Ko J, Seo, B (2005) FISH and GISH: Modern cytogenetic techniques. Indian J Biotechnol 4:307-315  

17. Formisano G, Paris HS, Frusciante I, Ercolano MR (2010) Commercial Cucurbita pepo squash hybrids carrying disease resistance introgressed from Cucurbita moschata have high genetic similarity. Plant Genet Resour 8:198-203. doi:10.1017/S1479262110000183  

18. Fuchs J, Brandes A, Schubert I (1995) Telomere sequence localization and karyotype evolution in higher plants. Plant Syst Evol 196:227-241. doi:10.1007/BF00982962  

19. Gao XF, Chen SK, Gu Z, Zhao JZ (1995) A chromosomal study on the genus Gynostemma (Cucurbitaceae). Acta Bot Yunnan 17:312-316  

20. Garcia-mas J, Benjak A, Sanseverino W, Bourgeois M, Mir G, Gonzalez VM, Henaff E, Camara F, Cozzuto L, et al (2012) The genome of melon (Cucumis melo L.). Proc Natl Acad Sci USA 109:11872-11877. doi:10.1073/pnas.1205415109  

21. Guo S, Zhang J, Sun H, Salse J, Lucas WJ, Zhang H, Zheng Y, Mao L, Ren Y, et al (2013) The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nat Genet 45:51-58. doi:10.1038/ng.2470  

22. Huang S, Li R, Zhang Z, Li L, Gu X, Fan W, Lucas WJ, Xie B, Ni P, et al (2009) The genome of the cucumber, Cucumis sativus L. Nat Genet 41:1275- 1281. doi:10.1038/ng.475  

23. Heslop-Harisson J (1991) The molecular cytogenetics of plants. J Cell Sci 100:5-21  

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

25. Jeffrey C (1967) On the classification of Cucurbitaceae. Kew Bull 20:417-426. doi:10.2307/4108235  

26. Jeffrey C (1980) A review of the Cucurbitaceae. Bot J Linean Soc 81:233-247. doi:10.1111/j.1095-8339. 1980.tb01676.x  

27. Jiang J, Friebe B, Gill B (1993) Recent advances in alien gene transfer in wheat. Euphytica 73:199-212. doi:10.1007/BF00036700  

28. Jiang LY, Qian ZQ, Guo ZG, Wang C, Zhao GF (2009) Polyploid origins in Gynostemma pentaphyllum (Cucurbitaceae) inferred from multiple gene sequences. Mol Phylogenet Evol 52:183-191. doi:10.1016/j.ympev.2009.03.004  

29. Jobst J, King K, Hemleben V (1998) Molecular evolution of the internal transcribed spacers (ITS1 and ITS2) and phylogenetic relationships among species of the family Cucurbitaceae. Mol Phylogenet Evol 9:204-19. doi:10.1006/mpev.1997.0465  

30. Kato A, Vega JM, Han F, Lamb JC, Birchler, JA (2005) Advances in plant chromosome identification and cytogenetic techniques. Curr Opin Plant Biol 8:148-54. doi:10.1016/j.pbi.2005.01.014  

31. Kato A, Albert PS, Vega JM, Birchler JA (2006) Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation. Biotech Histochem 81:71-78. doi:10.1080/105202290-600643677  

32. Kim DK (2010) Antioxidative constituents from the whole plant of Actinostemma lobatum Maxim. J Korean Soc Appl Biol Chem 53:746-751. doi:10.3839/jksabc.2010.113  

33. Kim DK, Seo SG, Kwon SB, Park YD (2016) Development of RAPD and SCAR markers related to watermelon mosaic virus and zucchini yellow mosaic virus resistance in Cucurbita moschata. Hortic Environ Biotechnol 57:61-68. doi:10.1007/s13580-016-0090-0  

34. Koo DH, Hur Y, Jin DC, Bang JW (2002) Karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) using C-banding and bicolor fluorescence in situ hybridization. Mol Cells 13:413-8  

35. Koo DH, Nam YW, Choi D, Bang JW, De Jong H, Hur Y (2010) Molecular cytogenetic mapping of Cucumis sativus and C. melo using highly repetitive DNA sequences. Chromosome Res 18:325-36. doi:10.1007/s10577-010-9116-0  

36. Khrustaleva LI, 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. doi:10.1046/j.1365-313x.2001. 00995.x  

37. Książczyk T, Taciak M, Zwierzykowski Z (2010) Variability of ribosomal DNA sites in Festuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH. J Appl Genet 51:449-460. doi:10.1007/BF03208874  

38. Lashermes P, Andrzejewski S, Bertrand B, Combes MC, Dussert S, Graziosi G, Trouslo P, Anthony F (2000) Molecular analysis of introgressive breeding in coffee (Coffea arabica L.). Theor Appl Genet 100:139-146. doi:10.1007/s001220050019  

39. Lee Tb (2003) Coloured flora of Korea. Vol. I, II. Hyangmunsa, Seoul, Korea, pp774-780  

40. Leitch I, Heslop-Harrison J (1992) Physical mapping of the 18S-5.8 S-26S rRNA genes in barley by in situ hybridization. Genome 35:1013-1018. doi:10.1139/g92-155  

41. Li WL, Berlyn GP, Ashton PMS (1996) Polyploids and their structural and physiological characteristics relative to water deficit in Betula papyrifera (Betulaceae). Am J Bot 83:15-20  

42. Li ZH, Liu ZL, Zhao P, Su HL, Zhao GF (2012) A review on studies of systematic evolution of Gynostemma Bl.[J]. Acta Bot Bore-Occi Sin 10:32  

43. Liao H, Zhao Y, Zhou Y, Wang Y, Wang X, Lu F, Song Z (2011) Microsatellite markers in the traditional Chinese medicinal herb Gynostemma pentaphyllum (Cucurbitaceae). Am J Bot 98:e61-e63. doi:10.3732/ajb.1000456  

44. Liao PC, Tsai CC, Chou CH, Chiang YC (2012) Introgression between cultivars and wild populations of Momordica charantia L. (Cucurbitaceae) in Taiwan. Int J Mol Sci 13:6469-6491. doi:10.3390/ijms13056469  

45. Liu B, Davis T (2011) Conservation and loss of ribosomal RNA gene sites in diploid and polyploid Fragaria (Rosaceae). BMC Plant Biol 11:157. doi:10.1186/1471-2229-11-157  

46. Macas J, Neumann P, Navratilova A (2007) Repetitive DNA in the pea (Pea sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula. BMC Genomics 8:427. doi:10.1186/1471-2164-8-427  

47. Marquioni V, Bertollo LAC, Diniz D, De Bello Cioffi M (2013) Comparative chromosomal mapping in Triportheus fish species. Analysis of synteny between ribosomal genes. Micron 45:129-135. doi:10.1016/j.micron.2012.11.008  

48. Martins C, Galetti Jr PM (1999) Chromosomal localization of 5S rDNA genes in Leporinus fish (Anostomidae, Characiformes). Chromosome Res 7:363-367. doi:10.1023/A:1009216030316  

49. Martins C, Galetti Jr PM (2001) Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers. Genome 44:903-910. doi:10.1139/g01-069  

50. Matoba H, Mizutani T, Nagano K, Hoshi Y, Uchiyama H (2007) Chromosomal study of lettuce and its allied species (Lactuca spp., Asteraceae) by means of karyotype analysis and fluorescence in situ hybridization. Hereditas 144:235-243. doi:10.1111/j.2007.0018-0661.02012x  

51. Meyers LA, Levin DA (2006) On the abundance of polyploids in flowering plants. Evolution 60:1198-1206. doi:10.1554/05-629.1  

52. Miao J, Frazier T, Huang L, Zhang XQ, Zhao B (2016) Identification and characterization of switchgrass histone H3 and CENH3 genes. Front Plant Sci 7:979. doi:10.3389/fpls.2016.00979  

53. Mishima M, Ohmido N, Fukui K, Yahara T (2002) Trends in site-number change of rDNA loci during polyploid evolution in Sanguisorba (Rosaceae). Chromosoma 110:550-558. doi:10.1007/s00412-001-0175-z  

54. Ng TJ (1993) New opportunities in the Cucurbitaceae. In J Janick, JE Simon, eds, New Crops. Wiley, New York, USA, pp 538-546  

55. Niu Y, Yan W, Lv J, Yao W, Yu L (2013) Characterization of a novel polysaccharide from tetraploid Gynostemma pentaphyllum Makino. J Agric Food Chem 61:4882-4889. doi:10.1021/jf400236x  

56. Ntuli, NR (2007) Genetic improvement of selected indigenous Cucurbitaceae species important for food and medicinal purposes in KwaZulu-Natal, South Africa. MS Thesis, University of Zululand, South Africa, pp 1-25  

57. Ordonez A, Gomez J, Vattuone M (2006) Antioxidant activities of Sechium edule (Jacq.) Swartz extracts. Food Chem 97:452-458. doi:10.1016/j.foodchem.2005.05.024  

58. Otto SP (2007) The evolutionary consequences of polyploidy. Cell 131:452-462. doi:10.1016/j.cell.2007.10.022  

59. Parisod C, Holderegger R, Brochmann C (2010) Evolutionary consequences of autopolyploidy. New Phytol 186:5-17. doi:10.1111/j.1469-8137.2009.03142.x  

60. Ramsey J (2011) Polyploidy and ecological adaptation in wild yarrow. Proc Natl Acad Sci 108:7096-7101. doi:10.1073/pnas.1016631108  

61. Raskin O, Barber J, Nevo E, Belyayev A (2008) Repetitive DNA and chromosomal rearrangements: speciation-related events in plant genomes. Cytogenet Genome Res 120:351-357. doi:10.1159/000121084  

62. Razmovski-Naumovski V, Huang THW, Tran VH, Li GQ, Duke CC, Roufogalis BD (2005) Chemistry and pharmacology of Gynostemma pentaphyllum. Phytochem Rev 4:197-219. doi:10.1007/s11101-005-3754-4  

63. Renner SS, Schaefer H, Kocyan A (2007) Phylogenetics of Cucumis (Cucurbitaceae): cucumber (C. sativus) belongs in an Asian/Australian clade far from melon (C. melo). BMC Evol Biol 7:1. doi:10.1186/1471-2148-7-58  

64. Rice A, Glick L, Abadi S, Einhorn M, Kopelman N, Salman-Minkov A, Mayzel J, Chay O, Mayrose I (2015) The Chromosome Counts Database (CCDB) - a community resource of plant chromosome numbers. New Phytol 206:19-26. doi:10.1111/nph.13191  

65. Sain RS, Joshi P, Divakara Sastry EV (2002) Cytogenetic analysis of interspecific hybrids in genus Citrullus (Cucurbitaceae). Euphytica 128:205-210. doi:10.1023/A:1020800113252  

66. Sax K, Sax HJ (1937) Stomata size and distribution in diploid and polyploid plants. J Arnold Arbor 18:164-172  

67. Schaefer H, Renner S (2010) Cucurbitaceae. In K Kubitzki, eds, Flowering Plants: Eudicots. The Families and Genera of Vascular Plants, Vol 10. Springer, Berlin, Heidelberg, Germany, pp 112-174. doi:10.1007/978-3-642-14397-7_10  

68. Schulze E, Beck E, Müller-Hohenstein K (2005) Environment as stress factor: stress physiology of plants. Plant Ecology. Springer-Verlag, Berlin, Germany, p 702  

69. Singh AK (1979) Cucurbitaceae and polyploidy. Cytologia 44:897-905. doi:10.1508/cytologia.44.897  

70. Snowdon RJ, Køhler W, Friedt W, Køhler A (1997) Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids. Theor Appl Genet 95:1320-1324. doi:10.1007/s001220050699  

71. Sousa A, Fuchs J, Renner SS (2012) Molecular cytogenetics (FISH, GISH) of Coccinia grandis: ca. 3 myr-old species of Cucurbitaceae with the largest Y/autosome divergence in flowering plants. Cytogenet Genome Res 139:107-118. doi:10.1159/000345370  

72. Sybenga J (2012) Cytogenetics in plant breeding. Springer Sci. Bus. Media. Dreijenlaan, Vol 7, HA Wageningen, The Netherlands, pp 1-10. doi:10.1007/978-3-642-84083-8  

73. Tong JM, Li LF, Song LP (2006) Study on the analgesic effect of the fruits of Thladiantha dubia Bunge [J]. J Chengde Méd Coll 1:001  

74. Tong J, Zhao B, Zhang, Y (2010) Anti-inflammatory effects and mechanism of fruit of Thladiantha dubia Bunge on adjuvant arthritis rats [J]. J Chengde Méd Coll 4:004  

75. (2017) Missouri Botanical Garden.  

76. Vrana, J, Simkova H, Kubalakova M, Cihalikova J, Dolezel J (2012) Flow cytometric chromosome sorting in plants: the next generation. Methods 57:331-337. doi:10.1016/j.ymeth.2012.03.006  

77. Waminal NE, Kim HH (2012) Dual-color FISH karyotype and rDNA distribution analyses on four Cucurbitaceae species. Hortic Environ Biotechnol 53:49-56. doi:10.1007/s13580-012-0105-4  

78. Waminal NE, Kim NS, Kim HH (2011) Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species. Genes Genom 33:521-528. doi:10.1007/s13258-011-0046-9  

79. Weng Y, Sun Z (2012) Major cucurbit crops. In Y-H Wang, TK Behera, C Kole, eds, Genetics, Genomics and Breeding of Cucurbits. Science Publishers, NH, USA, pp 1-16  

80. Whitaker TW (1933) Cytological and phylogenetic studies in the Cucurbitaceae. Bot Gaz 780-790. doi:10.1086/334347  

81. Xu Y, Yang F, Chen Y, Ma L, Wang J, Li L (2007) Comparative analysis of rDNA distribution in metaphase chromosomes of Cucurbitaceae species. Yi Chuan 29:614-620. doi:10.1360/yc-007-0614  

82. Zamir D (2001) Improving plant breeding with exotic genetic libraries. Nat Rev Genet 2:983-989. doi:10.1038/35103590  

83. Zhang YL, Zhao B, Chen JS, Liu YP, Tong JM (2010) Analgesic effect and effective fractions of the roots of Thladiantha dubia Bunge [J]. Lishizhen Med Mater Méd Res 10:029  

84. Zhou XH, Qian CT, Lou QF, Chen JF (2009) Molecular analysis of introgression lines from Cucumis hystrix Chakr. to C. sativus L. Sci Hortic 119:232-235. doi:10.1016/j.scienta.2008.08.011  

85. Zhuang FY, Chen JF, Staub JE, Qian CT (2006) Taxonomic relationships of a rare Cucumis species (C. hystrix Chakr.) and its interspecific hybrid with cucumber. HortScience 41:571-574