Article | 06. 2014 Vol. 32, Issue. 3
Chromosome Redundancy and Tree Phenotype Variation in Autotetraploid Trifoliate Orange

Facuity of Bioscience & Industry, Jeju National University1
Citrus Research Station, National Institute of Horticultural & Herbal Science2
Department of Tea Sciences, Zhejiang University3
Research Institute for Subtropical Agriculture & Biotechnology, Jeju National University4

2014.06. 366:374


The study was conducted to investigate the possibility that epigenetic DNA methylation causes tree phenotypic variation in autotetraploids through evaluating the phenotypic variation and DNA methylation in autotetraploids occurred spontaneously from diploid trifoliate orange. Chromosome analysis confirmed that fourteen trifoliate orange trees of selected by flow cytometry were tetraploids (2n = 4X = 36) without any aneuploids. Chromomycin A3 staining determined that these trees were all autotetraploid with doubled chromosome set. Tree phenotypes, such as tree height and width, branching number, length, and angle, internode length, and leaf characteristics, varied in the autotetraploids. Chlorophyll indices were diverse in the autotetraploids, but photosynthetic rates were not significantly different. In addition, a wide range of variation was observed in stomatal density and guard cell length. Analysis of global cytosine DNA methylation showed that there was a variation of the methylation level in autotetraploids. More than half of 14 autotetraploids had at least 2 times higher methylation level than diploid trifoliate orange. The results indicate that tree phenotypic variation in autotetraploids might be related to global DNA methylation for reducing gene redundancy.

1. Allario, T., J. Brumos, J.M. Colmenero-Flores, F. Tadeo, Y. Froelicher, M. Talon, L. Navarro, P. Ollitrault, and R. Morillon. 2011. Large changes in anatomy and physiology between diploid Rangpur lime (Citrus limonia) and its autotetraploid are not associated with large es in leaf gene expression. J. Exp. Bot. 62:2507-2519.  

2. Aleza, P., Y. Froelicher, S. Schwarz, M. Agusti, M. Hernandez, J. Juarez, F. Luro, R. Morillon, L. Navarro, and P. Ollitrault. 2011. Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment. Ann. Bot. 109:37-50.  

3. Aversano, R., I. Caruso, G. Aronne, V.D. Micco, N. Scognamiglio, and D. Carputo. 2013. Stochastic changes affect Solanum wild species following autopolyploidization. J. Exp. Bot. 64:625-635.  

4. Barros e Silva, A.E., A. Marques, K.G.B. dos Santos, and M. Guerra. 2010. The evolution of CMA bands in Citrus and related genera. Chromosome Res. 18:503-514.  

5. Befu, M., A. Kitajima, Y.X. Lin, and K. Hasegawa. 2000. Classification of ‘Tosa-Buntan’ pummelo (Citrus grandis [L.] Osb.), ‘Washington’ navel orange (C. sinensis [L.] Osb.), and trifoliate orange (Poncirus trifoliata [L.] Raf.) chromosomes using young leaves. J. Jpn. Soc. Hort. Sci. 69:22–28.  

6. Cameron, J.W. and H.B. Frost. 1968. Genetics, breeding, and nucellar embryony, p. 325-370. In: W. Reuther, L.D. Batcherlor, and H.J. Webber (eds.). The citrus industry. Vol. 2. Univ. of California Press, Berkeley, CA, USA.  

7. Demeulemeester, M.A.C., N. Van Stallen, and M.P. De Proft. 1999. Degree of DNA methylation in chicory (Cichorium intybus L.): Influence of plant age and vernalization. Plant Sci. 142:101-108.  

8. Dutt, M., M. Vasconcellos, K.J. Song, F.G. Gmitter, Jr., and J.W. Grosser. 2010. In vitro production of autotetraploid Ponkan mandarin (Citrus reticulate) using cell suspension culture. Euphytica 173:235-242.  

9. Ferrante, S.P., S. Lucretti, S. Reale, A. De Patrizio, L. Abbate, N. Tusa, and M.T. Scarano. 2009. Assessment of the origin of new citrus tetraploid hybrids (2n = 4x) by means of SSR markers and PCR based dosage effects. Euphytica 173:223-233.  

10. Food and Agricultural Organization (FAO). 2012. FAOSTAT.   

11. Grosser, J.W., E.S. Louzada, F.G. Gmitter, Jr., and J.L. Chandler. 1994. Somatic hybridization of complementary citrus rootstock: Five new hybrids. HortScience 29:812-813.  

12. Grosser, J.W., P. Ollitrault, and O. Olivares-fuster. 2000. Somatic hybridization in citrus: An effective tool to faciltate variety improvement. Biol. Plant. 36:434-449.  

13. Gunasekare, M.T.K. and M.A.B. Ranatunga. 2003. Polyploidy in tea (Camellia sinensis L.) and its application in tea breeding: A review. Sri Lanka J. Tea Sci. 68(2):14-26.  

14. Jia, F., Y.P. Fu, W.Q. Liu, Z. Du, and Y.D. Zhao. 2011. Quantitative determination of DNA Methylation in tobacco leaves by HPLC. Afr. J. Agri. Res. 6:1545-1548.  

15. Kaneyoshi, J., T. Furuta, M. Kurao, and S. Yamaguchi. 2008. Induced tetraploid by colchicine treatment in some monoembryonic citrus cultivars and triploidy production by using the tetraploid as seed parents. Hort. Res. 7:5-10.  

16. Lavania, U., S. Srivastava, S. Lavania, S. Basu, N.K. Misra, and Y. Mukai. 2012. Autopolyploidy differentially influences body size in plants, but facilitates enhanced accumulation of secondary metabolites, causing increased cytosine methylation. Plant J. 71:539-549.  

17. Medina-Urrutia, V., K.F.L. Madera, P. Serrano, G. Ananthakrishnan, J.W. Grosser, and W.W. Guo. 2004. New intergeneric somatic hybrids combining amblycarpa mandarin with six trifoloate/ trifoliate hybrid selections for lime rootstock improvement. HortScience 39:355-360.  

18. Miranda, M., F. Ikeda, T. Endo, T. Moriguchi, and M. Omura. 1997. Comparative analysis on the distribution of heterochromatin in Citrus, Poncirus, and Fortunella chromosomes. Chromosome Res. 5:86-92.  

19. Ministry for Food, Agriculture, Forestry, and Fisheries (MFAFF). 2011. Food, agriculture, forestry and fisheries statistical yearbook. p. 118-119.  

20. Oiyama, I. and S. Kobayashi. 1991. Citrus pentaploids from small seeds of diploid × diploid crosses. HortScience 26:292-293.  

21. Osborn, T.C., J.C. Pires, J.A. Birchler, D.L. Auger, Z.J. Chen, H.S. Lee, L. Comai, A. Madlung, R.W. Doerge, V. Colot, and R.A. Martienssen. 2003. Understanding mechanisms of novel gene expression in polyploids. Trends Genet. 19:141-147.  

22. Ranatunga, M.A.B. and M.T.K Gunasekare. 2003. A comparative assessment of some morphological and anatomical attributes to identify markers for screening polyploid genotypes of tea (Camellia sinensis L.). Sri Lanka J. Tea Sci. 68(1):12-19.  

23. Recupero, G.R., G. Russo, and S. Recupero. 2005. New promising citrus triploid hybrids selected from crosses between mono-embryonic diploid female and tetraploid male parents. HortScience 40:516-520.  

24. Romero-Aranda, R., B.R. Bondada, J.P. Syvertsen, and J.W. Grosser. 1997. Leaf characteristics and net exchange of diploid and autotetraploid citrus. Ann. Bot. 79:153-160.  

25. Sémon, M. and K.H. Wolfe. 2007. Consequences of genome duplication. 2007. Curr. Opin. Genet. Dev. 17:505-512.  

26. Song, K.J., S.B. Kim, J.H. Park, E.U. Oh, K.G. Lee, D.W. Kim, J.H. Kang, J.S. Kim, J.H. Oh, and F.G. Gmirrer. 2011. Frequency and growth characteristics of polyploids occurred spontaneously in some mandarin hybrid. Kor. J. Hort. Sci. Technol. 29:617-622.   

27. Usman, M., T. Saeed, M.M. Khan, and B. Fatima. 2006. Occurrence of spontaneous polyploids in Citrus. Hort. Sci. (Prague) 33:124-129.  

28. Wakana, A., N. Hanada, S.M. Park, I. Fukudome, and K. Kajiwara. 2005. Production of tetraploid forms of acid citrus cultivars by top grafting of shoots with sprouting axially buds treated with colchicines. J. Fac. Agr. Kyushu Univ. 50:93-102.  

29. Wang, C.G., H. Li, Z.Y. Xue, C.B. Chen, Y. Gu, D.L. Sun, and W.Q. Song. 2009. Marker-based analysis of genome structure and DNA methylation in a watermelon (Citrullus lanatus) ploidy series. Bot. Stud. 50:389-402.  

30. Yamamoto, M., A.A. Abkenar, R. Matsumoto, H. Nesumi, T. Yoshida, T. Kuniga, T. Kubo, and S. Tominaga. 2007. CMA banding patterns of chromosome in major Citrus species. J. Jpn. Soc. Hort. Sci. 76:36-40.  

31. Yamamoto, M. and S. Tominaga. 2004. CMA banding pattern of chromosomes is useful for the identification of chromosome doubling in haploid citrus. Breed. Sci. 54:351-354.  

32. Ye, Y.M., J. Tong, X.P. Shi, W. Yuan, and G.R. Li. 2010. Morphological and cytological studies of diploid and colchicine- induced tetraploid lines of crape myrtle (Lagerstroemia indica L.). Sci. Hortic. 124: 95-101.   

33. Yu, Z, G. Haberer, M. Matthes, T. Rattei, K.F.X. Mayer, A. Gierl, and R.A. Torres-Ruiz. 2010. Proc. Natl. Acad. Sci. USA 107:17809-17814.  

34. Zhu, S.P., J.-K. Song, Z.-Y. Hu, B. Tan, Z.-Z. Xie, H.-L. Yi, and X.-X. Deng. 2009. Ploidy variation and genetic composition of open-pollinated triploid citrus progenoies. Bot. Studies 50:319-324.