Citrus Research Institute, National Institute of Horticultural & Herbal Science RDA1
Planning and Coordination Division, National Institute of Horticultural & Herbal Science RDA2
Fruit Research Division, National Institute of Horticultural & Herbal Science RDA3
This study was carried out to develop molecular techniques to allow the selection of zygotic seedlings in the early stage of the plant development. We identified 37 pollen-specific molecular markers from RAPD analysis and successfully used them for identification of the zygotic seedlings from various hybrid crosses. Three Satsuma mandarin cultivars (‘Morita unshiu’, ‘Nangan 20’ and ‘Miyagawawase’) were used as mother parents and seven cultivars (‘Ponkan’, ‘Lee’, ‘Kinokuni’, ‘Shiranuhi’, ‘Tamnaneunbong’, ‘Shinyegam’, and ‘Sunburst’ mandarins) served as pollen parents. PCR analysis showed that 2 primers could identify zygotic hybrid seedlings. Among them, an UBC-27 primer was used to identify the zygotic seedlings from hybrid crosses of “‘Nangan 20’ × ‘Kinokuni’” mandarin, “‘Nangan 20 × Ponkan’” mandarin and “‘Miyagawawase × Sunburst’” tangerine. In total 29 out of 40 seedlings (73%), 9 out of 47 seedlings (19%), and 13 out of 45 (29%) were identified as zygotic seedlings, respectively. These results can show that the pollen-specific markers selected in this study can be used effectively for early identification of zygotic seedlings from Citrus hybrid crosses.
1. Ahmad, M., A. Javaid, H. ur-Rahman, S.I. Hussain, A. Ramzan, and A. Ghafoor. 2012. Identification of mandarin X orange hybrids using simple-sequence repeat markers. J. Agric. Res. 50:225-232.
2. AL-Anbari, A.K., N. Kanawapee, T.A. AL-Kazragi, A. Al-Mashhadani, S. Barusrux, P. Pornpongrungrueng, and P. Theerakulpisut. 2014. Genetic diversity of citrus (Rutaceae) in Iraq based on random amplified polymorphic DNA (RAPD) markers. Afr. J. Agric. Res. 9:1012-1019.
3. Andrade-Rodríguez, M., A. Villegas-Monter, G. Carrillo-Castañeda, and A. García-Velázquez. 2004. Polyembryony and identification of Volkamerian lemon zygotic and nucellar seedlings using RAPD. Pesq. Agropec. Bras. 39:551-559.
4. Ashari, S., D. Aspinall, and M. Sedgley. 1988. Discrimination of zygotic and nucellar seedlings of five polyembryonic citrus rootstocks by isozyme analysis and seedling morphology. J. Hortic. Sci. Biotechnol. 63:695-704.
5. Esen, A. and R.K. Soost. 1977. Adventive embryogenesis in citrus and its relation to pollination and fertilization. Am. J. Bot. 64:607-614.
6. Furr, J.R, P.C. Reece, and G. Hranciar. 1946. Identification of hybrid and nucellar citrus seedlings by a modification of the rootstock color test. Proc. Fla. State Hortic. Soc. 59:38-42.
7. Gmitter, F.G. Jr, C. Chen, M.A. Machado, A.A. Souza, P. Ollitrault, Y. Froethlicher, and T. Shimizu. 2012. Citrus genomics. Tree Genet. Genomes 8:611-626.
8. Golein, B., R. Fifaei, and M. Ghasemi. 2011. Identification of zygotic and nucellar seedlings in citrus interspecific crosses by inter simple sequence repeats (ISSR) markers. Afr. J. Biotechnol. 10:18965-18970.
9. Kepiro J.L. and M, L. Roose. 2007. Nuceller Embryony, p. 141-149. In: I.A. Khan (ed). Citrus Genetics Breeding and Biotechnology. CAB international, UK.
10. Khan I.A. and M, L. Roose. 1988. Frequency and characteristics of nucellar and zygotic seedlings in three cultivars of trifoliate orange. J. Am. Soc. Hortic. Sci. 113:105-110.
11. Lima, D.R., A.D. Vilarionnos, W. dos. Soares Filho, and C. de J. Barbosa. 2000. Identification of citrus zygotic seedlings by use of RAPD markers. Revista Brasileira de Fruiticultura 22:181-185.
12. Mondal, B. and R. Saha. 2014. Identification if zygotic and nucellar seedlings of Citrus reticulate and Citrus aurantifolia using RAPD. Int. J. Adv. Biotechnol. Res. 5:25-30.
13. Moon, D.K. and K.C. Ko. 1991. Isozymes as genetic markers in Citrus growing in Cheju and their use for identification of nucellar and zygotic seedlings. J. Kor. Soc. Hort. Sci. 32:59-65.
14. Moore, G.A. and W.S. Castle. 1988. Morphological and isozymic analysis of open-pollinated Citrus rootstock populations. J. Hered. 79:59-63.
15. Oliveira, A.C., A.N. Garcia, M. Cristofani, and M.A. Machado. 2002. Identification of citrus hybrids through the combination of leaf ape× morphology and SSR markers. Euphytica 128: 397-403.
16. Oliveira, R.P., C.I. Aguilar-Vildoso, and M.A. Machado. 2003. Selection processes in a citrus hybrid populationusing RAPD markers. Pesq. agropec. bras. Brasília. 38:1297-1302.
17. Pieringer, A.P. and G.J. Edwards. 1967. Identification of nucellar and zygotic citrus seedlings by infrared spectroscopy. J. Am. Soc. Hortic. Sci. 86:226-234.
18. Rao, M.N., J.R. Soneji, C. Chen, S. Huang, F.G. Gmitter. 2008. Characterization of zygotic and nucell-ar seedlings from sour orange-like citrus rootstock candidates using RAPD and EST-SSR markers. Tree Genet. Genomes 4:113-124.
19. Ruiz, C., M.P. Breto, and M.J. Asins. 2000. A quick methodology to identify sexual seedlings in citrus breeding programs using SSR markers. Euphytica 112:89-94.
20. Scarano, M.T., N. Tusa, L. Abbate, S. Lucretti, L. Nardi, and S. Ferrante. 2003. Flow cytometry, SSR and modified AFLP markers for the identification of zygotic plantlets in backcrosses between ‘Femminello’ lemon cybrids (2n and 4n) and a diploid clone of ‘Femminello’ lemon (Citrus limon L. Burm. F.) tolerant to malsecco disease. Plant Sci. 164:1009-1017.
21. Tatum, J., C.J. Hearn, and R.E. Berry. 1978. Characterization of citrus cultivars by chemical differentiation. J. Am. Soc. Hortic. Sci. 103:492-496.
22. Tusa, N., L. Abbate, S. Ferrante, S. Lucretti, and M.T. Scarano. 2002. Identification of zygotic and nucellar seedlings in citrus interploid crosses by means of isozymes, flow cytometry and ISSR-PCR. Cell Mol. Biol. Lett. 7:703-708.
23. Yoo, K.Y., K.H. Cho, I.S. Shin, J.H. Kim, S. Heo, J.H. Non, H.R. Kim. 2009. Analysis of Genetic Relationships of Grapevine Cultivars (Vitisssp.) in Korea Using RAPD Markers. Kor. J. Breed. Sci. 41:437-443.
24. Yun, S.H., Y.S. Moon, S.B. Jin, I.K. Kang, and D.H. Lee. 2011. Early Identification of Putative Zygo-tic Seedlings in Citrus Crosses between ‘Morita unshiu’(Citrus. unshiu Marc.) and ‘Ponkan’(C. reticulata Blanco) Using RAPD and SRAP. J. Life Sci. 21:502-508.
25. Weinaaum, S.A., E. Cohen, and P. Spiegel-Roy. 1982. Rapid screening of satsuma mandarin progeny to distinguish nucellar and zygotic seedlings. HortScience. 17:239-240.