Skip to main content
Log in

A quick methodology to identify sexual seedlings in citrus breeding programs using SSR markers

  • Published:
Euphytica Aims and scope Submit manuscript

Abstract

In citrus breeding and genetics, it is very important to distinguish between zygotic and nucellar seedlings in order to eliminate unwanted genotypes. Usually, isozyme marker shave been employed to determine the genetic origin of young plants. In this work we propose the use of SSR markers as an alternative methodology and compare them with isozymes in this kind of screenings. Two different populations were analysed: one derives from an interspecific cross and the other from selfing. We conclude that, in most cases, microsatellites are more efficient than isozymic markers to identify the sexual origin of citrus seedlings, given their higher level of polymorphism and the scarce number of polymorphic isozymes in some populations. We describe a quick and efficient methodology for SSR analysis, including a fast DNA extraction in microcentrifuge tubes, and visualization through silver staining, which eliminates the need for a labelling step.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Anderson, C.M., W.S. Castle & G.A. Moore, 1991. Isozymic identification of zygotic seedlings in Swingle citrumelo Citrus paradisi x Poncirus trifoliata nursery and field populations. J Amer Soc Hort Sci 116: 322-236.

    CAS  Google Scholar 

  • Ashari, S., D. Aspinall & M. Sedgley, 1988. Discrimination of zygotic and nucellar seedlings of five polyembryonic citrus rootstocks by isozyme analysis and seedling morphology. J Hort Sci 63: 695-703.

    Google Scholar 

  • Asíns, M.J., R. Herrero & L. Navarro, 1995. Factors affecting three isozyme-gene expression. Theor Appl Genet 90: 892-898.

    Article  Google Scholar 

  • Asíns, M.J., P.F. Mestre, R. Herrero, L. Navarro & E.A. Carbonell, 1998. Molecular markers: a continuously growing biotechnology area to help citrus improvement. Fruits 53: 293-302.

    Google Scholar 

  • Beidler, L.L., P.R. Hilliard & R.L. Rill, 1982. Ultrasensitive staining of nucleic acids with silver. Anal Biochem 126: 374-380.

    Article  PubMed  CAS  Google Scholar 

  • Cameron, J.W., 1979. Sexual and nucellar embriony in F1 hybrids and advanced crosses of Citrus and Poncirus. J Am Soc Hort Sci 104: 408-410.

    Google Scholar 

  • Cameron, J.W. & H.B. Frost, 1968. Genetics and nucellar embriony. In: W. Reuther, H.J. Webber & L.D. Batchelor (Eds.), The Citrus Industry. Vol II, pp. 327-371. University of California, Berkeley.

    Google Scholar 

  • Cameron, J.W. & R.K. Soost, 1969. Citrus. In: F.D. Ferweda & F. de Witt (Eds.), Outlines of Perennial Crop Breeding in the Tropics. pp. 129-162. Wageningen, H. Veenman & Zonen.

    Google Scholar 

  • Frost, H.B., 1943. Genetics and breeding. In: H.J. Webber & L.D. Batchelor (Eds.), The Citrus Industry. Vol. I, pp. 817-913. Univ California Press, Berkeley, Los Angeles.

    Google Scholar 

  • Herrero, R., M.J. Asíns, E.A. Carbonell & L. Navarro, 1996. Genetic diversity in the orange subfamily Aurantioidae. I. Intraspecies and intragenus genetic variability. Theor Appl Genet 92: 599-609.

    Article  CAS  Google Scholar 

  • Iglesias, L., H. Lima & J.P. Simon, 1974. Isozyme identification of zygotic and nucellar seedlings in Citrus. J Hered 65: 81-84.

    Google Scholar 

  • Karhu, A., P. Hulme, M. Karjalainen, P. Karvonen, K. Karkkainen, D. Neale & O. Savolainen, 1996. Do molecular markers reflect patterns of differentiation in adaptive traits of conifers? Theor Appl Genet 93: 215-221.

    Article  CAS  Google Scholar 

  • Khan, I.A. & M.L. Roose, 1988. Frequency and characteristics of nucellar and zygotic seedlings in three cultivars of trifoliate orange. J Am Soc Hort Sci 113: 105-110.

    Google Scholar 

  • Kijas, J.M.H., M.R. Thomas, J.C.S. Fowler & M.L. Roose, 1997. Integration of trinucleotide microsatellites into a linkage map of Citrus. Theor Appl Genet 94: 701-706.

    Article  CAS  Google Scholar 

  • Litt, M. & J.A. Luty, 1989. A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am J Hum Genet 44: 397-401.

    PubMed  CAS  Google Scholar 

  • Mestre, P.F., M.J. Asíns, J.A. Pina, E.A. Carbonell & L. Navarro, 1997. Molecular markers flanking citrus tristeza virus resistance gene from Poncirus trifoliata (L.) Raf. Theor Appl Genet 94: 458-464.

    Article  CAS  Google Scholar 

  • Moore, G.A. & W.S. Castle, 1988. Morphology and isozymic analysis of open-pollinated Citrus rootstock populations. J Hered 799: 59-63.

    Google Scholar 

  • Morgante, M. & A.M. Olivieri, 1993. PCR-amplified microsatellite as markers in plant genetics. The Plant J 3: 175-182.

    Article  CAS  Google Scholar 

  • Mullis, K., S. Fallona, S. Schraf, R. Saiki, G. Horn & H. Erlich, 1986. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant Biol 51: 263-273.

    PubMed  CAS  Google Scholar 

  • Orford, S.J., N.S. Scott & J.N. Timmis, 1995. A hypervariable middle repetitive DNA sequence from citrus. Theor Appl Genet 91: 1248-1252.

    Article  CAS  Google Scholar 

  • Raybould, A.F., R.J. Mogg, C. Aldam, C.J. Gilddon, R.S. Thorpe & R.T. Clarke, 1998. The genetic structure of sea beat (Beta vulgaris ssp. maritima) populations. III. Detection of isolation by distance at microsatellite loci. Heredity 80: 127-132.

    Article  Google Scholar 

  • Roose, M.L. & S.N. Traugh, 1988. Identification and performance of citrus trees on nucellar and zygotic rootstocks. J Am Soc Hort Sci 113: 100-105.

    Google Scholar 

  • Russell, J.R., J.D. Fuller, M. Macaulay, B.G. Hatz, A. Jahoor, W. Powell & R. Waugh, 1997. Direct comparison of levels of genetic variation among barley accessions detected by RFLPs, AFLPs, SSRs and RAPDs. Theor Appl Genet 95: 714-722.

    Article  CAS  Google Scholar 

  • Spiegel-Roy, P., A. Bardi & A. Shani, 1977. Peroxidase isozymes as a tool for early separation of nucellar and zygotic Citrus seedlings. Proc Int Soc Citriculture 2: 619-624.

    CAS  Google Scholar 

  • Tautz, D., 1989. Hypervariability of simples sequences as a general source for polymorphic DNA markers. Nucl Acid Res 17: 6463-6471.

    CAS  Google Scholar 

  • Torres, A.M., R.K. Soost & V. Diedenhofen, 1978. Leaf isozymes as genetic markers in citrus. Amer J Bot 65: 869-881.

    Article  Google Scholar 

  • Torres, A.M., R.K. Soost & T. Mau-Lastovicka, 1982. Citrus isozymes. Genetics and distinguishing nucellar from zygotic seedlings. J Hered 73: 335-339.

    CAS  Google Scholar 

  • Webber, H.J., 1932. Variation in citrus seedlings and their relation to rootstock selection. Hilgardia 7: 1-79.

    Google Scholar 

  • Weber, J. & P.E. May, 1989. Abundantt class of human DNA polymorphisms which can be typed using the Polymerase Chain Reaction. Am J Hum Genet 44: 388-396.

    PubMed  CAS  Google Scholar 

  • White, G. & W. Powell, 1997. Isolation and characterisation of microsatellite loci in Swietenia humilis (Meliaceae): an endangered tropical hardwood species. Molec Ecology 6: 851-860.

    Article  CAS  Google Scholar 

  • Xiang, C. & M.L. Roose, 1988. Frequency and characteristics of nucellar and zygotic seedlings in 12 citrus rootstocks. Sci Hort 37: 47-59.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ruiz, C., Paz Breto, M. & Asíns, M. A quick methodology to identify sexual seedlings in citrus breeding programs using SSR markers. Euphytica 112, 89–94 (2000). https://doi.org/10.1023/A:1003992719598

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1003992719598

Navigation