ORIGINAL PAPER Characterization of zygotic and nucellar seedlings from sour orange-like citrus rootstock candidates using RAPD and EST-SSR markers Madhugiri Nageswara Rao & Jaya R. Soneji & Chunxian Chen & Shu Huang & Fred G. Gmitter Jr. Received: 9 May 2006 / Revised: 24 March 2007 / Accepted: 7 May 2007 / Published online: 12 June 2007 # Springer-Verlag 2007 Abstract A series of crosses designed for introgression of mandarin (Citrus reticulata Blanco) and pummelo (C. maxima Merr.) germplasm, to develop an alternative rootstock to sour orange (C. aurantium L.), were carried out. It is necessary to identify those hybrids that yield nucellar seedlings for rootstock propagation. Rootstocks can be developed through traditional plant breeding methods; however, the ability to screen and select for economically important traits (such as production of true nucellar seedlings) in an efficient fashion is limited by the difficulties of screening techniques based on whole plant performance. To address these problems, we have used randomly amplified polymorphic DNA (RAPD) and fluorescently labeled expressed sequence tag simple sequence repeat (EST-SSR) molecular markers. A total of 204 individual seedlings obtained from 34 hybrid parental plants were successfully characterized using five RAPD primers. Ten hybrid parents and their progenies, found to be genetically similar among themselves, were selected for more scrutiny using eight EST-SSR primer pairs. The degrees of genetic similarity (nucellars) among progeny seedlings were determined and compared with that of their parents. The mean genetic similarity varied from 67–99% among the selected rootstock candidates screened. The genetic similarity relation- ship identified using RAPD and EST-SSR molecular markers was highly concordant (p =0.001). Two elite rootstock candidates (B6R5T56; B6R11T129) that seem to be ideal for future mandarin and pummelo derived rootstock breeding programs have been identified. Our results indicate that either RAPD or EST-SSR analyses could be equally successful in identifying true nucellars among the progenies obtained from introgression crosses of mandarin and pummelo, thus improving the accuracy of early selection in a citrus rootstock breeding program. Keywords Citrus . Genetic similarity . Molecular markers . Nucellar . Rootstock . Zygotic Introduction Citrus, one of the major fruit crops, is grown throughout the tropical and subtropical regions of the world. Most of the citrus cultivars are known to have resulted from natural hybridization and mutation (Hodgson 1967). Citrus is propagated by budding the desired scion onto rootstock seedlings. The rootstock is a major contributor to tree performance and longevity since it determines the tolerance to soil-borne pathogens and pests; it influences tree size, yield, fruit quality (Davies and Albrigo 1994), and also allows the scion to adapt to a variety of soil conditions. Sour orange (Citrus aurantium L.) has been used as a successful rootstock throughout the world; in fact, it is one of the few rootstocks that can tolerate salinity, high pH, or calcareous soils to some degree (Castle 1987; Grosser et al. 2004). However, trees of most commercial scions on sour orange rootstock are most vulnerable to the so-called “quick-decline” disease caused by certain strains of citrus tristeza virus (CTV; Bar-Joseph et al. 1981, 1989; Yokomi et al. 1994). CTV infections have destroyed entire citrus industries on sour orange in many countries, and most of Tree Genetics & Genomes (2008) 4:113–124 DOI 10.1007/s11295-007-0092-2 Communicated by A. Abbott M. Nageswara Rao : J. R. Soneji : C. Chen : S. Huang : F. G. Gmitter Jr. (*) University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA e-mail: fgg@crec.ifas.ufl.edu