Characterization of zygotic and nucellar seedlings from sour orange-like citrus rootstock candidates using RAPD and EST-SSR markers

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 relationship 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.

Citrus genomics

“…Dominant markers such as random amplified polymorphic DNA (RAPDs; Luro et al 1992;Higashi et al 2000), inter-simple sequence repeat Fang et al , 1998, and Amplified Fragment Length Polymorphisms (AFLPs; Liang et al 2006) have been useful for large-scale characterization of genomes for which previous genomic sequence information was not available. These marker types are still used in citrus for nucellar/zygotic differentiation (Rao et al 2007), genetic mapping (de Simone et al 1998;Sankar and Moore 2001;de Oliveira et al 2007;Gulsen et al 2010), germplasm studies (Krueger and Roose 2003;Pang et al 2007;Kumar et al 2010;Biswas et al 2010b;Yang et al 2010) and somatic hybrid genome characterization (Scarano et al 2002;Guo et al 2002;Fu et al 2004). More powerful single locus codominant markers such as restriction fragment length polymorphism (Federici et al 1998), sequence characterized amplified regions (SCARs; Nicolosi et al 2000) and cleaved amplified polymorphic sequences (CAPs) from ESTs have also been developed and utilized in citrus.…”

“…SSR markers have been included in citrus genetic maps (Chen et al 2008a;Lyon et al 2007;Luro et al 2007;Ollitrault et al 2008b;. In addition to genetic mapping, SSRs were used for: the analysis of genetic diversity Corazza-Nunes et al 2002;Barkley et al 2006), discriminating zygotic and nucellar seedlings (Ruiz et al 2000;Oliveira et al 2002;Rao et al 2007), control of the origin of plants obtained by induced gynogenesis (Froelicher et al 2007;Aleza et al 2009), molecular characterization of triploid cultivars (Aleza et al 2010) or somatic hybrids Bassene et al 2009a) and the analysis of the origin of 2n gametes (Luro et al 2004;Chen et al 2008c;Cuenca et al 2009;Ferrante et al 2010).…”

Caracterização molecular de uma progênie de tangerineira 'Clementina Fina' e 'Montenegrina'

“…Estudos dessa natureza têm sido frequentes em populações obtidas por cruzamentos dirigidos em citros (RAO et al, 2008); entretanto, esses trabalhos têm sido baseados no uso de marcadores dominantes, como RAPD (randon amplified polymorphic DNA) e AFLP (amplified fragment lenght polymorphism). A análise de similaridade em plantas perenes, assim como os citros, pode auxiliar programas de melhoramento genético, pois permite identificar precocemente genótipos mais ou menos semelhantes aos parentais e a formação de combinações gênicas diferentes das encontradas nos genitores.…”

Identificación de híbridos de limón mexicano mediante marcadores moleculares SSR

“…Otros autores, entre ellos Nageswara et al (2008) identificaron 77.8% plántulas nucelares y 22.2% plántulas cigóticas, con marcadores EST-SSR, en híbridos de introgresión de mandarina (C. reticulata Blanco) y pomelo (C. maxima Merr.). Otros autores como Mei-lian et al (2007) reportan en cruzas de tangerina roja (C. reticulata Blanco) X naranja trifoliada (P. trifoliata L.) 111 individuos híbridos a través de rescate de embriones y verificados por marcadores SSR y 44 plantas híbridas a partir de cruzas de mandarina Satsuma (C. unshiu Marc) X naranja trifoliada (P. trifoliata L.).…”