Identification of citrus hybrids through the combination of leaf apex morphology and SSR markers

Citrus breeding programs normally face several biological obstacles such as apomixis and polyembryony that result in a cumbersome identification of hybrid seedlings. The main purpose of this work is to describe the combined use of visual selection based on the leaf apex morphology and SSR analysis to differentiate hybrid from nucellar seedlings derived from the cross between the 'Murcott' tangor [Citrus reticulata Blanco x Citrus sinensis (L.) Osb.] and 'Pêra' sweet orange [Citrus sinensis (L.) Osb.]. A new morphological variable named leaf apex morphometric index is also described as the quantitative basis of the visual selection. The efficiency of visual selection of hybrids was tested under two growth conditions, seedlings germinated in seedbeds and in plastic tubes. Putative hybrid seedlings were also confirmed through the analysis of simple sequence repeats (SSR). The visual selection of hybrid seedlings resulted in an increase of 87.2% (p < 0.01) and 202.2% (p < 0.001) in the number of correctly identified hybrids when compared to the method of random picking of seedlings in seedbeds and plastic tubes, respectively. The results indicate that the combination of visual selection and SSR analysis for the identification of hybrids derived from the cross of polyembryonic citrus cultivars will improve the accuracy of the selection, save time, and reduce the costs involved in the use of molecular markers alone in citrus breeding programs.