A reliable and cost-effective method for the early identification of citrus rootstock hybrids using high resolution melting analysis.

Citrus rootstock breeding is hampered by the difficulty in discriminating between zygotic and nucellar seedlings if morphological traits are absent or not fully reliable. Here we describe an affordable and reliable method for an early identification of hybrids in progenies derived from polyembryonic female parents, based on a DNA isolation from pooled leaf samples (2 to 4 plants per sample) coupled to High Resolution Melting (HRM) analysis at two SNP loci. This strategy is particularly useful when crosses involve highly apomictic female parents, and when trifoliate hybrids (such as citranges or citrumelos) or unifoliate species are used as parents. The method was validated on 1,088 progenies derived from the crosses alemow (Citrus macrophylla Wester) × Swingle citrumelo [C. paradisi Macf. × Poncirus trifoliata (L.) Raf.] (A×S), and Cleopatra mandarin (C. reshni Hort. ex Tanaka) × Swingle citrumelo (C×S). Leaf pooling reduced the number of DNA isolations for population genotyping, while HRM avoided post-PCR handling of samples and identified unequivocally the bulks containing the unifoliate hybrids. A consistent difference was found between A×S and C×S populations in the frequency of uni- and multifoliate hybrids (69% unifoliate in A×S vs. 10% in C×S), suggesting that the combination between parents influenced the early multifoliate leaf expression.