Multilocus haplotyping by parallel sequencing to decipher the interspecific mosaic genome structure

Recent studies support the theory that four basic taxa (Citrus medica, Citrus maxima, Citrus reticulata and Citrusmicrantha) have generated all cultivated Citrus species. It is supposed that the genomes of most of the actual citrus cultivars are interspecific mosaics of large DNA fragments issued from a limited number of interspecificmeiotic events. In the present work, we analyzed how haplotypic multilocus study of closely linked SNPsallows phylogenetic assignment of DNA fragments for the main cultivated species. We have developed a newmethod based on universal primers to prepare the amplicons to be analyzed by 454 technology (Roche). Itwas applied for direct multilocus haplotyping of 12 gene fragments of 48 Citrus genotypes. Moreover, Sangersequencing was performed on a subset of these amplicons (seven gene fragments of 24 citrus genotypes) tovalidate the 454 results. Consensus haplotype sequences were successfully identified from 454 sequencing. Sanger and 454 results were mostly identical. C. reticulata was the most polymorphic basic taxa. The averagedifferentiation between the basic taxa was about 20 SNPs/kb. These polymorphisms were enough forunambiguous multilocus differentiation of the basic species and assignment of phylogenetic origin for eachhaplotype of the secondary species. Multilocus haplotyping by parallel sequencing will be a powerful tool todecipher the interspecific mosaic genome structure of cultivated citrus.