Multilocus SNPs analysis allows phylogenetic assignation of DNA fragments to decipher the interspecific mosaic genome structure of cultivated citrus

Abstract

All current studies seem to support the theory that four basic taxa (C. medica, C. maxima, C. reticulata and C. micrantha) have generated all cultivated Citrus species. It is supposed that the genomes of most of the modern Citrus cultivars, vegetatively propagated, are interspecific mosaic of large DNA fragments issued from a limited number of inter-specific meiosis. In the present work we analyse how multilocus study of closely linked SNPs allows a phylogenetic assignation of DNA fragments of the main cultivated species. Genomic fragments of 25 genes dispersed in the different chromosomes covering more than 12,5 Kb were amplified by PCR and sequenced (Sanger) for 24 accessions representative of 10 species. Moreover we checked the potential of parallel pyrosequencing (454 Roche) for direct multilocus haplotyping of heterozygous genotypes. Amplified fragments from 7 genes in 8 genotypes were obtained by using an original new method based on universal primers. C. clementina (Clementine) was used as model for secondary species. Citrus reticulata was the most polymorph basic taxa with an average of 4.2 SNPs/kb. The average differentiation between the basic taxa was about 20 SNPs/kb. For each amplified gene fragment, this polymorphism was enough for unambiguous multilocus differentiation of the basic species and assignation of a phylogenetic origin for the secondary species. A preliminary reconstitution of phylogenetic structure of chromosome 3 is proposed for sweet orange, sour orange, grapefruit, lemon and lime. Consensus haplotype sequences were successfully obtained from 454 sequencing with genotype sequence in total agreement with Sanger control. Each haplotype sequence of Clementine was univocally assigned to one of the haplotype clusters of the basic taxa. Phylogenetic origin of specific DNA fragments can be assigned from multilocus analysis of closely linked SNPs. Multilocus haplotyping by parallel sequencing of individual DNA molecule will be a very powerful tool to decipher the interspecific mosaic genome structure of cultivated citrus. (Resume d'auteur)