Multilocus Haplotyping by Parallel Sequencing to Decipher the Interspecific Mosaic Genome Structure of Cultivated Citrus

Abstract

The most important economic Citrus species originated from natural interspecific hybridization between four ancestral taxa (C. reticulata, C. maxima, C. medica and C. micrantha) with limited further interspecific recombination due to apomixis and vegetative propagation. Such reticulate evolution coupled with vegetative propagation results in genomes that are mosaics of large chromosome fragments of the basic taxa, in frequent interspecific heterozygosity. Breeding of these species is hampered by their complex heterozygous genomic structures. Haplotyping of multiple gene fragments along the genome should be a powerful approach to resolve the evolutionary history of the gene pools, to reveal the admixture genomic structure of current species and to develop innovative breeding schemes. We have analysed the efficiency of parallel sequencing with 454 methodology to decipher the hybrid structure of modern citrus species and cultivars along chromosome 2. Four hundred fifty four amplicon libraries were established with the fluidigm array system for 48 genotypes and 16 gene fragments of chromosome 2. Haplotypes were established from the reads of each accession and phylogenetic analyses were performed from the haplotypic data of each gene fragment. The length of 454 reads and the level of differentiation between the ancestral taxa of modern citrus allowed efficient haplotype phylogenetic assignations for 12 of the 16 gene fragments. The analysis of the mixed genomic structure of modern species and cultivars (i) revealed C. maxima introgressions in modern mandarins; (ii) was consistent with previous hypothesis regarding the origin of secondary species; and (iii) provided a new picture of the evolution of chromosome 2. Perspectives to rebuild the main secondary species from the basic taxa are discussed.