title: Next generation haplotyping to decipher nuclear genomic interspecific admixture in Citrus species: analysis of chromosome 2.
abstract: BACKGROUND
  The most economically important Citrus species originated by natural interspecific hybridization between four ancestral taxa (Citrus reticulata, Citrus maxima, Citrus medica, and Citrus micrantha) and from limited subsequent interspecific recombination as a result of apomixis and vegetative propagation. Such reticulate evolution coupled with vegetative propagation results in mosaic genomes with large chromosome fragments from the basic taxa in frequent interspecific heterozygosity. Modern breeding of these species is hampered by their complex heterozygous genomic structures that determine species phenotype and are broken by sexual hybridisation. Nevertheless, a large amount of diversity is present in the citrus gene pool, and breeding to allow inclusion of desirable traits is of paramount importance. However, the efficient mobilization of citrus biodiversity in innovative breeding schemes requires previous understanding of Citrus origins and genomic structures. Haplotyping of multiple gene fragments along the whole genome is a powerful approach to reveal the admixture genomic structure of current species and to resolve the evolutionary history of the gene pools. In this study, the efficiency of parallel sequencing with 454 methodology to decipher the hybrid structure of modern citrus species was assessed by analysis of 16 gene fragments on chromosome 2.
  
  
  RESULTS
  454 amplicon libraries were established using the Fluidigm array system for 48 genotypes and 16 gene fragments from chromosome 2. Haplotypes were established from the reads of each accession and phylogenetic analyses were performed using the haplotypic data for 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 hypotheses regarding the origin of secondary species, and (iii) provided a new picture of the evolution of chromosome 2.
  
  
  CONCLUSIONS
  454 sequencing was an efficient strategy to establish haplotypes with significant phylogenetic assignations in Citrus, providing a new picture of the mixed structure on chromosome 2 in 48 citrus genotypes.
authors.affiliation: UMR AGAP, Institut National de la Recherche Agronomique (Inra), Centre Inra de Corse, F-20230, San Giuliano, France. curk@corse.inra.fr.|Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113, Moncada, Valencia, Spain. curk@corse.inra.fr.
authors.forename: Franck
authors.initials: F
authors.lastname: Curk
authors.affiliation: Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113, Moncada, Valencia, Spain. ancillo@ivia.es.
authors.forename: Gema
authors.initials: G
authors.lastname: Ancillo
authors.affiliation: Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113, Moncada, Valencia, Spain. angarcia@ivia.es.
authors.forename: Andres
authors.initials: A
authors.lastname: Garcia-Lor
authors.affiliation: UMR AGAP, Institut National de la Recherche Agronomique (Inra), Centre Inra de Corse, F-20230, San Giuliano, France. luro@corse.inra.fr.
authors.forename: François
authors.initials: F
authors.lastname: Luro
authors.affiliation: UMR AGAP, Centre de coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), TA A-108/02, 34398, Montpellier, Cedex 5, France. xavier.perrier@cirad.fr.
authors.forename: Xavier
authors.initials: X
authors.lastname: Perrier
authors.affiliation: UMR AGAP, Centre de coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), TA A-108/02, 34398, Montpellier, Cedex 5, France. jean-pierre.jacquemoud-collet@cirad.fr.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Jacquemoud-Collet
authors.affiliation: Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113, Moncada, Valencia, Spain. lnavarro@ivia.es.
authors.forename: Luis
authors.initials: L
authors.lastname: Navarro
authors.affiliation: Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113, Moncada, Valencia, Spain. ollitrault@cirad.fr.|UMR AGAP, Centre de coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), TA A-108/02, 34398, Montpellier, Cedex 5, France. ollitrault@cirad.fr.
authors.forename: Patrick
authors.initials: P
authors.lastname: Ollitrault
mesh_terms: D001947:Breeding; D002467:Cell Nucleus / Q000235:genetics; D032461:Chromosomes, Plant / Q000235:genetics*; D002957:Citrus / Q000235:genetics*; D018745:Genome, Plant; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing; D010802:Phylogeny; D018515:Plant Leaves / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D013045:Species Specificity