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(1)op yr ig ht pr ot ec te d.. op yr ig ht pr ot ec te d. F. op yr ig ht pr ot ec te d. F. 1. 10 00. Po op yr ig ht pr ot ec te d. F. 10 00. Po st e 10 00. Po st er s. C. Po s 10 00. Po s 0 00 F1 d.. te. C op s.. ig yr op. yr. ht. ig. pr. ht. ot. pr. ec. ot. te. ec. d.. F1. d.. te. s. pr ig ht yr s. C op. er st. 00. F1. 0. Conclusion. 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.. ec. ot pr. Po. 0. 00. F1. d. te ec. ot. The Figure 6 (NJ tree of the locus 9.2) that one haplotype belongs to the Mandarin group and the other one to the Pumelo group.. Po 0 00. For each amplified fragment, the polymorphism was enough for multilocus differentiation of the basic species and assignation of a phylogenetic origin for the secondary species. A preliminary reconstitution of phylogenetic structure of the chromosome 3 is proposed for clementine, sweet orange, sour-orange, grapefruit, lime and lemon (figure 4). However only haplotype analysis would allow definitive assignment. A Parallel pyrosequencing (454 Roche) for multilocus haplotyping of heterozygous genotypes has been done…. ht. .F. ot ec pr ht ig yr op C s. er st. Po. 0 00 F1 d. te ec pr ot ig ht yr .C op rs. te. st. Figure 4: Citrus Phylogenetic Structure of the Chromosome 3. ig. As an example, we can see with the Figure 5 (NJ tree of the locus 5.5) that both haplotypes belong to the Mandarin group.. Figure 6: Neighbor Joining Tree of Locus 9.2 Haplotypes. Po s. s. er. te d. 10 00. .F te d ot ec pr. 00 d. te ec pr ot. C op. Locus 3.7. Po 0. 00. F1. ht. ig. yr. C. s.. er. st Po 0. Lemon. Lime. yr ig ht. Locus 3.6. te. d.. Locus 3.5. ec ot pr ht. Sour Orange Grapefruit. 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.. Figure 5: Neighbor Joining Tree of Locus 5.5 Haplotypes. F1. Citrus genome Chromosome 3. d.. ec te. ot pr. ht. ig. yr. op. C s. er st Po 0. 00. F1 d. te ec ot pr. Orange. Locus 3.8 Locus 3.9 Locus 3.10 Locus 3.11. C. clementina (clementine) was used as model for secondary species.. Figure 2: Fragment Amplification Using Universal M13 Primers. Clementine Locus 3.1 Locus 3.2 Locus 3.3. 0 00. F1. 00 F1 d. ct e te pr o ht ig yr. op. ig yr op C. s.. st Po. er. st Po 0. 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 of Citrus.. 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 of Citrus were obtained by using an original new method based on universal primers (Figure 2).. op. Mandarins. er. s.. op. Material and Methods. C. It was assumed that intermediary position between two species cluster corresponded to interspecific heterozygosity. Figure 3: ACP, Phylogenetic Assignation of the Sequenced Fragments of the Locus 5.5. C. yr. op. ig. yr. ht. ig. pr. ht. Figure 1: Origin of the Modern Cultivars of Citrus. Po st er s. C. te d ec ot ht. ot. ot. pr. Grapefruits (C. paradisi). ec. Citrons. The average SNP rate was about 20 SNPs/kb. Citrus reticulata was the most polymorph basic taxa with an average of 4.2 SNPs/kb. For each gene an ACP was performed from SNP data to analyze the genetic organization (e.g. Figure 3).. pr. Oranges (C. sinensis). Mandarin hybrids, Tangor (e.g. clementine), Tangelos…. C. 00. 00. F1. te. F1 Hybrides. yr. te. d.. Sour oranges (C. aurantium). ec. Lemons (C. limon). Pumelos. d.. 00 F1. Limes (C. aurantifolia). 0. Po. 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 (Figure 1). 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 analyzed how multilocus study of closely linked SNPs allows a phylogenetic assignation of DNA fragments of the main cultivated species.. Pumelos (C. maxima). 0. Mandarins (C. reticulata). Haplotype Assignation (454). Genotype Assignation (Sanger). st er s.. Introduction. C. Citrons (C. medica). Micrantha (C. micrantha). International Center of Agricultural Research for Development,. C. Instituto Valenciano de Investigaciones Agrarias. .F. French National Institute for Agricultural Research. (1): INRA, (French National Institute for Agricultural Research) UR1103 Génétique et Ecophysiologie de la Qualité des Agrumes, F-20230 San Giuliano, France; (2): IVIA, (Valencian Institute for Agricultural Research), Centro de Protección Vegetal y Biotecnología, Ctra. Moncada-Náquera km 4.5, E-46113 Moncada, Valencia, Spain; (3): CIRAD, (International Center of Agricultural Research for Development), Department BIOS, TGU AGAP, Avenue Agropolis, TA A-75/02. F-34398 Montpellier Cedex 5, France;. 10. ivia. Curk F. (1,2), Ancillo G. (2), Garcia-Lor A. (2), Luro F. (1), Navarro L. (2) and Ollitrault P. (3,2). Po st er s. C. op yr ig ht pr ot ec te d. F. Multilocus SNPs Analysis Allows Phylogenetic Assignation of DNA Fragments to Decipher the Interspecific Mosaic Genome Structure of Cultivated Citrus.

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