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Multilocus SNPs analysis allows phylogenetic assignation of DNA fragments to decipher the
interspecific mosaic genome structure of cultivated citrus
Franck Curk, Gema Ancillo, Andres Garcia-Lor, François Luro, Luis Navarro, Patrick Ollitrault
To cite this version:
Franck Curk, Gema Ancillo, Andres Garcia-Lor, François Luro, Luis Navarro, et al.. Multilocus SNPs analysis allows phylogenetic assignation of DNA fragments to decipher the interspecific mosaic genome structure of cultivated citrus. Plante genome evolution, Sep 2011, Amsterdam, Netherlands. 2011. �hal-02746454�
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Comment citer ce document :
Curk, F., Ancillo, G., Garcia-Lor, A., Luro, F., Navarro, L., Ollitrault, P. (2011). Multilocus SNPs analysis allows phylogenetic assignation of DNA fragments to decipher the interspecific mosaic genome structure of cultivated citrus. Presented at Plante genome evolution, Amsterdam,
NLD (2011-09-04 - 2011-09-06).
Multilocus SNPs Analysis Allows Phylogenetic Assignation of DNA Fragments to Decipher the
Interspecific Mosaic Genome Structure of Cultivated Citrus
Curk F.
(1,2), Ancillo G.
(2), Garcia-Lor A.
(2), Luro F.
(1), Navarro L.
(2)and Ollitrault P.
(3,2)(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;
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. Mandarins (C. reticulata) Citrons (C. medica) Pumelos (C. maxima) F1 Hybrides Oranges (C. sinensis) Sour oranges (C. aurantium) Lemons (C. limon) Micrantha (C. micrantha) Limes (C. aurantifolia) Grapefruits (C. paradisi)
Mandarin hybrids, Tangor (e.g. clementine), Tangelos…
Figure 1: Origin of the Modern Cultivars of Citrus
Introduction
International Center of Agricultural Research for Development, Instituto Valenciano de
Investigaciones Agrarias
ivia
Conclusion
Material and Methods
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).
C. clementina (clementine) was used as
model for secondary species. Figure 2: Fragment Amplification Using
Universal M13 Primers
Genotype Assignation (Sanger)
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.
Figure 5: Neighbor Joining Tree of Locus 5.5 Haplotypes
French National Institute for Agricultural Research
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. 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
Haplotype Assignation (454)
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). It was assumed that
intermediary position between two species
cluster corresponded to
interspecific heterozygosity
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…
Figure 3: ACP, Phylogenetic Assignation of the Sequenced Fragments of the Locus 5.5
Citrus
genome
Chr
omosome 3
Figure 4: Citrus Phylogenetic Structure of the Chromosome 3
Clementine Orange Sour Orange Grapefruit Lime Lemon
Locus 3.1 Locus 3.2 Locus 3.3 Locus 3.5 Locus 3.6 Locus 3.7 Locus 3.8 Locus 3.9 Locus 3.10 Locus 3.11
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.
Pumelos
Citrons
