title: Haplotype Detection from Next-Generation Sequencing in High-Ploidy-Level Species: 45S rDNA Gene Copies in the Hexaploid Spartina maritima.
abstract: Gene and whole-genome duplications are widespread in plant nuclear genomes, resulting in sequence heterogeneity. Identification of duplicated genes may be particularly challenging in highly redundant genomes, especially when there are no diploid parents as a reference. Here, we developed a pipeline to detect the different copies in the ribosomal RNA gene family in the hexaploid grass Spartina maritima from next-generation sequencing (Roche-454) reads. The heterogeneity of the different domains of the highly repeated 45S unit was explored by identifying single nucleotide polymorphisms (SNPs) and assembling reads based on shared polymorphisms. SNPs were validated using comparisons with Illumina sequence data sets and by cloning and Sanger (re)sequencing. Using this approach, 29 validated polymorphisms and 11 validated haplotypes were reported (out of 34 and 20, respectively, that were initially predicted by our program). The rDNA domains of S. maritima have similar lengths as those found in other Poaceae, apart from the 5'-ETS, which is approximately two-times longer in S. maritima. Sequence homogeneity was encountered in coding regions and both internal transcribed spacers (ITS), whereas high intragenomic variability was detected in the intergenic spacer (IGS) and the external transcribed spacer (ETS). Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH) revealed the presence of one pair of 45S rDNA signals on the chromosomes of S. maritima instead of three expected pairs for a hexaploid genome, indicating loss of duplicated homeologous loci through the diploidization process. The procedure developed here may be used at any ploidy level and using different sequencing technologies.
authors.affiliation: UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l'Univers de Rennes), University of Rennes 1, Bât 14A Campus Scientifique de Beaulieu, 35 042 Rennes Cedex, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Boutte
authors.affiliation: UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l'Univers de Rennes), University of Rennes 1, Bât 14A Campus Scientifique de Beaulieu, 35 042 Rennes Cedex, France.
authors.forename: Benoît
authors.initials: B
authors.lastname: Aliaga
authors.affiliation: UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l'Univers de Rennes), University of Rennes 1, Bât 14A Campus Scientifique de Beaulieu, 35 042 Rennes Cedex, France.
authors.forename: Oscar
authors.initials: O
authors.lastname: Lima
authors.affiliation: UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l'Univers de Rennes), University of Rennes 1, Bât 14A Campus Scientifique de Beaulieu, 35 042 Rennes Cedex, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Ferreira de Carvalho
authors.affiliation: UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l'Univers de Rennes), University of Rennes 1, Bât 14A Campus Scientifique de Beaulieu, 35 042 Rennes Cedex, France.
authors.forename: Abdelkader
authors.initials: A
authors.lastname: Ainouche
authors.affiliation: Biology Centre ASCR, Institute of Plant Molecular Biology, Branišovská 31, České Budějovice, CZ-37005, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Macas
authors.affiliation: UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l'Univers de Rennes), University of Rennes 1, Bât 14A Campus Scientifique de Beaulieu, 35 042 Rennes Cedex, France UMR Institut de Génétique, Environnement et Protection des Plantes, Institut National de la Recherche Agronomique, BP35327, 35653 Le Rheu Cedex, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Rousseau-Gueutin
authors.affiliation: UMR Institut de Génétique, Environnement et Protection des Plantes, Institut National de la Recherche Agronomique, BP35327, 35653 Le Rheu Cedex, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Coriton
authors.affiliation: UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l'Univers de Rennes), University of Rennes 1, Bât 14A Campus Scientifique de Beaulieu, 35 042 Rennes Cedex, France.
authors.forename: Malika
authors.initials: M
authors.lastname: Ainouche
authors.affiliation: UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l'Univers de Rennes), University of Rennes 1, Bât 14A Campus Scientifique de Beaulieu, 35 042 Rennes Cedex, France armel.salmon@univ-rennes1.fr.
authors.forename: Armel
authors.initials: A
authors.lastname: Salmon
mesh_terms: D019295:Computational Biology / Q000379:methods; D004275:DNA, Ribosomal; D017343:Genes, Plant; D018745:Genome, Plant; D023281:Genomics / Q000379:methods; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing; D017404:In Situ Hybridization, Fluorescence; D058977:Molecular Sequence Annotation; D016366:Open Reading Frames; D010802:Phylogeny; D006109:Poaceae / Q000145:classification* / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011123:Polyploidy; D012335:RNA, Ribosomal / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; C088179:RNA, ribosomal, 45S
keywords: bioinformatics; duplication; paralogy; poaceae; polyploidy