title: Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia.
abstract: BACKGROUND
  Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes.
  
  
  RESULTS
  Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30% of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome.
  
  
  CONCLUSIONS
  Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20%.
authors.affiliation: Institute of Integrative Biology (IBZ), ETH Zurich, Zurich, Switzerland.|Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc - Holice, Czech Republic.
authors.forename: N
authors.initials: N
authors.lastname: Blavet
authors.affiliation: Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc - Holice, Czech Republic.|Department of Plant Developmental Genetics, Institute of Biophysics of the CAS, v.v.i., Brno, Czech Republic.
authors.forename: H
authors.initials: H
authors.lastname: Blavet
authors.affiliation: Laboratoire de Biométrie et Biologie Evolutive (UMR 5558), CNRS/Université Lyon 1, Villeurbanne, France.
authors.forename: A
authors.initials: A
authors.lastname: Muyle
authors.affiliation: Laboratoire de Biométrie et Biologie Evolutive (UMR 5558), CNRS/Université Lyon 1, Villeurbanne, France.
authors.forename: J
authors.initials: J
authors.lastname: Käfer
authors.affiliation: Department of Plant Developmental Genetics, Institute of Biophysics of the CAS, v.v.i., Brno, Czech Republic.
authors.forename: R
authors.initials: R
authors.lastname: Cegan
authors.affiliation: Pole Rhone-Alpes de Bioinformatique (PRABI), Villeurbanne, France.
authors.forename: C
authors.initials: C
authors.lastname: Deschamps
authors.affiliation: Institute of Integrative Biology (IBZ), ETH Zurich, Zurich, Switzerland.
authors.forename: N
authors.initials: N
authors.lastname: Zemp
authors.affiliation: Laboratoire de Biométrie et Biologie Evolutive (UMR 5558), CNRS/Université Lyon 1, Villeurbanne, France.
authors.forename: S
authors.initials: S
authors.lastname: Mousset
authors.affiliation: Unité de Recherche en Génomique Végétale (UMR 1165), INRA/Université d'Evry-Val-d'Essonne - ERL CNRS 8196, Evry, France.
authors.forename: S
authors.initials: S
authors.lastname: Aubourg
authors.affiliation: Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: R
authors.initials: R
authors.lastname: Bergero
authors.affiliation: Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: D
authors.initials: D
authors.lastname: Charlesworth
authors.affiliation: Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc - Holice, Czech Republic.|Department of Plant Developmental Genetics, Institute of Biophysics of the CAS, v.v.i., Brno, Czech Republic.
authors.forename: R
authors.initials: R
authors.lastname: Hobza
authors.affiliation: Institute of Integrative Biology (IBZ), ETH Zurich, Zurich, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Widmer
authors.affiliation: Laboratoire de Biométrie et Biologie Evolutive (UMR 5558), CNRS/Université Lyon 1, Villeurbanne, France. gabriel.marais@univ-lyon1.fr.
authors.forename: G A B
authors.initials: GA
authors.lastname: Marais
mesh_terms: D001483:Base Sequence; D032461:Chromosomes, Plant / Q000235:genetics*; D019143:Evolution, Molecular; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D012730:Sex Chromosomes / Q000235:genetics; D019849:Sex Determination Processes; D029751:Silene / Q000235:genetics* / Q000254:growth & development