title: Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.
abstract: BACKGROUND
  In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution.
  
  
  RESULTS
  In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members.
  
  
  CONCLUSIONS
  The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a Ruby gem for this class of analyses.
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain. seoanezonjic@hotmail.com.
authors.forename: Pedro
authors.initials: P
authors.lastname: Seoane-Zonjic
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain. racanas@uma.es.
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Cañas
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain. rociobm@scbi.uma.es.
authors.forename: Rocío
authors.initials: R
authors.lastname: Bautista
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain. pgomez@uma.es.
authors.forename: Josefa
authors.initials: J
authors.lastname: Gómez-Maldonado
authors.affiliation: Departamento de Biología Vegetal, Facultad de Farmacia, ERI Biotecmed, Universidad de Valencia, Avda. Vicent Andrés Estellés s/n, 46100, Burjassot, Valencia, Spain. isabel.arrillaga@uv.es.
authors.forename: Isabel
authors.initials: I
authors.lastname: Arrillaga
authors.affiliation: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY, 14853, USA. nf232@cornell.edu.
authors.forename: Noé
authors.initials: N
authors.lastname: Fernández-Pozo
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain. claros@uma.es.
authors.forename: M Gonzalo
authors.initials: MG
authors.lastname: Claros
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain. canovas@uma.es.
authors.forename: Francisco M
authors.initials: FM
authors.lastname: Cánovas
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain. cavila@uma.es.
authors.forename: Concepción
authors.initials: C
authors.lastname: Ávila
mesh_terms: D022202:Chromosomes, Artificial, Bacterial; D018744:DNA, Plant / Q000235:genetics; D005091:Exons; D015723:Gene Library; D018745:Genome, Plant; D023281:Genomics / Q000379:methods; D007438:Introns; D008957:Models, Genetic; D028223:Pinus / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D018744:DNA, Plant