title: Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes.
abstract: BACKGROUND
  BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library.
  
  
  RESULTS
  This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight.
  
  
  CONCLUSIONS
  Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.
authors.affiliation: Clemson University Genomics Institute, Clemson University, 51 New Cherry Street, Clemson, SC 29634, USA. ffeltus@clemson.edu
authors.forename: Frank A
authors.initials: FA
authors.lastname: Feltus
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Saski
authors.forename: Keithanne
authors.initials: K
authors.lastname: Mockaitis
authors.forename: Niina
authors.initials: N
authors.lastname: Haiminen
authors.forename: Laxmi
authors.initials: L
authors.lastname: Parida
authors.forename: Zachary
authors.initials: Z
authors.lastname: Smith
authors.forename: James
authors.initials: J
authors.lastname: Ford
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Staton
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Ficklin
authors.forename: Barbara P
authors.initials: BP
authors.lastname: Blackmon
authors.forename: Chun-Huai
authors.initials: CH
authors.lastname: Cheng
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Schnell
authors.forename: David N
authors.initials: DN
authors.lastname: Kuhn
authors.forename: Juan-Carlos
authors.initials: JC
authors.lastname: Motamayor
mesh_terms: D002099:Cacao / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial; D018745:Genome, Plant; D015698:Genomic Library; D040641:Quantitative Trait Loci; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA