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Title: Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

Author
item FELTUS, F - Clemson University
item SASKI, CHRIS - Clemson University
item MOCKAITIS, KEITHANNE - Indiana University
item HAIMINEN, NIINA - International Business Machines Corporation (IBM)
item PARIDA, LAXMI - International Business Machines Corporation (IBM)
item SMITH, ZACHARY - Indiana University
item FORD, JAMES - Indiana University
item STATON, MARGARET - Clemson University
item FICKLIN, STEPHEN - Clemson University
item BLACKMON, BARBARA - Clemson University
item Schnell Ii, Raymond
item Kuhn, David
item MOTAMAYOR, JUAN - Mars, Inc

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/27/2011
Publication Date: 7/27/2011
Citation: Feltus, F.A., Saski, C.A., Mockaitis, K., Haiminen, N., Parida, L., Smith, Z.M., Ford, J., Staton, M., Ficklin, S.P., Blackmon, B.P., Schnell II, R.J., Kuhn, D.N., Motamayor, J.C. 2011. Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes. Biomed Central (BMC) Genomics. 12:379. DOI: 10.1186/1471-2164-12-379.

Interpretive Summary: Theobroma cacao, whose seeds are the source of cocoa, the raw material for the multi-billion dollar US chocolate industry, is an important tropical agriculture commodity that is affected by a number of fungal pathogens, including frosty pod (Moniliophthora roreri H.C. Evans, Stalpers, Samson & Benny) and witches’ broom (Moniliophthora perniciosa (Aime and Phillips-Mora comb. nov.). We are trying to find molecular genetic markers that are linked to disease resistance in Theobroma cacao to aid in a marker assisted selection (MAS) breeding program to ensure a reliable supply of cocoa for the US confectionary industry. By sequencing a QTL rich region of the cacao genome, as described in this paper, we can identify molecular genetic markers tightly associated with disease resistance and other horticultural traits. Our results are important to scientists trying to understand the mechanism of disease resistance and, eventually, to cacao farmers who will benefit from superior disease resistant cultivars produced through our MAS breeding program.

Technical Abstract: Background: BAC-based physical maps provide for sequencing across an entire genome or selected sub-genome regions of biological interest. Using the minimum tiling path as a guide, it is possible to select specific BAC clones from prioritized genome sections such as a genetically defined QTL interval and sequence those clones using a pooled shotgun sequencing approach. This pooled BAC approach was taken to sequence a QTL-rich region, of ~3Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Results: Using various mixtures of read coverages from paired and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the 454-based assembly with a subset of ten BACs individually sequenced and assembled using Sanger reads. An optimal mixture of reads for assembly was identified. We also found that a quality reference assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Conclusions: Our results and other pooled BAC studies suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genome 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 pooled-BAC by pooled-BAC entire genome assembly is completed.