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Title: Construction of genomic library of Theobroma cacao using genotyping by sequencing (GBS)

Author
item Takrama, Jemmy - Cocoa Research Institute Of Ghana
item Kuhn, David
item Gutierrez, Osman
item Schnell, Raymond - Mars, Inc
item Fulton, Theresa - Cornell University - New York
item Elshire, Robert - Cornell University - New York
item Buckler, Edward - Ed
item Mitchell, Sharon - Cornell University - New York

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/11/2012
Publication Date: 1/11/2012
Citation: Takrama, J., Kuhn, D.N., Gutierrez, O.A., Schnell, R.J., Fulton, T., Elshire, R.J., Buckler Iv, E.S., Mitchell, S.E. 2012. Construction of genomic library of Theobroma cacao using genotyping by sequencing (GBS). Meeting Abstract. Available: https://pag.confex.com/pag/xx/webprogram/Paper2082.html.

Interpretive Summary: Theobroma cacao, the source of cocoa beans for chocolate, is an important tropical agriculture commodity that is affected by a number of fungal pathogens and insect pests, as well as concerns about yield and quality. We are trying to find molecular genetic markers that are linked to disease resistance and other important economic traits to aid in a marker assisted selection (MAS) breeding program for cacao to ensure a reliable supply of cocoa for the US confectionary industry. Recently a highly multiplexed genotype-by-sequencing (GBS) procedure that is suitable for population studies, germplasm characterization, breeding, and trait mapping was developed and applied to maize and barley recombinant inbred populations at the Institute for Genetic Diversity (Cornell University, New York) (ref: A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. Elshire, R. J., Glaubitz, J. C., Qi San, Poland, J. A., Kawamoto, K., Buckler, E. S.,& Mitchell S. E., (2011) PloS ONE 6(5):e19379 doi:10.1371/journal.pone.0019379). GBS is based on high-throughput, next-generation sequencing (NGS) technology (Illumina, Inc.) of genomic subsets targeted by a restriction enzyme, ApeK1(GCW|GC, where W is A or T). The procedure involves a single-well digestion of genomic DNA and adapter ligation without fragment size selection. Although the procedure was developed for maize (2.3 Gbp) it was successfully applied to Theobroma cacao (430 Mbp) after preliminary tests showed that ApeK1 satisfactorily restricted the cacao genome. Samples ran comprised 48 F1 mapping population from CATIE, 48 F2 mapping population from Brazil and 96 samples from the germplasm collection of the Cocoa Research Institute of Ghana (CRIG). In all cases, DNA was extracted by the CTAB method and further purified by the QIAgen Kit. In this study we present the results of the preparation of a genomic library of T. cacao using the GBS procedure. Data analysis is in progress. 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 and more productive cultivars produced through our MAS breeding program.

Technical Abstract: Recently a highly multiplexed genotype-by-sequencing (GBS) procedure that is suitable for population studies, germplasm characterization, breeding, and trait mapping was developed and applied to maize and barley recombinant inbred populations at the Institute for Genetic Diversity (Cornell University, New York) (ref: A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. Elshire, R. J., Glaubitz, J. C., Qi San, Poland, J. A., Kawamoto, K., Buckler, E. S.,& Mitchell S. E., (2011) PloS ONE 6(5):e19379 doi:10.1371/journal.pone.0019379). GBS is based on high-throughput, next-generation sequencing (NGS) technology (Illumina, Inc.) of genomic subsets targeted by a restriction enzyme, ApeK1(GCW|GC, where W is A or T). The procedure involves a single-well digestion of genomic DNA and adapter ligation without fragment size selection. Although the procedure was developed for maize (2.3 Gbp) it was successfully applied to Theobroma cacao (430 Mbp) after preliminary tests showed that ApeK1 satisfactorily restricted the cacao genome. Samples ran comprised 48 F1 mapping population from CATIE, 48 F2 mapping population from Brazil and 96 samples from the germplasm collection of the Cocoa Research Institute of Ghana (CRIG). In all cases, DNA was extracted by the CTAB method and further purified by the QIAgen Kit. In this study we present the results of the preparation of a genomic library of T. cacao using the GBS procedure. Data analysis is in progress.