Construction of Genomic Library of Theobroma cacao using Genotyping by Sequencing (GBS)

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.