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Title: A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species

item ELSHIRE, ROBERT - Cornell University
item GLAUBITZ, JEFFREY - Cornell University
item SUN, QI - Cornell University
item Poland, Jesse
item KAWAMOTO, KEN - Cornell University
item Buckler, Edward - Ed
item MITCHELL, SHARON - Cornell University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/4/2011
Publication Date: 5/4/2011
Citation: Elshire, R.J., Glaubitz, J.C., Sun, Q., Poland, J.A., Kawamoto, K., Buckler IV, E.S., Mitchell, S.E. 2011. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One. 123:307-326.

Interpretive Summary: Rapid developments in DNA sequencing technology are quickly reducing the cost of sequencing in many species. To utilize this new technology in diverse crop species with large genomes, we developed a simple procedure for making multiplex libraries for DNA sequencing. By multiplexing many samples the sequencing cost can be reduced to the point where many samples can be evaluated for genetics and breeding. We used a DNA restriction enzyme to target a limited portion of the genome in maize and barley for sequencing. Using DNA barcoded adapters, unique to each sample we were able to sequence 48 samples in single run. From this data, we identified 200,000 molecular markers in maize and 25,000 molecular markers in barley at a relatively low cost per sample. Using a simple protocol as described here, this genotyping-by-sequencing method can rapidly produce hundreds of thousands of molecular markers in many diverse species, enabling new genetics studies in under-researched crops, novel germplasm or thousands of samples in a plant breeding program.

Technical Abstract: Advances in next-generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolf Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tags becomes the reference. Alternatively, for phylogenetic analyses in the absence of a reference genome, the reads can simply be treated as dominant markers. Future application of GBS to breeding, conservation and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.