Reduced Representation Sequencing for Rapidly Genotyping Diverse Species.

While most money and effort in the last decade have focused on genotyping humans, there are now tremendous opportunities to explore the vast amounts of genetic diversity found in the rest of the world’s species. Whether maize, flies, or bacteria, many organisms are 10-50 times more diverse than humans. While abundant diversity is a challenge to assays that rely on scoring fixed positions (microarrays or single base extension), it is a boon to direct sequencing approaches because sequencing efficiency for genotyping scales directly with genetic diversity. We have been developing sequence-based approaches in diverse organisms that could be used for population studies, germplasm characterization, breeding, and trait mapping. These methods can be generalized to any species and will be particularly efficient for surveys involving diverse germplasm. Key to the approaches are: (1) reducing genome representation through restriction enzymes (REs); (2) choosing REs that avoid the main repetitive classes in a clade; (3) generating sticky ends to permit the use of lower quality DNA; (4) producing a very inexpensive barcoding system that reduces sample handling and allows single tube digestion and adapter ligation; (5) modulating the barcode length so that sequence quality is not compromised by reading through RE cut-sites; and (6) reducing genome coverage so that samples can be scored very inexpensively (missing SNPs in haplotype blocks are imputed).