rAmpSeq: Using repetitive sequences for robust genotyping

Abstract

AbstractRepetitive sequences have been used for DNA fingerprinting and genotyping for more than a quarter century. Now, with our knowledge of whole genome sequences, repetitive sequences can be used to identify polymorphisms that can be mapped and scored in a systematic manner. We have developed a simple, robust platform for designing primers, PCR amplification, and high throughput cloning that allows hundreds to thousands of markers to be scored for less than $5 per sample. Conserved regions were used to design PCR primers for amplifying thousands of middle repetitive regions of the maize (Zea mays ssp. mays) genome. Bioinformatic scans were then used to identify DNA sequence polymorphisms in the low copy intervening sequences. When used in conjunction with simple DNA preps, optimized PCR conditions, high multiplex Illumina indexing and a bioinformatic marker calling platform tailored for repetitive sequences, this methodology provides a cost effective genotyping strategy for large-scale genomic selection projects. We show detailed results from four maize primer sets that produced between 1,335-3,225 good coverage loci with 1056 that segregated appropriately in a bi-parental family. This approach could have wide applicability to breeding and conservation biology, where hundreds of thousands of samples need to be genotyped for very minimal cost.