Maize Genotyping Using Rapid-Seq (Randomly-Amplified Polymorphic DNA Sequencing)

Genotyping based on sequencing (GBS) has become standard for maize genomic analysis. GBS approaches typicaly use restriction digestions of DNA, ligation of adaptors and PCR amplification, followed by next-generation sequencing and alignment. GBS is limited to the portion of the genome sampled by existing types of restriction enzymes and involves several enzymatic steps. Also, large numbers of low-depth reads are observed, requiring extensive data imputation and increasing error rate. We assessed an alternative to GBS – Randomly-Amplified Polymorphic DNA sequencing (RAPiD-Seq), which is based on a single PCR reaction using primers containing a combination of degenerate and specific bases. RAPiD-Seq needs less than 100 ng of DNA, and modifications to the specific sequences in the primer permit evaluation of a nearly unlimited number and distribution of genomic regions. To evaluate RAPiD-seq in maize, we genotyped the reference genome B73, 25 parents from the NAM population, 70 NILs derived from those parents plus 12 randomly repeated samples. A total of 59,554 SNPs were identified in the population with median sequencing depth of 31× and a reproducibility of genotypic call of 0.99 when 12 random samples were independently repeated. Markers were equally distributed with a median interval of 56Kbp. A panel with 5K and 20K markers were selected and consistently genotyped with an estimated multiplexing of 144 and 96 samples per HiSeq lane. NIL samples had a median heterozygosis of 0.0047 and genotypic data was used to identify introgression blocks. Results suggest that RAPiD-Seq is a viable and flexible method for maize genotyping.