Targeted Resequencing in Peanuts Using the Fluidigm Access Array

Presence of homoeologous gene copies in allopolyploid peanut makes it challenging to select for homologous SNPs differentiating two or more cultivars. An integrated approach of improved bioinformatics and targeted resequencing to select homologous SNPs in tetraploid peanut is utmost important. In this study, we tested several methods to separate homeologous SNPs and then performed the SNP analysis. 1) Raw transcriptome reads were mapped to the synthetic tetraploid genome reference generated by combining A- and B -genome scaffolds to separate homologous SNPs among 13 cultivars and using the GATK pipeline and python scripts. 2) SNPs were identified between A -genome diploids and tetraploids using SWEEP which is a sliding window protocol and filters SNPs based on the haplotype. 3) The OLin de_novo transciptome assembly was used as a reference to identify SNPs. Approximately 10,500 polymorphic SNPs were obtained, of which 48 targets of around 400 bp length were selected for validation on Fluidigm Access Array as an proof of concept, followed by mass sequencing on an Illumina MiSeq. Some of these targets consisted of adjacent SNPs that differentiated both A- and B- genome copies together. We believe this approach will benefit tetraploid breeding programs by reducing the cost of genotyping of QTL mapping populations and selection of favorable alleles in both sub- genomes.