Target enrichment sequencing in cultivated peanut (Arachis hypogaea L.) using probes designed from transcript sequences

Enabled by the next generation sequencing, target enrichment sequencing (TES) is a powerful method to enrich genomic regions of interest and to identify sequence variations. The objective of this study was to explore the feasibility of probe design from transcript sequences for TES application in calling sequence variants in peanut, an important allotetraploid crop with a large genome size. In this study, we applied an in-solution hybridization method to enrich DNA sequences of seven peanut genotypes. Our results showed that it is feasible to apply TES with probes designed from transcript sequences in polyploid peanut. Using a set of 31,123 probes, a total of 5131 and 7521 genes were targeted in peanut A and B genomes, respectively. For each genotype used in this study, the probe target capture regions were efficiently covered with high depth. The average on-target rate of sequencing reads was 42.47%, with a significant amount of off-target reads coming from genomic regions homologous to target regions. In this study, when given predefined genomic regions of interest and the same amount of sequencing data, TES provided the highest coverage of target regions when compared to whole genome sequencing, RNA sequencing, and genotyping by sequencing. Single nucleotide polymorphism (SNP) calling and subsequent validation revealed a high validation rate (85.71%) of homozygous SNPs, providing valuable markers for peanut genotyping. This study demonstrated the success of applying TES for SNP identification in peanut, which shall provide valuable suggestions for TES application in other non-model species without a genome reference available.