Genotyping Association Panel using SLAF-seq

SNP genotyping of the association panel was performed using a SLAF-seq approach^{17 (link)}. Construction of the peanut DNA libraries and Illumina sequencing of the plants were performed at Biomarker Technologies Corporation in Beijing, China. Through restriction enzymes HaeIII and Hpy166II (New England Biolabs, NEB, USA) that digest peanut genomic DNA into DNA fragments of 364–464 bp^{23 (link)}, the sequencing libraries of 202 peanut accession were constructed. The physical position of the markers were identified by aligning the sequence of a 125 bp paired-end reads attached to each marker with the ‘pseudomolecules’ genome sequences of diploid peanut (Arachis duranensis-AA and Arachis ipaensis-BB, https://www.peanutbase.org) using local BLASTn (BLAST: Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/Blast.cgi). If the reads matched two or more locations in the reference genome of peanut, the markers were regarded as non-specific markers and discarded. Accurate markers were selected throught three steps. First, all candidate markers must be called in the mixed reads from parents and all the F₆ samples using GATK (https://software.broadinstitute.org/gatk/). Second, all candidate markers must be called less than 20%. Third, some hemi-SNPs which showed polymorphism in sub-genomes were used for polymorphism markers in genetic linkage map in RIL population.