Primer design and test:
Allele-specific forward primers and a common reverse primer were designed for use in KASP assays (LGC Genomics Ltd. Hoddesdon, UK; http://www.lgcgenomics.com/kasp-genotyping-reagents) using BatchPrimer3 (http://probes.pw.usda.gov/batchprimer3/) with the “Allele specific primers and allele flanking primers” option. The parameters used were 60−120 bp in size, Tm between 58 and 60°, and GC content between 30 and 80%. The alternative alleles were marked with 6-FAM and reference alleles with VIC. For each SNP, two allele-specific forward primers and one common reverse primer were designed. A schematic diagram of SNP discovery and primer design is shown in Figure 1, A and B. Primer information is listed in File S1.
Figure 1  A schematic diagram of single-nucleotide polymorphism (SNP) discovery and Kompetitive allele-specific polymerase chain reaction (KASP) primer design. A. ipaënsis K 30076 is used as proxy for the B-genome of A. hypogaea. (A) Alignment of A. ipaënsis K 30076, A. magna K 30097, and A. batizocoi K 9484 paired-end cDNA reads onto A. ipaënsis K 30076 genomic sequence, Pseudomolecule Araip.B08 (where rust resistance QTL reside) and the identification of SNPs. (B) Example of design of allele-specific and site specific (common) primers for the SNPs identified. KASP assays were performed with the following genotypes: the diploids A. ipaënsis K 30076, A. batizocoi K9484 and A. magna K 30097, the induced allotetraploids (A. magna K 30097 x A. stenosperma V15076)4x (here called MagSten) and (A. batizocoi K9484 × A. stenosperma V10309)4x (here called BatSten1) and six A. hypogaea cultivars (Tifrunner, Tifguard, GA-06G, NC3033, ICVG 88145, and SPTG_06). Reactions consisted of 2 μL of KASP 2X reaction mix, 0.055 μL of assay primer mix (12 mM of each allele-specific primer and 30 mM of common primer) and 20 ng of genomic DNA, in a 4-µL volume. A C1000 Thermal Cycler (Bio-Rad) was used with the following cycling conditions: 94° for 15 min, nine cycles of 94° for 20 sec, touchdown starting at 65° for 60 sec (decreasing 0.8° per cycle), 29 cycles of 94° for 20 sec, and 57° for 60 sec (http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB/PDFs/KASP_SNP_Genotyping_Manual.pdf). To improve the results, a second KASP program was run as following: 9 cycles of 94° for 20 sec and 57° for 60 sec. Fluorescence was read by a The LightCycler 480 Instrument II (Roche Life Science) and analyzed using the LightCycler 480 Software (V.1.5.1).