KASP primer design and validation on a tetraploid background
Of a total of 24 assays designed, 22 worked well with the samples tested. Nineteen of them successfully distinguished A. ipaënsis and A. hypogaea from A. magna and A. batizocoi. Four cluster configurations were observed: (1) with nine assays, two clusters were present: one with A. ipaënsis plus the six cultivars of A. hypogaea and another with A. magna, A. batizocoi, and their derived induced allotetraploids MagSten and BatSten1 (noted in File S1 as Ah = Ai≠(Ab = Am = MagSten = BatSten)). An example is shown in Figure 5A; (2) with eight assays, one extra cluster was observed: A. hypogaea formed a different cluster intermediate in position (noted in File S1 as Ah≠Ai≠(Ab = Am = MagSten = BatSten))(Figure 5B); (3) with two assays, a different extra cluster was observed: MagSten was distinguished from all other genotypes (noted as (Ah = Ai)≠(Ab = Am = BatSten)≠MagSten)); (4) and finally, in three assays, A. ipaënsis formed an isolated cluster, being the other cluster formed by all the other genotypes (noted as Ai≠(Ah = Ab = Am = MagSten = BatSten)).
Figure 5  Screenshots of the two most common examples of Arachis B-genome single-nucleotide polymorphism genotyping using Kompetitive allele-specific polymerase chain reaction assays. Both patterns show differentiation between A. ipaënsis K 30076 and the B-genome of A. hypogaea from the wild species A. magna K 30097 and A. batizocoi K 9484 and the induced allotetraploids MagSten and BatSten1. In (A), two clusters are present: one with A. ipaënsis and all A. hypogaea cultivars, and another with the wild species and induced allotetraploids (noted in File S1 as Ah = Ai≠(Ab = Am = MagSten = BatSten)). In (B), three clusters are present. In these cases, A. hypogaea forms a different cluster, intermediate in position [noted in File S1 as Ah≠Ai≠(Ab = Am = MagSten = BatSten)]. All genotypes with A. batizocoi derived rust-resistant cluster in different groups to the susceptible genotypes.