S-genotyping of old apple cultivars from the Carpathian basin: methodological, breeding and evolutionary aspects

Abstract

Apple exhibits self-incompatibility controlled by the multiallelic S-locus. Twenty-three old apple cultivars were S-genotyped using three different approaches (allele-specific polymerase chain reaction (PCR) + cleaved amplified polymorphic sequences (CAPS), consensus PCR + sequencing and consensus PCR + CAPS) to compare the robustness and reliability of these techniques and characterise genotypes from the Carpathian basin that might be useful in resistance breeding. Best results were obtained using the ASPF3 and ASPR3S consensus primer pair that detected 96% of all alleles carried by the 23 cultivars tested. Flow cytometry analysis was also needed to control the completeness of the genotypes as was seen in case of a tetraploid cultivar with only three assigned S-alleles. The genetic disparity between the old Carpathian basin and modern apple cultivars was indicated by differences in allele frequency data (S 9, S 24 and S 26) as well as single nucleotide polymorphisms in S 1, S 2, S 7 S 24 and S 26 and indels in S 20 and S 26 alleles. An alignment of partial genomic sequences indicated trans-specific and trans-generic evolution of S-ribonuclease alleles in the Maloideae subfamily (S 26 and S 28) and a possibly recent introgression event (S 1) between Malus × domestica and Malus sylvestris. These data suggest that the genome of old cultivars from the Carpathian basin was enriched by several Malus taxa and are free from the consequences of modern breeding. These cultivars may contribute to the widening of the genetic basis of cultivated apple and prevent genetic erosion in future commercial cultivars.