Molecular basis of off-type microsatellite markers in papaya

Papaya (Carica papaya L.) is a major fruit crop in the tropics and is a model system for studying sex chromosome evolution in plants. From a simple sequence repeat (SSR) mapping project in papaya, 37 SSRs displayed non-parental alleles in the F₂ population, known as off-type markers. They all belong to Class I (≥20 bp, hypervariable markers). To investigate the molecular basis of these non-parental alleles, seven SSRs with off-type alleles were cloned and sequenced from two parents and selected F₂ individuals. SSR repeat motif variation was the major cause for the generation of off-type alleles and single nucleotide polymorphisms (SNPs) and Insertion/deletion also contributed to off-type polymorphism. Off-type SSRs arose after one meiosis at 3 % frequency. Two of the seven markers had no polymorphisms between the parents but non-parental alleles segregated among the F₂ individuals. Four of the seven markers exhibited SNPs between parents with transition and transversion at 2:1 ratio. Six markers showed additional SNPs between F₂ offspring and parents with transition and transversion at about 1:1 ratio, suggesting higher than expected level of transversion mutations at the F₂ population after one meiosis event between the parental genomes. These off-type SSRs were analyzed in a second F₂ population and 43 % of them were conserved. The major cause of off-type SSRs in papaya is the variation of repeat length in Type I SSRs, and the underlining mechanism might be DNA replication slippage of repeat motifs. The presence and high conservation of these off-type markers demonstrated that hypervariable class I SSRs could generate genetic variation in one generation and increase genetic diversity for natural selection to enhance fitness.