Using high-throughput sequencing to investigate the evolution of self-incompatibility genes in the Brassicaceae: strategies and challenges

Background: The high polymorphism of genes under long-term balancing selection makes efficient genotyping of large numbers of individuals difficult, particularly when they are members of a gene family. For the Major Histocompatibility Complex (MHC) in animals, 454 amplicon sequencing has been used to obtain sequences from duplicated loci in multiple individuals, as an alternative to traditional cloning and sequencing approaches. Aims: The purpose of this study was to assess the potential for using 454 sequencing to study other loci under balancing selection: genes involved in plant self-incompatibility (SI). As a test case, we focus on diploids and tetraploids in the genus Arabidopsis. Methods: We used four previously developed primer combinations that amplify a range of alleles of the pistil gene, SRK, and its previously recognised paralogs. The amplification products from duplicate polymerase chain reactions were separately pooled from each individual, tagged and sequenced. Reads were clustered and classified using a reference database of SRK and its known paralogs. Duplicate sets of reactions and individuals with known genotypes were used to assess error rates and the efficiency of clustering. Results: After preprocessing, the few remaining sequencing errors and chimeric sequences did not affect the resolution of genotypes. Optimal clustering was obtained with a 90% sequence similarity criterion and excluding sequences present at a frequency of less than 7% of the total reads for an individual. Conclusions: The protocol is promising for efficiently genotyping large numbers of individuals for highly variable gene families.