Application of an Ion-Torrent sequencing approach for genotyping Leguminosae species

Abstract

The genetic diversity of Pisum sativum and Vicia faba has to be investigated. That is done by conducting a phylogenetic analysis based upon the sequence of single nucleotide polymorphisms (SNP's), linked to possible resistance genes against high salinity and the fungal disease Ascochyta blight. To conduct the analysis a method has to be developed and optimised to determine the sequence of chosen SNP's, which is the aim of this thesis. The method for sequencing contains five procedures. First SNP's are chosen equally divided over the linkage groups. Secondly primer sets are designed to amplify DNA fragments containing the SNP's. A genomic PCR is performed for amplification and a second one to introduce a barcode and an adaptor for sequencing. To prepare the fragments for sequencing an emulsion PCR and enrichment are necessary. Finally the sequencing is performed with Ion Torrent next generation sequencing. To optimise the first two PCR's three thermocyclers are compared: GeneAmp PCR System 9700, Robocycler Gradient 96 and TProfessional thermocycler, as well as two DNA polymerase kits: OneTaq DNA Polymerase and PrimeSTAR GXL. The optimisation of the sequencing preparation consists of finding the right concentration of the fragments. 71.7 % of the fragments were amplified for Vicia faba and 64.4 % for Pisum sativum, using the developed primer sets. The combination of the TProfessional thermocycler and OneTaq DNA Polymerase turns out to be the most optimal. The sequencing preparation works best for a concentration of 13 pM.