Development of a high-throughput PCR & sequencing approach for SNP identification in Leguminosae species

Abstract

Genotypes of Pisum sativum and Vicia faba with high protein content and resistance against diseases, heat and drought need to be found. The genotypes may differ because of point mutations in the genome, single nucleotide polymorphisms (SNP's). 88 genotypes of Pisum sativum and 116 of Vicia faba should be examined to find the ones corresponding to the desired phenotype. This work can be minimalized by making a phylogenetic analysis based on SNP's. The goal of this master's thesis is to optimize PCR and sequencing conditions to identify SNP's. Three thermocyclers are tested: TProfessional Thermocycler (Biometra), GeneAmp PCR System 9700 (Applied Biosystems), and Stratagene Robocycler (Agilent technologies). Three PCR kits are tested: MyTaq DNA Polymerase (Bioline), Primestar GXL (Clontech), and OneTaq DNA Polymerase (New England Biolabs). 60 SNP sequences for Vicia faba and 45 for Pisum sativum are selected and primers are designed. In a first PCR, these DNA fragments are amplified. A second PCR is done to elongate the DNA fragment with an adaptor sequence, as a preparation for sequencing. SNP's are identified using Ion Torrent sequencing, which also includes library preparation and an emulsion PCR (emPCR). The ideal concentration of the amplicon library for emPCR is determined. Optimal PCR conditions are obtained with the TProfessional Thermocycler and OneTaq DNA Polymerase kit. 35,5 % of the primers for Pisum sativum and 28,3 % for Vicia faba did not work and should be redesigned. The ideal concentration for emPCR is 1