S. TSONEV, M. VELICHKOVA, E. TODOROVSKA, V. AVRAMOVA and N. K. CHRISTOV
AgroBioInstitute, BG – 1164 Sofia, Bulgaria
TSONEV, S., M. VELICHKOVA, E. TODOROVSKA, V. AVRAMOVA and N. K. CHRISTOV, 2013. Development of multiplex primer sets for cost efficient SSR genotyping of maize (Zea mays) mapping populations on a capillary sequencer. Bulg. J. Agric. Sci., Supplement 2, 19: 5–9
Because of their accessibility, genomic abundance and high level of polymorphism SSRs are often markers of choice in many QTL mapping studies. To obtain a good mapping resolution one have to genotype large number of individuals in a large number of marker loci, which is both labour-intensive and expensive task. The multiplex-ready PCR technique (MRT™) provides possibility for fast optimisation with simultaneous amplification and fluorescent labelling of several applicants in one reaction tube. In this study, we propose a low-cost protocol for optimising high-throughput genotyping of maize mapping populations using SSR markers and capillary electrophoresis. The method is based on MRT™ and consists of three stages.
In the first stage, the parental lines and F1 were screened in 176 public SSR loci. Fifty-four of the analysed loci were found to be polymorphic between parental lines. In the second stage, primer concentrations were optimised in uniplex reactions. In the third stage, the polymorphic markers were grouped in 17 multiplex reactions containing 2 to 5 primer pairs and tested for successful amplification in multiplex PCR reactions. Using the optimised protocol, a maize DH population consisting of 192 individuals was genotyped. The cost and effort benefits of the proposed method are discussed.