A VERSATILE FLUORESCENCE-BASED MULTIPLEXING ASSAY FOR COMBINED GENOTYPING OF SSR AND INSERTION-DELETION POLYMORPHISM (IDP) MARKERS ON CAPILLARY ELECTROPHORESIS SYSTEMS

E. G. Todorovska, N. K. Christov, S. Tsonev

Abstract: During the last decade the number of the IDP markers and their utilization in laboratory practice are constantly growing. Their combination with SSR markers can facilitate saturation of genetic maps. The main disadvantage of both types of markers is the low degree of automation. Although, SSR and IDP markers share similar properties and way of detection, information on multiplexing both types of markers in a single PCR reaction has not been described. Therefore, the present study aimed at increased throughput of the PCR marker assays on a capillary sequencer by simultaneous amplifcation and fluorescent labeling of SSRs and redundant set of IDP markers in a single closed tube format. Ninety six co-dominant markers, with allele sizes suitable for multiplexing with SSRs, were selected from a public set of 1229 maize IDP markers. Twenty seven of them were polymorphic between the parental lines of a double-haploid population derived from F1 cross of a mutant maize inbred with its progenitor line. Seventeen of the polymorphic IDPs together with nineteen SSRs were used to develop a versatile fluorescence-based multiplexing assay for combined genotyping on capillary electrophoresis systems. Both IDP and SSR markers were successfully co-amplifed and fluorescently labeled in 11 multiplex PCR reactions that included up to 4 primer pairs. The robustness of the method was validated by genotyping and map construction in the DH mapping population. The proposed multiplexing method will provide advantages for enrichment of target regions in fine mapping and map-based cloning projects as well as in marker assisted selection. With the increased availability of genome information and genome sequences of multiple varieties, the method could be successfully applied in other model and crop plant species.

Keywords: IDP; maize; mapping; multiplex PCR; SSR

Date published: 2017-09-18