Multiplex PCR (mPCR) analyzes several markers simultaneously in a single reaction. The development of an SSR multiplex system requires estimation of range of allele sizes of different markers for their grouping into multiplex sets. In the present investigation duplex, triplex, quadruplex, and pentaplex combinations of SSR markers were optimized for mPCR using four different soybean genotypes namely, JJS 335, Durga, MACS 124 and JS 90-41.Twenty SSR markers distributed across all linkage groups of soybean were used to develop 22 multiplex sets of markers. These 22 multiplex sets comprised of twelve duplex, five triplex, four quadruplex and one pentaplex assays. Each band of mPCR was considered a single genetic marker and using all mPCR marker sets bands in the size range of 100-300bp were observed. The methodology used was quite systematic, rapid, reliable, and appropriate for qualitative detection and can be extended for multiplex development in other plant species having SSR marker technology for efficient plant genetic resources management including genetic diversity analysis, cultivar identification and evolutionary studies in plants.

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