Development of multiplex SSR marker systems of soybean (Glycine max (L.) Merr.) for certification and identification of cultivars

Background. Soybean (Glycine max (L.) Merr.) is a strategically important crop, and expanding its cultivated diversity requires reliable and reproducible methods of identification and control in seed production. Microsatellite markers are widely used for certification and identification of cultivars, but to improve the accuracy and productivity of genotyping, it is necessary to develop multiplex systems on their basis.

Materials and methods. DNA samples from individual soybean plants of 15 cultivars of various origin were studied. As a marker base, we used 22 informative microsatellite loci with trinucleotide repeats distributed throughout the soybean genome. Using bioinformatic approaches, we designed new primer pairs for 19 loci, adapted for specific amplification of target DNA fragments under unified conditions of multiplex PCR. For experimental validation, forward primers were labeled with different fluorophores. When distributing fluorophores among SSR markers, we took into account the size ranges of amplified DNA fragments, thus avoiding signal overlap of different markers during the fragment analysis. PCR products were separated by capillary electrophoresis using a genetic analyzer. Optimization of multiplex PCR included stepwise selection of marker combinations, primer concentration ratios, and thermal cycling conditions.

Results. Two multiplex systems were developed, each including 11 informative SSR markers. The optimized multiplex PCR conditions ensured specific, stable, and reproducible amplification of DNA fragments corresponding to the loci. Based on the results, panel files were created in fragment analysis software and adjusted versus a positive control, which ensured correct interpretation and comparability of results between analytical runs.

Conclusion. The developed multiplex SSR marker systems improve genotyping accuracy and reduce its duration, thereby enabling their application in genetic certification and identification of soybean cultivars.

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