| Summary: | Background: Red clover (Trifolium pratense L.) is an important forage crop throughout the world due to its high forage quality, nitrogen fixation capacity and beneficial effects on the soil fertility. But aluminum (Al) toxicity limits significantly red clover production in acid soils, which represent more than one third of the world's agricultural lands. Natural variation for Al3+ ions resistance has been identified in many crop species so development of tolerant accessions and varieties is a promising approach for red clover breeding. In this context the objectives of this article were to select in vitro and evaluate using different DNA markers the tolerant to toxic aluminum breeding samples of red clover. Methods: Seeds of the experimental hybrid population were germinated under various aluminum concentrations, including control without aluminum. Epicotyls of seedlings without roots and with roots not less than 4–5 mm were subcultivated on agar's medium of Gamborg B5 with 2 mg/L of 6-benzylaminopurine and 100 mg/L of Al3+ and then planted in the cassettes with soil. Seedlings and adult plants F2, tolerant to 50 and 100 mg/L of Al3+ were selected, grown in vegetative pots and used further for molecular analyses. Genetic variability between tolerant and susceptible red clover genotypes was evaluated based on DNA markers: sequence-related amplified polymorphism (SRAP), retrotransposon microsatellite amplified polymorphism (REMAP) and inter-primer binding site polymorphism (iPBS). Results: Aluminum-tolerant red clover samples were obtained by in vitro selection on the medium with toxic aluminum ions. F2 seedlings in the variants with 50 mg/L and 100 mg/L of Al3+ were characterized by longer length and roots size compared with F1 seedlings and variety-standard at the same aluminum concentration. Subsequent molecular analysis showed that REMAP and iPBS were efficient markers to detect distinguishes among red clover accessions. The average level of polymorphism was identified as 45.8 using REMAP and 68.2% with iPBS; the average values of polymorphism information content (PIC) were 0.764 and 0.746 accordingly, higher compared to SRAP (0.741). Conclusions: Combination of the biotechnology methods and the current DNA-technologies based on REMAP and iPBS markers is effective approach to improve precision and reliability of selection and assessing of red clover genotypes with tolerance to toxic aluminum ions (Al3+). Breeding samples identified in this study, can be used as a promising initial material for development the new varieties with stable inheritability of the aimed trait.
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