Two-Step Identification of N-, S-, R- and T-Cytoplasm Types in Onion Breeding Lines Using High-Resolution Melting (HRM)-Based Markers
High-resolution melting (HRM) analysis is a powerful detection method for fast, high-throughput post-PCR analysis. A two-step HRM marker system was developed for identification of the N-, S-, R- and T-cytoplasms of onion. In the first step for the identification of N-, S- and R-cytoplasms, one forwa...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-01-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/2/1605 |
| Summary: | High-resolution melting (HRM) analysis is a powerful detection method for fast, high-throughput post-PCR analysis. A two-step HRM marker system was developed for identification of the N-, S-, R- and T-cytoplasms of onion. In the first step for the identification of N-, S- and R-cytoplasms, one forward primer was designed to the identical sequences of both <i>cox1</i> and <i>orf725</i> genes, and two reverse primers specific to the polymorphic sequences of <i>cox1</i> and <i>orf725</i> genes were used. For the second step, breeding lines with N-cytoplasm were evaluated with primers developed from the <i>orfA501</i> sequence to distinguish between N- and T-cytoplasms. An amplicon with primers to the mitocondrial <i>atp9</i> gene was used as an internal control. The two-step HRM marker system was tested using 246 onion plants. HRM analysis showed that the most common source of CMS, often used by Russian breeders, was S-cytoplasm; the rarest type of CMS was R-cytoplasm; and the proportion of T-cytoplasm among the analyzed breeding lines was 20.5%. The identification of the cytoplasm of a single plant by phenotype takes from 4 to 8 years. The HRM-based system enables quick and easy distinguishing of the four types of onion cytoplasm. |
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| ISSN: | 1661-6596 1422-0067 |