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Developing PCR-RFLP and SNP based markers for determining cytoplasmic male sterile factors in the genus Amaranthus (Amaranthaceae)

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Developing PCR-RFLP and SNP based markers for determining cytoplasmic male sterile factors in the genus  amaranthus. (Amaranthaceae)

* Selvaraj Dhivya and Ramalingam Sathishkumar

Amaranthus L. is a herbaceous plant comprising approximately 70 species, with three subgenera, which contains both cultivated and wild types. The cultivated species are used for food grains, leafy vegetables, potential forages and ornamentals. Genetic diversity analysis in amaranths is important for development germplasm of core set with a widely diverse population and effective utilization of plant genetic resources. Cytoplasmic male sterility (CMS) is one of the most important traits in crop breeding, which has been used for commercial seed production by F1 hybrid cultivars of Amaranthus species. Our aim is to develop a polymerase chain reaction-based restriction fragment length polymorphism (PCR -RFLP) marker to distinguish male-fertile (N) and male-sterile (S) cytoplasm in Amaranthus species by examining the distribution of the haplotypes in diverse breeding lines, cultivars, and wild. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm (either N/S)-containing varieties with restriction enzyme revealed and distinguished the N and S with functional and substitution cytoplasmic site. The developed PCR-RFLP marker was validated for cytoplasmic male sterile factors in 15 samples belonging to the wild and cultivated cultivars, which showed CMS-specific sequence-characterized amplified region (SCAR) marker. The PCR-RFLP marker can identify N- or S-cytoplasms in DNA sample mixtures in which they are 10-fold less, indicating that the use of the marker has diagnostic precision. We also confirmed the efficacy of the SNP detected in the psbA gene for high throughput discrimination of CMS factors using Real-time PCR. This approach is useful for the identification of CMS factors in large amaranthus breeding populations which facilitate in crop's improvement.

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