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DNA Extraction from a Single Seed for Marker-Assisted Selection in Squash

American Journal of Plant Sciences 2021年第12期12卷 1912-1925页

作者： Isabella Martinez Vincent N. Michael Yuqing Fu swati shrestha Geoffrey Meru Horticultural Sciences Department and Tropical Research & Education Center University of Florida Homestead FL USA

Marker-assisted selection is an important tool in squash (Cucurbita species) breeding. A seed-based genotyping system would not only allow selection of desirable individuals prior to planting, but also reduce the cost associated with leaf-derived DNA genotyping, such as the need for greenhouse facilities and ultra-low-temperature storage freezers. A robust seed-based genotyping system requires a non-destructive sampling method and DNA of sufficient quantity and quality for marker-assisted selection. In the current study, six cultivars representing Cucurbita pepo (Black Beauty and Yellow Crookneck), C. moschata (Butterbush and Fairytale), and C. maxima (Buttercup and Big Max) were used to develop a suitable seed-based genotyping system for squash. Seed chips for DNA extraction were sampled by removing 1/3 of the distal end, while the remnant seed-embryos were sowed to assess germination potential. Four extraction methods including two column-based commercial kits (CTAB and ENZA) and two detergent-based conventional methods (CTAB and SDS) were assessed for DNA quality and quantity. Utility of extracted DNA for downstream applications was tested by genotyping with SSR and SNP markers. There was no significant difference in germination percentage between whole and cut seeds across the six cultivars. The average DNA concentration across methods ranged from 11.6 ng/μL to 62.6 ng/μl, while the DNA quality (A260/280) ranged from 0.89 to 1.95. Although DNA was obtained for all the extraction methods, only EZNA and Favorgen methods yielded DNA of sufficient quality for marker-assisted selection.

关键词： Cucurbita Genotyping DNA Markers SNP SSR

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