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

作　　者：Isabella Martinez Vincent N. Michael Yuqing Fu Swati Shrestha Geoffrey Meru 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)

机构地区：[1]Horticultural Sciences Department and Tropical Research & Education Center, University of Florida, Homestead, FL, USA

出　　处：《American Journal of Plant Sciences》2021年第12期1912-1925,共14页美国植物学期刊（英文）

摘　　要：Marker-assisted selection is an important tool in squash (<i>Cucurbita</i> 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 <i>Cucurbita</i> <i>pepo</i> (Black Beauty and Yellow Crookneck), <i>C</i>. <i>moschata</i> (Butterbush and Fairytale), and <i>C</i>. <i>maxima</i> (Buttercup and Big <span>Max) were used to develop a suitable seed-based genotyping system for squash.</span> Seed chips for DNA extraction were sampled by removing </span><span style="font-family:"">1/3</span><span style="font-family:""> of the distal end, while the remnant seed-embryos were sowed to assess germination potential. <span>Four extraction methods including two column-based commercial kits (CTAB</span> 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 (A_260/280) 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.Marker-assisted selection is an important tool in squash (<i>Cucurbita</i> 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 <i>Cucurbita</i> <i>pepo</i> (Black Beauty and Yellow Crookneck), <i>C</i>. <i>moschata</i> (Butterbush and Fairytale), and <i>C</i>. <i>maxima</i> (Buttercup and Big <span>Max) were used to develop a suitable seed-based genotyping system for squash.</span> Seed chips for DNA extraction were sampled by removing </span><span style="font-family:"">1/3</span><span style="font-family:""> of the distal end, while the remnant seed-embryos were sowed to assess germination potential. <span>Four extraction methods including two column-based commercial kits (CTAB</span> 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 (A_260/280) 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 

分 类 号：R73[医药卫生—肿瘤]