TITLE:
DNA Extraction from a Single Seed for Marker-Assisted Selection in Squash
AUTHORS:
Isabella Martinez, Vincent N. Michael, Yuqing Fu, Swati Shrestha, Geoffrey Meru
KEYWORDS:
Cucurbita, Genotyping, DNA Markers, SNP, SSR
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.12 No.12,
December
28,
2021
ABSTRACT: 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.