Screening of sex in asparagus at early growth stages

Abstract

To determine the sex of asparagus (Asparagus officinalis) at the seedling stage, an easy, economical, and reliable method was developed. We used a modified single-step DNA extraction protocol, which resulted in a crude extract containing sufficient genomic DNA for use as a template. The male-specific marker (Asp1-T7sp) is a dominant marker and may lead to false negatives caused by an incomplete reaction; therefore, a multiplex polymerase chain reaction (PCR) was developed using a ribosomal RNA gene marker. The resulting banding pattern distinguished males from females without false negatives. To determine the best tissue for extraction of template DNA, phylloclades (a specialized stem that resembles and functions like a leaf) or root tips of individual asparagus plants were collected and weighed. A 4.0-mg phylloclade sample or a 0.8-mg root sample provided sufficient DNA for PCR analysis of asparagus. Root excision at day 19 did not affect subsequent growth of asparagus seedlings after 28 days. The method can determine the sex of asparagus at day 19 after seeding. A combination of single-step DNA extraction from root tips and multiplex PCR made for a simple and reliable screening method.