Asparagus is an economically important perennial, dioecious vegetable crop. Male asparagus plants usually produce greater spear yields than females (Ellison, 1986; Moon, 1976; Sinton and Wilson, 1999). Male plants do not create the weed problem that results from asparagus seedlings from female plants (Ellison, 1986). The volunteer asparagus seedlings reduce the F1 genetic purity of a production field, often resulting in long-term reductions in quality and vigor (Walker et al., 1999). On the other hand, female plants produce thicker spears (Uesugi et al., 1992; Uragami, 1988). However, the geographic isolation of males and females can produce the desired quality of spears and prevent seed production. Seedlings must, however, be grown for 1–2 years after transplantation until flowering to distinguish male from female plants (Sneep, 1953). The sex-determining locus in asparagus is referred to as M (the male-determining gene) and m (the female-determining gene). Females are homogametic (mm), and males are heterogametic (Mm). Supermales (MM) are desirable for the production of all-male asparagus progeny and to ensure that female plants are not transplanted into the field. Self-pollination of andromonoecious plants (Sneep, 1953) or doubled haploid lines produced by anther and/or microspore culture (Falavigna, 1979; Falavigna et al., 1990, 1996; Inagaki et al., 1980; Shiga et al., 2009; Torrey and Peirce, 1983) have made it possible to breed supermale asparagus plants. Thus, the practical application of an all-male hybrid cultivar has progressed from possibility to actuality, for example the cultivars Gijnlim (bred in The Netherlands) and Zuiyu (Uragami et al., 2011). The limited number of all-male cultivars remains a problem because those available lack adaptations to a cropping type for white or green asparagus, have reduced quality, lack resistance to local pathogens, and lack the ability to adapt to a variety of climates. Indeed, a mix of male and female plants is still planted in most of the asparagus fields.
The development of molecular markers linked to sex-determining chromosome segments would enable the simplification and promotion of the breeding of supermale individuals and the cultivation of males only. Many attempts to identify genetic markers linked to sex determination in asparagus have been undertaken. Such studies have used isoenzyme markers (Maestri et al., 1991); restriction fragment length polymorphism markers (Biffi et al., 1995); randomly amplified polymorphic DNA (RAPD) markers (Gebler et al., 2007); RAPD and sequence-characterized amplified region (SCAR) markers (Jiang and Sink, 1997); and amplified fragment length polymorphism, SCAR markers, or both (Jamsari et al., 2004; Nakayama et al., 2006; Reamon-Büttner et al., 1998; Reamon-Büttner and Jung, 2000). The male-specific marker (Asp1-T7) was first detected as being closely linked to the sex-determining locus by Jamsari et al. (2004). It was developed as a common marker, Asp1-T7sp, in many types of cultivars by Nakayama et al. (2006). Furthermore, this marker was applied to a loop-mediated isothermal amplification method (Shiobara et al., 2011). However, the practical use of primers for Asp1-T7sp is sometimes reduced by the presence of false negatives, where a male plant is wrongly scored as female. This misrecognition is mainly the result of an incomplete PCR reaction, caused by the quality and/or quantity of DNA template, and/or contamination by inhibitors. To overcome this, a multiplex PCR reaction would be a useful way to amplify the target fragment together with a reaction control. Multiplex PCR has been developed for the detection of different pathogens (Fraaije et al., 2001; Pastrik, 2000; Winton and Hansen, 2001). Multiplex PCR was used to determine the sex of papaya (Carica papaya) plants (Parasnis et al., 2000; Urasaki et al., 2002). We also tried to develop an improved, rapid, and easy method of DNA extraction from asparagus. In almost all studies of asparagus sex markers, a cetyltrimethyl ammonium bromide (CTAB) method, with or without modification (Gebler et al., 2007; Jiang and Sink, 1997; Nakayama et al., 2006; Reamon-Büttner et al., 1998), or a commercial kit based on benzyl chloride method (Shiobara et al., 2011) has been used for DNA preparation. These methods are time-consuming and involve high costs for some reagents and kits. This problem could be solved using a small-scale crude extraction because PCR screening does not require purified DNA. Thomson and Henry (1995) reported single-step plant DNA extraction for PCR. We applied this method to save cost and time. Previously, samples for DNA extraction have been limited to phylloclades or stalks in asparagus because aboveground parts could be collected easily from potted or field-grown plants. We examined the use of roots for crude DNA samples to hasten the date for sex determination before the production of seedlings.
In this study, a practical screening of sex in asparagus at early growth stages was established by the application of simple DNA extraction and multiplex PCR from root tips of young seedlings.
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