300 Echenard et al.: Sex Identification in Ginkgo biloba Arboriculture & Urban Forestry 2008. 34(5):300–307. A New and Improved Automated Technology for Early Sex Determination of Ginkgo biloba Vincent Echenard, François Lefort, Gautier Calmin, Robert Perroulaz, and Lassaad Belhahri Abstract. Random amplified polymorphic DNA (RAPD) technique with male associated decamer primer S1478 was used to amplify DNA from 72 leaf samples collected from Ginkgo biloba trees with known sexual determinism in the canton of Geneva, Switzerland. This marker was found to be male-specific and was lacking in all female plants. Automated random polymorphic DNA analysis (ARPA), a new automated technology developed in the frame of this work, proved highly effective in distinguishing males and females with 100% efficiency and successful in male and female discrimination from a collection of young seedlings derived from a sexual cross. Our findings provide unambiguous evidence that ARPA combined with the male-associated decamer primer S1478 could be considered an efficient, rapid, and easy method to make an early sex determination in the dioecious tree Ginkgo biloba. The maidenhair tree Ginkgo biloba is a widely popular decidu- ous and dioecious gymnosperm species that is native to China and has been cultivated for well over a millennium. Ginkgo biloba has been described as a “living fossil” because it is known to have existed early in the Jurassic period and is believed to be the sole survivor of the ancient family of Ginkgoaceae (Carrier et al. 1998; Zhou and Zheng 2003). In Asia, G. biloba is used in traditional medicine and its seeds, also known as Ginkgo nuts, are considered a delicacy and are also a popular cuisine item (Kiple and Ornelas 2000). Asian people systematically planted the tree and many living ginkgoes are known to be more than five centuries old in China and for approximately 1,000 years in Japan. Ginkgo was introduced to Europe in the 18th century where it was very popular in large cities like London and Paris (Rohr 1989). The oldest European ginkgo would have been sown in 1730 in the Utrecht Botanical Garden. Most of the earlier trees raised in Europe appear to have been males. The first recorded female tree was found in Bourdigny near Geneva in 1814 of which scions were grafted on a male tree in the Botanic garden of Montpellier where the first perfect seed has grown (Michel 1985). G. biloba had an ability to survive pests, drought, storms, ice, and city soils (Aoki 1997; Handa et al. 1997; Honda 1997; Kim et al. 1997). Because it does not have invasive roots and requires minimum maintenance, it could be the perfect urban tree with a large potential planting range in temperate and subtropical cli- mates in both hemispheres. The ginkgo is dioecious. That simply means that there are separate male and female plants (Laurain 2000). Female trees produce seeds that emit a noxious, foul odor on falling to the ground. The smell’s description ranges from “rancid butter” to “vomit” and therefore female trees are con- sidered to be undesirable (Wada and Haga 1997). The slippery pulp can also be a liability. These features caused city govern- ments to actually remove and ban the female from being planted. Male ginkgoes do not produce a fruit and are selected as the main cultivars used to transplant in urban communities. This male cultivar of Ginkgo casts dense shade and the dense crown makes it suitable as a screen or noise buffer. It makes a durable ©2008 International Society of Arboriculture street tree where there is enough overhead vertical space to ac- commodate the large size. The availability of male and female associated random am- plified polymorphic DNA (RAPD) markers such as the decamer primer S1478 (Jiang et al. 2003) prompted us to validate a reli- able marker by testing it over a large collection of trees in the Canton of Geneva that originates from China as well as Japan. Jiang et al. (2003) screened 1200 random decamers, which gen- erated 8,372 RAPD primers among which they found two male- linked markers RAPD markers. One of these markers, S1478, was validated for sex determination in Ginkgo trees from two Chinese regions The need for a more rapid and reproducible method of early sexual determinism in G. biloba led us to de- velop an automated version of RAPD, which previously relied on manual agarose gel electrophoresis and DNA detection by ethidium bromide staining. Automated random polymorphic DNA analysis (ARPA) has several advantages over the former method, thus increasing its usefulness as a technique for assess- ing the sexual determinism in G. biloba. The new method was found to be highly reproducible at all levels of replication tested. In this study, a capillary electrophoresis system was used to resolve large (up to 682 bp) ARPA–polymerase chain reaction size fragments from plant genomic DNA, representing a new application for this automated system. Therefore, ARPA was shown to be an efficient, easy, and cost-effective means for processing a large collection of G. biloba seeds for early sex determination. MATERIALS AND METHODS Plant Material A total of 72 G. biloba trees of identified sex collected from Geneva, Carouge, Chêne-Bougeries, Choulex, Cologny, Corsier, Grand-Lancy, Grand-Saconnex, Jussy, Meinier, Meyrin, Nyon, Onex, Petit-Lancy, Petit-Saconnex, Vandoeuvre (Canton, Geneva) were used in this study. These are trees planted in different streets in the listed localities (Figure 1) and were lo- cated before the flowering period. A picture of each tree was taken on four occasions in each season from 11 November 2006
September 2008
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