Townsend Et Al.: Aflp Analysis of Dna from Dried Hop Cones

technique to identify accurately hop cultivars. Traditionally, hops cultivar identification has been verified Hop (Humulus lupulus L.) cones are used extensively in beer by either a chemical analysis of lupulin glands, or by brewing to enhance flavor and impart bittering. Verifying hops cultivar identity has traditionally been accomplished by morphological characmorphological characteristics. Both traits are influenced teristics or a chemical analysis of lupulin glands but these traits may by environmental conditions during cone development vary according to environmental influences. The objective of this which, in turn, can lead to difficulty in cultivar identificaresearch was to develop an AFLP (amplified fragment length polytion (Hartl and Seefelder, 1998). Since hop cultivars are morphism) protocol for analyzing DNA extracted from dried hop clonally propagated and all individuals within a cultivar cones. The DNA was extracted from dried cones of six hop genotypes are genetically identical, DNA fingerprinting using a by a technique published for grapes (Vitis spp). The reagents, MseI molecular marker approach would be an ideal way to primers, and protocol were part of a commercially available kit, while distinguish among hop cultivars. the 6-carboxyfluorescein-labeled EcoRI primers were purchased separately. Eleven primer combinations amplified an average of 546.5 The AFLP (amplified fragment length polymorscorable fragments with an average of 49.7 fragments per primer phism) technique (Vos et al., 1995) is a recent innovation combination. All genotypes were differentiated with the primer comthat has proven both powerful and reliable. This techbinations studied. Average genetic similarity estimates ranged benique has several advantages over earlier marker techtween 0.956 and 0.995 among the six hop genotypes studied. This nologies: very little genomic DNA is required, many research provides the hops industry with a powerful technique to polymorphisms can be generated per reaction, prior verify accurately hops cultivar identity and purity through an analysis knowledge of specific DNA sequences is not needed, of dried cone DNA. and the technique has high reproducibility (Vos et al., 1995). In a comparison among AFLP, RFLP (restriction fragment length polymorphism), and RAPD (random H are used in beer brewing to impart bittering, amplified polymorphic DNA) analysis of 14 soybean enhance flavor, and as a preservative. The plant [Glycine max (L.) Merr.] cultivars, Lin et al. (1996) organs of interest to brewers are the female flowers, or reported AFLP to average six polymorphic bands in cones, which contain lupulin glands with a chemical 60 of the 64 primer pairs tested, RAPD produced a profile desirable for brewing. The wide variation in polymorphic band with only 35% of the primers tested, chemical profiles found among hop cultivars gives the and more than 50% of the RFLP probes failed to distinbrewer flexibility to impart specific flavor and aroma guish a polymorphic band. In other work, Lu et al. characteristics to beer (Neve, 1991). (1996) found that AFLP generated more polymorphic One of the challenges faced by both the brewing inbands per primer or probe than other molecular dustry and hop breeders has been obtaining a reliable marker technologies. The AFLP technique has been successfully used to M.S. Townsend, Dep. of Crop and Soil Sci. Oregon State Uni., Corvallis, OR 97331-3002; J.A. Henning and D.L. Moore, USDA-ARS, differentiate genetically populations or individuals Corvallis, OR 97331-3002. Contribution of the USDA-ARS. Received within numerous species including tea (Camellia sinensis 15 Nov. 1999. *Corresponding author ([email protected]). L.) (Paul et al., 1997), cotton (Gossypium barbadense L.) (Feng et al., 1997), tef (Eragrostis tef (Zucc) Trotter) Published in Crop Sci. 40:1383–1386 (2000). 1384 CROP SCIENCE, VOL. 40, SEPTEMBER–OCTOBER 2000 Table 1. Primer sequences used for AFLP analysis of DNA extracted from dried hop cones.