Genetic Characterization of an Unknown Chinese Bulbous Leek-Like Accession and Its Relationship to Similar Allium Species
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HORTSCIENCE 40(6):1690–1694. 2005. similar to leek. We concentrated on an unusual accession received in Shanxi province, China, where it is seed propagated but used in diets as Genetic Characterization of an garlic. Studies included the majority of mor- phologically similar alliums, using karyotype Unknown Chinese Bulbous analysis, genomic in situ hybridization (GISH), pollination, genome size determination and Leek-like Accession and its molecular markers using nuclear rDNA and plastid cpDNA probes. Relationship to Similar Allium Species Material and Methods Borut Bohanec,1 Marijana Jakše, and Predrag Šesek University of Ljubljana, Biotechnical Faculty, Centre for Plant Biotechnology Plant material. In Shanxi province, China, in 1996, local agronomists gave B. Bohanec and Breeding, Jamnikarjeva 101, 1111 Ljubljana, Slovenia and M. Jakše two accessions of an Allium Michael J. Havey described as seed-forming garlic. No further information on the origin of these accessions Agricultural Research, U.S. Department of Argiculture, Department of Horticulture, could be obtained. These accessions obtained 1575 Linden Drive, University of Wisconsin, Madison, WI 53706 in Shanxi province in China will be referred to as chinese bulbous leek. Plants were raised Additional index words. Allium ampeloprasum, bulbous leek, great-headed garlic, karyotype, from seeds and maintained in the experimental fl ow cytometry, GISH, DNA polymorphism fi eld of the Biotechnical Faculty in Ljubljana. Abstract. Bulbous leek-like plants are a poorly defi ned group usually assigned to the Allium Sensory evaluation was done by 10 panelists, ampeloprasum complex. Studies were initiated to determine the origin of an unusual bul- each of whom was given bulbs of garlic and bous accession received in Shanxi province in China, where it was used in diet as garlic but chinese bulbous leek. The taste attributes propagated by seeds, and to genetically compare this accession with morphologically similar (aroma intensity, crispness, and pungency plants from Europe. Genetic analyses included karyotypes and genomic in situ hybridiza- (scale 1 = low, 5 = high intensity) were as- tion, pollination to leek, genome size determination and nuclear rDNA and plastid DNA sessed immediately after peeling and cutting polymorphisms. Results revealed that this agriculturally interesting accession from China samples. is a so far unknown variant within tetraploid A. ampeloprasum cultivated taxa. We also Leaves and bulb tissues were obtained from observed that great-headed garlic did not share derived states in the chloroplast with leek, A. polyanthum—two accessions (the fi rst from revealing that this cultivated plant does not possess the cytoplasm of leek or garlic, while its St. Mandrier sur Mer, France and the second 1C genome size was 17% bigger than those of studied leek and bulbous-leek accessions. from Ile d’Yeu, France—for the second, deter- mination of species was noted as not certain), The genus Allium L. contains about 700 headed garlic, is cultivated for its bulbs and A. commutatum (from Corsica), two accessions species, several of which are edible and are asexually propagated. Mathew (1996) states of great-headed garlic (from Spain and from among man’s most ancient cultivated crops. that great-headed garlic closely resembles A. India), and three forms of bulbous leeks—one The edible alliums and their areas of cultivation ampeloprasum and does not warrant classifi - from Ile d’Yeu (France), one from Apt (France) have been recently reviewed by Fritsch and cation as a separate species. Mathew (1996) and one from Guadeloupe were kindly provided Friesen (2002). The large number of Allium also described A. commutatum Guss. and by C.M. Messiaen (Le Teil, France), from his species and the diversity among the cultivated A. bourgeaui Rech. as closely related to A. personal collection. Additional samples that forms, which are spread across diverse climates ampeloprasum. Similarly Brewster (1994), were included in studies were bulb onion, throughout the Northern hemisphere, permits following the classifi cation proposed by Hanelt leek, garlic, bunching onion and A. tuberosum for the occasional discovery of new cultivated (1990), cites two major crop species within Rottl., which were either commercial varieties forms, not yet well described. Most botanical subgenus Allium section Allium: A. sativum or accessions used in other genetic studies at classifi cations of species were based on mor- (garlic) and A. ampeloprasum classifi ed into the University of Wisconsin. phological characterizations, however modern the four horticultural groups of leek, kurrat, Chromosome preparations. The root tips genetic techniques can provide new insights great-headed garlic and pearl onion. Based were excised from 1- to 2-cm-long roots ob- regarding phylogenetic relationships. on their use, Messiaen et al. (1993) classifi ed tained after germinating on moist fi lter paper Major crops within subgenus Allium section leek-like plants as A. polyanthum Schultes, at 25 °C. They were then treated by a 1% Allium are A. sativum L. (garlic) and the various A. ampeloprasum (sensu stricto), A. com- (v/v) solution of monobromonaphtalene for cultivated forms of A. ampeloprasum L. Ac- mutatum and A. scorodoprasum L.. These 24 h at 4 °C or by 2 mM 8-hydroxyquinoline cording to Mathew (1996), A. ampeloprasum authors mention that wild A. ampeloprasum for 0.5 to 2 h at room temperature followed is widespread through out the Mediterranean var. bulbiferum (from Yeu Island, off Western by storing for a further 0.5 to 2 h at 4 °C. The region and Middle Eastern countries; however, France) and Great Headed Garlic are chemi- tissue was fi xed in Farmer’s fi xative at for at populations have been recorded in other areas cally closer to garlic. least 24 h at 4 °C. Root tips were digested in possibly due to ancient introductions. Using The ploidy levels of bulbous leek-like plants 4% cellulase (Onozuka) and 1% pectolyase the botanical terminology of Matthew (1996), are highly variable. As recently reviewed by (Y23) for 45 min before squashing in 45% wild A. ampeloprasum is thought to be the Mathew (1996), the chromosome numbers of acetic acid. Cover slips were removed after progenitor form of crop plants A. porrum L. A. ampeloprasum ranged from 16 to 56, A. freezing at –80 °C and the slides were dried. For (leek) and A. kurrat Schweinf., both cultivated porrum from 32 to 48, A. kurrat at 32, A. com- karyotype analysis chromosomes were stained for their leaves. A third variant, termed great- mutatum from 16 to 48, and A. polyanthum at with 2 µg·mL–1 4'-6-diamidino-2-phenylindole 32. The predominant ploidy level among these (DAPI). The best quality preparations were species is tetraploid (32), while other ploidies used for chromosome counting and arranging Received for publication 7 Apr. 2005. Accepted are less frequent and may be associated with into karyograms on the basis of increasing for publication 21 June 2005. We are grateful to Lin vegetative propagation. Allium ampeloprasum, Rufa, Shanxi Academy of Agricultural Sciences and chromosome length. Measurements of chromo- Charles-Marie Messiaen (Le Teil, France) for provid- A. porrum and A. commutatum may possess some length were performed using the program ing accessions used in this study. This research was variable numbers of B chromosomes. Image-Pro Express 4.0. supported in part by project P4-0077. Our studies focused on the genetic char- Genomic in situ hybridization. Total ge- 1Corresponding author; e-mail borut.bohanec@bf. acterization of economically useful forms nomic DNA was isolated from young leaves uni-lj.si. of bulbous alliums with leaf morphologies using standard cationic detergent cetyl-tri- 1690 HORTSCIENCE VOL. 40(6) OCTOBER 2005 OOctober.indbctober.indb 11690690 88/11/05/11/05 99:00:16:00:16 AAMM methylammonium bromide (CTAB) method. et al., 1998) genome size for onion (33.5 pg using CTAB procedure as described by Saghai- Total genomic DNA from leek, bulbous leek, per nucleus) was used as standard from which Maroof et al. (1984). Digestions, transfers to garlic and great-headed garlic was labeled others including Vicia faba were calculated. fi lters, hybridizations, autoradiography, and with biotin using BioNick labeling kit (Life For measurements, samples of the studied scoring of polymorphisms were as previously Technologies) according to the supplier’s in- accession were mixed with species used as reported (Havey, 1991, 1992). structions. Total genomic DNA of garlic, leek standard, data are based on four repetitions, and and of the undetermined Allium species was analysis was completed within 3 to minimize Results fragmented by autoclaving for 6 min and used seasonal variation effect. as blocking DNA. Following standard RNase Hybridizations using nuclear rDNA and Morphological characteristics of an acces- and pepsin treatments, in situ hybridization cpDNA probes. Variation was assessed at sion of chinese bulbous leek. Chinese bulbous and probe detection were performed according previously characterized polymorphisms in leek formed, partially depending on growth to a procedure modifi ed from Puizina et al. the chloroplast and nuclear 45 s ribosomal (r) conditions, more or less typical leek-like (1999). Briefl y, chromosomes were denatured DNAs, which can distinguish A. ampelopra- pseudostems. At the end of the fi rst vegetation in 70% formamide in 2× SSC for 2 min at 70 sum and A. sativum. The nuclear 45s rDNAs year a single bulb (round) was formed (Fig. °C, dehydrated through a cold ethanol series were distinguished using polymorphisms at 1A). At the end of the second year of vegeta- and air dried. The probe mixture, containing or between DraI, EcoRI, EcoRV, and XbaI tive growth (Fig. 1B), bulbs were composed of 40 to 200 ng of biotinylated genomic DNA, (Havey, 1992). The cytoplasms were differenti- several cloves (Fig. 1C and D). Flower stalks 20 to 60 times that amount of blocking DNA ated using chloroplast characters 7, 9, and 13 were formed in the second growth year and (when used), 50% deionized formamide, 10% (Havey, 1991).