Assessment of Genetic Relationships Among Sexual and Asexual Forms of a Ilium Cepa Using Morphological Traits and RAPD Markers

Heredity 78 (1997) 403—409 Received 4 April 1996

Assessment of genetic relationships among sexual and asexual forms of A ilium cepa using morphological traits and RAPD markers

MAUD LE THIERRY DENNEQUIN*, OLIVIER PANAUD, THIERRY ROBERT AND AGNES RICROCH Laboratoire Evolution et Systématique, bâtiment 362, Université Paris-Sud. URA CNRS 2154, 91405 Orsay cedex, France

Thespecies Allium cepa includes two major crops on the basis of morphological traits and typical reproduction mode: sexually reproduced biennial onions and vegetatively propagated perennial shallots which rarely flower. In addition, the seed-propagated shallot, a recently released variety with intermediate phenotype for life history, has been described and used by breeders. A joint analysis using molecular markers (random amplified polymorphic DNA, RAPD) and morphological characters was undertaken for the assessment of genetic diversity and crop classification among Allium cepa genotypes. A morphological study describing growth. and development was carried out on a nested sampling (34 accessions) from two geographical origins (European or tropical) and of different plant status (improved varieties or local ecotypes). Four primers generated 24 reproducibly scorable DNA bands which gave individual fingerprinting for representative accessions of both onions and shallots. Our results indicate that the seed-propagated shallot is more closely related to onions than to vegetatively propagated shallots and, moreover, reveal a geographical structure of genetic diversity. The evolutionary significance of these data is discussed.

Keywords:Alliumcepa, diversity, onion, RAPD, reproductive strategy, shallot.

Introduction of the species into three botanical groups (Hanelt et al., 1992): the Common Onion group, constituted by Thedomestication process of Allium cepa is very sexual reproductive onions, the Aggregatum group, ancient, as shown by the presence of onions in Egypt formed with vegetatively propagated shallots, and 5000 years ago. According to Zeven & Zhukovsky the Proliferum group, which presents vegetatively (1975), there is either a single centre of origin for propagated onions producing bulblets (topset). both onions and shallots located in central Asia or Asexual shallots have been placed in the species two different centres located in the same area. A. ascalonicum; however, its interfertility with A. Nowadays selection and isolated cultivation have cepa (Atkin, 1953) was the reason for considering produced a large spectrum of locally adapted vari- both as the same species (A. cepa). The existence of eties with respect to daylength response for bulbing. those two interfertile forms suggests the presence of Local ecotypes display a high genetic variability for an evolutionary continuum between a sexual form traits such as photoperiod, precocity of bulbing and and a vegetatively reproduced form selected against bulb dormancy (Rouamba et al., 1993). However, flowering during the domestication process. Onions the influence of geographical distribution in respect and asexual shallots can be differentiated by of daylength response on the structure of genetic morphological characters (single, large bulb for diversity is insufficiently studied. onions vs. bulblets for shallots), by their reproduc- The species A. cepa displays different reproductive tive mode (sexual reproduction vs. asexual reproduc- modes: sexual reproductive onions (allogamous and tion) and by their life cycle (biennial for onions vs. protandrous) and vegetatively propagated shallots. perennial for shallots). Recent classifications of A. cepa suggest a partition Taxonomy and genetic diversity within the genus Allium have already been studied by isozymes, *Correspondence. E-mail: [email protected] rDNA, restriction fragment length polymorphism 1997 The Genetical Society of Great Britain. 403 404 M. LE THIERRY D'ENNEQUIN ETAL.

(RFLP) and RAPD markers. Studies using isozymes per accession were analysed. For vegetatively propa- have measured genetic variability of between- and gated shallots three individuals per clone were within-population variation (Peffley & Orozco- studied. Among 21 shallot accessions, 14 accessions Castillo, 1987; Rouamba et at., 1993). Taxonomic were used in the morphological study. No morpho- studies carried out with RFLP markers revealed logical data were recorded on seven accessions that high differentiation between species, especially for flowered. For the seed-propagated shallot, 10 indi- species belonging to the section Cepa (Bradeen & viduals (both from seeds and bulblets) were Havey, 1995). RFLP markers have been used at an analysed. intraspecific level in order to analyse genetic similar- Characteristics describing growth and develop- ities between onions with different kinds of ment listed in Table 2 were chosen because they daylength response (Bark & Havey, 1995). Genetic have been shown to be valuable descriptors of onion relationships between A. cepa and A. fistulosum were and shallot life history (Messiaen et al., 1993). Every reported by Ricroch et at. (1992) using in situ hybrid- week, bulb and neck diameters of onions were meas- ization of rDNA sequences. ured during the development cycle in order to deter- RAPD markers are useful for determining the mine a bulb ratio. When maximal bulb diameter is distribution of genetic variability within and among twice minimal neck diameter (R2), it indicates the taxonomic units and have been applied for this onset of bulbing (Brewster, 1977). The number of purpose in different species, especially on clones days from sowing to R2 was calculated. Leaf number (Castiglione et al., 1993). They have been used in A. of onions and shallots was scored every week until sativum (MaaB & Klaas, 1995) and in A. cepa harvesting. The other characteristics were deter- (Wilkie et al., 1993; Bohanec et aL, 1995). mined at maturity. A plant was determined as In this study, we assessed the efficiency of using mature when 50 per cent of leaves wilted and were chosen morphological characters and RAPD recumbent on the ground. At harvest onion bulbs markers to examine the distribution of genetic diver- and shallot bulblets were dried at 80°C for 48 h and sity among several accessions of sexual and asexual the dry matter content was measured. forms of A. cepa representing a wide geographical range. Moreover, we addressed the question of Data analysis The data recorded from the 33acces- whether the seed-propagated shallot belongs to the sions of onions and vegetatively propagated shallots onion or shallot groups. were submitted to multivariate statistical analysis. The combined procedure described by Sarr & Materials and methods Pernès (1988) was used. This procedure involvesa principal component analysis, a clustering algorithm Growth conditions using the Euclidian distance calculated from the data matrix, and a discriminant analysis. It allows Seeds of 19 accessions of onions and bulbiets of 21 an accurate description of phenotypic variation and its shallot accessions were planted between 2 and 10 organization within the gene pool studied. February and cultivated under standard conditions in greenhouses at Paris-Sud University(France) (Table 1). During the period of growth and develop- Molecularstudy ment, the mean minimum and maximum tempera- Plantmaterial Eight European and tropical onion ture ranged from 15 to 32°C. For each form, accessions were chosen. They represented the ecotypes (coming from germp!asm collections) and daylength response spectrum of local ecotypes and improved varieties (currently under selection or improved varieties. Per accession, 1—10 individuals commercial varieties) from tropical and European were scored for RAPD analysis. This sampling origins were chosen. The seed-propagated shallot was the French commercial variety 'Cuisse de scheme ensured representation of a largegeographi- Poulet', cultivated in the western region of France. cal and ecotypic range. For shallots, the 14acces- Both seeds and bulblets were cultivated. sions representative of ecotypes and commercial clones were chosen. Because the shallotsample contained only 14 accessions (17 genetic individ- Morphologicalanalysis uals), the sample size was too small to assess the geographical distribution of the genetic variability. Plantmaterial The number of analysed individuals per accession was chosen according to reproductive For the seed-propagated shallot, eight of the 10 individuals tested in the morphologicalanalysis were mode. For sexually reproduced onions 10 individuals examined.

The Genetical Society of Great Britain, Heredity, 78, 403—409. ASSESSMENT OF DIVERSITY IN ALLIUM CEPA 405

Table1 Plantstatusand geographical origin of Allium cepa accessionsstudied ina morphological analysis (M) and in a molecular study (RAPD) Onions Ecotypes European origin Rosé de Roscoff 33A (France)', OP* 0-01 M RAPD Rosé de Roscoff 33B (France), OP 0-02 M Ralolette des Cévennes (France)a, OP 0-03 M Museau de lièvre 47A (France), OP 0-04 M RAPD Tropical origin Tintilou (BurkinaFaso)h, OP 0-05 M RAPD 4Ni (Niger)", OP 0-06 M Improved varieties European origin Jaune hâtif de Valence (Spain), OP 0-07 M Jaune espagnol (Spain), OP 0-08 M RAPD Rouge espagnol (Spain)d, OP 0-09 M Blanc de Paris (France)c, OP 0-10 M Auxone (France)e, OP 0-11 M RAPD Tropical origin Short day hybrid (USA)1, F,** 0-12 M Violet de Galmi Ma027 (Mauritania)1, OP 0-13 M RAPD Violetde Galmi 1634 (Ivory Coast)1, OP 0-14 M RAPD Violetde Galmi No5 (Senegal)1, OP 0-IS M Short day white (USA)1, OP 0-16 M Rio Solo (USA)", F, 0-17 M RAPD Rio Enrique (USA)h, F, 0-18 M Rio Radji red (USA)", F, 0-19 M Vegetatively propagated shallots Ecotypes European origin RAPD E2 (France) S-Ui M M E9 globe (France)' S-02 E14 (France) S-03 M M RAPD E15 long (France) S-04 M E18 globe (France) S-OS Tropical origin M Guinée (Guinea)' S-06 S-07 M Abidjan (Ivory Coast)1 M RAPD Brazzaville (Congo)1 S-08 M OAEG (Guinea)1 S-09 Improved varieties European origin RAPD El (France) S-lU S-li M RAPD E3 (France) RAPD E4 (France)' S-12 S-13 RAPD E7 (France) RAPD E10 S-14 globe (France)' M RAPD Eli globe (France)' S-iS S-16 M RAPD E13 long and 'grise'(France)' RAPD E16 experimental, globe (France)' S-17 S-18 RAPD E17 globe (France)' RAPD E19 experimental, globe (France)' S-19 S-20 M RAPD Echalotegrise (France)" Tropical origin M Oignon blanc (Ivory Coast)" S-21 Seed-propagated shallots European origin M RAPD Cuisse de poulet (France)', OP SP-1 Theorigins of accessions are indicated in brackets. Ecole National Supérieure d'Horticulture de Versailles (France); "Institut Nationald'Etudes en Recherche Agronomique (Burkina-faso); cClause (France);dVilmorin (France); Coopd'or (France); 'd palmers seed (USA); 1Tropicasem (Senegal); hRioColorado (USA); Institut National de Recherche Agronomique de Ploudaniel (France); 'Centre de cooperation Internationale en Recherche Agronomique pour le Développement de La Reunion (France); "Rungis market (France); 'Les jardiniers d'Anjou et du Poitou a Allonnes (France). *Open pollinated; **F, hybrid. The Genetical Society of Great Britain, Heredity, 78, 403—409. 406 M. LE THIERRY D'ENNEQUIN ETAL.

Table2Listof traits measured on onions and vegetatively propagated shallots

Onions Shallots

Leaf number (LN) Leaf number (per cluster of bulbiets) (LN) Leaf number at R2 (LNR2) Bulbiet diameter (BD) Bulb diameter (BD) Bulbiet height (BH) Bulb height (BH) Bulbiet fresh matter content (BFMC) Fresh matter content (FMC) Bulbiet dry matter content (BDMC) Dry matter content (DMC) Buiblet plate length (BPL) Plate length (PL) Buiblet number (BN) Axillary buds number (ABN) Days number from sowing to R2 (DNR2)

RAPD amplification only the dendrogram based on the Nei and Li distance is shown. The Boolean matrix was also DNAwas isolated from fresh leaf material (0.7 g) by submitted to factorial correspondence analysis a CTAB method based on that of Saghai-Maroof et (Morrison, 1990). This multivariate procedure is a!. (1984). One of four lO-mer oligonucleotides: similar to a principal component analysis except for OPA-04 (5'-AATCGGGCTG-3'), OPA-19 (5'- computation of coordinates along axes which is CAAACGTCGG-3'),OPA-20 (5'-GTTGCGATCC- based on a x2distance. 3') and OPB-11 (5'-GTAGACCCGT-3') (Operon technologies Inc., Alameda, California, USA) were used for each amplification. Conditions reported by Results Bohanec etal.(1995) were improved. Each reaction (251tL)consisted of 1 m of MgCI2, 4 m each of Morphology and life history dATP, dCTP, dGTP, dTTP (Boehringer-Manheim, The life history of the seed-propagated shallot was Germany), 400 flM primer, 0.5 U of Taq DNA poly- observed from sowing seeds or from planting bulbs. merase (Appligene-Oncor, France), 1 x reaction From seed, one large bulb was obtained which buffer (1 mrvi MgCI2, 20 mrvi Tris HCI pH 8.0, ethyle- rapidly divided to produce a cluster of small bulbs. nediaminetetraacetic acid (EDTA) 1 mi, dithio- When one bulb was planted it further divided to threitol 1 m, glycerol 50 per cent). The reaction form a cluster of small bulbs, followed by flowering mix was overlaid with a drop of mineral oil and in the second year. incubated in a thermal cycler (thermal cycler 480, The principal component analysis based on Perkin Elmer-Cetus, USA) programmed as follows: morphologicalcharacters 48 cycles of 1 mm denaturation at 94°C, 1 mm (datanot shown) annealing at 37°C and 1.5 mm extension at 72°C, suggested that phenotypic diversity was organized in two and three major clusters for onions andvege- followed by final extension at 72°C followed bya tatively propagated shallots, respectively. This struc- cooling at 4°C. Tubes containing all reaction ture was confirmed by a clustering algorithm products except template DNA were used as followed by a discriminant analysis which alloweda control. Ten millilitres of amplification products final classification (Table 3). were electrophoresed in 3 per cent agarose gels in In onions, the most discriminant variableswere 0.5 x TBE. We used qX DNA digested with HaeIH bulb fresh matter content (FMC) and bulb as a molecular weight marker. height (BH), both descriptors of bulb yield. Moreover, BH is a morphological character reflecting selection for Dataanalysis bulb shape. Geographical distinction was less obvious for vegetatively propagated shallots than for Eachamplification was repeated three times. The onions. Three clusters were discriminated by three visible and reproducible bands were scored as major variables: bulbiet plate length (BPL), leaf present (1) or absent (0). Similarity indices (F) from number (LN) and buiblet fresh matter content Jaccard (1908) and Nd & Li (1979) were calculated. (BFMC). Clusters 1 and 2 included all the European Genetic distances (1-F) were used to build a dendro- accessions (both improved varieties or local gram according to the UPGMA clustering procedure. ecotypes); the first one was composed of accessions Because results using the two indices were similar, that presented a very good yield, and the secondone

The Genetical Society of Great Britain, Heredity, 78, 403—409. ASSESSMENT OF DIVERSITY IN ALLIUM CEPA 407 was formed by low yield accessions which have high Bohanec et a!., 1995). These four primers generated values for variables depicting life history (LN, BPL). a total of 24 reproducible bands scored for statistical Cluster three contained all the tropical accessions. analysis. An example of patterns using primer OPA They were characterized by low yield values similar 19 for different accessions of onion shows polymor- to the values of cluster 2. Variables (LN and BFMC) phism within and between accessions (Fig. 1). involved in the discriminant analysis of vegetatively RAPD analysis did not identify any band specific to propagated shallots describe carbohydrate allocation one of the three forms (onions, vegetatively propa- from leaves to storage organs. Concerning the seed- gated shallots or the seed-propagated shallot). propagated shallot, we noticed the emergence of However, some bands were more frequent in one both seeds and bulblets (topsets) within umbels. form than in another. The dendrogram (Fig. 2) and the two factorial component analyses (data not shown) from analysis Differentiationof sexual and asexual forms based of RAPD bands showed a differentiation between on RAPD markers the 'echalote grise' (S-16 and S-20), the vegetatively Thechoice of the four primers in this present study propagated shallots and the onions. The seed-propa- was based on the polymorphism revealed in previous gated shallot is closer to onions than to vegetatively studies on Allium species (Wilkie et al., 1993; propagated shallots. The differentiation is mainly in

Table 3 Results of discriminant analysis for onions and shallots based on morphology and life cycle traits

Onions Shallots

Cluster number 2 3 100 % of well-classified individuals 100 Discriminant power of the canonical variables 0.93 0.95; 0.73 The more discriminant variables FMC, BH* BPL, LN, BFMC* S-01, S-02, S-03, Cluster 1 0-01, 0-02, 0-03, 0-04, 0-09, 0-10, S-is, S-16 0-11 S-04, S-il, S-OS, Cluster 2 0-05, 0-06, 0-07, 0-08, 0-12, 0-13, S-20 0-14, 0-15, 0-16, 0-17, 0-18, 0-19 S-06, S-07, S-08, Cluster 3 S-09, S-21

*See Table 2 for abbreviations. 1 2 3 4 5 6 78 910 11121314151617

1353 Fig. 1 Patterns obtained with one 1078 primer (OPA 19) for individuals of 872 three accessions of onions. Lane 1: ladder; lanes 2—6: DNA from five indi- 603 viduals of accession 'Jaune espagnol'; lanes 7—11: DNA from five individuals of accession 'Violet de Galmi 1634'; lanes 12—17 from six individuals of accession 'Violet de Galmi Ma027'.

The Genetical Society of Great Britain, Heredity, 78, 403—409. 408 M. LE THIERRY D'ENNEQUIN ETAL.

S-is e S-20 e mined by the bulb ratio (R2) could not be scored. The sexual characteristic of its life history prevented s-oat I: S-lie S-14 any comparison with the vegetative reproduction of 5-12 S-Ia the asexual form. However, this first part of the s-al S-lie study confirmed geographical differentiation both s-Is 8 S-04e S-I9e I for onions and vegetatively propagated shallots. S-13e Within onions the two clusters reflected European 0-lieS-18e 0-171 and tropical origins, with the exception of two Euro- 0-131 pean varieties ('Jaune hâtif de Valence' and 'Jaune 0-01 e - espagnol') within the tropical cluster. The latter are short-day varieties with a low daylength requirement for bulbing that can be cultivated in tropical areas

sp-1 and in southern Europe. Differences between clusters probably reflect variation in selection for yield characteristics such as bulb shape. A similar phenomenon was observed within the asexual form, 0-080 which may have either a large number of small bulblets or a small number of large bulbiets. Concerning 0-04e the seed-propagated shallot, the presence of bulbiets (topsets) was probably caused by stressed growth 0-131 conditionsin greenhouses. This reproductive

0-04e strategy is a trait observed in many wild taxa such as 0-131 A. vineale, rarely in the Common Oniongroup but still frequently in the Proliferum group. 0-14th The use of neutral markers allowed the seed- 0-04e propagated shallot to be compared with onions and 0-1411: vegetatively propagated shallots. Not surprisingly, 0.08e the dendrogram (Fig. 2) showed that differentiation 0-051 in relation to reproductive mode was more ancient than the geographical differentiation. The 'echalote Fig.2 Dendrogram showing the differentiation of onions, grise' (S-16 and S-20)neverflowers in natural condi- seed- and vegetatively propagated shallots (see Table 1 tions, although flowering can be induced by severe for accession details) built based on the Nei and Li stress. Its divergence from the other vegetatively distance (e, european origin; t, tropical origin). propagated shallots has already been suggested from morphological characteristics such as bulb skin, umbel morphology and root type (Messiaen et a!., 1993). Our results are in agreement with this obser- relation to the reproductive mode; but within each vation. The molecular study suggests that the seed- form differentiation by geographical origin (tropical propagated shallot should be classified in the onion or European) was observed. cluster. Furthermore the dendrogram revealed that this sexual form was closer to European onions than Discussion to tropical ones. The The geographical differentiation found in the morphological study showed that the seed-prop- morphological study was confirmed by RAPD agated shallot constitutes an intermediate form with polymorphism. Rouamba (1992) has proposed that life history of both sexual and asexual forms. The from the primary centre of diversity located in life cycle of the seed-propagated shallot is biennial central Asia two independent pathways of migration and characterized by bulblet (topsets) production to Europe and to Africa led to adaptive radiation of (similar to vegetative growth of vegetatively propa- onions. If this hypothesis is valid, our results show gated shallots) followed by seed production (similar that gene flow has probably occurred between Euro- to onions). The presence of a transient stage in its pean and tropical gene pools after their initial life history characterized by the formation of a single differentiation. large bulb is not comparable to the onset of bulbing Further studies on the diversity found in wild of onions. Consequently, the onset of bulbing deter- species (such as A. pskemense or A. vavilovii) will

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