Genetic Diversity of Ornamental Allium Species and Cultivars Assessed with Isozymes
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J Appl Genet 49(3), 2008, pp. 213–220 Original article Genetic diversity of ornamental Allium species and cultivars assessed with isozymes Agnieszka Krzymiñska1, Magdalena Gaw³owska2, Bogdan Wolko2, Jan Bocianowski3 1Department of Ornamental Plants, University of Life Sciences, Poznañ, Poland 2Institute of Plant Genetics, Polish Academy of Sciences, Poznañ, Poland 3Department of Mathematical and Statistical Methods, University of Life Sciences, Poznañ, Poland Abstract. The aim of the study was to verify the similarity of 13 species and 5 cultivars of ornamental alliums and classify them into groups based on morphological and isozyme variation. The work embraced: Allium aflatunense, A. caeruleum, A. christophii, A. giganteum, A. karataviense, A. moly, A. nigrum, A. pyrenaicum, A. rosenbachianum, A. schubertii, A. siculum (syn. Nectaroscordum siculum), A. sphaerocephalon, A. strictum, A. stipitatum ‘Album’, A. ‘Ivory Queen’, A. ‘Lucy Ball’, A. ‘Mont Blanc’, and A. ‘Purple Sensation’. Scape length, inflorescence diameter, and flowering period were recorded. Isozyme marker polymorphism was assessed by starch gel electrophoresis. Eight polymorphic isozyme systems (AAT, GPI, PGM, ALAT, ACP, DIAP, ALDO, PGD) were selected from 16 analysed in the taxa. Besides the differences between the taxa, the isozymes revealed intraspecific polymorphism in 5 systems. A total of 37 markers were obtained and used for dendrogram construction. The most similar taxa were A. karataviense with A. ‘Ivory Queen’, and A. karataviense with A. christophii (similarity level 0.78). A high similarity of 11 taxa belonging to one group (A. aflatunense, A. christophii, A. giganteum, A. karataviense, A. nigrum, A. schubertii, A. ‘Ivory Queen’, A. ‘Lucy Ball’, A. stipitatum ‘Album’, A. ‘Mont Blanc’, A. ‘Purple Sensation’) suggested that this group could be identified with the subgenus Melanocrommyum. Keywords: genetic similarity, intraspecific polymorphism, isozyme variation, ornamental alliums, phylogenetic analysis Introduction morphism: apart from Allium and Amerallium also Caloscordum, Melanocrommyum and Rhizirideum The genus Allium encompasses 700 species and is (Samoylov et al. 1999). The subgenus Melano- distributed all over the Northern Hemisphere. crommyum was divided into 16 sections (Fritsch Apart from the familiar agricultural crops, many 1997). ornamental species belong to this genus, but its Three of the species of the subgenus taxonomy is uncertain (Hong et al. 1996). Melanocrommyum included in this study were Three subgenera were separated on the basis of classified earlier by using GISH and RAPD morphological and anatomical characters: Allium, (Friesen et al. 1997). The RAPD technique was Amerallium and Nectaroscordum (Traub 1968, applied also by Hong et al. (1996) in the analysis 1972). A closely related species, A. siculum (syn. of ornamental alliums, including 5 species from Nectaroscordum siculum), having a characteristic the set of taxa evaluated in this study. In 1997, smell, is in some classifications excluded from the Dubouzet et al. analysed alliums from the subge- genus Allium because of the leaf vascular bundle nus Rhizirideum (not present in our analysis) ac- pattern. Relatively recently, 5 subgenera were dis- cording to dot blot hybridization with randomly tinguished on the basis of chloroplast DNA poly- amplified DNA probes. Dubouzet and Shinoda Received: February 20, 2007. Accepted: February 15, 2008. Correspondence: A. Krzymiñska, Department of Ornamental Plants, University of Life Sciences, D¹browskiego 159, 60–594 Poznañ, Poland; e-mail: [email protected] 214 A. Krzymiñska et al. (1999) grouped Old and New World alliums ac- planted in rows in the first half of October. Each cording to ITS DNA sequence. Three species from taxon was planted in 3 rows, each containing this study were analysed in the above paper. 10 bulbs, so a total of 540 bulbs were planted. Isozymes were applied in Allium sativum L. by The bulbs originated from the Horticultural Farm Pooler and Simon (1993), Maass and Klaas of Mr Bogdan Królik (Poland), the Dutch com- (1995), and Ipek and Simon (2002). pany Toolen, and the Polish company Haubitz The aim of this study was to verify the similar- Polska. Cultivation conditions were equal for all ity of 13 species and 5 ornamental cultivars, and to plants. During the growing period, both morpho- classify them into particular groups based on mor- logical and phenological characters were ob- phological and isozyme variation. We report on served. The paper presents only selected the usefulness of isozymes for assessment of simi- characters, which are most important: scape larity of species and cultivars, in comparison to length, inflorescence diameter, and flowering morphological estimation. date. The first 2 characters were subjected to a one-way analysis of variance, while means were clustered by using Duncan’s test at a significance Materials and methods level of a = 0.05. GenStat software (GenStat 1993) was used for statistical analysis. Plant material Isozyme electrophoresis Our investigations covered 13 species and 5 cultivars of ornamental alliums: with purple flow- For the genetic analysis, 5 plants were chosen ran- ers, Allium aflatunense, A. christophii, A. giganteum, domly among the 30 plants of each taxon, which A. rosenbachianum, A. sphaerocephalon, A. ‘Lucy had been evaluated morphologically and Ball’, A ‘Purple Sensation’; with light pink flowers phenologically. The top sections (10-cm-long) of A. pyrenaicum, A. strictum, A. karataviense; with young leaves (2–4-week-old) were collected in pink-purple flowers A. schubertii; with blue flowers 2003. Electrophoresis was run on 11% starch gels A. caeruleum; with yellow flowers A. moly; with (Gottlieb 1973). Fresh leaf materials for AAT white flowers A. nigrum, A. stipitatum ‘Album’, (aspartate aminotransferase), ALAT (alanine amino- A. ‘Ivory Queen’, A. ‘Mont Blanc’; and with yel- transferase), EST (esterase), GPI (glucosephosphate Table 1. Taxonomic classification of evaluated Allium species and cultivars Species/cultivar Subgenus Section References A. aflatunense Melanocrommyum Megaloprason Linne von Berg et al. 1996 A. caeruleum Allium Caerulea Klass and Friesen 2002 A. christophii Melanocrommyum Kaloprason Linne von Berg et al. 1996 A. giganteum Melanocrommyum Megaloprason Dubouzet and Shinoda 1999 A. karataviense Melanocrommyum Miniprason Klass and Friesen 2002 A. moly Amerallium Molium Linne von Berg et al. 1996 A. nigrum Melanocrommyum Melanocrommyum Klass and Friesen 2002 A. pyrenaicum A. rosenbachianum Melanocrommyum Friesen et al. 1997 A. schubertii Melanocrommyum Kaloprason Hong et al. 1996 A. siculum Nectaroscordum Dubouzet and Shinoda 1996 A. sphaerocephalon Allium Allium Linne von Berg et al. 1996 A. strictum A. stipitatum 'Album' Melanocrommyum Megaloprason Linne von Berg et al. 1996 A. 'Ivory Queen' A. 'Lucy Ball' Melanocrommyum Friesen et al. 1997 A. 'Mont Blanc' A. 'Purple Sensation' Melanocrommyum Friesen et al. 1997 low-green flowers A. siculum. Table 1 presents their isomerase), LAP (leucine aminopeptidase), MDH classification according to published literature. (malate dehydrogenase), ME (malic enzyme), PGM Alliums were grown in the field in the (phosphoglucomutase), and PRX (peroxidase) anal- 2001/2002 and 2002/2003 seasons. Bulbs were yses were extracted in tris-maleate buffer. Isozymes in ornamental alliums 215 The potassium phosphate extraction buffer was used enzyme activity staining can be found at Wolko for ACP (acid phosphatase), ALDO (aldolase), and Œwiêcicki (1987). DIAP (diaphorase NADH), FUM (fumarase), IDH Polymorphism was interpreted as an absent or (isocitrate dehydrogenase), PGD (6-phospho- present band. The results were combined in a 0/1 gluconate dehydrogenase), and SKDH (shikimate data matrix and analysed by using GenStat soft- dehydrogenase). Fresh tissue was crushed, the crude ware (GenStat 1993). The coefficient of genetic extract was absorbed by filter paper wicks similarity was determined in the 18 ornamental (Whatman 31ET) and applied to the starch gel. Paper Allium taxa on the basis of the variability of 37 wicks were removed from the gel after 10 min of zones of enzymatic activity, defined as markers. electrophoresis. Four different buffer systems were The coefficients were calculated from the equation applied in order to optimise electrophoresis condi- (Nei and Li 1979): GAB =2NAB /(NA +NB), where tions for each analysed enzyme system. Samples N is the number of bands shared by objects A extracted by using tris-maleate buffer (Tris-amino- AB and B; N is the number of scored bands of object methane, maleic anhydride, polyvinylpyrrolidone A A; and N is the number of scored bands of b B (PVP-40), glycerol, TritonX-100, -merkapto- object B. The coefficients were used for hierarchi- ethanol) were placed on both A/B and T system. cal clustering by using the unweighted pair group The potassium phosphate extraction buffer method with arithmetic means (UPGMA). The re- (dipotassium hydrogen orthophosphate, hydro- lationships among species and cultivars were pre- chloric acid, contained the same components like sented in the form of a dendrogram. tris-maleate buffer) was used for samples sub- jected to electrophoresis on both H and C systems (Wolko 1995). System A/B was applied according Results to Selander et al. (1971), system T according to Weeden (1987), system C according to Clayton and Tretiak (1972), and system H according to The analysed Allium species and cultivars differed in Cardy et al. (1980). Electrophoresis