Bot. Bull. Acad. Sin. (2002) 43: 63-68

Hao et al. Taxonomical relationships in Allium sect. Sacculiferum

A study of taxonomical relationships among species of Korean Allium sect. Sacculiferum (Alliaceae) and related species using inter-simple sequence repeat (ISSR) markers

Gang Hao1, Dong-Hee Lee2, June Seung Lee2, and Nam Sook Lee2,*

1South China Institute of Botany, The Chinese Academy of Sciences, Guangzhou 510650, P. R. China

2Department of Biology, Ewha Womans University, Seoul 120-750, Korea

(Received July 2, 2001; Accepted September 13, 2001)

Abstract. Morphological differences among Korean Allium subgenus Rhizirideum sect. Sacculiferum and some related species are minor, making species delimitation difficult. Inter-simple sequence repeat (ISSR) was employed to assess genetic diversity and relationships. Thirty-four accessions, representing six taxa of A. thunbergii, A. sacculiferum, A. deltoides-fistulosum, A. cyaneum var. cyaneum, A. cyaneum var. deltoides, A. anisopodium, were sampled in this study. The ISSR markers revealed high polymorphism among taxa studied. In the phenetic analysis, accessions of A. sacculiferum and A. deltoides-fistulosum are nested within A. thunbergii accession group; A. cyaneum var. cyaneum and A. cyaneum var. deltoides are separated from the above sect. Sacculiferum group; the status of putative A. anisopodium is less clear, with a position between sect. Sacculiferum and A. cyaneum. We propose that A. deltoides-fistulosum and A. sacculiferum be sunk into A. thunbergii and that the putative A. anisopodium be recognized as A. cyaneum var. deltoides.

Keywords: Allium; Genetic diversity; Korea; Phenetic analysis; Sect. Sacculiferum.

Introduction

Section Sacculiferum is small in the large subgenus Rhizirideum of Allium, but poorly delimited. When Gritzenko (1979) proposed this section, it included two species, A. sacculiferum Maxim. and A. komarovianum Vved, both characterized by globose to ovate bulbs, simple coriaceous bulb coats, 3-angular or keeled flat leaves, subglobose rose or violet flowers, simple stamens much longer than the tepals, deep nectary grooves at the base of the ovary covered by hood-like projections, and finally flat obovate seeds (Hanelt and Fritsch, 1994). Gritzenko's (1979) two species were subsequently sunk into A. thunbergii, a rather broadly circumscribed species (Xu, 1980; Xu et al., 1990). Xu (1980) erroneously placed A. thunbergii in sect. Haplostemen, together with the commonly cultivated East Asiatic species A. chinense, a very close relative. In the most recent infrageneric classification of Allium, Hanelt and Fritsch (1994) broadened the concept of sect. Sacculiferum by including A. chinense, and A. virgunculae, a related Japanese species, within it.

In the revision of Korean Allium (Yu et al., 1981), eight species and one variety were recognized in subgenus Rhizirideum, viz: A. thunbergii, A. sacculiferum, A. deltoides-fistulosum (sect. Sacculiferum), A. cyaneum var. cyaneum, A. cyaneum var. deltoides, A. splendens (sect.

Reticulato-bulbosa), A. anisopodium (sect. Anisopodia), A. senescens (sect. Rhizirideum), A. victorialis (sect. Anguinum). Morphologically A. splendens, with its tooth-like scale of the inner filament, and the latter two species, A. senescens with stalk before flowering, and A. victorialis having flat ovate shaped leaf, are distinct from others in the Korean taxa of subgen. Rhizirideum. For sect. Sacculiferum, the species status of A. sacculiferum was maintained, and A. deltoides-fistulosum was newly described (Yu et al., 1981). The morphological differences among most of those species, however, are minor, making species identification difficult. For example, some populations occurring in the Mt. Dukyu region were labeled A. cyaneum var. cyaneum with interstamenous appendage and wax powders on the leaf surfaces, and cross section of leaves semi-circular and hollow, while populations in Mt. Kaya and Isl. Cheju were proposed as A. cyaneum var. deltoides without interstamenous appendage and wax-powders on the leaf surfaces, and cross section of leaves triangular and hollow. Populations distributed in Mt. Sorak were recognized as A. anisopodium with leaf cross-section narrow and flat and solid (Yu et al., 1981), despite the fact that they resemble A. cyaneum var. deltoides in many other respects (Woo, 2000).

Molecular markers have proven useful in clarifying genetic relationships within subgenus Rhizirideum (Dubouzet et al., 1997; Raamsdonk et al., 200), but sect. Sacculiferum remains intact in those studies. Until recently, a preliminary phylogenetic study of sect.

*Corresponding author. Tel: 82-2-3277 2366; Fax: 82-2-3277 2385; E-mail: namsook@mm.ewha.ac.kr


Botanical Bulletin of Academia Sinica, Vol. 43, 2002

Ge and Sun, 1999), and the procedures are simpler than AFLPs. ISSR markers may offer considerable variation among varieties and have been widely used in cultivated species (reviewed in Wolfe and Liston, 1998). The aims of the present study are: (1) to demonstrate the utility of ISSR markers for taxonomical relationship studies among closely related species; (2) to clarify the specific status of taxa in sect. Sacculiferum and related taxa, and (3) to elucidate the identity of some uncertain taxa by ISSR analysis.

Materials and Methods

Plant Materials

Thirty four accessions, representing six taxa as recognized by Yu et al. (1981) (A. thunbergii, A. sacculiferum, A. deltoides-fistulosum, A. cyaneum var. cyaneum, A. cyaneum var. deltoides, A. anisopodium), were sampled in this study (Table 1). Allium spendens of sect. Reticulato-bulbosa, A. senescens of sect. Rhizirideum, and A. victorialis of sect. Anguisnum were not included due to their relative distinctiveness. Sect. Sacculiferum has been chosen asthe core group, as well as some other

Sacculiferum and related species employing ITS sequences (Woo, 2000) revealed that A. thunbergii, A. sacculiferum, A. deltoides-fistulosum and the Dukyu accession of A. cyaneum var. cyaneum were closely related and indistinguishable in the ITS tree; they constituted a polytomy, with A. cyaneum var. deltoides as the sister group, and the Bisundae accession of putative A. anisopodium furthermore became their sister group, whereas the Russian accession of A. anisopodium (material from Royal Botanical Garden, Edinburgh) deviated notably and was placed on another clade. A succinct taxonomy based on morphological characters and ITS sequence data is not available for sect. Sacculiferum and related species.

In this study we report an inter-simple sequence repeat (ISSR) analysis on the genetic diversity and relationships of sect. Sacculiferum and some related species. ISSR analysis is a PCR- based technique, similar to RAPDs and AFLPs (Wolfe and Liston, 1998). With primers composed of microsatellite sequences and using higher annealing temperature, ISSR markers have better reproducibility than RAPDs (Fang and Roose, 1997; Ratnaparke et al., 1998;


Hao et al. Taxonomical relationships in Allium sect. Sacculiferum

polymorphism at the intraspecific level was calculated as the proportion of polymorphic loci to the total number of loci scored in all accessions of the same taxon. The computer program POPGENE (Yeh et al., 1997) was used to estimate the expected heterozygosity (HE) (Nei, 1973) and Nei's (1972) genetic identity (I). The UPGMA (unweighted pair-group method with arithmetical averages) dendrogram of Nei's (1972) genetic distance was constructed using POPGENE.

Results

ISSR Diversity

A total of 93 loci were scored for the 15 SSR primers in the 34 accessions of Allium surveyed. The locus number varied from 3 to 10 per primer (averaging 6.6), with fragment size ranging from 300 to 1,500 bp. Among the taxa studied, A. thunbergii has the highest expected heterozygosity (HE = 0.268) and ISSR polymorphism (82%), while A. deltoides-fistulosum has the lowest (HE = 0.159; ISSR polymorphism = 50.9%) (Table 2). Nei's (1972) genetic identities (I) between taxa range from 0.650 ( A. deltoides-fistulosum vs. A. cyaneum var. cyaneum 1) to 0.868 ( A. thunbergii vs. A. sacculiferum ), with a mean identity of 0.7810.061 (Table 3).

Genetic Relationships

In the phenetic analysis based on genetic distances, the 34 accessions of Allium cluster into two main groups (Figure 1): one includes 23 accessions and is furthermore divided into two subgroups, one for the three taxa of sect. Sacculiferum (A. thunbergii, A. sacculiferum and A. deltoides-fistulosum), the other one for the five accessions

closely related species in subgen. Rhizirideum. Wherever possible 5-6 accessions were sampled for each taxon. Taking into account the grouping of Dukyu accession of A. cyaneum var. cyaneum with sect. Sacculiferum in the ITS tree (Woo, 2000), one accession of A. cyaneum var. cyaneum originating from China was also sampled from Royal Botanical Garden, Edinburgh. Korean plant materials including bulbs were collected in the wild and cultivated in the greenhouse of Ewha Womans University, while A. cyaneum var. cyaneum from Royal Botanical Garden, Edinburgh was germinated by seeds.

Genomic DNA Extraction and PCR Amplification

Total DNA was extracted from fresh leaf tissue, following the 2 CTAB method (Doyle and Doyle, 1987). One hundred SSR primers from the Biotechnology Laboratory, University of British Columbia (UBC primer set no. 9) were screened, and 15 primers (Nos. 807, 808, 809, 810, 811, 817, 825, 835, 841, 850, 851, 857, 861, 864, 891) were finally used. Amplification was performed in a volume of 20 l containing 1.5 mM MgCl2, 2% formamide, 200 nM primer, 0.75 units of Taq polymerase, and 20 ng of genomic DNA. The following cycle program was adopted: an initial 5 min at 94C, followed by 30 s at 94C, 45 s at 50-55C (depending on primers used), 1.5 min at 72C for 35 cycles, and 7 min at 72C for a final extension. The amplified products were separated on 1.5% agarose gel and detected by staining with ethidium bromide. The gels were photographed under UV light with Polaroid film 667.

Data Analysis

The amplified unambiguous bands were scored manually to compile a presence/absence matrix. Percentage of


Botanical Bulletin of Academia Sinica, Vol. 43, 2002

of A. anisopodium; the other main group consists of 11 accessions of A. cyaneum var. cyaneum and A. cyaneum var. deltoides, in which the Chinese accession of A. cyaneum var. cyaneum groups with the Korean accessions of A. cyaneum var. cyaneum.

Discussion

ISSR Markers

Essentially, ISSR markers make possible the genome-wide estimation of genetic diversity of individuals, allowing us to produce a large amount of data in a short time. ISSR techniques have been widely applied to assess genetic diversity in several economically important crop and fruit plants, and in biological conservation. Occasionally, it has been used to study relationships at the interspecific level (Huang and Sun, 2000). The present study demonstrates that ISSR can provide a clear portrayal of relationships among closely related congeneric species. In a previous study of sect. Sacculiferum using ribosomal

DNA ITS gene sequence data (Woo, 2000), the relationships of the three species of sect. Sacculiferum, A. thunbergii, A. sacculiferum, A. deltoides-fistulosum, were not completely resolved, largely due to the insufficiency of nucleotide substitution, although ITS sequences have proven useful for resolving relationships at the generic and infrageneric levels (Baldwin et al., 1995). That ISSR markers should be used with caution in systematic study is pointed up by the fact that while ISSR always offers a great deal of variation within and among populations, a high level of polymorphisms may also introduce a high level of homoplasies in some genetically divergent species. A compromise approach is to reduce the number of polymorphic loci per primer by optimizing the amplification condition and to sample a large number of accessions per species.

Genetic Relationships

The naturalness of the species group of A. thunbergii-A. sacculiferum-A. deltoides-fistulosum was somewhat supported by the phenetic analyses (Figure 1). The highest ISSR diversity was found in A. thunbergii (Table 2), a species widely distributed in China, Japan, Korea, and Mongolia. The highest diversity of A. thunbergii is concordant with the fact that a high genetic diversity value might be related to wide distribution (Hamrick, 1989). ISSR markers indicate that species of A. sacculiferum and A. deltoides-fistulosum, as now recognized, originated from within A. thunbergii. Given that these three taxa have slight morphological differences, this is not unexpected. Allium sacculiferum occurs in Korea, Mongolia, and eastern Siberia, differing from A. thunbergii by its short leaf sheath, cross section of leaf laminar, and keel. Xu (1980) and Xu et al. (1990) merged A. sacculiferum into A. thunbergii, although a thorough study of the infraspecific variability was appealed.

Allium deltoides-fistulosum was described by Yu et al. (1981) with the diagnostic characters of a cross section of leaf triangular and the leaf upright. It has apparent resemblance with A. thunbergii, and Woo (2000) reduced it to a variety of A. thunbergii (although not yet formally described). The ISSR markers provided additional information to clarify taxonomical relationships among the three taxa of sect. Sacculiferum. In the phenetic analysis (Figure 1), each of the six accessions of A. sacculiferumand A. deltoides-fistulosum constitute a division respectively, and are nested within the A. thunbergii accession group. ISSR markers indicated that A. sacculiferum and A. deltoides-fistulosum are not sufficiently divergent to merit species status. The value of genetic identity (I = 0.868) between A. sacculiferum and A. thunbergii is higher than that of a congeneric level (I = 0.65-0.70), but lower than a conspecific value (I = 0.950) (Crawford, 1990), although the ISSR estimates may not be directly comparable to those obtained with isozymes. The genetic identity between A. thunbergii and A. deltoides-fistulosum is almost the same as that between A. sacculiferum and A. thunbergii. We are inclined to

Figure 1. UPGMA dendrogram for 34 accessions representing six taxa in this study based on ISSR data. Taxon abbreviations are given in Table 1.


Hao et al. Taxonomical relationships in Allium sect. Sacculiferum

amplified DNA probes. Theor. Appl. Genet. 95: 1223-1228.

Fang, D.Q. and M.L. Roose. 1997. Identification of closely related citrus cultivars with inter-simple sequence repeat markers. Theor. Appl. Genet. 95: 408-417.

Ge, X.J. and M. Sun. 1999. Reproductive biology and genetic diversity of a cryptoviviparous mangrove Aegiceras corniculatum (Myrsinaceae) using allozyme and intersimple sequence repeat (ISSR) analysis. Mol. Ecol. 8: 2061-2069.

Gritzenko, P.P. 1979. Novye taksony v rode Allium L. Nauchno-Tekhn. Byull. Vsesoyusn. Ordena Lenina Ordena Druzhby Narodov Nauchno-Issl. Inst. Rasteniev. N. I. Vavilova 87: 22-24.

Hamrick, J.L. 1989. Isozymes and the analysis of genetic structure in plant populations. In D.E Soltis and P.S. Soltis (eds.), Isozymes in Plant Biology. Dioscorides Press, Portland, Oregon, pp. 87-105.

Hanelt, P. and R. Fritsch. 1994. Notes on some infrageneric taxa in Allium L. Kew Bull. 49: 559-564.

Huang, J.C. and M. Sun. 2000. Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA. Theor. Appl. Genet. 100: 1050-1060.

Nei, M. 1972. Genetic distance between populations. Amer. Natu. 106: 283-292.

Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proc. Nat. Acad. Sci. USA 70: 3321-3323.

Raamsdonk, L.W.D van, M.V. van, Ginkel, and C. Kik. 2000. Phylogeny reconstruction and hybrid analysis in Allium subgenus Rhizirideum. Theor. Appl. Genet. 100: 1000-1009.

Ratnaparke, M.B., M. Tekeoglu, and F.J. Muehlbauer. 1998. Inter-simple-sequence- repeat (ISSR) polymorphisms are useful for finding markers associated with disease resistance gene clusters. Theor. Appl. Genet. 97: 515-519.

Wolfe, A.D. and A. Liston. 1998. Contributions of PCR-based methods to plant systematics and evolutionary biology. In D.E. Soltis, P.S. Soltis, and J.J. Doyle (eds.), Plant Molecular Systematics II. Kluwer, Boston, pp. 43-86.

Woo, H.K. 2000. A Taxonomy of Korean Allium Based on the Morphology and ITS Sequences. M. Phil Thesis. Department of Biology, Ewha Womans University, Seoul, Korea.

Xu, J-M. 1980. Allium L. In F-T. Wang and T. Tang (eds.), Fl. Reipubl. Popularis Sin. Science Press, Beijing, Vol.14, pp. 170-272. (in Chinese)

Xu, J-M., P, Hanelt, and C-L. Long. 1990. Key to the Alliums of China. Herbertia 46: 140-164.

Yeh, F.C., R.C. Yang, T.B.J. Boyle, Z.H. Ye, and J.X. Mao. 1997. POPGENE, the User-friendly Shareware for Population Genetic Analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Alberta.

Yu, S.O., S.T. Lee, and W.T. Lee. 1981. A taxonomic study of the Allium species in Korea. J. Korean Pl. Taxonomy 11: 21-41.

maintain one species for this group of taxa, echoing the treatment of Xu (1980) and Xu et al. (1990). While A. deltoides-fistulosum could be reduced to variety status (Woo, 2000), the same delimitation is plausible for A. sacculiferum.

While in the ITS study (Woo, 2000) A. cyaneum var. cyaneum is not distinctive from the species group of sect. Sacculiferum, an obvious boundary divides them in the ISSR analysis (Figure 1). The Chinese accession of A. cyaneum var. cyaneum unexceptionally groups with Korean accessions, and in turn groups with the division of accessions of A. cyaneum var. deltoides. The status of putative A. anisopodium, however, is less clear. In the phenetic analysis (Figure 1) its position is intermediate between sect. Sacculiferum and A. cyaneum (sect. Reticulato-bulbosa). Morphologically, Korean A. anisopodium resembles A. cyaneum var. deltoides with respect to ratio of leaf and scape lengths, ratio of perianth and filament lengths, and pedicel. Allium anisopodium is well presented in China, central Asia, Mongolia, Siberia, and North Korea. The occurrence of A. anisopodium in South Korea was likely to be misidentified. The distinction of ITS sequences between Russian and Korea accessions (Woo, 2000) lent support to this judgment. The putative A. anisopodium populations in the Bisundae region may appropriately be recognized as A. cyaneum var. deltoides. More analyses of the genetic diversity of unambiguous materials of A. anisopodium from outside Korea would be desirable before any conclusion is drawn.

Acknowledgements. This research was supported by grants from Korea Research Foundation (KRF-1999-015-DI 0085) and Women Universities of the National RND Project (98-N6-01-01-A-09). We are grateful to: Dr. Reinhard Fritsch for providing two of the reference papers; Dr. Nikolai Friesen, Dr. Xue-Jun Ge and Dr. Jun-Chao Huang for invaluable advice; and Royal Botanical Garden, Edinburgh, for providing some Allium samples.

Literature Cited

Baldwin, B.G., M.J. Sanderson, J.M. Porter, M.F. Wojciechowski, C.S. Cambell, and M.J. Donoghue. 1995. The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Ann. Missouri Bot. Gard. 82: 247-277.

Crawford, D.J. 1990. Plant Molecular Systematis. John Wiley and Sons, New York.

Doyle, J.J. and J.L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11-15.

Dubouzet, J.G., K. Shinoda, and N. Murata. 1997. Phylogeny of Allium L. subgenus Rhizirideum (G. Don ex Koch) Wendelbo according to dot blot hybridization with randomly


Botanical Bulletin of Academia Sinica, Vol. 43, 2002