2009, vol. 62, 63–70 Miłosz Smolik, Marcin Kubus ISSR analysis of chosen Gleditsia accessions obtained from Polish collections Received: 25 June 2009; Accepted: 22 October 2009 Abstract: Species of Gleditsia show considerable morphological variability that makes them difficult to distin- guish using either vegetative or floral characters. Honeylocusts, especially the thornless cultivars, are popular ornamental, shade, street, attractive landscape trees. In this study the ISSR technique was used to evaluate the range of genetic variability between seven genotypes of Gleditsia cultivated in Polish dendrological collec- tions [Gleditsia caspica Desf., Gleditsia japonica Miq., Gleditsia japonica Miq. var. korainensis (= G. korainensis Nakai), Gleditsia triacanthos L., Gleditsia triacanthos L. (bulk), Gleditsia triacanthos f. inermis (L.) Zabel (bulk). Forty ISSR primers were tested and 18 were selected for their ability to produce clear and reproducible pat- terns of multiple bands. A total of 177 loci of 260–2600 bp were amplified, of which 89 (50%) were polymor- phic, 14 (8%) monomorphic and 74 (42%) were accession-specific. Accession-specific ISSR loci were ob- tained for all of the seven accessions tested. A dendrogram generated using the UPGMA, based on a similarity measure of total character difference, showed that the Gleditsia accessions were clustered into two main groups (‘a’ and ‘b’). The first grup – ‘a’ – included: Gleditsia triacanthos L., Gleditsia triacanthos L. (bulk) and Gleditsia triacanthos f. inermis (L.) Zabel (similarity 0.61–0.75), the second – ‘b’ – included 2 species: Gleditsia ja- ponica and Gleditsia japonica var. korainensis (similarity 0.43). Analysis of the phylogenetic similarity dendrogram has shown wide range of diversity between studied accessions. The clustering pattern obtained in our experiment was in agreement with the data based on morphological, allozyme and ITS analysis. Additional key words: Gleditsia, genetic variability, fingerprinting, ISSR Address: M. Smolik, Department of Horticultural Plant Breeding, M. Kubus Department of Dendrology and Landscape Architecture, West Pomeranian University of Technology, Janosika 8, 71-424 Szczecin, Poland, e-mail: [email protected] Introduction dial symmetry (rarely encountered in this family) and fruits in the form of pods. The Gleditsia genus (locust trees), belonging to the The North American honey locust (Gleditsia tria- Caesalpinioideae subfamily of the legume (pea) family canthos L.), its thornless form [Gleditsia triacanthos f. Fabaceae (=Leguminosae), encompasses approximately inermis (L.) Zabel] as well as a few thornless cultivars twelve (14) species with natural stands in North are cultivated throughout Poland, but they are not America (2), South America (1), northern India (1), very common (Seneta 1996, Czekalski and Danie- along the Caspian Sea in Azerbaijan and Iran (1) and lewicz 1997). several species in east and southeast Asia (Schnabel Under the conditions existing in Poland, this tree et al. 2003). These are trees (rarely shrubs) with char- goes through the full cycle of vegetative and genera- acteristic twice-pinnate or pinnately compound tive development ending with the formation of seeds leaves, thorns, polygamous dioecious flowers with ra- capable of germination (Czekalski and Danielewicz 64 Miłosz Smolik, Marcin Kubus 1997, Kubus 1999). Other Gleditsia species, i.e., Cas- al. 2002, Rajesh et al. 2002, Sudupak 2004), Morus pian locust (Gleditsia caspica Desf.), Japanese honey lo- species (Vijayan and Chatterju 2003), lilacs of various cust (Gleditsia japonica Miq.) and Korean honey locust morphologies and phenotypes (Rzepka-Plevneš et al. (Gleditsia japonica Miq. var. korainensis), are rarely cul- 2006) or maples from the Przelewice Arboretum col- tivated in Poland, mostly in botanical gardens and ar- lection (Rzepka-Plevneš et al. 2007). boreta, and no reference to their blossoming and fruit In the present study, ISSR was used to determine bearing can be found in the literature. variability, fingerprints and phylogenetic dependencies The morphological features of Gleditsia genus taxa between locust tree taxa from Polish dendrological col- are strongly diversified (Rehder 1960, Krüssmann lections. Furthermore, attempts were made to deter- 1960). However, unequivocal determination of the mine the genetic relationship between the specimen species affiliation of trees that have not undergone described by Bojarczuk and Zieliński (1980) and the generative development is difficult, as in the case of remaining genotypes in question and to compare the the locust trees (Gleditsia sp.) described by Bojarczuk honey locust and its thornless form (G. triacanthos f. and Zieliński (1980), which have a characteristic co- inermis) on the basis of descriptive genotyping of bulk lumnar growth habit. Instability of sex expression is samples of their DNA. The presented research repre- observed in honey locust (Michener 1986, Kubus sents a preliminary attempt to address these topics. 1999), and approximately 60% of seedlings of thorn- less forms (G. triacanthos f. inermis) do not develop the Materials and methods thorns characteristic of the species if they are propa- gated vegetatively (Fowells 1965, Blair 1990). Plant material representing seven locust trees was In this study, an attempt was made to determine obtained from various Polish collections, including: the scope of genotypic variability within selected Gleditsia caspica Desf., Gleditsia japonica Miq., Gleditsia specimens of the Gleditsia genus from various regions japonica Miq. var. korainensis (= G. korainensis Nakai), of the world that are cultivated in Poland (excluding Gleditsia triacanthos L. – pure species obtained from cultivars). The ISSR technique described by Zietkie- Arboretum of Polish Academy of Science in Kórnik wicz et al. (1994) was used. This technique amplifies (central part of Poland), Gleditsia triacanthos L. (bulk), DNA sequences located between tandem repetitive Gleditsia triacanthos f. inermis (L.) Zabel (bulk) and microsatellite sequences. Gleditsia sp. In the experiment in question, bulk DNA These sequences, or more precisely the variability samples were formed by mixing equal amounts of within them and their occurrence in introns, exons, DNA isolated separately from young leaves of five centromeres, and telomeres (Condit et al. 1991), trees identified as Gleditsia triacanthos L. and four trees have become in recent years the objects of a number identified as Gleditsia triacanthos f. inermis (L.) Zabel of studies and were used to define the genetic varia- growing in various stands in the city of Szczecin tion and phylogenetic relationships between acces- (north-west Poland). Morphological characteristics sions of Douglas-fir of various origins (Tsumura et al. and origin of the aforementioned accessions were 1996), eucaplyptus germplasm (Van der Nest et al. listed in the Table 1. 2000), tea (Mondal 2002), Cicer germplasm (Iruela et Table 1. Origin of the plant’s material Parameters: high The age of tree Accesion Origin Place of groving/appearance [m]/circuit of strump [years] [cm] at 1.3 m high Gleditsia caspica Desf. the south coasts of Caspian Sea Arboretum of WULS-SGGW 48 11.5/43 Erewan Kanaker, Botanical Gar- in Rogów near Łódź den, Armenia (central Poland) Gleditsia japonica Miq. Japan Kasukabe, Kanagawa Arboretum of WULS-SGGW 32 2.8/5.5 Botanical Garden in Rogów near Łódź the shrub – with no (central Poland) central trunk Gleditsia japonica Miq. var. North Korea Pyong-Yang H.B. Arboretum of WULS-SGGW 37 2/6 korainensis (= G. Botanical Garden in Rogów near Łódź the shrub – with no korainensis Nakai) (central Poland) central trunk Gleditsia triacanthos L. Canada, Toronto Arboretum in Glinna near 16 4.2/17 (the natural position) Szczecin (northwest Poland) Gleditsia triacanthos f. USA (the provenience Since 1926 in Botanical Garden – about 85 17/213 inermis L.(Zabel) of the seeds is unknown) Szczecin (northwest Poland) Gleditsia sp. (the provenience of the seeds Arboretum of PAS in Kórnik about 75 13/124 is unknown) near Poznań (central Poland) WULS – Warsaw University of Life Science (SGGW) PAS – Polish Academy of Science ISSR analysis of chosen Gleditsia accessions obtained from Polish collections 65 The total genomic DNA from about 200 mg of the character ‘1’ means the presence of a specific band fresh leaf material was extracted using the Genomic and ‘0’ represents its absence (Gelquant/MiniBis Pro Mini AX Plant kit (A&A Biotechnology). ISSR-PCR – Bio Imaging Systems – USA). The Nei and Li (1979) mixtures (25 µl) contained: 2.0 mM MgCl2, 100 mM algorithm contained in the TREECON computer KCl, 20 mM Tris-HCl pH 8.3, 0.1% Triton X-100, 1 package software was used to calculate the genetic out of 40 different 0.2 µM primers (UBC – University distances between the Gleditsia accessions (Van de of British Columbia primer sets), 0.2 mM of each Peer and De Wachter 1994). Rooted phenograms dNTP (Fermentas), 1.0 units of Taq DNA polymerase were constructed by the UPGMA (unweighted pair (Fermentas) and 100 ng template genomic DNA. The group with arithmetic mean) method, and the robust- contaminating RNA was removed by digestion with ness of the tree topology was assessed by 2,000 boot- RNase A (20 mg · cm–3). DNA was amplified using a strap resamplings (Felsenstein 1985). Mastecycler 5333 (Eppendorf) thermal cycler using the following programme: initial denaturation at Results 94°C for 7 min, 40 cycles of 30s at 94°C, 50 s at an- nealing temperature, 2 min at