Variabilities in Karyotype and Molecular ISSR in Gonospermum Fruticosum (C

Variabilities in karyotype and molecular ISSR in Gonospermum fruticosum (C. Smith ex Link) Less., Argyranthemum coronopifolium (Willd.) Humphries and two strains of A. foeniculaceum (Willd.) Webb ex Schultz-Bip. (Asteraceae the tribe Anthemideae) collected in the Canary Islands

Irina V. Tatarenko1, Makiko Nishimura2, Masahiro Morikawa2, Harue Shinoyama3, Katsuhiro Suzuki2, Shino Kimura4, Ekaterina Tatarenko2, Tsuyoshi Motohashi2 and Katsuhiko Kondo2, 5

1Research and Education Center of Biodiversity and Ecology, Moscow Pedagogical State University, Kibalchicha 6, kor.5, Moscow, Russia; 2Laboratory of Plant Genetics and Breeding Science, Department of Agriculture, Faculty of Agriculture, Tokyo University of Agriculture, 1737 Funako, Atsugi City 243-0034, Japan; 3Laboratory of Plant Breeding and Biotechnology, Fukui Agricultural Experiment Station, 52-21, Ryo-Machi, Fukui 918-8215, Japan; 4Laboratory of Plant Chromosome and Gene Stock, Graduate School of Science, Hiroshima University, 1-4-3 Kagamiyama, Higashi-Hiroshima City 739-8526, Japan

5Author for correspondence: ([email protected]) Received July 4, 2013; accepted August 3, 2013

ABSTRACT. Gonospermum fruticosum (C. Smith ex Link) Less, Argyranthemum coronopifolium (Willd.) Humphries and two strains of A. foeniculaceum (Willd.) Webb ex Schultz-Bipl all placed in the family Asteraceae, the tribe Anthemideae, were collected in Tenerife, the Canary Islands, Spanish Territory, Africa. They were analyzed in order to clarify their relationships and variabilities by karyotypes and molecular ISSR (inter simple sequence repeat). Gonospermum fruticosum and Argyranthemum coronopifolium studied showed the common karyotype different from that of two strains of A. foeniculaceum. Among the 54 ISSR bands obtained in the four species studied 46 were polymorphic, and 22 were species-specific. Gonospermum and Argyranthemum showed quite different band patterns from each other. Two strains of A. foeniculaceum showed different, large polymorphic patterns. Thus, the ISSR must be useful tool to isolate and evaluate intergeneric, interspecific, down to intraspecific genic differences, furthermore, introgressive hybridization within the members of the tribe Anthemideae, the Asteraceae.

KEYWORDS: Argyranthemum coronopifolium, Argyranthemum foeniculaceum, Asteraceae, Canary Islands, Gonospermum fruticosum, Inter Simple Sequence Repeat (ISSR), Tribe Anthemideae,

Twenty-three species of Argyranthemum revised by Dendranthema) and Glebionis (Trehane 1998) together Humphries (1976) plus some more newly recorded species with other taxonomic groups such as Ismelia and (Santos personal communication) grow in Macaronesia in Heteranthemis by Humphries (1976) and Bremer and the Canary Islands, Madeira and the Salvage Island, Humphries (1993). Argyranthemum frutescens (L.) Spanish Territory in Africa (Bremer and Humphries 1993). Schultz-Bip can be artificially hybridized with Glebionis In contrast, Gonospermum consisted of four species in the carinatum (Schousb.) Tzvelev and with G. coronaria (L.) Canary Islands and has close relatives such as Inulanthera Cassini ex Spach. with aid of the ovule culture (Ohtsuka and Lugoa (Bremer and Humphries 1976). They are taxo- and Inaba 2008). nomically placed in the Asteraceae, the tribe Anthemideae Inter-Simple Sequence Repeats (ISSR) are partially Cass., the subtribe Chrysantheminae Less. (Bremer and inserted into the microsatellite region in the genome. The Humphries 1993) or Gonosperminae (Francisco-Ortega et ISSR is amplified by using the sequence of an adjacent al. 2001). The majority of the species of Argyranthemum microsatellite region for a primer (Zietkiewicz et al. 1994; such as A. foeniculaceum (Willld.) Webb ex Schultz-Bip. Kondo et al. 2003), and is applicable to polymorphism and A. frutescens (L.) Schultz-Bip. have crowded to non- analysis. Since the ISSR is a random primer, it does not crowded succulent leaves with two to 14 deeply filiform need the sequence information for a primer design, and its to linear-lanceolate lobes but some species have leaves operation is simple and easy like the RAPD (random with primary leaf-lobes ovate to obovate in outline, cypselas amplified polymorphic DNA) method (Hiraoka et al. creamish or have leaves oblong to obovate. Leaves of 2009), and it is used for phylogeography analyses or Argyranthemum adauctum (Link in Buch) Humphries and identification of individual species. It can be applied in those of Gonospermum fruticosum are very similar in many studies involving genetic identity, parentage, clone morphological character to each other. and strain identification, and taxonomic studies of closely Argyranthemum was seemed to be morphologically related species (Kondo et al. 2003; Racharak et al. 2007). and karyotypically similar to Chrysanthemum (formerly 54 TATARENKO ET AL.

MATERIALS AND METHODS covered by cover slips and were tapped by stick to spread Plant materials. Seeds of Gonospermum fruticosum (C. chromosomes in cells. Smith ex Link) Less., Argyranthemum coronopifolium Karyotype formulas of Gonospermum fruticosum, (Willd.) Humphries and two strains of A. foeniculaceum Argyranthemum coronopifolium, and two strains of A. Webb ex Sch. were collected in the Canary Islands, Spain foeniculaceum (Table 1) were prepared on the basis of and were sown, germinated and grew up in the greenhouse mean based on the size data of chromosome characters of (Table 1; Fig. 1), to analyze and compare their relationships ten somatic metaphase cells. Position of the primary of karyotypes and molecular ISSR. constrction in chromosome followed Levan et al. (1964): Arm ratio was calculated by long arm/short arm: 1.0-1.7 Karyotype analysis. After young plants of the species was grouped as the median centromeric chromosome, (Table 1) grew well (Fig. 1), their growing root tips ca 5 3.1-7.0 the subterminal centromere, and 7.1 or more as the mm long were collected and pretreated in 2 mM terminal centromere. 8-hydroxyquinoline at 18°C for 2 h. Then, they were fixed in 45% acetic acid at 4°C for a few min., before, they were DNA extractions. Total genomic DNA of each species of macerated in the 2:1 mixture of 1N HCl and 45% acetic Argyranthemum studied was extracted from fresh leaves by acid at 60°C. Root tips less than 1 mm long were cut and using the CTAB method described by Doyle and Doyle stained by 1% aceto-orcein on glass slides for 30 min. in a (1987) . chamber moistened by 45% acetic acid. Then, they were

Table 1. Accession numbers of the plants of Gonospermum fruticosum, Argyranthemum coronopifolium and two strains of A. foeniculaceum collected in Tenerife, the Canary Islands Accession Locality in the Canary Species name number* Islands 1 Gonospermum fruticosum (C. Smith ex Link) Less. Anaga Area, Tenerife 3 Argyranthemum coronopifolium (Willd.) Humphries Anaga Area, Tenerife 12 A. foeniculaceum Webb ex Sch. Mt. Teide, Tenerife 15 A. foeniculaceum Webb ex Sch. Mt. Teide, Tenerife *I.V. Tatarenko’s accession number for seed collection

Fig. 1. Inter- and intra-specific variation of leaves. A. Gonospermum fruticosum (C. Smith ex Link) Less. (Accession No. 1). B. Argyranthemum coronopifolium (Willd.) Humphries. (Accession No. 3). C. and D. A. foeniculaceum Webb ex Sch. (Accession Nos. 12 and 15). KARYOTYPE AND MOLECULAR ISSR VARIABILITIES IN GONOSPERMUM AND ARGYRANTHEMUM 55

DNA amplification. Five out of 21 primers were selected by the primary screening and were selected for analysis. The 10µl reaction mixture included 5µl of 2 X KOD FX neo buffer, 1.25 µl of 2 mM dNTP, 10 pmol of ISSR primer, 0.1µl of KOD FX Neo (TOYOBO), 10 ng of template DNA. PCR was performed using Gene Amp PCR System 9700 (PE Applied Biosystems) and TaKaRa PCR Thermal Cycler Dice (TAKARA BIO Inc.) and DNA amplification were carried out with one cycle of 94°C for 5 min, followed by 40 cycles of denature temperature 94°C for 30sec, annealing temperature 52°C, 54°C, 58°C and 60°C (depending on primers used, Table 1) for 30 sec, and 72°C for 1.5 min, and concluded by one cycle of 72°C for 10 min. PCR products was electrophoresed on 1.5% agarose gel with DNA size marker (200bp DNA Ladder, TaKaRa) at 100 V for 30 min in Mupid-2 plus (ADVANCE) filled by 1 X TAE Buffer. After staining of the electrophoresed gel with Ethidium Bromide solution for 20 min observation and photography of the band were performed using Printgraph AE-6933 FXCF-U (ATTO) under the ultraviolet exposure. Fig. 2. Karyotypes of mitotic-metaphase chromosome RESULTS AND DISCUSSION sets in Gonospermum fruticosum (A), Argyranthemum The chromosome numbers in Gonospermum fruticosum, coronopifolium (B), and A. foeniculaceum (C and D). A. sat Argyranthemum coronopifolium, and A. foeniculaceum K(2n=18)=16m+2m in Gonospermum fruticosum (Accession No. 1). B. K(2n=18)=16m+2smsat in A. Accession numbers 12 and 15 studied were commonly coronopifolium (Accession No. 3). C. K(2n=18)=14m+ 2n=18 (diploid; the basic chromosome number of X=9 is 4smsat in A. foeniculaceum (Accession No. 12). D. K(2n= sat common to the members of the tribe Anthemideae) 18)=14m+4sm in A. foeniculaceum (Accession No.15). (Ardevol-Gonzales et al. 1993; Kondo et al. 2003) and Bar = 10µm. relatively big chromosome sizes and the largest chromosomes were bigger than 10 µm and the smallest Argyranthemum coronopifolium and A. foeniculaceum chromosomes were less than 3.8µm (see Fig. 2). These Accession numbers 12 and 15 studied showed very less chromosome characters in Gonospermum and differences in karyotypes from those of the majority of the Argyranthemum studied were different from those of diploid species of Asiatic polyploid Chrysanthemum such Asiatic members in the tribe Anthemideae (Ardevol- as K(2n=18)=14m or sm+4st of which 2-4 chromosomes Gonzales et al. 1993; Kondo et al. 2003) and relatively have satellites (e.g., Borgen et al. 2003; Suda et al. 2003; big chromosome sizes and the largest chromosomes were Tatarenko et al. 2011; Kondo et al. 2012). Moreover, bigger than 10 µm and the smallest chromosomes were hybridization and introgression might be common to the less than 3.8µm (see Fig. 2). These chromosome charac- species of Argyranthemum in Tenerife after certain teristics in Gonospermum and Argyranthemum studied isolation mechanisms would be broken and they might were different from those of Asiatic members in the tribe have homoploid hybrid speciation and repeatedly species Anthemideae, especially in Chrysanthemum, although complexity (Fjellheim et al. 2009) in compared to Asiatic they have common basic chromosome numbers of X=9 Chrysanthemum such as polyploidization from diploid up (Bramwell et al. 1976; Ortega and Navarro 1977; to much higher ploids such as hexaploid (2n=54; Tanaka Brockmann 1987; Febles et al. 1989; Kondo et al. 2003). and Watanabe 1972; octoploid (2n=72; 2n=72; Nakata Thus, the karyotypes of Gonospermum fruticosum, and Kumagai 1999) and aneuploidization in high-ploids

Table 2. Karyotypes obtained in Gonospermum fruticosum, Argyranthemum coronopifolium and two strains of A. foeniculaceum Accession Chromosome Species name Karyotype number* Number 1 Gonospermum fruticosum (C. Smith ex Link) Less 18 16m+2smsat 3 A. coronopifolium (Willd.) Humphries 18 16m+2smsat 12 A. foeniculaceum Webb ex Sch. 18 14m+4smsat 15 A. foeniculaceum Webb ex Sch. 18 14m+4smsat *Tatarenko’s number of seed collection 56 TATARENKO ET AL. in Asiatic Chrysanthemum especially hybridization of foeniculaceum Accession number 15 had four specific cultivars by artificial cross-hybridization by artificial bands (7.4%). Three species such as A. coronopifolium hybridization in high-ploids in Asiatic Chrysanthemum, and A. foeniculaceum Accession numbers 12 and 15 had especially hybridization of cultivars by artificial eight common bands, and Gonospermum fruticosum had hybridization (Endo 1969; Endo 1994; Aoyama et al. only two common bands of other three species. Two 1997). Concerning Canary’s species of the tribe strains of A. foeniculaceum showed nine common bands. Anthemideae, the Asterceae, currently much more human On the other hand, two strains of A. foeniculaceum had activities including tourisms and floricultural, artificial respectively different band patterns and two distinctively hybridizations would make more diversified flowering common bands which had the great common feature of Argyranthemum. For instance, triploid cultivars were 76.0%. found in cultivars of Argyranthemum frutescens. The result of PCR amplification of the ISSR region in ISSR profiles. In order to evaluate the genetic relativity Argyranthemum confirmed the polymorphic bands of among the four species, five ISSR markers (Table 3) were Argyranthemum including specific bands of each species used. Out of 54 bands generated, 46 were found to be obtained were reproducible and sharp. Gonospermum polymorphic (86.7% polymorphism). The band pattern of fruticosum showed specific banding pattern different from four Argyranthemum species with ISSR primer 2, 5, and 6 those of the Argyranthemum species studied here. However, was shown in Fig. 5 and the primer 11 and 15 was shown they had two bands common to those two genera studied. in Fig. 4. Gonospermum fruticosum had 12 specific bands They must be somewhat closely related to each other. (22.2%) out of 54 bands, A. coronopifolium had five Additionally, respective taxa studied had own specific specific bands (9.3%), A. foeniculaceum Accession bands. Thus, the ISSR must be important tool to identify number 12 had one specific band (1.9%) and A. estimation of genetic diversity.

Table 3. Selected primers and genetic variations among Gonospermum fruticosum, Argyranthemum coronopifolium and two stains of A. foeniculaceum Accession numbers 12 and 15 in the Canary Islands in ISSR analysis

Primer Sequence Tm/Ta Total number Number of polymorphic bands Number of specific (5’~3’) (°C) of bands Number % bands for species

ISSR2 (GA)8C 52/54 10 6 60.0 6

ISSR5 (AG)8C 52/54 12 12 100.0 3

ISSR6 (AG)8A 50/52 9 9 100.0 3

ISSR11 (AC)8CTG 58/60 15 11 73.3 5

ISSR15 (AC)8GG 56/58 8 8 100.0 5 Total number of bands 54 46 - 22 Average 10.8 9.2 86.7 4.4

Fig. 3. ISSR banding pattern produced by each ISSR primer 2, 5 and 6. Lane M is 200bp DNA Ladder. Lane 1, Gonospermum fruticosum; lane3, A. coronopifolium; lane 12, A. foeniculaceum Accession number 12; lane 15, A. foeniculaceum Accession number 15. A thick, white arrow point the bands pointing respective common bands seen in the four species. KARYOTYPE AND MOLECULAR ISSR VARIABILITIES IN GONOSPERMUM AND ARGYRANTHEMUM 57

Fig. 4. ISSR banding pattern produced by each ISSR primer 11 and 15. Lane M is 200bp DNA Ladder. Lane 1, Gonospermum fruticosum: Lane 3, A. coronopifolium: Lane 12, A. foeniculaceum Accession number 12; Lane 15, A. foeniculaceum Accession number 15. A thick white arrow pointing respective common bands seen in the four species.

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