Originated from Argyranthemum Frutescens Parentages
Chromosome Botany (2014) 9: 97-112 © Copyright 2014 by the International Society of Chromosome Botany
Identifying, discriminating and isolating cultivars of ‘Marguerites’ originated from Argyranthemum frutescens parentages and their intergeneric and interspecific hybridities by DNA markers amplified by RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat)*
Masahiro Morikawa1,4, Takahiro Ｍuto2, Arnoldo Santos-Guerrra3 and Katsuhiko Kondo1,5
1Laboratory of Plant Genetics and Breeding Science, Department of Agriculture, Faculty of Agriculture, Tokyo University of Agriculture, 1737 Funako, Atsugi City, Kanagawa Prefecture 243-0034, Japan; 2Plant Cultivation and Breeding Section, Izu Agricultural Research Center, Shizuoka Prefectural Reserch Institute of Agriculture and Forestry, Izu Peninsula 3012 Inatori, Izuchou, Kamo-Gun, Shizuoka Prefecture 413-0411, Japan; 3Head of Botany Department, Orotava Botanical Garden, Tenerife, Canary Island, Spain; 4The Present Address: Saitama Genshu Ikuseikai Co., Ltd., 2616 Niibori, Kuki City, Saitama Prefecture 346-0105, Japan
5Author for Correspondence: [email protected] Received May 5, 2014; accepted September 30, 2014
ABSTRACT. Abilities of the polymorphic DNA markers amplified by RAPD (Randomly Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeat) were studied, detected and systematized breeding lines and expanding molecular variabilities and molecular phylogenetic lines of the cultivarietal ‘Marguerites’ of the Argyranthemum lineage and especially the intergeneric hybrids of ‘Marguerite’ X Glebionis carinata Tzvelev or G. coronaria (L.) Spach. The reproducible polymorphic bands were generated by RAPD-PCR using RAPD primer OPC-04, in respective species of Glebionis and the intergeneric hybrids of the ‘Marguerites.’ The primer OPC-04 made it possible to identify either two intergeneric hybrids of ‘Marguerite’ X G. carinata or ‘Marguerite’ X G. coronaria. The polymorphic bands were amplified by ISSR-PCR in the breeding lines and the cultivars of the ‘Marguerite,’ while they were not amplified by RAPD. A specific band was confirmed by ISSR-5 primer in the ‘Marguerite’ cultivar strain of ‘Zairai Shiro.’ This primer ISSR-5 detected and identified certain specific band among the band pattern of ‘Southern Elegance White,’ this cultivar has cv. ‘Early White’ as the paternal parent. Similar polymorphic band pattern was confirmed by ISSR-PCR, in the breeding lines and the cultivars of the ‘Marguerite’ which have similarity in flower color, flowering type and share the hybrid parents.
KEYWORDS: Argyranthemum, Glebionis carinata, Glebionis coronaria, ISSR, Marguerites, RAPD
Argyranthemum is taxonomically placed in the Anthemideae, their commercial purposes. Asteraceae and grows sympatrically or allopatrically with Argyranthemum can be cross-hybridized easily with 24 species in Macaronesia in the Canary Islands of Spain Glebionis (Tsukamoto 1986; Ohtsuka and Inaba 2008). and in Madeira and the Salvage Islands of Portugal Up to the present, numerous hybrids among many (Bramwell and Bramwell 1974; Bremer and Humphries combinations of close relatives might have been occurred 1993). Argyranthemum is closely related to and easily artificially directly or indirectly to make cultivar makes hybrids with Glebionis (Trehane 1995), Ismelia complexity (Aoyama 2000) to perform multi-different and Heteranthemis (Bremer and Humphries 1993). A coloration and inflorescence heads. It was made question member of A. frutescens (L.) Schultz-Bip. has been well- that “What Marguerites are” according to his detection known as the common name of the ‘Marguerite’ group result by chromosome analysis of 38 cultivars and their with more than 100 cultivars imported to Japan from origin of so-called the commercialized A. frutescense that France since the Meiji Era (1868-1912) for the purpose of numerous closely related species and cultivars could be decolative ornamental plants in greenhouses and later cut- involved with hybridization and morphological types of flower products and potted flower products in the warmer cultivars from yellow to deep red-colored as well as white- sunny outdoor seashores step-by-step. Royal Horticultural colored ligulate as well as double flowers. Japanese Society of UK has listed more than 100 cultivars of cultivar. ‘Zairai Shiro’ showed the basic characteristics of ‘Marguerites’ (Cheek 1993). They have met so many white color-pigment and the ligulate flowers around the chances to face and hybridize with other genera of edge of the head inflorescence to perform the common chrysanthemums closely related to Argyranthemum character of the single flower head, but some species such during their long term cultivation mainly in Izu Peninsula, as A. maderense (D. Don) Humphries has originally natural Shizuoka Prefecture and Kagawa Prefecture, in Japan for yellow-colored flowers. Moreover, some ‘Margarite’ cultivars have been much more cross-hybridized to amplify *Based on the Master’s thesis of M. Morikawa submitted to Graduate diversifications by hybridization; for instance, ‘Margarite’ School of Agriculture, Tokyo University of Agriculture cv. ‘Izu Yellow’ might be a result of hybridization of 98 MORIKAWA ET AL.
Glebionis coronaria to extract yellow color in the work of carinatum (Schousb.) Tzvelev were rather easily to 1970’s in the Izu area (Tsukamoto 1986). On the other hand, hybridize with Argyranthemum frutescence ‘Marguerites’ cv. ‘Margarites’ was cross-hybridized with G. carinata by (Zietkiewicz et al. 1994) since three species of Glebionis works of the Plant Cultivation and Breeding Section, Izu were closely related to the latter taxon. On the other hand, Agricultural Research Center, Shizuoka Prefectural 32 cultivars of ‘Marguerites’ were expected not only by Research Institute of Agriculture and Forestry, Izu breeders in France and other Europian nations but also Peninsula, Shizuoka Prefecture and made various hybrid many Japanese growers’ communities as well as staff cultivars such as ‘Canaria Queen,’ ‘Carnival Queen,’ members of the Plant Cultivation and Breeding Section, ‘Fu-Ren-Ka,’ ‘Garnet Queen,’ ‘Peach Queen,’ ‘Queen Izu Agricultural Research Center, Shizuoka Prefectural Mice,’ and so on as the member of ‘Margarite’ (Ohtsuka Recerch Institute of Agriculture and Forestry, Izu and Inaba 2008). Among them cv. ‘Fu-Ren-Ka’ was bred Agricultural Research Center, Izu Peninsula, Japan. and developed for some different purposes and different Additionally, 25 strains for new cultivars amplified were usefulness such as fragrance, decorative cut-flowers, studied here during the course of investigation by ISSR potmums (potted chrysanthemums), and so on and may (Inter Simple Sequence Repeat) and RAPD methods. bred to extract for many more different purposes. ISSR is a random primer and does not need the sequence Observing and concidering the appearances of such a big information for a primer design, and its operation is flower variant in the ‘Margarite’ could appear not only simple and easy like RAPD method and it is used for intraspecific hybridization within Argyranthemum phylogenetic analyses or identification of individual frutescence but also interspecific hybridization within the cultivars. It can be applied in many studies involving genus and some intergeneric hybridization. Thus, genetic identity, parentage, clone and strain identification, molecular analyses by RAPD by PCR and ISSR were and taxonomic studies of closely related species (Kondo applied to identify phylogenetic lineages (Williams et al. et al. 2003; Racharak and Eiadthong 2007). 1990). Comparing those amplified polymorphisms with DNA extractions. Total genomic DNA’s of the species RAPD can be used for identifying cultivars as well as of Argyranthemum and Glebionis, of their hybrids, 32 hybridities. The ISSR is amplified by using the sequence cultivars of Argyranthemum frutescence ‘Marguerites’ of an adjacent microsatellite region for a primer and 25 newly progressed strains for new cultivars studied (Zietkiewicz et al. 1994; Kondo et al. 2003; Tatarenko et were extracted from fresh leaves by using the CTAB al. 2013). Analyses of polymorphism with ISSR are method described by Doyle and Doyle (1987). Three partially inserted into the microsatellite region in the hundreds to 500 mg frozen leaf samples were homogenized genome. with liquid nitrogen and put in individual 2 ml tubes and Certain distinct characteristics of the flower heads of washed once with washing buffer [0.1 M Tris-HCl pH 8.0, the ‘Maguerites’ such as flower color in white, yellow, 2% 2-mercaptoethanol, 1% polyvinylpyrrolidone (PVP) pink and so on, single-ray flower-type, double-ray flower and 0.05 M ascorbic acid]. Two percent CTAB extraction type, anemone-type, inglewick tipped flower type, and buffer containing 2% 2-mercapthoethanol and 2% some other flower types and the fragrant-flower types polyvinylpyrrolidone were then added and incubated at should be originated not only from ‘Marguerites’ themselves 60°C for at least 30 min. After incubation, the samples but also from a lot of unrecorded interspecific cross- were centrifuged and the supernatant was collected. The hybridizations with many other species of Argyranthemum DNA was then isolated with 500µl 24:1 chloroform- by insect pollination, besides well-recorded man-made, isoamyl alcohol (CIA), precipitated with equal volume of hand pollinations. Thus, origin of numerous types of 2-propanol, and washed with 500 µl 24:1 chloroform- ‘Marguerites’ should be analyzed by molecular-based isoamyl alcohol (CIA), precipitated with equal volume of RAPD and ISSR not only for detecting their parentages 2-propanol, and washed with 500 µl 70% ethanol. The but also for finding new combinations for developing and DNA pellet was briefly dried at room temperature, systematizing newly expected cultivars with high resuspended in 500 ml sterilized distilled water, added efficiency. with RNase for a final concentration of 10 µg/ml and incubated for 30 min at 37°C. The solution was then MATERIALS AND METHODS washed with 50:50 volume of Phenol:Chroroform to Plant materials. A plant of Argyranthemum cornopifolium remove the proteins and followed by 24:1 CIA wash to (Willd.) Humphries originally collected in Anega Area, remove the proteins and followed by 24:1 CIA was to Tenerife, the Canary Islandand, two plants of A. remove the remaining phenol. The final precipitation was foeniculaceum Webb ex Sch. (A and B) originally done by adding 1/10 volume of 7.5M Ammonium acetate collected in Anega Area (Tatarenko et al. 2013) were used and 2.5 volumes of 99.5% ethanol, and incubated at -80°C for the present study. Not only with those species of for 15 minutes. The DNA pellet was collected by Argyranthemum but also the other species of Argyranthemum centrifuge, washed with 500 µl 70% ethanol and was could hybridize with each other. Additionally, Glebionis completely dried before resuspended in appropriate coronaria (L.) Spach, G. segetum (L.) Fourr and G. amount of sterilized distilled water. The DNA IDENTIFYING AND DISCRIMINATING CULTIVARS OF ‘MARGUERITES’ 99 concentration was quantified using a spectrophotometer 1-0 data matrices those data were analyzed by the and a working sample solution, with concentration of 100 computer software (http://aoki2.si.gunmau.ac.jp/Black ng/µl, was prepared and stored at -20°C. Nineteen readily Box) to make phylogenetic dendrogram by the Ward’s available ISSR primers: (AC)8GG, (TC)8C, (TG)8G, method for a claster analysis.
(AC)8GA, (GA)8C, AC8C, (AG)8G, (CT)8A, (TG)8A, Repetitive sequences occur repeatedly in genomes and
(AC)8GA, (GA)8C, (AC)8C, (AG)8G, (CT)8A, (TG)8A, dispersed and thus, sequre highly frequently DNA
(AG)8(CT)C, (AG)8(CT)A, (GA)8(CT)T, (GA)8(CT)T, polymorphism (Huang et al. 2003) and even closely
(GA)8(CT)G, (CA)8(AG)C, (AC)8G, (AC)8(CT)G, (ATG)6, related genetic distances of quite close relatives such as
(GAA)6 and (AGT)(ACG)(AGT)(TC)7 were initially between cultivars have been reported their validities screened and 11 of that produced reproducible and clear (Huang et al. 2003, 2004; Maeda et al., 2008) fragments were selected (Table 2) and used to provide polymorphic markers for genetic diversity. RESULTS AND DISCUSSION Polymerase chain reaction (PCR) amplifications were RAPD-PCR The result of initial screening using total 60 performed in 20µl reactions, consisted of 1.25 U Taq RAPD primers of OPA-01~20, OPB-01~2 and OPC- DNA polymerase (Takara Ex Taq), 2µl 10x reaction buffer 01~20, some polymorphic bands of ‘Marguerite’ and containing 20 mM MgCl2, 1.6µl of dNTP mixture (2.5 intergeneric hybrid cultivars detected by OPC-04 primer. mM each), 0.25 µM primer, and 100 ng template DNA. Clear and reproducible polymorphic bands were detected Amplification was performed. The amplification products all 70 individuals by RAPD-PCR using OPC-04 (Figs. were separated on 2% agarose gels buffered with 1x TAE 1-5). Two clear bands of 1300bp and 1500bp were and stained with ethidium bromide to visualize the bands. detected on 56 individuals of breeding lines and cultivars Band size was estimated using 200 bp Takara DNA ladder. originated in Argyranthemum frutescens. Three wild DNA amplification. Five out of 21 primers were selected species of Argyranthemum also has these bands. by the primary screening and were selected for analysis. The cultivars of ‘Zairai Ki’ and ‘Izu Yellow’ that are The 10µl reaction mixture included 5 µl of 2XKOD FX intergeneric hybrid cultivars of ‘Marguerite’ and Glebionis neo buffer, 1.25 µl of 2 mM dNTP, 10 pmol of ISSR coronaria had a common band with the marguerite and a primer, 0.1µl of KOD FX Neo (TOYOBO), 10 ng of band of 750bp. The cultivars of ‘Queen Mice’, ‘Fu-Ren- template DNA. PCR was performed using Gene Amp Ka’, ‘Carnival Queen’ and ‘Peach Queen’ were PCR System 9700 (PE Applied Biosystems) and TaKaRa intergeneric hybrid-cultivars of ‘Marguerite’ x G. carinata PCR Thermal Cycler Dice (TAKARA BIO Inc.) and DNA while ‘Canaria Queen’ and ‘Garnet Queen’ originated amplification were carried out with one cycle of 94°C for from ‘Peach Queen’ exposed by X-ray radiation had two 5 min, followed by 40 cycles of denature temperature common bands to those of G. coronaria and a 550bp 94°C for 30 sec, annealing temperature 52°C, 54°C, 58°C band. Glebionis segetum had a band of 1200bp. These and 60°C (depending on primers used, Table 1) for 30 sec, three clear bands were reproducible well. However, in and 72°C for 1.5 min, and concluded by one cycle of 72°C RAPD-PCR by total 60 RAPD primers, polymorphic for 10 min. PCR products was electrophoresed on 1.5% banding pattern between breeding lines and cultivars of agarose gel with DNA size marker (200 bp DNA Ladder, ‘Marguerite’ and some wild species of Argyranthemum TaKaRa) at a 100 V for 30 min.in Mupid-2 plus could not be detected. However, RAPD-PCR by OPC-04 (ADVANCE) filled by 1XTAE Buffer. After staining of primer identified two intergeneric hybrid cultivars of the electrophoresed gel with Ethidium Bromide solution ‘Marguerite’ x G. coronaria and six intergeneric hybrid for 20 min., observation and photography of the band cultivars of ‘Marguerite’ x G. carinata. Since RAPD-PCR were performed using Printgraph AE-6933 FXCF-U with OPC-04 primer identified some intergeneric hybrid (ATTO) under the ultraviolet exposure. cultivars that had each origin of Grebionis, making Electrophoresis Electrophoresis gel at the concentration investigation of hybridity in intergeneric cross breeding of 1.5% was prepared with TAE buffer (prepared by 40 more efficiency by this primer was expected. Each of the mM Tris-acetate and 1 mM EDTA) and 1.5% agarose gel specific banding patterns was confirmed in three species (Takara: L03 TAKARA 5003). PCR product added of Grebionis, so OPC-04 was effective in identification of loading buffer (Sigma; G-7654) and DNA sized marker hybrid if G. segetum used for a parentage. In order to find 200 bp DNA ladder 2 µl. Mupid-2 plus (Advance) as an RAPD marker that detect a polymorphic in wild species electrophoresis equipment was used with electrophoresis of Argyranthemum or breeding lines and cultivars of tub TAE buffer filled out before 100V electrified for 30 Argyranthemum, more RAPD-PCR with RAPD primers min., and then, the gel was put and shaken in ethidium were necessary. bromide for 30 min in dark and stained. Photography was ISSR-PCR Six ISSR primers such as ISSR-5, 6, 7, 11, made under UV exposure by Printgraph AE-6933FXCF-U 48 and 49 screened from a total 49 ISSR primers detected (Atto Co.). clear polymorphic bands in the breeding lines and the Cluster analysis Affter appearance (1) or not (0) of cultivars of ‘Marguerite’. Especially, reproducible bands bands by ISSR-PCR were checked and obtained to make were obtained by ISSR-5, 6, 11 and 49 (Figs. 6-25). A 100 MORIKAWA ET AL.
Table 1. Growing strains, cultivars within Argyranthemum frutescens ‘Margarites’, and other species of Argyranthemum and Glebionis studied. ID number Breeding line Flower color Flower type 1 08-15-7 Yellow Single 2 10-3-4 Red Single 3 10-3-11 Red Single 4 10-4-1 White Single 5 10-9-2 White Single 6 10-9-3 White Single 7 c12-2-1 White Single 8 c12-3-1 White Single 9 c12-4-1 White Single 10 c12-13-1 Yellow Single 11 c12-14-1 Yellow Single 12 c12-21-1 Yellow Single 13 c12-21-2 White Single 14 c12-21-3 Yellow Single 15 c12-21-4 White Single 16 c12-21-5 White Single 17 c12-21-6 White Single 18 c12-21-7 White Single 19 p12-45-1 White, Yellow at the base of corolla Anemone 20 p12-45-2 White Single 21 p12-45-3 White, Pink at the base of corolla Single 22 p12-45-4 White Single 23 p12-46-1 White and Pink in combination Single 24 p12-48-1 White Single 25 p12-81-1 Pink Single Cultivars 26 ‘Zairai Shiro’ White Single 27 ‘Unzen Zairai’ White Single 28 ‘Early White’ White Single 29 ‘Faery White’ White Single 30 ‘Pink Southern Candle’ Pink Doube 31 ‘Peach Southern Candle’ Deep Pink Single 32 ‘White Ripple’ White Single 33 ‘Princess Little White’ White Double 34 ‘Faery Light Pink’ Pink Single 35 ‘Sweet Ripple’ White Double-Anemone 36 ‘Sunday Ripple’ White Single 37 ‘Angel Mice’ Pink Single 38 ‘Cheryl Mice’ Pink Anemone-shaped 39 ‘Lady Mice’ ① Pink Anemone-shaped 40 ‘Lady Mice’ ② Pink Anemone-shaped 41 ‘Princess Lemonade’ Pale Yellow Single 42 ‘Silk Ball’ White Double 43 ‘White Ripple Pure’ White Single 44 ‘White Jewel’ White Single 45 ‘Candy Mice’ ① Deep Pink Single 46 ‘Candy Mice’ ② Deep Pink Single 47 ‘Sour Ripple’ White Single 48 ‘Southern Elegance White’ White Single 49 ‘Cutie Mice’ Whitish Pink Anemone 50 ‘Moon Light’ Pale Yellow Single 51 ‘Super Lemonade’ Yellow Single 52 ‘Brown Eye’ White Single 53 ‘Lovely Friend’ Pink Single 54 ‘Oboro-Zuki’ Pale Yellow Anemone 55 ‘Hot Berry’ Pink Red Single 56 ‘Fire Cracker’ Pink Red Double IDENTIFYING AND DISCRIMINATING CULTIVARS OF ‘MARGUERITES’ 101
ID number Breeding line Flower color Flower type ‘Marguerite’ x Glebionis coronaria 57 ‘Zairai Ki’ Yellow Single 58 ‘Izu Yellow’ Yellow Single ‘Marguerite’ x Glebionis carinatum 59 ‘Queen Mice’ Mixture of Deep Pink and Yellow Single 60 ‘Fu-Ren-Ka’ White Single 61 ‘Carnival Queen’ Yellow Single 62 ‘Peach Queen’ Complexed Pale Deep-Orange and Yellow Single 63 ‘Canaria Queen’ Light Yellow Single 64 ‘Garnet Queen’ Deep Red Single Argyranthemum 65 A. coronopifolium Willd. White Single 66 A. foeniculaceum Willd. (12) White Single 67 A. foeniculaceum Willd. (15) White Single Glebionis 68 G. coronaria L. Yellow-only or Mixture of Yellow and White Single 69 G. segetum (L.) Fourr. Yellow or Mixture of Yellow and White Single 70 G. carinata (Schousb.) Tzvelev White, Yellow and Red or their Mixture Single Experimental strains of ‘ Margarites’ were bred and preserved by the Plant Cultivation and Breeding Section, Izu Agricultural Research Center, Shizuoka Prefectural Research Institute of Agriculture and Forestry, Izu Agricultural Research Center. *All of the plants listed here for our present research are conserved in the Plant Cultivation and Breeding Section, Izu Agricultural Research Center, Shizuoka Prefectural Research Institute of Agriculture and Forestry.
Table 2. Selected RAPD primer used in this study Number of Polymorphic Numbers of polymorphic bands in Primer Sequences Band numbr bands intergeneric cultivars of Marguerite OPC-4 CCGCATCTAC 4 3 3
Table 3. ISSR primers selected Number of polymorphic Annealing Polymorphic band Primers base sequences Band number bands in ‘Marguerite’s temperature number and their cultivars ISSR-5 (AG)8+CTC 54°C 5 5 1 ISSR-6 (AC)8+CTA 58°C 10 9 7 ISSR-11 (AC)8+CTG 60°C 11 11 8 ISSR-49 (AG)8+TC 56°C 14 14 13 total 41 amplified fragments were detected by four ISSR primers were investigated, but banding pattern by either primers and 40 amplified fragments were polymorphic. primers in breeding lines and Cultivars had high Thirty-eight polymorphic bands in the breeding lines and homologous, assay with farther more ISSR primers is the cultivars of ‘Marguerite’ were obtained and length of needed to obtain more specific bands. bands was to 4,000bp from 500bp. On the other hand, Cluster Analysis. A 1-0 data matrix based on polymorphic 1400bp bands that were specific to lines of ‘Zairai Shiro’ banding pattern was obtained by each primer, and made a were detected by ISSR-5. This specific bands were dendrogram that indicated a similarity of polymorphic common to only ‘Zairai Shiro’, ‘Unzen Zairai’, ‘Early banding pattern by the Ward method (Fig. 26). Six out of White’ and ‘Southern Elegance White’. ‘Zairai Shiro’ was seven breeding lines and the cultivars that have yellow introduced to Japan for the first time. ‘Unzen Zairai’ was flower belonged to the group ①. All of the breeding lines originated from ‘Zairai Shiro’. ‘Early White’ is one of the and the cultivars that have breeding lines 08-15-7 as seed old cultivars selected from ‘Southern Elegance White’ parent or pollen parent belonged to the group ①, and which was generated from ‘White Jewel’ as the maternal white flower that have 08-15-7 as parent also belonged to parent and ‘Early White’ as the paternal parent by artificial this group. Parental origin of the cultivar ‘Super Lemonade’ cross hybridization. Thus, this cultivar could have a that has yellow flower in this group ① unknown parents, common sequence with ‘Early White’ as the paternal but this cultivar could have genetically closely related parent. Ability of ISSR primers detecting these specific with the breeding line of 08-15-7. Numerous cultivars bands allow to investigate child and parent relation in with pink flowers were in the group ②. ‘Lady Mice ②’ some cultivars. Polymorphic bands obtained by another and ‘Lady Mice ①’ were flower color variant cultivars of 102 MORIKAWA ET AL.
M 1 2 3 4 5 6 7 8 901 11 12 13 14 15 M
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Fig. 1. Band patterns shown by RAPD primer OPC-04. M: 200bp DNA Ladder. 1 : 08-15-7, 2 : 10-3-4, 3 : 10-3-11, 4 : 10-4-1, 5 : 10-9-2①, 6 : 10-9-3①, 7 : c12-2-1, 8 : c12-3-1, 9 : c12-4-1, 10 : c12-13-1, 11 : c12-14-1, 12 : c12-21-1, 13 : c12-21-2, 14 : c12-21-3, 15 : c12-21-4.
M 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M
4000bp
2000bp
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Fig. 2. Band patterns shown by RAPD primer OPC-04. M: 200bp DNA Ladder. 16 : c12-21-5, 17 : c12-21-6, 18 : c12-21-7, 19 : p12-45-1, 20 : p12-45-2, 21 : p12-45-3, 22 : p12-45-4, 23 : p12-46-1, 24 : p12-48-1, 25 : p12-81-1, 26 : ‘Zairai Shiro’, 27 : ‘Unzen Zairai’, 28 : ‘Early White’, 29 : ‘Faery White’, 30 : ‘Pink Southern Candle’
M 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 M
4000bp
2000bp
1000bp
200bp
Fig. 3. Band patterns shown by RAPD primer OPC-04. M: 200bp DNA Ladder. 31 : ‘Peach Southern Candle’, 32 : ‘White Ripple’, 33 : ‘Princess Little White’, 34 : ‘Faery Light Pink’, 35 : ‘Sweet Ripple’, 36 : ‘Sunday Ripple’, 37 : ‘Angel Mice’, 38 : ‘Cheryl Mice’, 39 : ‘Lady Mice’ ①, 40 : ‘Lady Mice’ ②, 41 : ‘Princess Lemonade’, 42 : ‘Silk Ball’, 43 : ‘White Ripple Pure’, 44 : ‘White Jewel’, 45 : ‘Candy Mice’ ① IDENTIFYING AND DISCRIMINATING CULTIVARS OF ‘MARGUERITES’ 103
M 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 M
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2000bp
1000bp 750bp 550bp
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Fig. 4. Band patterns shown by RAPD primer OPC-04. M: 200bp DNA Ladder. 46 : ‘Candy Mice’ ②, 47 : ‘Sour Ripple’, 48 : ‘Southern Elegance White’, 49 : ‘Cutie Mice’, 50 : ‘Moon Light’, 51 : ‘Super Lemonade’, 52 : ‘Brown Eye’, 53 : ‘Lovely Friend’, 54 : ‘Oboro-Zuki’, 55 : ‘Hot Berry’, 56 : ‘Fire Cracker’, 57 : ‘Zairai Ki’, 58 : ‘Izu Yellow’, 59 : ‘Queen Mice’, 60 : ‘Fu-Ren-Ka’.
M 61 62 63 64 65 66 67 68 69 70 M
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2000bp
1000bp 750bp 550bp
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Fig. 5. Band patterns shown by RAPD primer OPC-04. M: 200bp DNA Ladder. 61 : ‘Carnival Queen’, 62 : ‘Peach Queen’, 63 : ‘Canaria Queen’, 64 : ‘Garnet Queen’, 65 : Argyranthemum coronopifolium, 66 : A. foeniculaceum (12), 67 : A. foeniculaceum (15), 68 : Glebionis coronaria. 69 : G. segetum. 70 : G. carinata.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M
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2000bp
1000bp
200bp
Fig. 6. Band patterns shown by ISSR primer ISSR-5. M: 200bp DNA Ladder. 1 : 08-15-7, 2 : 10-3-4, 3 : 10-3-11, 4 : 10-4-1, 5 : 10-9-2①, 6 : 10-9-3①, 7 : c12-2-1, 8 : c12-3-1, 9 : c12-4-1, 10 : c12-13-1, 11 : c12-14-1, 12 : c12-21-1, 13 : c12-21-2, 14 : c12-21-3, 15 : c12-21-4. 104 MORIKAWA ET AL.
M 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M
4000bp
2000bp 1400bp
1000bp
200bp
Fig. 7. Band patterns shown by ISSR primer ISSR-5. M: 200bp DNA Ladder. 16 : c12-21-5, 17 : c12-21-6, 18 : c12-21-7, 19 : p12-45-1, 20 : p12-45-2, 21 : p12-45-3, 22 : p12-45-4, 23 : p12-46-1, 24 : p12-48-1, 25 : p12-81-1, 26 : ‘Zairai Shiro’, 27 : ‘Unzen Zairai’, 28 : ‘Early White’, 29 : ‘Faery White’, 30 : ‘Pink Southern Candle’
M 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 M
4000bp
2000bp
1000bp
200bp
Fig. 8. Band patterns shown by ISSR primer ISSR-5. M: 200bp DNA Ladder. 31 : ‘Peach Southern Candle’, 32 : ‘White Ripple’, 33 : ‘Princess Little White’, 34 : ‘Faery Light Pink’, 35 : ‘Sweet Ripple’, 36 : ‘Sunday Ripple’, 37 : ‘Angel Mice’, 38 : ‘Cheryl Mice’, 39 : ‘Lady Mice’ ①, 40 : ‘Lady Mice’ ②, 41 : ‘Princess Lemonade’, 42 : ‘Silk Ball’, 43 : ‘White Ripple Pure’, 44 : ‘White Jewel’, 45 : ‘Candy Mice’ ①
M 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 M
4000bp
2000bp 1400bp 1000bp
200bp
Fig. 9. Band patterns shown by ISSR primer ISSR-5. M: 200bp DNA Ladder. 46 : ‘Candy Mice’ ②, 47 : ‘Sour Ripple’, 48 : ‘Southern Elegance White’, 49 : ‘Cutie Mice’, 50 : ‘Moon Light’, 51 : ‘Super Lemonade’, 52 : ‘Brown Eye’, 53 : ‘Lovely Friend’, 54 : ‘Oboro-Zuki’, 55 : ‘Hot Berry’, 6 : ‘Fire Cracker’, 57 : ‘Zairai Ki’, 58 : ‘Izu Yellow’, 59 : ‘Queen Mice’, 60 : ‘Fu-Ren-Ka’. IDENTIFYING AND DISCRIMINATING CULTIVARS OF ‘MARGUERITES’ 105
M 61 62 63 64 65 66 67 68 69 70 M
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Fig. 10. Band patterns shown by ISSR primer ISSR-5. M: 200bp DNA Ladder 61 : ‘Carnival Queen’, 62 : ‘Peach Queen’, 63 : ‘Canaria Queen’, 64 : ‘Garnet Queen’, 65 : Argyranthemum coronopifolium, 66 : A. foeniculaceum (12), 67 : A. foeniculaceum (15), 68 : Glebionis coronaria, 69 : G. segetum, 70 : G. carinata.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M
4000bp
2000bp
1000bp
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Fig. 11. Band patterns shown by ISSR primer ISSR-6. M: 200bp DNA Ladder M: 200bp DNA Ladder. 1 : 08-15-7, 2 : 10-3-4, 3 : 10-3-11, 4 : 10-4-1, 5 : 10-9-2①, 6 : 10-9-3①, 7 : c12-2-1, 8 : c12-3-1, 9 : c12-4-1, 10 : c12-13-1, 11 : c12-14-1, 12 : c12-21-1, 13 : c12-21-2, 14 : c12-21-3, 15 : c12-21-4.
M 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M
4000bp
2000bp
1000bp
200bp
Fig. 12. Band patterns shown by ISSR primer ISSR-6. M: 200bp DNA Ladder. 16 : c12-21-5, 17 : c12-21-6, 18 : c12-21-7, 19 : p12-45-1, 20 : p12-45-2, 21 : p12-45-3, 22 : p12-45-4, 23 : p12-46-1, 24 : p12-48-1, 25 : p12-81-1, 26 : ‘Zairai Shiro’, 27 : ‘Unzen Zairai’, 28 : ‘Early White’, 29 : ‘Faery White’, 30 : ‘Pink Southern Candle’ 106 MORIKAWA ET AL.
M 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 M
4000bp
2000bp
1000bp
200bp
Fig. 13. Band patterns shown by ISSR primer ISSR-6. M: 200bp DNA Ladder. 31 : ‘Peach Southern Candle’, 32 : ‘White Ripple’, 33 : ‘Princess Little White’, 34 : ‘Faery Light Pink’, 35 : ‘Sweet Ripple’, 36 : ‘Sunday Ripple’, 37 : ‘Angel Mice’, 38 : ‘Cheryl Mice’, 39 : ‘Lady Mice’ ①, 40 : ‘Lady Mice’ ②, 41 : ‘Princess Lemonade’, 42 : ‘Silk Ball’, 43 : ‘White Ripple Pure’, 44 : ‘White Jewel’, 45 : ‘Candy Mice’ ①
M 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 M
4000bp
2000bp
1000bp
200bp
Fig. 14. Band patterns shown by ISSRprimer ISSR-6. M: 200bp DNA Ladder. 46 : ‘Candy Mice’②, 47 : ‘Sour Ripple’, 48 : ‘Southern Elegance White’, 49 : ‘Cutie Mice’, 50 : ‘Moon Light’, 51 : ‘Super Lemonade’, 52 : ‘Brown Eye’, 53 : ‘Lovely Friend’, 54 : ‘Oboro-Zuki’, 55 : ‘Hot Berry’, 56 : ‘Fire Cracker’, 57 : ‘Zairai Ki’, 58 : ‘Izu Yellow’, 59 : ‘Queen Mice’, 60 : ‘Fu-Ren-Ka’.
M 61 62 63 64 65 66 67 68 69 70 M
4000bp
2000bp
1000bp
200bp
Fig. 15. Band patterns shown by ISSR primer ISSR-6. M: 200bp DNA Ladder. 61 : ‘Carnival Queen’, 62 : ‘Peach Queen’, 63 : ‘Canaria Queen’, 64 : ‘Garnet Queen’, 65 : A. coronopifolium, 66 : A. foeniculaceum (12), 67 : A. foeniculaceum (15), 68 : G. coronaria, 69 : G. segetum, 70 : G. carinata. IDENTIFYING AND DISCRIMINATING CULTIVARS OF ‘MARGUERITES’ 107
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M
4000bp
2000bp
1000bp
200bp
Fig. 16. Band patterns shown by ISSR primer ISSR-11. M: 200bp DNA Ladder. 1 : 08-15-7, 2 : 10-3-4, 3 : 10-3-11, 4 : 10-4-1, 5 : 10-9-2①, 6 : 10-9-3①, 7 : c12-2-1, 8 : c12-3-1, 9 : c12-4-1, 10 : c12-13-1, 11 : c12-14-1, 12 : c12-21-1, 13 : c12-21-2, 14 : c12-21-3, 15 : c12-21-4.
M 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M
4000bp
2000bp
1000bp
200bp
Fig. 17. Band patterns shown by ISSRprimer ISSR-11. M: 200bp DNA Ladder. 16 : c12-21-5, 17 : c12-21-6, 18 : c12-21-7, 19 : p12-45-1, 20 : p12-45-2, 21 : p12-45-3, 22 : p12-45-4, 23 : p12-46-1, 24 : p12-48-1, 25 : p12-81-1, 26 : ‘Zairai Shiro’, 27 : ‘Unzen Zairai’, 28 : ‘Early White’, 29 : ‘Faery White’, 30 : ‘Pink Southern Candle’
M 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 M
4000bp
2000bp
1000bp
200bp
Fig. 18. Band patterns shown by ISSR primer ISSR-11. M: 200bp DNA Ladder. 31 : ‘Peach Southern Candle’, 32 : ‘White Ripple’, 33 : ‘Princess Little White’, 34 : ‘Faery Light Pink’, 35 : ‘Sweet Ripple’, 36 : ‘Sunday Ripple’, 37 : ‘Angel Mice’, 38 : ‘Cheryl Mice’, 39 : ‘Lady Mice’ ①, 40 : ‘Lady Mice’ ②, 41 : ‘Princess Lemonade’, 42 : ‘Silk Ball’, 43 : ‘White Ripple Pure’, 44 : ‘White Jewel’, 45 : ‘Candy Mice’ ① 108 MORIKAWA ET AL.
M 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 M
4000bp
2000bp
1000bp
200bp
Fig. 19. Band patterns shown by ISSRprimer ISSR-11. M: 200bp DNA Ladder. 46 : ‘Candy Mice’ ②, 47 : ‘Sour Ripple’, 48 : ‘Southern Elegance White’, 49 : ‘Cutie Mice’, 50 : ‘Moon Light’, 51 : ‘Super Lemonade’, 52 : ‘Brown Eye’, 53 : ‘Lovely Friend’, 54 : ‘Oboro-Zuki’, 55 : ‘Hot Berry’, 56 : ‘Fire Cracker’, 57 : ‘Zairai Ki’, 58 : ‘Izu Yellow’, 59 : ‘Queen Mice’, 60 : ‘Fu-Ren-Ka’.
M 61 62 63 64 65 66 67 68 69 70 M
4000bp
2000bp
1000bp
200bp
Fig. 20. Band patterns shown by ISSR primer ISSR-11. M: 200bp DNA Ladder. 61 : ‘Carnival Queen’, 62 : ‘Peach Queen’, 63 : ‘Canaria Queen’, 64 : ‘Garnet Queen’, 65 : A. coronopifolium, 66 : A. foeniculaceum (12), 67 : A. foeniculaceum (15), 68 : G. coronaria, 69 : G. segetum, 70 : G. carinata.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M
4000bp
2000bp
1000bp
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Fig. 21. Band patterns shown by ISSR primer ISSR-49. M: 200bp DNA Ladder. 1 : 08-15-7, 2 : 10-3-4, 3 : 10-3-11, 4 : 10-4-1, 5 : 10-9-2①, 6 : 10-9-3①, 7 : c12-2-1, 8 : c12-3-1, 9 : c12-4-1, 10 : c12-13-1, 11 : c12-14-1, 12 : c12-21-1, 13 : c12-21-2, 14 : c12-21-3, 15 : c12-21-4. IDENTIFYING AND DISCRIMINATING CULTIVARS OF ‘MARGUERITES’ 109
M 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M
4000bp
2000bp
1000bp
200bp
Fig. 22. Band patterns shown by ISSR primer ISSR-49. M: 200bp DNA Ladder. 16 : c12-21-5, 17 : c12-21-6, 18 : c12-21-7, 19 : p12-45-1, 20 : p12-45-2, 21 : p12-45-3, 22 : p12-45-4, 23 : p12-46-1, 24 : p12-48-1, 25 : p12-81-1, 26 : ‘Zairai Shiro’, 27 : ‘Unzen Zairai’, 28 : ‘Early White’, 29 : ‘Faery White’, 30 : ‘Pink Southern Candle’
M 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 M
4000bp
2000bp
1000bp
200bp
Fig. 23. Band patterns shown by ISSRprimer ISSR-49. M: 200bp DNA Ladder. 31 : ‘Peach Southern Candle’, 32 : ‘White Ripple’, 33 : ‘Princess Little White’, 34 : ‘Faery Light Pink’, 35 : ‘Sweet Ripple’, 36 : ‘Sunday Ripple’, 37 : ‘Angel Mice’, 38 : ‘Cheryl Mice’, 39 : ‘Lady Mice’ ①, 40 : ‘Lady Mice’ ②, 41 : ‘Princess Lemonade’, 42 : ‘Silk Ball’, 43 : ‘White Ripple Pure’, 44 : ‘White Jewel’, 45 : ‘Candy Mice’ ①
M 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 M
4000bp
2000bp
1000bp
200bp
Fig. 24. Band patterns shown by ISSR primer ISSR-49. M: 200bp DNA Ladder ISSR. 46 : ‘Candy Mice’ ②, 47 : ‘Sour Ripple’, 48 : ‘Southern Elegance White’, 49 : ‘Cutie Mice’, 50 : ‘Moon Light’, 51 : ‘Super Lemonade’, 52 : ‘Brown Eye’, 53 : ‘Lovely Friend’, 54 : ‘Oboro-Zuki’, 55 : ‘Hot Berry’, 56 : ‘Fire Cracker’, 57 : ‘Zairai Ki’, 58 : ‘Izu Yellow’, 59 : ‘Queen Mice’, 60 : ‘Fu-Ren-Ka’. 110 MORIKAWA ET AL.
M 61 62 63 64 65 66 67 68 69 70 M
4000bp
2000bp
1000bp
200bp
Fig. 25. Band patterns shown by ISSR primer ISSR-49. M: 200bp DNA Ladder ISSR. 61 : ‘Carnival Queen’, 62 : ‘Peach Queen’, 63 : ‘Canaria Queen’, 64 : ‘Garnet Queen’, 65 : A. coronopifolium, 66 : A. foeniculaceum (12), 67 : A. foeniculaceum (15), 68 : G. coronaria, 69 : G. segetum, 70 : G. carinata.
‘Cheryl Mice’, these three cultivars have more closely confirmed in breeding lines of ‘Zairai Shiro’ in this study. related. Three out of five cultivars that have deep peach color which have been belonged to the group. The SUMMARY cultivars and the breeding lines with this flower color The DNA markers amplified by RAPD and ISSR were have 07-30-1 as the origin. Since ‘Fire Clacker’ was investigated to get know identification abilities of the belonged to the group ③ that have deep peach color in breeding lines as well as certain cultivars of the marguerite flower, this cultivar seemed to have the same origin as the and the intergeneric hybrids of marguerite x two species 07-30-1. Three species of Argyranthemum, two species of of Glebionis. The reproducible polymorphic bands were Glebionis and intergeneric hybrids performed the cultivar generated by RAPD-PCR using RAPD primer OPC-04, in ‘Fu-Ren-Ka’ of marguerite x G. carinata belonged to the respective species of Glebionis and the intergeneric group ④. Four intergeneric hybrid-cultivars of ‘Marguerite’ hybrids of ‘Marguerite’ and two species of Glebionis. and G. carinata belonged to the group ⑤. Basic marguerite The primer OPC-04 clearly identified either the two cultivars such as ‘Zairai Shiro’, ‘Unzen Zairai’ and ‘Early intergeneric hybrids of ‘Marguerite’ x G. coronaria or White’ were placed in the group ⑥. Two cultivars of ‘Marguerite’ x G. carinata. The polymorphic bands were ‘Sweet Ripple’ and ‘Silk Ball’ having double-flowered amplified by ISSR-PCR in the breeding lines and the type of unique character among the ‘Marguerite’ cultivars cultivars of the ‘Marguerite’, while they were not belonged to the group ⑦ which had the same morphological amplified by RAPD. A specific band was confirmed by character and detected molecular-systematically. These ISSR-5 primer in the ‘Marguerite’ cultivar strains of two cultivars were made by natural crosses in stead of ‘Zairai Shiro’. This primer ISSR-5 detected and identified hand-crosses, and thus, the parental origin has been the certain specific band among the band pattern of unknown, but these may have a common origin. Also, ‘Southern Elegance White’, this cultivar has cultivar ‘White Ripple Pure’ that belonged to the group ⑦ was ‘Early White’ for the paternal parent. Similar polymorphic natural hybrid seedling and ‘Silk Ball’ could one of the band pattern were confirmed by ISSR-PCR, in the two parents. All ‘Marguerite’ and two intergeneric hybrid breeding lines and the cultivars of the ‘Marguerite’ which cultivars with G. coronaria belonged to group ⑧. As a have similar flower color, flowering type, and share the result, that have nearly characteristic in traits like flower hybrid parents. color, flower type and parental origin indicated similar polymorphic banding pattern in ISSR. These were LITERATURE CITED generally decided to multiple groups by on a dendrogram, Aoyama, M. 2000. Observations of chromosome numbers and but mixed of breeding lines and cultivars was confirmed, cytological features in marguerite varieties (Argyranthemum frutescens L.). Soc. Hort. Sci. 69 (Suppl. 2): 201 (In clearly classification was difficult. It could be caused by Japanese). Abstracts for the Autumn Meeting of the bleeding lines and cultivars of the ‘Marguerite’ were very Japanese Society for Horticultural Science in Hiroshima. close genetically, and genome structures had been Bramwell, D. and Bramwell, Z. 1974. Wild Flowers of the complex by repeat of artificial hybrid in breeding process. Canary Islands. Thornes, London. p. 437. Bremer, K. and Humphries, C. J. 1993. Generic monograph of However, more polymorphic analysis in basic cultivars the Asteraceae-Anthemideae. Bull. Nat. Hist. Mus. such as ‘Zairai Shiro’ and wild species that may be London (Botany) 23(2): 71-177. concerned in breeding of marguerite could allow to detect Cheek, R. 1993. La Belle Marguerite. Royal Horticulture Society, London. effective DNA marker that can identify a origin of Doyle, J. J. and Doyle, J. L. 1987. A rapid DNA isolation breeding lines and cultivars just like specific bands was procedure for small quantities of fresh leaf tissue. IDENTIFYING AND DISCRIMINATING CULTIVARS OF ‘MARGUERITES’ 111
08-15-7 c12-21-6 c12-21-3 c12-14-1 ‘Super Lemonade’ c12-21-7 c12-21-2 10-9-2① c12-4-1 ① c12-21-5 ‘Oboro-Zuki’ 10-9-3① c12-21-1 c12-21-4 c12-13-1 ‘White jewel’ ‘White ripple’ ‘Princess Little white’ 10-3-11 c12-2-1 c12-3-1 ‘Angel mice’ ② ‘Princess lemonade’ ‘Lovely friend’ ‘Sunday ripple’ ‘Cheryl mice’ ‘Lady mice’① ‘Lady mice’② 10-3-4 p12-45-1 p12-81-1 ③ ‘Moon light’ p12-46-1 ‘Sour ripple’ ‘Firecracker’ ‘Fu-Ren-Ka’ A.coronopifolium A.foeniculaceum (12) A.foeniculaceum (15) ④ G.coronaria G.segeta G.carinata ‘Faery white’ ‘Faery light pink’ ‘Peachqueen’ ⑤ ‘Canaria queen’ ‘Garnet queen’ ‘Carnival queen’ 10-4-1 ‘Hot berry’ p12-45-2 ‘Zairai Shiro’ ⑥ ‘Brown eye’ p12-45-4 ‘Unzen Zairai’ ‘Early white’ ‘Peach southern candle’ ‘White ripple pure’ ⑦ ‘Sweet ripple’ ‘Silk ball’ p12-45-3 ‘Cutie mice’ ‘Southern elegance white’ ‘Queen mice’ p12-48-1 ⑧ ‘Pink southern candle’ ‘Candy mice’① ‘Candy mice’② ‘Zairai Ki’ ‘Izu yellow’
Fig. 26. Phylogenetic trees of genetic relationships among the members of ‘Marguerite’ based on the Polymorphic bands prepared by ISSR-PCR determined by the Ward method. 112 MORIKAWA ET AL.
Phytochem. Bull. 19: 11-15. japonica of East Asia and Pogonia ophioglossoides of Hiraoka, Y., Kuramoto, N., Okamura, M., Ohira, M., Southeastern North America. Proc. APOC7: 91-92. Taniguchi, T., and Fujisawa, Y. 2009. Clone identification Racharak, P. and Eiadthong, W. 2007. Genetic relationship and genetic relationship among candidates for superior among subspecies of Musa acuminata Colla and trees in Rhus succedanea L. using ISSR, AFLP, and A-genome consisting edible cultivated bananas assayed RAPD markers. Journ. Jpn. Hort. Soc. 91: 246-252. with ISSR markers Songklanakarin. Journ. Sci. Technol., Huang, J., Tanabe, K. and Itai, A. 2003. Identification of 29: 1479-1489. flowering Lotus cultivars by ISSR (inter-simple sequence Takahashi, C. and Kondo, K. 2004. A comparison of katyotypes repeat) markers. Hort. Res. Japan 2: 259-264. in two artificial reciprocal hybrids between Pogonia Huang, J., Tanabe, K. and Itai, A. 2004. Identification of minor and Pogonia ophioglossoides (Orchidaceae). parent-offspring correlation and presumption of pollen Chrom. Sci. 8: 11-16. parent in Lotus with ISSR markers. Hort. Res. Japan 3: Tatarenko, I. V., Nishimura, M., Morikawa, M., Shinoyama, 251-256. H., Suzuki, K., Kimura, S., Tatarenko, E., Motohashi, T. Kondo, K., Abd El-Twab, M. H., Idesawa, R., Kimura, S. and and Kondo, K. 2013. Variabilities in karyotype and Tanaka, R. 2003. Chapter 6. Genome phylogenetics in molecular ISSR in Gonospermum fruticosum (C. Smith et Chrysanthemum sensu lato. pp. 117-200. In; Sharma, A. Link) Less., Argyranthemum cornopifolium (Willd.) K. and Sharma, A., Eds. Plant genome, Biodiversity and Humphries and two strains of A. foeniculaceum (Willd.) evolution. Science Publishers, Inc., Plymouth, United Webb ex Schultz-Bip. (Asteraceae the tribe Anthemideae) Kingdom, pp.386. collected in the Canary Islands. Chrom. Bot. 8: 55-60. Maeda, F., Tsugane, T., Suzuki, S. and Aoki, K. 2008. Genetic Trehane, P. 1995. Proposal to conserve Chrysanthemum L. homogeneity of ‘Koitozairai’, a local varietiy of soybean, with conserved type (Compositae). Taxon 44: 439-444. by ISSR analysis of polymorphism. Research Report of Tsukamoto, Y. 1986. Cv. ‘Izu yellow’. Asahi Engei Hyakka Chiba Prefectural Integrated Agriculture Research Center 24: VII: 79 (In Japanese). 7: 69-73 (In Japanese). Williams, J. G. K., Kublick, A. R., Livak, K. J., Refalsky, J. Ohtsuka, H. and Inaba Z. 2008. Intergeneric hybridization of and Tingey, S. V. 1990. DNA polymorphism amplified by marguerite (Argyranthemum frutescens) with annual arbitrary primers are useful as genetic markers. Nucl. chrysanthemum (Glebionis carinatum) and crown daisy Asids Res. 18: 6531-6536. (G. coronaria) using ovule culture. Plant Biotechnology Zietkiewicz, E., Rafalski, A. and Labuda, D. 1994. Genome 25: 535-539. fingerprinting by simple sequence repeat (SSR)-anchored Oryu, M. and Kondo, K. 2001. Relationship between Pogonia polymerase chain amplification. Genomics 20: 176-183.