Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2005
Volume 8
Issue 2
Topic:
Horticulture
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Lema-Rumińska J. , Zalewska M. , Sadoch Z. , Jerzy M. 2005. IDENTIFICATION OF CHRYSANTHEMUM (Dendranthema grandiflora Tzvelev) MUTANTS OF NERO AND WONDER GROUPS USING RAPD MARKERS, EJPAU 8(2), #04.
Available Online: http://www.ejpau.media.pl/volume8/issue2/art-04.html

IDENTIFICATION OF CHRYSANTHEMUM (DENDRANTHEMA GRANDIFLORA TZVELEV) MUTANTS OF NERO AND WONDER GROUPS USING RAPD MARKERS

Justyna Lema-Rumińska1, Małgorzata Zalewska2, Zbigniew Sadoch3, Marek Jerzy4
1 Department of Ornamental Plants and Vegetable Crops, University of Technology and Agriculture in Bydgoszcz, Poland
2 Department of Ornamental Plants and Vegetable Crops, University of Technology and Life Sciences in Bydgoszcz, Poland
3 Plant Breeding and Acclimatization Institute in Bydgoszcz
4 Department of Ornamental Plants, Agricultural University of Poznan, Poland

 

ABSTRACT

The present study investigated chrysanthemum radiomutants (Dendranthema grandiflora Tzvelev) and their original cultivars of two cultivar groups; Nero and Wonder. The Ney similarity index revealed a high diversity between the cultivars and in all of the cases was less than 0.829. The cluster analysis separated cultivars into three groups, with the one of Nero group, the other of Wonder group and the cultivar 'Bronze Wonder'.

The aim of the present study was to identify cultivars using RAPD markers. Eight of twenty primers resulted in polymorphic bands which allowed for distinguishing between the chrysanthemum cultivars examined. A single primer or set of two primers resulted in a specific fixed and repeatable pattern of bands in each original cultivar and in each mutant - DNA-fingerprinting.

Key words: Dendranthema grandiflora, radiomutants, RAPD, DNA-fingerprinting, identification.

INTRODUCTION

Dendranthema grandiflora Tzvelev is a one of the important ornamental plants in production and breeding of horticulture. Today a source of new cultivars of chrysanthemums is to make a cross between two cultivars, the appearance of sports or transgenic plants. The other efficient method of obtaining new cultivars is also mutation breeding [1, 2, 3, 17]. The first stage of estimation of new cultivars is identifying them and distinguishing between them. Occasionally, identification has been based on phenotypical characters, but a better method involves DNA markers e. g. RAPD (Random Amplified Polymorphic DNA), utilizing polymerase chain reaction [13]. The first study on the identification of chrysanthemums with the application of RAPD markers was carried out by Wolff and Peters-Van Rijn [14].

The aim of the present study using the RAPD method was distinguishing between the chrysanthemum radiomutants of groups, Nero and Wonder, and their identification.

MATERIAL AND METHODS

Plant materials. The plant material consisted of a chrysanthemum (Dendranthema grandiflora Tzvelev) cultivars groups, Nero and Wonder (table 1 and figure 1). The `Mini Nero´ mutant was obtained as a result of X radiation of 25 Gy on the `Red Nero´ leaf cuttings in vivo but `Bronze Wonder´ and `Red Wonder´ mutants were obtained as a result of gamma radiation of 15 Gy on the `Lilac Wonder´ leaf explants in vitro. All the mutants were vegetatively propagated in vivo by shoot cuttings in the greenhouse.

Table 1. Inflorescence characteristics for original cultivars `Red Nero´ and `Lilac Wonder´ and its mutants

Cultivar

Code

Inflorescence

colour

size

type

Red Nero

RN

dark red with yellow eye

small-flower

semifull

Mini Nero1)

MN

orange red with yellow eye

small-flower

semifull

Lilac Wonder

LIW

violet pink

middle-flower

full, semiball

Bronze Wonder2)

BW

reddish brown

middle-flower

full, semiball

Red Wonder2)

RW

red

middle-flower

full, semiball

1)The mutant was written in Polish Cultivar Register in 1995.
2)These mutants were written in Polish Cultivar Register in 1996.

Figure 1. The Nero and Wonder groups

DNA isolation. Total DNA was isolated according to the method described by Davis et al. [4]. Five grams of fresh matter of leaves and leaf buds obtained from young plants, grown in the glasshouse were used for DNA preparation. Bulk DNA samples consisted of ten individual plants. The CTAB-based (hexadecyltrimethylammonium bromide, Sigma-Aldrich, St. Louis, USA) extraction procedure was used for additional DNA purification [9].

RAPD analysis. Seven decanucleotides and one nine-mer oligonucleotide selected after screening 20 primers in preliminary experiments were used in PCR reactions. The primers are listed in table 2 (IDT, IA, USA). PCR amplification reactions were performed in a final volume of 25 µL containing 0.5 unit of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania), 1 × reaction buffer, 3.0 mM MgCl2, 200 µM each of dATP, dCTP, dGTP and dTTP, 1 µM primer and 20 ng genomic DNA. Amplifications were performed with a Biometra Uno-Thermoblock (Biometra GmbH, Germany) programmed as follows: 4 min at 94°C for the first cycle, 45 cycles at a denaturation temperature of 94°C for 1 min, annealing temperature 36°C during 1 min, and extension temperature 72°C for 2 min. After the last cycle samples were incubated for 4 min at 72°C. RAPD analyses were run in duplicate for all materials.

The PCR products were separated on 1.5% agarose gels stained with ethidium bromide. The size of the DNA bands was identified by using the "Gel Doc" gel documentation system and computer program "Quantity One" (Bio-Rad, Hercules, USA). The genetic similarity index was calculated according to the formula given by Nei and Li [8]. Only the bands that did not vary between replicates were used for the calculations. Cluster analysis was done with the Statistica Computer Package Version 5, `97 Edition.

RESULTS

From among of the twenty primers examined eight (OPB-04, OPB-18, OPF-06, OPM-09, OPM-18, RAPD-01, RAPD-09 and RAPD-12) resulted in polymorphic bands which were the basis for distinguishing the chrysanthemum cultivars (table 2). Eight primers amplified a total of 67 bands including polymorphic fragments. The number of polymorphic bands varied between 2 and 16 per primer, with amplification products varying between 212 - 2730 bp.

Table 2. Primers nucleotide sequences and the number of RAPD polymorphic fragments per primer

Primer

Nucleotide sequences

No. of polymorphic fragments/primer

OPB-04

5´-GGA/CTG/GAG/T-3´

11

OPB-18

5´-CCA/CAG/CAG/T-3´

7

OPF-06

5´-GGG/AAT/TCG/G-3´

16

OPM-09

5´-GTC/TTG/CGG/A-3´

7

OPM-18

5´-CAC/CAT/CCG/T-3´

2

RAPD-01

5´-TCC/TAC/GCA/C-3´

5

RAPD-09

5´-GAC/CGC/TTG/T-3´

10

RAPD-12

5´-GGG/CTC/ATA/-3´

9

Primer OPF-06 (figure 2A, table 3), primer RAPD-09 (figure 2B, table 4), or primer sets: RAPD-01 (figure 2C) and OPF-06; RAPD-12 and OPF-06; OPB-04 and OPF-06; OPB-18 and OPF-06 gave a specific fixed and repeatable (maintaining the reaction conditions) set of bands, the DNA fingerprinting in the original cultivars or mutants.

OPF-06 and RAPD-09 primers distinguished 5 cultivars examined. The other remaining primers distinguished from 2 to 3 cultivars.

Figure 2. RAPD analysis of the original chrysanthemum cultivars `Red Nero´ and `Lilac Wonder´ and its radiomutants using primer: A - OPF-06, B - RAPD-09,
C - RAPD-01 (M - molecular marker DNA Ladder Mix, the code of cultivar: see table 1)

Table 3. DNA fingerprinting of original chrysanthemum cultivars `Red Nero´ and `Lilac Wonder´ and ist mutants using OPF-06 primer

Primer

Molecular weight
(bp)

Cultivar

RN

MN

LIW

RW

BW

OPF-06

1578

0

0

0

0

1

1277

1

1

0

0

0

1112

1

1

1

0

1

948

0

0

1

0

1

911

1

0

0

0

0

The band: present (1), absent (0)
The code of cultivar: see table 1.

Table 4. DNA fingerprinting of original chrysanthemum cultivars `Red Nero´ and `Lilac Wonder´ and its mutants using RAPD-09 primer

Primer

Molecular weight
(bp)

Cultivar

RN

MN

LIW

RW

BW

RAPD -09

2107

0

0

0

0

1

999

1

1

0

1

1

931

1

1

0

0

0

877

1

0

0

0

0

755

0

1

0

0

0

710

0

0

1

1

0

Explanation: see table 2.

Table 5. Similarity index (Nei and Li 1979) for the original cultivars - `Red Nero´ and `Lilac Wonder´ and its radiomutants
 

RN

MN

LIW

RW

BW

RN

1

0.696

0.603

0.533

0.640

MN

0.696

1

0.600

0.511

0.581

LIW

0.603

0.600

1

0.829

0.643

RW

0.533

0.511

0.829

1

0.566

BW

0.640

0.581

0.643

0.566

1

The code of cultivar: see table 1.

The Ney similarity index values given in table 5 showed that the highest value of the similarity index out of all the cultivars estimated which amounted to 0.829 was observed between the original cultivar `Lilac Wonder´ and its mutant `Red Wonder´, while the lowest (0.511) between mutants `Red Wonder´ and `Mini Nero´. Relative a high similarity index was also recorded between cultivars: ´Red Nero´ and `Mini Nero´ (0.696). The genetic similarity of Nero and Wonder groups revealed by cluster analysis based on RAPD markers is shown on the dendrogram (figure 3). The cluster analysis readily separated the accessions we analysed into three groups, with the one of Nero group, the other of Wonder group and the cultivar - `Bronze Wonder´.

Figure 3. Dendrogram of the original chrysanthemum cultivars `Red Nero´ and `Lilac Wonder´ and its radiomutants revealed by cluster analysis based on the genetic similarity indices (the code of cultivar: see table 1)

DISCUSSION

Wolff et al. [15] used RAPD markers to identify chrysanthemums. They managed to distinguish between chrysanthemum cultivars which did not belong to a single group using two or three primers. However Wolff et al. [15] suggested, that no possibility on a DNA level to distinguish the family cultivars derived vegetatively from one original cultivar.

The present study of the two original cultivars and their mutants of the Nero and Wonder groups and the study of the Lady group [6], obtained due to ionizing radiation, confirmed a possibility of distinguishing between them at molecular level by RAPD markers. Eight primers resulted in polymorphic bands, which point to differences in the genotypes studied. Two of the primers OPF-06 and RAPD-09 made they possible to identify all cultivars. The other primers were grouped into sets composed of two primers which made it possible to identify also all the genotypes examined. The Ney similarity index shown relative a high similarity between cultivars `Red Nero´ and `Mini Nero´. It was interesting because `Mini Nero´ mutant had a most morphologically changes (a dwarf growth with brighter and smaller inflorescences) in relation to the original cultivar. Possible causes of the lack of distinct differences of RAPD patterns between `Mini Nero´ and `Red Nero´ could be that polymorphic bands resulted from the amplification of both coding and non-coding genome regions. Arbitrary primers used in RAPD methods could produced predominantly DNA fragments, which did not reflect changes in genes involved in expression of characteristic `Mini Nero´ and `Red Nero´ phenotypes. A low similarity was observed between mutant `Bronze Wonder´ and `Red Wonder´, and `Bronze Wonder´ mutant was also separated from the Wonder group on the dendrogram. May be it was connected with the ploidy level, because the `Bronze Wonder´ mutant had 54 chromosomes number but others Wonder cultivars only 45 [5].

According to Wolff [16], artificially induced mutants showed polymorphic bands which distinguished them from the others and suggested greater changes in the genome than those in sports. The changes could have concerned point mutations in genes as well as greater chromosomal aberrations, e.g. a loss of a part or the entire chromosome [11, 16]. The study on the evaluation of ploidy in chrysanthemum cultivars of Nero and Wonder groups shown change in the chromosome number only in one of the mutant, `Bronze Wonder´. The number of chromosomes by nine differed from that of the original cultivar [5].

RAPD markers were also used for characterisation of fifteen nonrelated commercial varieties of chrysanthemum by Martin et al. [7]. The most unrelated variety, from the 15 studied by Martin et al. [7], has less than 60 % similarity with the others. In our study of closely related genotypes of Nero and Wonder groups, we observed similarity on a little higher level within groups. This fact suggested, that the changes could have concerned of great chromosomal aberrations in mutants genes.

Scott et al. [10] and Trigiano et al. [12] used DAF and ASAP techniques for distinguishing closely related cultivars of chrysanthemum, and they used few primer combination to identification all members of groups, but those methods are laborious and expensive in relation to RAPD markers.

The present study confirm the applicability of the RAPD method to identify chrysanthemum cultivars. The fact, that it was sufficient, to analyse all genotypes studied, only 1 or 2 primers confirms a greater applicability of this method to identify closely related cultivars as early as at the cutting stage, which is important in mutation breeding.

REFERENCES

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  2. Broertjes C., Roest S., Bokelmann G. S., 1976. Mutation breeding of Chrysanthemum morifolium Ram. Using in vivo and in vitro adventitious bud techniques. Euphytica 25, 11-19.

  3. Datta S. K., Gupta M. N., 1981. Cytomorphological, palynological and biochemical studies on control and gamma-induced mutants of chrysanthemum cultivar E-13. Sabrao Journal 13 (2), 136-148.

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  5. Lema-Rumińska J., Zalewska M., 2002. Ocena ploidalności mutantów chryzantemy wielkokwiatowej (Dendranthema grandiflora Tzvelev) uzyskanych w wyniku mutagenezy indukowanej in vitro i in vivo promieniowaniem jonizującym [Evaluation of ploidy in chrysanthemum mutants (Dendranthema grandiflora Tzvelev) obteined in mutagenesis induced in vitro and in vivo by ionizing radiation]. Acta Sci. Pol., Hortorum Cultus 1 (2), 43-48 [in Polish].

  6. Lema-Rumińska J., Zalewska M., Sadoch Z., 2004. Radiomutants of chrysanthemum (Dendranthema grandiflora Tzvelev) of the Lady group: RAPD analysis of the genetic diversity. Plant Breeding 123, 1-4.

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  15. Wolff K., Zietkiewicz E., Hofstra H., 1995. Identification of chrysanthemum cultivars and stability of DNA fingerprint patterns. Theor. Appl. Genet. 91, 439-447.

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Justyna Lema-Rumińska
Department of Ornamental Plants and Vegetable Crops,
University of Technology and Agriculture in Bydgoszcz, Poland
6 Bernardynska Street, 85-029 Bydgoszcz, Poland
tel. (+48 52) 374 95 22
fax (+48 52) 374-95-05
email: lem-rum@atr.bydgoszcz.pl

Małgorzata Zalewska
Department of Ornamental Plants and Vegetable Crops,
University of Technology and Life Sciences in Bydgoszcz, Poland
Bernardyńska 6, 85-029 Bydgoszcz, Poland
Phone: (+48) 52 374 95 36
email: zalewska@utp.edu.pl

Zbigniew Sadoch
Plant Breeding and Acclimatization Institute in Bydgoszcz
10 Powstańców Wielkopolskich Street, 85-950 Bydgoszcz, Poland

Marek Jerzy
Department of Ornamental Plants,
Agricultural University of Poznan, Poland
al. Dąbrowskiego 159, 60-594 Poznan, Poland

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