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Molecular classification of wild roses using organelle DNA probes
SCIENTIA HORTlCULTuRR ELSEXIER
Scientia Horticulturae 68 (1997) 191-196
Molecular classification of wild roses using organelle DNA probes S. Matsumoto a,*, H. Wakita a, H. Fukui b aDepurrmenr of Biology, Faculty ofEducution, Gifi University, Gi& 501 -I 1, Japun b Luborutory #Horticulture, Faculty r$Agriculrure. Gifu University, Gific 501 -I I, Jupun Accepted 23 August 1996
Abstract Chloroplast and mitochondrial DNA probes were used to deduce molecular systematics of 17 wild rose species by restriction fragment length polymorphism. Phylogenetic trees divided these genotypes into two to three groups of cytoplasmic relatedness depending upon the probe. The data indicated differential maternal inheritance of mitochondrial and chloroplast DNA in rose. The species forming a clade on phylogenetic trees also differed from one another phenotypically and belonged to different sections. 0 Elsevier Science B.V. Keywords:
Rose; RFLP; Organelle;
Phylogeny
1. Introduction The genus Rosa contains more than 140 species that are widely distributed in Europe, Asia, the Middle East and North America. Only seven species have contributed to the modem commercial rose. The genetic background of modem roses is therefore narrow compared with the available Rosa species. Plant systematics has been usually based on morphological characters, as an expression of the genetic phenotype. In contrast, DNA polymorphisms offer direct observation of the plant genotype. Although some rose cultivars have been identified using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) analyses of nuclear DNA (Hubbard et al., 1992; Rajapakse et al., 1992; Tortes et al., 1993), the degree of heterogeneity of organelle DNAs among rose species is unknown. RFLP analysis has been used to compare organelle DNAs from a wide range of other plant species (Pring and Lonsdale, 1985; Palmer, 1987). These comparisons
’ Corresponding
author. Tel.: + 81-58-293-2257;
fax: + 81-58-293-2207.
0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(96)00970-3
192
S. Matsumoto et al./ Scientia Horticulturae
68 (1997) 191-196
have proven particularly useful in the survey of cytoplasmic variation, and to trace the maternal lineages during evolution. Since some useful characteristics of higher plants, such as cytoplasmic male sterility and variegation in Epilobium show cytoplasmic inheritance, the identification of cytoplasmic diversity is a first step in preservation of genetically diverse germplasm. In this study, we describe the cytoplasmic diversity among wild roses on the basis of chloroplast and mitochondrial DNA analyses.
2. Materials and methods 2.1. Plant material
Most rose species used in this study were grown either in a greenhouse or the field at the Experimental Farms, Faculty of Agriculture, Gifu University, Japan. Rosa canina 1 and 2 samples were collected from different areas in the field, and R. canina ‘Inermis’ 1 and 2 samples were collected from the greenhouse and the field, respectively. Young leaves were collected and stored at -80°C until use. 2.2. Isolation and Southern blot analyses of rose DNA Total DNA from leaves of individual plants was isolated as described by Thomas et al. (1993). Five micrograms of total DNA were digested with a restriction enzyme, electrophoresed on 0.7% agarose gels, and transferred to a nylon membrane (Nylon membrane, positively charged, Boehringer Mannheim). The filter was hybridised with DNA probes labelled with a DIG DNA Labelling kit (Boehringer Mannheim). DNA probes were from sugar beet mitochondrial DNA; 1.5 kbp EcoRI fragment containing coxl gene (Senda et al., 1991) 4.3 kbp EcoRI fragment containing atp6 gene and tobacco chloroplast DNA; Bal, Ba2, B22, B25, B28 and IRB fragments (Sugiura et al., 1986). 2.3. Phylogenetic analysis The maximum parsimony method in the computer software PAUP version 3.1 .l (Swofford, 1993) was used to find the most parsimonious trees. For bootstrap analysis, 100 replications were conducted for each branch (Felsenstein, 1985).
Notes to Table 1: a A, Caninae; B, Gallicanae; C, Synstylae; D, Banksianae; E, Carolinae; F, Cassiorhodon; G, Pimpinellifoliae. b Additional fragments of 10.0 kbp, 7.5 kbp and 1.6kbp are present in all rose species. ’ Additional fragment of 5.9 kbp is present in all rose species. d Additional fragment of 2.5 kbp is present in all rose species. ’ Additional fragments of 3.6 kbp and 1.7 kbp are present in all rose species. f Additional fragments of 10.3 kbp, 9.2 kbp, 5.6 kbp, 1.0 kbp and 0.8 kbp are present in all rose species. g Additional fragment of 2.7 kbp is present in all rose species.
1
I
R. pimpinellifoliu
‘Stanwell Perpetual’ M&s domesticu
20
‘ plena’
R. pimpinellifolia
R. dumetorum ‘laxa’
R. moyesii ‘Geranium’
R. culifornica
R. rugosu
R. oirginiunu
R. hunksiue
R. x dupontii
R. mult$oru
R. x richurdii
R. mucrunthu
R. ruhiginosu
R. gluucu
‘Brags Stachellose’
R. cuninu
R. cuninu ‘Inermis’ 2
R. cuninu ‘Inermis’
R. cuninu 2
Rosu cuninu I
Species
6 7 8 9 10 II 12 13 14 I5 16 17 18 19
Sample no.
A A B B C C D E F F F F G G
Section a
10.0, 6.2, 5.7, 2.9
5.2 5.2 5.2 5.2 5.2 5.2 5.2 3.6 3.6 5.2 3.6 5.2 3.6 5.2 10.4
8.9 8.9 8.9 8.9 8.9 8.9 8.9 10.2 10.2 8.9 10.2 8.9 10.2 8.9 9.4, 2.6,
I .9
6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 10.4, 6.5 9.5, 6.5 6.7, 3.7, 2.8 9.5, 6.5 6.7, 3.7, 2.8 9.5, 6.5 6.7, 3.7, 2.8 10.0, 6.0
5.6 5.6 5.6 5.6 5.6 5.6 5.6 2.0 2.0 5.6 2.0 5.6 2.0 5.6
5.6 5.6 5.6 5.6 5.6
2.8 2.8 2.8 2.8 2.8
6.7. 6.7, 6.7, 6.7, 6.7,
8.9 8.9 8.9 8.9 8.9
5.2 5.2 5.2 5.2 5.2
3.7, 3.7, 3.7, 3.7, 3.7,
B22 BumHI ’
Hind111 d
Ba2
Ba2 BumHI ’
Bal BumHI b
in rose species and all fragments
fragments
Polymorphic
from chloroplast
10.0, 7.8, 2.8, 2.2, 0.9, 0.7
2.5
2.5
2.5 _
2.5 2.5 2.5 2.5 2.5 2.5 2.5
2.5 2.5 2.5 2.5 2.5
IRB Hind111 f
3.2 3.2 3.2 4.6 4.6 3.2 4.6 3.2 5.1 3.2
2.1 2. I 2.1 4.0 4.0 2.1 4.7 2.1 4.7 2.1
5.9, 3.7
3.2 3.2
2.1 2.1
6.9, 0.8
3.2 3.2
3.2 3.2 3.2 3.2 3.2
2.1 2.1 2.1 2. I 2.1 2. I 2.1
Hind111
COXI
coxl EcoRI
10.2, 8.6
5.8 _
5.8 5.8
_
_
_
_
BumHI g
utp6
DNA, cuxf and urp6 fragments
in M. domesficu detected with:
Summary of RFLP analyses of wild rose species and Mulus domesticu using Bal, Ba2, B22 and IRB fragments from mitochondrial DNA as probes. Fragment sizes are indicated in kbp
Table
3k
P
194
S. Matswnoto et al./Scientia
Horticulrurae
68 (1997) 191-196
3. Results and discussion 3.1. RFLP analyses The species were classified into two groups (Table l), when the DNA of 17 rose species was digested with BamHI, and hybridised with tobacco chloroplast DNA probe Bal . A 5.2 kbp fragment was missing in samples 13, 14, 16 and 18, with a 3.6 kbp fragment being present. A restriction site mutation may have occurred in the original 5.2 kbp fragment leaving a 1.6 kbp fragment. The same grouping was generated on the BamHI-digested DNA with the Ba2 probe from the small-single copy (SSC) region, the B22 probe from the LSC, and Hi&III-digested DNA with the IRB probe from the inverted repeat region (Table 1). A 10.2 kbp fragment of samples 13, 14, 16 and 18 on BamHI-digested/Ba:! probe might have been generated from an 8.9 kbp and a 1.3 kbp fragment missing on this blot by the restriction site mutation. Similarly, a 5.6 kbp fragment of samples 1-12, 15, 17 and 19 on BamHI_digested/B22 probe might have been generated from a 2.0 kbp and a 3.6 kbp fragment. Moreover, a 2.5 kbp fragment on HindIII-digested/IRB might have been generated from a 1.O kbp and a 1.5 kbp or a 0.8 kbp and a 1.7 kbp fragment, but the latter fragments were missing on this blot. Hybridisation of the HindIII-digested DNA with the Ba2 probe showed a similar classification which was explained by a restriction site mutation with the probe distinguishing Rosa virginiana (no. 13) from the Rosa rugosa (no. 14), Rosa moyesii ‘Geranium’ (no. 16) and Rosa pimpinellifolia (no. 18) (Table 1). In this case, a 6.5 kbp fragment of samples 13, 14, 16 and 18 might have been generated from a 3.7 kbp and a 2.8 kbp fragment, a 9.5 kbp fragment of samples 14, 16 and 18 might have been generated from a 6.7 kbp and a 2.8 kbp fragment, and a 10.4 kbp fragment of sample 13 might have been generated from a 9.5 kbp and a 0.9 kbp fragment missing on this blot. No polymorphism was detected when the B25 and B28 from LSC region were used as probes (data not shown). Reproducibility of the molecular classification was confirmed with DNA probes from mitochondrial DNA. Polymorphic fragments were detected on EcoRI and HindIII-digested DNA with the sugar beet mitochondrial DNA probe coxl, and the species classified into three groups (Table 1). Hybridisation of the BamHI-digested DNA with sugar beet mitochondrial DNA probe atp6 also showed single polymorphic fragments, and the species classified into two groups. This is the first report on cytoplasmic diversity within the Rosa genus by RFLP analyses. All of the polymorphic fragments in R. pimpinellifolia ‘Stanwell Perpetual’, the progeny of the cross R. X a’amascena bifkra and R. pimpinellifolia were not found in R. pimpinellifolia. Conversely, R. X dupontii (Rosa gallica X Rosa moschata) exhibited the same fragment patterns as Rosa macrantha, of R. gallica origin. These results support the maternal inheritance of cytoplasmic organelles in rose. 3.2. Phylogenetic analyses All of the bands detected in the rose species and Malus domestica were used in the maximum parsimony analysis with Malus domestica used as the out group. The bands
S. Matsumoto et al./Scientia
Horticulturae
68 (1997) 191-196
-1
(Al
-2
(A)
-3
(Al (A) (A) (A) (A) (BI (Bl (Cl (C) ID) IF1 IF1
-4
-5 -6 -7 -100.0%
6 -9 -
-9,.7%-c6,o%f
10 11 12 15
i
;:;
16 20
(G)
195
Fig. 1. Phylogenetic tree calculated from chloroplast DNA probe data using the maximum parsimony method. Numbers and letters in parentheses correspond to sample numbers and sections listed in Table 1. Bootstrap confidence values (%) are indicated on the branches.
were aligned in order of size for each blot, and the data matrix was constructed for each species according to the presence and absence of the bands. Presence and absence were indicated by 1 and 0, respectively, in the data matrix. R. cunina was designated as 1100100100110001011010001101101010101010 from the data using chloroplast DNA probes of Bal, Ba2/BamHI, Ba2/HindIII, B22, IRB, in that order. A single most parsimonious tree with bootstrap values was constructed from the data matrix of chloroplast DNA probes (Fig. 1). Seventeen rose species were divided into two groups; one was composed of all species in sections Caninae, Gallicanae, Synstylae and Banksianae, with some species from sections Cassiorhodon and Pimpinellifoliae, and the other group was composed of species from section Carolinae and some species from sections Cassiorhodon and Pimpinellifoliae (samples 13, 14, 16 and 18). A similar tree
Fig. 2. Phylogenetic tree calculated from mitochondrial DNA probe data using the maximum parsimony method. Numbers and letters in parentheses correspond to sample numbers and sections listed in Table 1. Bootstrap confidence values (%) are indicated on the branches.
1%
S. Mutsumoto et ul. / Scientiu Horticulturue
68 (1997) 191- I96
with bootstrap values was also constructed from the data matrix of mitochondrial DNA probes (Fig. 2). Rose species were mainly divided into four groups. One group corresponded to the chloroplast group including sections Caninae, Gallicanae, Synstylae and Banksianae, with some species from sections Cassiorhodon and Pimpinellifoliae in Fig. 1, and the remaining chloroplast group (samples 13, 14, 16 and 18) was further subdivided into nos. 16, 18 and 13 + 14 (Fig. 2). While the molecular classification was in general agreement with the traditional classification, there were some anomalies. For example, R. virginiana and R. rugosa formed a clade on phylogenetic trees of chloroplast and mitochondrial DNA with 98.7% and 8 1.6% of bootstrap replications, respectively, although they differ from one another phenotypically and were not in the same section. Further investigations using sequence analysis of the matK and rbcL loci, are in progress, examining a large number of wild roses in order to elucidate organelle diversity and its evolutionary implications.
Acknowledgements
We thank Dr H. Kato and T. Tsuchiya for computer analysis, Dr M. Thomas for critical reading of the manuscript, Dr T. Mikami for donating the mitochondrial DNA probes and Dr M. Sigiura of the Centre for Gene Research, Nagoya University for also donating the tobacco chloroplast DNA clones. This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture and Science of Japan (No. 07660032 and 07238210).
References Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39: 783-791. Hubbard, M., Kelly, J., Rajapakse, S., Abbott, A. and Ballard, R., 1992. Restriction fragment length polymorphisms in rose and their use for cultivar identification. HortScience, 27: 172- 173. Palmer, J.D., 1987. Chloroplast DNA evolution and biosystematic uses of chloroplast DNA. Am. Nat., 130: S6-S29. Pring, D.R. and Lonsdale, D.M., 1985. Molecular biology of higher plant mitochondrial DNA. Int. Rev. Cytol., 97: l-46. Rajapakse, S., Hubbard, M., Kelly, J.W., Abbott, A.G. and Ballard, R.E., 1992. Identification of rose cultivars by restriction fragment polymorphism. Sci. Hortic., 52: 237-245. Senda, M., Harada, T., Mikami, T., Sugiura, M. and Kinoshita, T., 1991. Genome organization and sequence analysis of the cytochrome oxidase subunit 11 gene from normal and male-sterile mitochondria in sugar beet. Curr. Genet., 19: 175-181. Sugiura, M., Shinozaki, K., Zaita, N., Kusuda, M. and Kumano, M., 1986. Clone bank of the tobacco (Nicotiunu tubucum) chloroplast genome as a set of overlapping restriction endonuclease fragments: mapping of eleven ribosomal protein genes. Plant Sci., 44: 21 l-216. Swofford, D., 1993. PAUP: Phylogenetic Analysis using Parsimony, Version 3.11. Computer program distributed by the Illinois Natural History Survey, Champaign, IL. Thomas, M.R., Matsumoto, S., Cain, P. and Scott, N.S., 1993. Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification. ‘Iheor. Appl. Genet., 86: 173-180. Tortes, A.M., Millan, T. and Cubero, J.I., 1993. Identifying rose cultivars using random amplified polymorphic DNA markers. HortScience, 28: 333-334.
Scientia Horticulturae 68 (1997) 191-196
Molecular classification of wild roses using organelle DNA probes S. Matsumoto a,*, H. Wakita a, H. Fukui b aDepurrmenr of Biology, Faculty ofEducution, Gifi University, Gi& 501 -I 1, Japun b Luborutory #Horticulture, Faculty r$Agriculrure. Gifu University, Gific 501 -I I, Jupun Accepted 23 August 1996
Abstract Chloroplast and mitochondrial DNA probes were used to deduce molecular systematics of 17 wild rose species by restriction fragment length polymorphism. Phylogenetic trees divided these genotypes into two to three groups of cytoplasmic relatedness depending upon the probe. The data indicated differential maternal inheritance of mitochondrial and chloroplast DNA in rose. The species forming a clade on phylogenetic trees also differed from one another phenotypically and belonged to different sections. 0 Elsevier Science B.V. Keywords:
Rose; RFLP; Organelle;
Phylogeny
1. Introduction The genus Rosa contains more than 140 species that are widely distributed in Europe, Asia, the Middle East and North America. Only seven species have contributed to the modem commercial rose. The genetic background of modem roses is therefore narrow compared with the available Rosa species. Plant systematics has been usually based on morphological characters, as an expression of the genetic phenotype. In contrast, DNA polymorphisms offer direct observation of the plant genotype. Although some rose cultivars have been identified using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) analyses of nuclear DNA (Hubbard et al., 1992; Rajapakse et al., 1992; Tortes et al., 1993), the degree of heterogeneity of organelle DNAs among rose species is unknown. RFLP analysis has been used to compare organelle DNAs from a wide range of other plant species (Pring and Lonsdale, 1985; Palmer, 1987). These comparisons
’ Corresponding
author. Tel.: + 81-58-293-2257;
fax: + 81-58-293-2207.
0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(96)00970-3
192
S. Matsumoto et al./ Scientia Horticulturae
68 (1997) 191-196
have proven particularly useful in the survey of cytoplasmic variation, and to trace the maternal lineages during evolution. Since some useful characteristics of higher plants, such as cytoplasmic male sterility and variegation in Epilobium show cytoplasmic inheritance, the identification of cytoplasmic diversity is a first step in preservation of genetically diverse germplasm. In this study, we describe the cytoplasmic diversity among wild roses on the basis of chloroplast and mitochondrial DNA analyses.
2. Materials and methods 2.1. Plant material
Most rose species used in this study were grown either in a greenhouse or the field at the Experimental Farms, Faculty of Agriculture, Gifu University, Japan. Rosa canina 1 and 2 samples were collected from different areas in the field, and R. canina ‘Inermis’ 1 and 2 samples were collected from the greenhouse and the field, respectively. Young leaves were collected and stored at -80°C until use. 2.2. Isolation and Southern blot analyses of rose DNA Total DNA from leaves of individual plants was isolated as described by Thomas et al. (1993). Five micrograms of total DNA were digested with a restriction enzyme, electrophoresed on 0.7% agarose gels, and transferred to a nylon membrane (Nylon membrane, positively charged, Boehringer Mannheim). The filter was hybridised with DNA probes labelled with a DIG DNA Labelling kit (Boehringer Mannheim). DNA probes were from sugar beet mitochondrial DNA; 1.5 kbp EcoRI fragment containing coxl gene (Senda et al., 1991) 4.3 kbp EcoRI fragment containing atp6 gene and tobacco chloroplast DNA; Bal, Ba2, B22, B25, B28 and IRB fragments (Sugiura et al., 1986). 2.3. Phylogenetic analysis The maximum parsimony method in the computer software PAUP version 3.1 .l (Swofford, 1993) was used to find the most parsimonious trees. For bootstrap analysis, 100 replications were conducted for each branch (Felsenstein, 1985).
Notes to Table 1: a A, Caninae; B, Gallicanae; C, Synstylae; D, Banksianae; E, Carolinae; F, Cassiorhodon; G, Pimpinellifoliae. b Additional fragments of 10.0 kbp, 7.5 kbp and 1.6kbp are present in all rose species. ’ Additional fragment of 5.9 kbp is present in all rose species. d Additional fragment of 2.5 kbp is present in all rose species. ’ Additional fragments of 3.6 kbp and 1.7 kbp are present in all rose species. f Additional fragments of 10.3 kbp, 9.2 kbp, 5.6 kbp, 1.0 kbp and 0.8 kbp are present in all rose species. g Additional fragment of 2.7 kbp is present in all rose species.
1
I
R. pimpinellifoliu
‘Stanwell Perpetual’ M&s domesticu
20
‘ plena’
R. pimpinellifolia
R. dumetorum ‘laxa’
R. moyesii ‘Geranium’
R. culifornica
R. rugosu
R. oirginiunu
R. hunksiue
R. x dupontii
R. mult$oru
R. x richurdii
R. mucrunthu
R. ruhiginosu
R. gluucu
‘Brags Stachellose’
R. cuninu
R. cuninu ‘Inermis’ 2
R. cuninu ‘Inermis’
R. cuninu 2
Rosu cuninu I
Species
6 7 8 9 10 II 12 13 14 I5 16 17 18 19
Sample no.
A A B B C C D E F F F F G G
Section a
10.0, 6.2, 5.7, 2.9
5.2 5.2 5.2 5.2 5.2 5.2 5.2 3.6 3.6 5.2 3.6 5.2 3.6 5.2 10.4
8.9 8.9 8.9 8.9 8.9 8.9 8.9 10.2 10.2 8.9 10.2 8.9 10.2 8.9 9.4, 2.6,
I .9
6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 6.7, 3.7, 2.8 10.4, 6.5 9.5, 6.5 6.7, 3.7, 2.8 9.5, 6.5 6.7, 3.7, 2.8 9.5, 6.5 6.7, 3.7, 2.8 10.0, 6.0
5.6 5.6 5.6 5.6 5.6 5.6 5.6 2.0 2.0 5.6 2.0 5.6 2.0 5.6
5.6 5.6 5.6 5.6 5.6
2.8 2.8 2.8 2.8 2.8
6.7. 6.7, 6.7, 6.7, 6.7,
8.9 8.9 8.9 8.9 8.9
5.2 5.2 5.2 5.2 5.2
3.7, 3.7, 3.7, 3.7, 3.7,
B22 BumHI ’
Hind111 d
Ba2
Ba2 BumHI ’
Bal BumHI b
in rose species and all fragments
fragments
Polymorphic
from chloroplast
10.0, 7.8, 2.8, 2.2, 0.9, 0.7
2.5
2.5
2.5 _
2.5 2.5 2.5 2.5 2.5 2.5 2.5
2.5 2.5 2.5 2.5 2.5
IRB Hind111 f
3.2 3.2 3.2 4.6 4.6 3.2 4.6 3.2 5.1 3.2
2.1 2. I 2.1 4.0 4.0 2.1 4.7 2.1 4.7 2.1
5.9, 3.7
3.2 3.2
2.1 2.1
6.9, 0.8
3.2 3.2
3.2 3.2 3.2 3.2 3.2
2.1 2.1 2.1 2. I 2.1 2. I 2.1
Hind111
COXI
coxl EcoRI
10.2, 8.6
5.8 _
5.8 5.8
_
_
_
_
BumHI g
utp6
DNA, cuxf and urp6 fragments
in M. domesficu detected with:
Summary of RFLP analyses of wild rose species and Mulus domesticu using Bal, Ba2, B22 and IRB fragments from mitochondrial DNA as probes. Fragment sizes are indicated in kbp
Table
3k
P
194
S. Matswnoto et al./Scientia
Horticulrurae
68 (1997) 191-196
3. Results and discussion 3.1. RFLP analyses The species were classified into two groups (Table l), when the DNA of 17 rose species was digested with BamHI, and hybridised with tobacco chloroplast DNA probe Bal . A 5.2 kbp fragment was missing in samples 13, 14, 16 and 18, with a 3.6 kbp fragment being present. A restriction site mutation may have occurred in the original 5.2 kbp fragment leaving a 1.6 kbp fragment. The same grouping was generated on the BamHI-digested DNA with the Ba2 probe from the small-single copy (SSC) region, the B22 probe from the LSC, and Hi&III-digested DNA with the IRB probe from the inverted repeat region (Table 1). A 10.2 kbp fragment of samples 13, 14, 16 and 18 on BamHI-digested/Ba:! probe might have been generated from an 8.9 kbp and a 1.3 kbp fragment missing on this blot by the restriction site mutation. Similarly, a 5.6 kbp fragment of samples 1-12, 15, 17 and 19 on BamHI_digested/B22 probe might have been generated from a 2.0 kbp and a 3.6 kbp fragment. Moreover, a 2.5 kbp fragment on HindIII-digested/IRB might have been generated from a 1.O kbp and a 1.5 kbp or a 0.8 kbp and a 1.7 kbp fragment, but the latter fragments were missing on this blot. Hybridisation of the HindIII-digested DNA with the Ba2 probe showed a similar classification which was explained by a restriction site mutation with the probe distinguishing Rosa virginiana (no. 13) from the Rosa rugosa (no. 14), Rosa moyesii ‘Geranium’ (no. 16) and Rosa pimpinellifolia (no. 18) (Table 1). In this case, a 6.5 kbp fragment of samples 13, 14, 16 and 18 might have been generated from a 3.7 kbp and a 2.8 kbp fragment, a 9.5 kbp fragment of samples 14, 16 and 18 might have been generated from a 6.7 kbp and a 2.8 kbp fragment, and a 10.4 kbp fragment of sample 13 might have been generated from a 9.5 kbp and a 0.9 kbp fragment missing on this blot. No polymorphism was detected when the B25 and B28 from LSC region were used as probes (data not shown). Reproducibility of the molecular classification was confirmed with DNA probes from mitochondrial DNA. Polymorphic fragments were detected on EcoRI and HindIII-digested DNA with the sugar beet mitochondrial DNA probe coxl, and the species classified into three groups (Table 1). Hybridisation of the BamHI-digested DNA with sugar beet mitochondrial DNA probe atp6 also showed single polymorphic fragments, and the species classified into two groups. This is the first report on cytoplasmic diversity within the Rosa genus by RFLP analyses. All of the polymorphic fragments in R. pimpinellifolia ‘Stanwell Perpetual’, the progeny of the cross R. X a’amascena bifkra and R. pimpinellifolia were not found in R. pimpinellifolia. Conversely, R. X dupontii (Rosa gallica X Rosa moschata) exhibited the same fragment patterns as Rosa macrantha, of R. gallica origin. These results support the maternal inheritance of cytoplasmic organelles in rose. 3.2. Phylogenetic analyses All of the bands detected in the rose species and Malus domestica were used in the maximum parsimony analysis with Malus domestica used as the out group. The bands
S. Matsumoto et al./Scientia
Horticulturae
68 (1997) 191-196
-1
(Al
-2
(A)
-3
(Al (A) (A) (A) (A) (BI (Bl (Cl (C) ID) IF1 IF1
-4
-5 -6 -7 -100.0%
6 -9 -
-9,.7%-c6,o%f
10 11 12 15
i
;:;
16 20
(G)
195
Fig. 1. Phylogenetic tree calculated from chloroplast DNA probe data using the maximum parsimony method. Numbers and letters in parentheses correspond to sample numbers and sections listed in Table 1. Bootstrap confidence values (%) are indicated on the branches.
were aligned in order of size for each blot, and the data matrix was constructed for each species according to the presence and absence of the bands. Presence and absence were indicated by 1 and 0, respectively, in the data matrix. R. cunina was designated as 1100100100110001011010001101101010101010 from the data using chloroplast DNA probes of Bal, Ba2/BamHI, Ba2/HindIII, B22, IRB, in that order. A single most parsimonious tree with bootstrap values was constructed from the data matrix of chloroplast DNA probes (Fig. 1). Seventeen rose species were divided into two groups; one was composed of all species in sections Caninae, Gallicanae, Synstylae and Banksianae, with some species from sections Cassiorhodon and Pimpinellifoliae, and the other group was composed of species from section Carolinae and some species from sections Cassiorhodon and Pimpinellifoliae (samples 13, 14, 16 and 18). A similar tree
Fig. 2. Phylogenetic tree calculated from mitochondrial DNA probe data using the maximum parsimony method. Numbers and letters in parentheses correspond to sample numbers and sections listed in Table 1. Bootstrap confidence values (%) are indicated on the branches.
1%
S. Mutsumoto et ul. / Scientiu Horticulturue
68 (1997) 191- I96
with bootstrap values was also constructed from the data matrix of mitochondrial DNA probes (Fig. 2). Rose species were mainly divided into four groups. One group corresponded to the chloroplast group including sections Caninae, Gallicanae, Synstylae and Banksianae, with some species from sections Cassiorhodon and Pimpinellifoliae in Fig. 1, and the remaining chloroplast group (samples 13, 14, 16 and 18) was further subdivided into nos. 16, 18 and 13 + 14 (Fig. 2). While the molecular classification was in general agreement with the traditional classification, there were some anomalies. For example, R. virginiana and R. rugosa formed a clade on phylogenetic trees of chloroplast and mitochondrial DNA with 98.7% and 8 1.6% of bootstrap replications, respectively, although they differ from one another phenotypically and were not in the same section. Further investigations using sequence analysis of the matK and rbcL loci, are in progress, examining a large number of wild roses in order to elucidate organelle diversity and its evolutionary implications.
Acknowledgements
We thank Dr H. Kato and T. Tsuchiya for computer analysis, Dr M. Thomas for critical reading of the manuscript, Dr T. Mikami for donating the mitochondrial DNA probes and Dr M. Sigiura of the Centre for Gene Research, Nagoya University for also donating the tobacco chloroplast DNA clones. This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture and Science of Japan (No. 07660032 and 07238210).
References Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39: 783-791. Hubbard, M., Kelly, J., Rajapakse, S., Abbott, A. and Ballard, R., 1992. Restriction fragment length polymorphisms in rose and their use for cultivar identification. HortScience, 27: 172- 173. Palmer, J.D., 1987. Chloroplast DNA evolution and biosystematic uses of chloroplast DNA. Am. Nat., 130: S6-S29. Pring, D.R. and Lonsdale, D.M., 1985. Molecular biology of higher plant mitochondrial DNA. Int. Rev. Cytol., 97: l-46. Rajapakse, S., Hubbard, M., Kelly, J.W., Abbott, A.G. and Ballard, R.E., 1992. Identification of rose cultivars by restriction fragment polymorphism. Sci. Hortic., 52: 237-245. Senda, M., Harada, T., Mikami, T., Sugiura, M. and Kinoshita, T., 1991. Genome organization and sequence analysis of the cytochrome oxidase subunit 11 gene from normal and male-sterile mitochondria in sugar beet. Curr. Genet., 19: 175-181. Sugiura, M., Shinozaki, K., Zaita, N., Kusuda, M. and Kumano, M., 1986. Clone bank of the tobacco (Nicotiunu tubucum) chloroplast genome as a set of overlapping restriction endonuclease fragments: mapping of eleven ribosomal protein genes. Plant Sci., 44: 21 l-216. Swofford, D., 1993. PAUP: Phylogenetic Analysis using Parsimony, Version 3.11. Computer program distributed by the Illinois Natural History Survey, Champaign, IL. Thomas, M.R., Matsumoto, S., Cain, P. and Scott, N.S., 1993. Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification. ‘Iheor. Appl. Genet., 86: 173-180. Tortes, A.M., Millan, T. and Cubero, J.I., 1993. Identifying rose cultivars using random amplified polymorphic DNA markers. HortScience, 28: 333-334.