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Chloroplast DNA probes as an aid in the molecular classification of Malus species
SCIENTIA HORTlCULnJRR ELSEVIER
Scientia Horticulturae 70 ( 1997) 8 l-86
Chloroplast DNA probes as an aid in the molecular classification of Mdus species S. Matsumoto ‘3* , H. Wakita a, J. Soejima b a Department b Fruit
of Biology, Faculty of Education, Gifu Unic;ersity, Gifu 501-I I, Japan Tree Research Station, Morioka Branch, Mot-i&z 020-11, Japan Accepted 13 March 1997
Abstract Chloroplast DNA probes were used to deduce molecular systematics of 19 M&s species by RFLP analysis. Phylogenetic trees divided these genotypes into 3 groups of cytoplasmic relatedness depending upon the probe. The data indicated maternal inheritance of chloroplast DNA in apple. The species from one series belonged to different clades on phylogenetic trees. 0 1997 Elsevier Science B.V. Keywords:
Apple; RFLP; Phylogeny
1. Introduction Apples are one of the most widely grown deciduous fruit crops in the world. Although a number of Malus species(from 25 to 74) have been designatedas primary species,the taxonomy of Mulus is not yet precisely defined. Plant systematicshas been usually basedon morphological characters, as an expressionof the genetic phenotype. In contrast, DNA polymorphisms offer direct observation of the plant genotype. Recently, it has been suggestedthat Ml3 phage DNA (Nybom, 1990a; Nybom, 1990b; Nybom et al., 1990) a cDNA clone from apple (Watillon et al., 1991) rDNA (Nybom et al., 1992), randomly amplified polymorphic DNAs (RAPD) (Koller et al., 1993, Mulcahy et al., 1993) and S-RNase-like genomic sequencesfrom apple (Matsumoto et al., 1995) might be a suitable molecular marker for identification of Mulus cultivars. In addition to these analysesof nuclear DNA, the degree of heterogeneity of organelle DNAs mainly
* Corresponding author. Tel.: + 81-58-293-2257; fax: + 81-58-293-2207; e-mail: [email protected]. 0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(97)00061-7
82
S. Matsumoto
et al. / Scientia
Horticulturae
70 (19971
81-86
among M&s domestica cultivars also has been investigated (Ishikawa et al., 1992). This analysis has proven particularly useful in the survey of cytoplasmic variation, and to trace the maternal lineages during evolution. However, little is known about M&s primary speciesto date. In this study, we describe the cytoplasmic diversity among M&s specieson the basisof chloroplast DNA analysis.
2. Materials
and methods
2.1. Plant material
All M&s plants used in this study were from collections at the Morioka Branch. Fruit Tree Research Station. Young leaves were collected, and stored at - 80°C until use. 2.2. isolation
and southern
blot analyses
of Malus
DNA
Total DNA from leaves of individual plants was isolated as described(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 hybridized with DNA probes labeled with a DIG DNA Labeling kit (Boehringer Mannheim), and the signalsdetected with the DIG LuminescentDetection kit (Boehringer Mannheim). DNA Probes were from tobacco chloroplast DNA; Bal, Ba2, B22, B25, B28 and IR, fragments (Sugiura et al., 1986). 2.3. Phylogenetic
analysis
The maximum parsimony method in the computer software PAUP version 3.1.1 (Swofford, 1993) was usedto find the most parsimonioustrees. For bootstrap analysis, 100 replications were conducted for each branch (Felsenstein, 1985).
3. Results and discussion 3.1. RFLP
analyses
The specieswere classified into 2 groups, when the DNA of 19 Malus specieswas digested with BarnHI, and hybridized with tobacco chloroplast DNA probe Bal located in large-singlecopy (LSC) region (Table 1). A 5.2-kbp fragment of samplenos. 1, 3-7, 17 and 19 might have been generated from a 3.3-kbp and a 1.9-kbp fragment by restriction site mutation. The same grouping was generated on EcoRI-digested DNA. with the Ba2 probe from the small-single copy (SSC) region (Table 1). A 2.5kbp fragment of samplenos. 1, 3-7, 17 and 19 might have been generated from a 1.3-kbp and a 1.2-kbp fragment by restriction site mutation.
a 1. Mnlus; h Additional ’ Additional ’ AdditionsI e Additional ’ Additmnal e Additmnal h Additmnal ’ Additional ’ Addaional ’ Additional
9 IO II 12 13 14 15 16 17 I8 19 20
8
4 5 6
2
Sample no.
Table I Summary Bal,
I7.
5.2, I .6 1.0
1.3, 2.5 I 3, I 3, 1.3, I 3, 1.3, 25 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 1.3, 2.5 1.3, 2.9,
52 33, I Y 5.2 5.2 5.2 5.2 5.2 3.3. 1.9 3 3, I 9 3 3, 1.9 3.3, I 9 3.3, 1.9 3.3, 1.9 3.3, I9 3.3. 1.9 3.3, 1.9 52 3 3. 1.9 s2 10.0, 7.5 I.1
1.2 I .9
1.2
1.2 I 2 12 I 2 1.2
1.2
3.2, 2.7. 2.1
I 2
1.2 I .2
1.2
1.2
0.88, 0.7. 0.56
1.9. 1.6, 1.3
5.8. 5.8, 5.8, 5 8, 5.8, 5.8, 5.4. 5.8, 5.8, 5.8, 5.8. 5.8. 5.x. 5 8, 5.8, 5.8, 5.8, 5.8, 1.8 3.4,
1.7, 1.7 1.7, 1.7 I 7, I 7, 1.7 1.7 1.7 I.7 1.7 I .7 1.7 1.7 I .7 1.7 1.7, I 7
Bal ’ bc*Rl
R28 ’
I .5, 0.8
6.2 6.2 6.2 6.2 6.2 6.2 6.2 6.2 62 62 6.2 6.2 6.2 6.2 6.2 6.2 6.2 6.2 42 4.0, 3.5
EcoRI
1.4
7.0.4.9
2.9 2.9
2.9
ElXlRl
DNA wtth
B2.5 =
detected
frown chloroplast
in R. conbro h
10.1
90, I.1
HtndIII
Bal
as probes
species.
rpecie% kbp are present m 811 apple species. 1.5 kbp. I I kbp, 0 8 khp, 0.66 kbp and 0.58 kbp are precent in all apple 0.8 kbp, 0.65 kbp and 0.55 kbp are present in all apple speaes. species. kbp are present in all apple species.
4 9, 4.2, 3.5 2.0, 1.5, 1.2 0.8, 0.66, 0.54
5.8 5.8 5.8 5.8 5.8 5.8 5.4 5.8 58 5.8 5.8 5.x 5.8 5.8 5.8 5.8 5.8 5.R
IR,’ ECORI
Ba2 ’ EroRl
Bal h
and 811 fragments
Ba2, B22 and IRB fragments
in apple specxa
825, 828.
fragments
u\tng
Ba1nHI
Polymorphic
II, ,Sorbomalus: 111, Eriolokur; IV, Cklorvmelus; V, Docynropsis. fragments of 8.8 kbp, 5.8 khp and 2.7 kbp arc present in all apple fragments of 3.0 kbp. 2.6 kbp. 1.X kbp, I .I kbp, 0.94 kbp and 0.64 fragmentc of 5.2 kbp. 4.2 kbp. 3 9 khp, 2.2 kbp. 2.0 kbp, I.6 kbp, fragments of 4.2 kbp, 3.0 kbp, 2.0 kbp, I .6 kbp, 1.5 kbp. 0.9 kbp, fragmcnta of 3 7 kbp, 1.5 kbp and 0.85 kbp are present tn all apple fragments of 2.4 kbp, 1.9 kbp, I .2 kbp, 1 .fl kbp, 0.84 kbp and 0.8 fragment of 10.1 kbp is present in all apple cpecles. fragment!, of IO I kbp and 3.0 kbp arc present ,n all apple apeucs. fragments of IO I kbp and I .3 kbp are present m all apple specie% fragments of 7.0 kbp, 2.1 kbp and 2 5 kbp a~ present m all apple
1” I IV V HI 1 I I 1 I I I I 1 I I II I I
Mnlus angurfi/oliu M rormgordes M. iosnsrs M rsrkonoskli M. rrdobatu M. ““nnanmrr.~ M. pumila M. asiolica M. rieboldii M. baccatu M. transmria M. knlliana M x atrosannuineo M x robusro M. mandskur~co M sarpnrii M. florenrina M. pratlii M. pwnifoliu Rosa caninu
0
ul weld apple \pec~es and Roro canon semen
analysis
Species
of RFLP
IO I, 10.0
I.1
1.5 I .4 1.5 14 IS I5 I .4 I .4 I.4 1.4 I.4 I.4 1.4 14 I .4 I .4 1.5 1.4 1.4 10.1. I5
Hmdlli
B28 J
sores are mdlcated
Hind111
B22 ’
(fragment
’
6 2 6, I.4
8.3 1.3 8.3 1.3 8.3 8.3 1.3 13 1.3 1.3 13 1.3 1.3 1.3 1.3 1.3 83 I3 13 80.6
HlndIII
825
in kbp)
84
S. Matsumoto
et al. / Scientia
Horticulturae
70 f 1997) 81-86
A 5.8-kbp fragment of sample numbers l-6 and 8- 18 on EcoRI-digested DNA with the IR, probe from the inverted repeat region might have been generated from a 5.4-kbp and a 0.4-kbp fragment missing on this blot by restriction site mutation. A 5.8-kbp fragment was also detected on the EcaRI-digested/Bal probe, and the samples having the fragment corresponded completely to those of EcoRI-digested/IR, probe. Since the Bal region is adjacent to the IR, region on the tobacco chloroplast DNA, the 5.8-kbp fragment might be derived from the junction of both regions. Instead of the 5.8-kbp fragment, the 5.4-kbp fragment of sample no. 7 on the EcoRI-digested/IR, probe was also detected on the same sample of EcoRI-digested/Bal probe. A 1.8-kbp fragment of sample no. 19 on EcoRI-digested/Bal probe might have been generated from a 1.7-kbp and a O.l-kbp missing on this blot by restriction site mutation. A 1.2-kbp fragment of sample numbers I, 3, 5, 6 and 17 on EcoRI-digested/Bal probe might have been generated from a 0.65-kbp and a 0.55-kbp fragment. A 6.2-kbp fragment of sample numbers 1- 18 on EcoRI-digested/B28 probe from inverted repeat region IR, might have been generated from a 4.2-kbp and a 2.0-kbp fragment missing on this blot by restriction site mutation. A 2.9-kbp fragment of sample numbers 3, 5 and 6 on EcoRI-digested/B25 probe from LSC region might have been generated from a 1.9-kbp and l.O-kbp fragment. The same classification has been shown between the HindIII-digested DNA with the Bal probe and the HindIII-digested DNA with the B22 probe from LSC. The Bal and the B22 region are linked together in tobacco chloroplast DNA, and at sample no. 7 on both blots, a lO.l-kbp fragment possibly from the junction of the Bal and B22 region divided into a 9.0-kbp and a l.l-kbp fragment by restriction site mutation. A 1.5-kbp fragment of sample nos. I, 3. 5, 6 and 17 on HindIII-digested/B28 probe might have been generated from a 1.6kbp and a O.l-kbp fragment missing on this blot. The same classification has been shown on HindIII-digested/B25 probe, and a 8.3-kbp fragment of sample numbers 1, 3, 5, 6 and 17 might have been generated from a 7.0-kbp and a 1.3-kbp fragment. M. X robusta, the progeny of the cross between M. buccata and M. prunifolia, exhibited the identical fragment patterns as M. baccata. Conversely, except for the 1.4-kbp and the 1.3-kbp fragments on HindIII-digested DNA/B28 probe and HindIIIdigested DNA B25 probe, respectively, none of the polymorphic fragments of M. X robusta were found in M. prunifolia. The 1.4-kbp and the 1.3-kbp fragments of M. x robustu might be derived from M. baccata. These results support the maternal inheritance of chloroplast DNA in M&s. 3.2. Phylogenetic analyses All of the bands detected in the Malus species and Rosa canina were used in the maximum parsimony analysis with R. canina used as the out group. The bands were aligned in the 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 ‘O’, respectively, in the data matrix. Mulus angustifolia was designatedas‘010110100101001011101110101101111011110101001001001110110101 0110010110000110111110010101101101010100’ from the data using chloroplast DNA
S. Matsumoto
et al. /Scientia
Horticulturae
70 (1997)
6 17 2 8 9 10 11
12 13 14 15
16 18 4 :9 20
81-86
85
i1) (II) fI1 iii (1) (1) (1) (I) iIj (1) (1) (1) (1) (V) I:;
Fig. 1. Phylogenetic tree calculated from chloroplast DNA probe data using the maximum parsimony method. Number and alphabet letters in parentheses correspond to No. and Sections listed in Table 1. The bootstrap confidence values (%l are indicated on the branches.
probes of Bal /BumHI, Ba2/EcoRI, IR,/EcoRI, Bal /EcoRI, B28/EcoRI, B25/EcoRI, Bal/HindIII, B22/HindIII, B28/HindIII and B25/HindIII, in that order. A single most parsimonious tree with bootstrap values was constructed from the data matrix of chloroplast DNA probes (Fig. 1). Nineteen Mulus species were divided into 3 groups; the first group was composed of species in Sorbomalus, Eriolobus, Chloromeles and M. yunnanensis from Malus, the secondgroup of speciesfrom Malus and the third group of Docyniopsis, M. pumila and M. prunifolia of Malus. While the molecular classification conformed closely to the traditional classification, there were someanomalies.For example, M. asiatica, M. pumilu and M. prun$lia are classified not only in the same sections, but in the same series, while M. asiatica is found in a different clade of M. pumila and M. prunifolia. Further investigations using sequenceanalysis of the matK and rbcL loci, are in progress, so as to examine a large number of Mulus species in order to elucidate organelle diversity and its evolutionary implications.
Acknowledgements We thank Dr. M. Sugiura of the Center for Gene Research,Nagoya University for donating the tobacco chloroplast DNA clones. This research was supported by a Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Culture, Sports and Science of Japan (Nos. 07660032 and 07238210). References Felsenstein, J., 1985. Confidence 783-791. Ishikawa, S., Kato, S., Imakawa, apple cultivars and rootstocks.
limits
on phylogenies:
an approach
using
the bootstrap.
S., Mikami, T., Shimamoto, Y., 1992. Organelle Theor. Appl. Genet. 83, 963-967.
DNA
Evolution
39,
polymorphism
in
86
S. Matsumoro
et al. / Scientia
Horticulturae
70 (1997)
81-86
Koller, B., Lehmann, A., McDermott, J.M., Gessler, C.. 1993. Identification of apple cultivars using RAPD markers. Theor. Appl. Genet. 85, 901-904. Matsumoto, S., Hoshi, N., Tsuchiya. T., Soejima, J., Komori, S., Ejiri, S., 1995. S-RNase like genomic sequences in apple for DNA fingerprinting. Acta Horticulturae 392, 265-274. Mulcahy, D.L., Cresti, M., Sansavini, S., Douglas, G.C., Linskens, H.F., Mulcahy, G.B., Vignani, R., Pancaldi, M., 1993. The use of random amplified polymorphic DNAs to fingerprint apple genotypes. Scientia Horticulturae 54, 89-96. Nybom, H., 1990a. Genetic variation in ornamental apple trees and their seedlings (M&s, Rosaceae) revealed by DNA ‘fingerprinting’ with the Ml3 repeat probe. Hereditas 113, 17-28. Nybom, H., 1990b. DNA fingerprints in sports of ‘Red Delicious’ apples. HortScience 25, 1641-1642. Nybom, H., Rogstad, S.H., Schaal, B.A., 1990. Genetic variation detected by use of the Ml3 ‘DNA fingerprint’ probe in M&s, Prunus, and Rubus (Rosaceae). Theor. Appl. Genet. 79. 153-156. Nybom, H., Gardiner, S., Simon, C.J., 1992. RFLPs obtained from an rDNA probe and detected with enhanced chemiluminescence in apples. HortScience 27, 355-356. Sugiura, M., Shinozaki, K., Zaita, N., Kusuda, M., Kumano, M., 1986. Clone bank of the tobacco (Nicotiana tabucum) chloroplast genome as a set of overlapping restriction endonuclease fragments: mapping of eleven ribosomal protein genes. Plant Sci. 44, 211-216. 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., Scott, N.S., 1993. Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification. Theor. Appl. Genet. 86, 173-180. Watillon, B., Druart, P., Jardin, P.D., Kettmann. R., Boxus, P., Bumy, A., 1991. Use of random cDNA probes to detect restriction fragment length polymorphisms among apple clones. Scientia Horticulturae 46, 235-243.
Scientia Horticulturae 70 ( 1997) 8 l-86
Chloroplast DNA probes as an aid in the molecular classification of Mdus species S. Matsumoto ‘3* , H. Wakita a, J. Soejima b a Department b Fruit
of Biology, Faculty of Education, Gifu Unic;ersity, Gifu 501-I I, Japan Tree Research Station, Morioka Branch, Mot-i&z 020-11, Japan Accepted 13 March 1997
Abstract Chloroplast DNA probes were used to deduce molecular systematics of 19 M&s species by RFLP analysis. Phylogenetic trees divided these genotypes into 3 groups of cytoplasmic relatedness depending upon the probe. The data indicated maternal inheritance of chloroplast DNA in apple. The species from one series belonged to different clades on phylogenetic trees. 0 1997 Elsevier Science B.V. Keywords:
Apple; RFLP; Phylogeny
1. Introduction Apples are one of the most widely grown deciduous fruit crops in the world. Although a number of Malus species(from 25 to 74) have been designatedas primary species,the taxonomy of Mulus is not yet precisely defined. Plant systematicshas been usually basedon morphological characters, as an expressionof the genetic phenotype. In contrast, DNA polymorphisms offer direct observation of the plant genotype. Recently, it has been suggestedthat Ml3 phage DNA (Nybom, 1990a; Nybom, 1990b; Nybom et al., 1990) a cDNA clone from apple (Watillon et al., 1991) rDNA (Nybom et al., 1992), randomly amplified polymorphic DNAs (RAPD) (Koller et al., 1993, Mulcahy et al., 1993) and S-RNase-like genomic sequencesfrom apple (Matsumoto et al., 1995) might be a suitable molecular marker for identification of Mulus cultivars. In addition to these analysesof nuclear DNA, the degree of heterogeneity of organelle DNAs mainly
* Corresponding author. Tel.: + 81-58-293-2257; fax: + 81-58-293-2207; e-mail: [email protected]. 0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(97)00061-7
82
S. Matsumoto
et al. / Scientia
Horticulturae
70 (19971
81-86
among M&s domestica cultivars also has been investigated (Ishikawa et al., 1992). This analysis has proven particularly useful in the survey of cytoplasmic variation, and to trace the maternal lineages during evolution. However, little is known about M&s primary speciesto date. In this study, we describe the cytoplasmic diversity among M&s specieson the basisof chloroplast DNA analysis.
2. Materials
and methods
2.1. Plant material
All M&s plants used in this study were from collections at the Morioka Branch. Fruit Tree Research Station. Young leaves were collected, and stored at - 80°C until use. 2.2. isolation
and southern
blot analyses
of Malus
DNA
Total DNA from leaves of individual plants was isolated as described(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 hybridized with DNA probes labeled with a DIG DNA Labeling kit (Boehringer Mannheim), and the signalsdetected with the DIG LuminescentDetection kit (Boehringer Mannheim). DNA Probes were from tobacco chloroplast DNA; Bal, Ba2, B22, B25, B28 and IR, fragments (Sugiura et al., 1986). 2.3. Phylogenetic
analysis
The maximum parsimony method in the computer software PAUP version 3.1.1 (Swofford, 1993) was usedto find the most parsimonioustrees. For bootstrap analysis, 100 replications were conducted for each branch (Felsenstein, 1985).
3. Results and discussion 3.1. RFLP
analyses
The specieswere classified into 2 groups, when the DNA of 19 Malus specieswas digested with BarnHI, and hybridized with tobacco chloroplast DNA probe Bal located in large-singlecopy (LSC) region (Table 1). A 5.2-kbp fragment of samplenos. 1, 3-7, 17 and 19 might have been generated from a 3.3-kbp and a 1.9-kbp fragment by restriction site mutation. The same grouping was generated on EcoRI-digested DNA. with the Ba2 probe from the small-single copy (SSC) region (Table 1). A 2.5kbp fragment of samplenos. 1, 3-7, 17 and 19 might have been generated from a 1.3-kbp and a 1.2-kbp fragment by restriction site mutation.
a 1. Mnlus; h Additional ’ Additional ’ AdditionsI e Additional ’ Additmnal e Additmnal h Additmnal ’ Additional ’ Addaional ’ Additional
9 IO II 12 13 14 15 16 17 I8 19 20
8
4 5 6
2
Sample no.
Table I Summary Bal,
I7.
5.2, I .6 1.0
1.3, 2.5 I 3, I 3, 1.3, I 3, 1.3, 25 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 1.3, 2.5 1.3, 2.9,
52 33, I Y 5.2 5.2 5.2 5.2 5.2 3.3. 1.9 3 3, I 9 3 3, 1.9 3.3, I 9 3.3, 1.9 3.3, 1.9 3.3, I9 3.3. 1.9 3.3, 1.9 52 3 3. 1.9 s2 10.0, 7.5 I.1
1.2 I .9
1.2
1.2 I 2 12 I 2 1.2
1.2
3.2, 2.7. 2.1
I 2
1.2 I .2
1.2
1.2
0.88, 0.7. 0.56
1.9. 1.6, 1.3
5.8. 5.8, 5.8, 5 8, 5.8, 5.8, 5.4. 5.8, 5.8, 5.8, 5.8. 5.8. 5.x. 5 8, 5.8, 5.8, 5.8, 5.8, 1.8 3.4,
1.7, 1.7 1.7, 1.7 I 7, I 7, 1.7 1.7 1.7 I.7 1.7 I .7 1.7 1.7 I .7 1.7 1.7, I 7
Bal ’ bc*Rl
R28 ’
I .5, 0.8
6.2 6.2 6.2 6.2 6.2 6.2 6.2 6.2 62 62 6.2 6.2 6.2 6.2 6.2 6.2 6.2 6.2 42 4.0, 3.5
EcoRI
1.4
7.0.4.9
2.9 2.9
2.9
ElXlRl
DNA wtth
B2.5 =
detected
frown chloroplast
in R. conbro h
10.1
90, I.1
HtndIII
Bal
as probes
species.
rpecie% kbp are present m 811 apple species. 1.5 kbp. I I kbp, 0 8 khp, 0.66 kbp and 0.58 kbp are precent in all apple 0.8 kbp, 0.65 kbp and 0.55 kbp are present in all apple speaes. species. kbp are present in all apple species.
4 9, 4.2, 3.5 2.0, 1.5, 1.2 0.8, 0.66, 0.54
5.8 5.8 5.8 5.8 5.8 5.8 5.4 5.8 58 5.8 5.8 5.x 5.8 5.8 5.8 5.8 5.8 5.R
IR,’ ECORI
Ba2 ’ EroRl
Bal h
and 811 fragments
Ba2, B22 and IRB fragments
in apple specxa
825, 828.
fragments
u\tng
Ba1nHI
Polymorphic
II, ,Sorbomalus: 111, Eriolokur; IV, Cklorvmelus; V, Docynropsis. fragments of 8.8 kbp, 5.8 khp and 2.7 kbp arc present in all apple fragments of 3.0 kbp. 2.6 kbp. 1.X kbp, I .I kbp, 0.94 kbp and 0.64 fragmentc of 5.2 kbp. 4.2 kbp. 3 9 khp, 2.2 kbp. 2.0 kbp, I.6 kbp, fragments of 4.2 kbp, 3.0 kbp, 2.0 kbp, I .6 kbp, 1.5 kbp. 0.9 kbp, fragmcnta of 3 7 kbp, 1.5 kbp and 0.85 kbp are present tn all apple fragments of 2.4 kbp, 1.9 kbp, I .2 kbp, 1 .fl kbp, 0.84 kbp and 0.8 fragment of 10.1 kbp is present in all apple cpecles. fragment!, of IO I kbp and 3.0 kbp arc present ,n all apple apeucs. fragments of IO I kbp and I .3 kbp are present m all apple specie% fragments of 7.0 kbp, 2.1 kbp and 2 5 kbp a~ present m all apple
1” I IV V HI 1 I I 1 I I I I 1 I I II I I
Mnlus angurfi/oliu M rormgordes M. iosnsrs M rsrkonoskli M. rrdobatu M. ““nnanmrr.~ M. pumila M. asiolica M. rieboldii M. baccatu M. transmria M. knlliana M x atrosannuineo M x robusro M. mandskur~co M sarpnrii M. florenrina M. pratlii M. pwnifoliu Rosa caninu
0
ul weld apple \pec~es and Roro canon semen
analysis
Species
of RFLP
IO I, 10.0
I.1
1.5 I .4 1.5 14 IS I5 I .4 I .4 I.4 1.4 I.4 I.4 1.4 14 I .4 I .4 1.5 1.4 1.4 10.1. I5
Hmdlli
B28 J
sores are mdlcated
Hind111
B22 ’
(fragment
’
6 2 6, I.4
8.3 1.3 8.3 1.3 8.3 8.3 1.3 13 1.3 1.3 13 1.3 1.3 1.3 1.3 1.3 83 I3 13 80.6
HlndIII
825
in kbp)
84
S. Matsumoto
et al. / Scientia
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A 5.8-kbp fragment of sample numbers l-6 and 8- 18 on EcoRI-digested DNA with the IR, probe from the inverted repeat region might have been generated from a 5.4-kbp and a 0.4-kbp fragment missing on this blot by restriction site mutation. A 5.8-kbp fragment was also detected on the EcaRI-digested/Bal probe, and the samples having the fragment corresponded completely to those of EcoRI-digested/IR, probe. Since the Bal region is adjacent to the IR, region on the tobacco chloroplast DNA, the 5.8-kbp fragment might be derived from the junction of both regions. Instead of the 5.8-kbp fragment, the 5.4-kbp fragment of sample no. 7 on the EcoRI-digested/IR, probe was also detected on the same sample of EcoRI-digested/Bal probe. A 1.8-kbp fragment of sample no. 19 on EcoRI-digested/Bal probe might have been generated from a 1.7-kbp and a O.l-kbp missing on this blot by restriction site mutation. A 1.2-kbp fragment of sample numbers I, 3, 5, 6 and 17 on EcoRI-digested/Bal probe might have been generated from a 0.65-kbp and a 0.55-kbp fragment. A 6.2-kbp fragment of sample numbers 1- 18 on EcoRI-digested/B28 probe from inverted repeat region IR, might have been generated from a 4.2-kbp and a 2.0-kbp fragment missing on this blot by restriction site mutation. A 2.9-kbp fragment of sample numbers 3, 5 and 6 on EcoRI-digested/B25 probe from LSC region might have been generated from a 1.9-kbp and l.O-kbp fragment. The same classification has been shown between the HindIII-digested DNA with the Bal probe and the HindIII-digested DNA with the B22 probe from LSC. The Bal and the B22 region are linked together in tobacco chloroplast DNA, and at sample no. 7 on both blots, a lO.l-kbp fragment possibly from the junction of the Bal and B22 region divided into a 9.0-kbp and a l.l-kbp fragment by restriction site mutation. A 1.5-kbp fragment of sample nos. I, 3. 5, 6 and 17 on HindIII-digested/B28 probe might have been generated from a 1.6kbp and a O.l-kbp fragment missing on this blot. The same classification has been shown on HindIII-digested/B25 probe, and a 8.3-kbp fragment of sample numbers 1, 3, 5, 6 and 17 might have been generated from a 7.0-kbp and a 1.3-kbp fragment. M. X robusta, the progeny of the cross between M. buccata and M. prunifolia, exhibited the identical fragment patterns as M. baccata. Conversely, except for the 1.4-kbp and the 1.3-kbp fragments on HindIII-digested DNA/B28 probe and HindIIIdigested DNA B25 probe, respectively, none of the polymorphic fragments of M. X robusta were found in M. prunifolia. The 1.4-kbp and the 1.3-kbp fragments of M. x robustu might be derived from M. baccata. These results support the maternal inheritance of chloroplast DNA in M&s. 3.2. Phylogenetic analyses All of the bands detected in the Malus species and Rosa canina were used in the maximum parsimony analysis with R. canina used as the out group. The bands were aligned in the 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 ‘O’, respectively, in the data matrix. Mulus angustifolia was designatedas‘010110100101001011101110101101111011110101001001001110110101 0110010110000110111110010101101101010100’ from the data using chloroplast DNA
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12 13 14 15
16 18 4 :9 20
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85
i1) (II) fI1 iii (1) (1) (1) (I) iIj (1) (1) (1) (1) (V) I:;
Fig. 1. Phylogenetic tree calculated from chloroplast DNA probe data using the maximum parsimony method. Number and alphabet letters in parentheses correspond to No. and Sections listed in Table 1. The bootstrap confidence values (%l are indicated on the branches.
probes of Bal /BumHI, Ba2/EcoRI, IR,/EcoRI, Bal /EcoRI, B28/EcoRI, B25/EcoRI, Bal/HindIII, B22/HindIII, B28/HindIII and B25/HindIII, in that order. A single most parsimonious tree with bootstrap values was constructed from the data matrix of chloroplast DNA probes (Fig. 1). Nineteen Mulus species were divided into 3 groups; the first group was composed of species in Sorbomalus, Eriolobus, Chloromeles and M. yunnanensis from Malus, the secondgroup of speciesfrom Malus and the third group of Docyniopsis, M. pumila and M. prunifolia of Malus. While the molecular classification conformed closely to the traditional classification, there were someanomalies.For example, M. asiatica, M. pumilu and M. prun$lia are classified not only in the same sections, but in the same series, while M. asiatica is found in a different clade of M. pumila and M. prunifolia. Further investigations using sequenceanalysis of the matK and rbcL loci, are in progress, so as to examine a large number of Mulus species in order to elucidate organelle diversity and its evolutionary implications.
Acknowledgements We thank Dr. M. Sugiura of the Center for Gene Research,Nagoya University for donating the tobacco chloroplast DNA clones. This research was supported by a Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Culture, Sports and Science of Japan (Nos. 07660032 and 07238210). References Felsenstein, J., 1985. Confidence 783-791. Ishikawa, S., Kato, S., Imakawa, apple cultivars and rootstocks.
limits
on phylogenies:
an approach
using
the bootstrap.
S., Mikami, T., Shimamoto, Y., 1992. Organelle Theor. Appl. Genet. 83, 963-967.
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