岐阜大学教育学部研究報告（自然科学）第２９巻，７―１
２，２００
５年３月
２，３.２００５
Sci. Rep. Fac. Educ. Gifu Univ.（Nat. Sci.）２
９，７―１

Genomic DNA sequence of S16c(=16)-RNase in apple: re-numbering ofS16(=27a)-and S22(=27b)-allele to S16a and S16b
Shogo Matsumoto and Yui Furusawa

Summary
We determined a genomic DNA sequence including a hypervariable HVa（RHV）region and an intronof the S16-RNase in ‘Bohnapfel’ apple. The deduced amino acid sequence of the S16-RNase in１）
completely matched that of S22(=27b)-RNase in ‘Alkmene’（S5S22=27b geno‘Bohnapfel’（S6bS9S16 genotype）
２）
and S16(=27a)-RNase in ‘Baskatong’（S16=27aS26 genotype）,２） however, the nucleotide sequencestype）

among them were different. We re-numbered S16(=27a) in ‘Baskatong’, S22(=27b) in ‘Alkmene’ and S16 in‘Bohnapfel’ as S16a, S16b and S16c, respectively.
Key words: Apple; S -allele; S -RNase; Malus x domestica Borkh.

Apple（Malus x domestica Borkh．
）has a S -RNase-based gametophytic self-incompatibility controlled by the３）S -RNases were detected in the style of apple, and cDNAs of１０and１１S -RNases frommulti-allelic S -locus．
１１−１４）
respectively. Knowledge of the S corresponding S -alleles have been cloned in Japan ４−１０） and in Europe，

genotypes of apple is very important for selection of compatible pollinators and facilitation of breeding programs. A PCR-digestion method for S -allele identification has been developed using each S -RNase gene５−７，９−１５）
specific primer set．

S -allele designations are confusing; for instance, the S -genotype of ‘Bohnapfel’ was identified as S9SxSy; x, y１６）
respectively. Recently, Broothaerts２） found＞１
１and S9S16S19 by cross pollination３） and stylar protein analysis，

that the S27a and S28 were present in ‘Bohnapfel’ and corresponded to S16 and S19，respectively. From the results,
２）
１）
However, Matsumoto et al ．
found that the S19
S27a and S28 were re-numbered to S16 and S19，respectively．

and S28 were different alleles, and the S6b instead of S19 was present in ‘Bohnapfel’. From the results, S28 was left
１）
This confusion was caused by inadequate analysis; i.e., the correunchanged and S19 was re-numbered to S6b．
２）
Asspondence between S28 and S19 was from S -allele-specific PCR-digestion analysis, not sequence analysis．

the correspondence between S28 and S19 by PCR analysis was contradicted, we determined the partial genomic
DNA sequences of S16-RNase in ‘Bohnapfel’ to confirm the correspondence between S27a and S16．
Alignment of the nucleotide and deduced amino acid sequences for the exons of S16-RNase in ‘Bohnapfel’
１４）
showed extremely high（AB１２６３２２）with those of S16(=27a)-and S22(=27a)-RNase determined by Van Nerum et al ．

similarity（Fig．
１）． Deduced amino acid sequences of S16，S16=27a and S22=27b are identical, whereas sequencecomparison revealed that the S16 only deviates at three and two point mutations from S16=27a（nucleotide positions
１
８０from A to G，３０
３from T to C and４０５from T to G）and S22=27b（nucleotide positions１８０from A to G and
３２
１from G to T），
respectively（Fig．
１）．All S -RNases in apple contains one intron at the same location within
１７）
the RHV region（Fig．
１；Matsumoto et al ．
）． The S16-RNase also contained an intron deduced from the pres-Department of Biology, Faculty of Education, Gifu University７

Shogo Matsumoto and Yui Furusawa

ence of plant５ and３ splice site consensus sequences at corresponding sites（Fig．
１）． The S16 intron size was２２
１８bp（Fig．
２），
which was longest within apple S-RNases. Previously, apple S-alleles were

MDS16cMDS16aMDS16b
MDS16cMDS16aMDS16b

CCGGCTGTCTGCAACTTTAATCCTACTCCATGTAAGGATCCTACTGACAAGTTGTTTACGCCGGCTGTCTGCAACTTTAATCCTACTCCATGTAAGGATCCTACTGACAAGTTGTTTACGCCGGCTGTCTGCAACTTTAATCCTACTCCATGTAAGGATCCTACTGACAAGTTGTTTACGP A V C N F N P T P C K D P T D K L F TP A V C N F N P T P C K D P T D K L F TP A V C N F N P T P C K D P T D K L F T

60

MDS16cMDS16aMDS16b
MDS16cMDS16aMDS16bGTTCACGGTTTGTGGCCTTCAAACAACGTAGGAGGTGACCCAGAATCTTGCAAGATAAGGGTTCACGGTTTGTGGCCTTCAAACAACGTAGGAGGTGACCCAGAATCTTGCAAGATAAGGGTTCACGGTTTGTGGCCTTCAAACAACGTAGGAGGTGACCCAGAATCTTGCAAGATAAGGV H G L W P S N N V G G D P E S C K I RV H G L W P S N N V G G D P E S C K I RV H G L W P S N N V G G D P E S C K I R

MDS16cMDS16aMDS16b
MDS16cMDS16aMDS16b▼ RHV
AATCATCGAACGAGAGCAAAAGCACTCGAACCCCAGTTGGAAATTATTTGGCCGAACGTGAATCATCGAACGAGAGCAAAAGCACTCGAACCCCAGTTGGAAATTATTTGGCCGAACGTAAATCATCGAACGAGAGCAAAAGCACTCGAACCCCAGTTGGAAATTATTTGGCCGAACGTAN H R T R A K A L E P Q L E I I W P N VN H R T R A K A L E P Q L E I I W P N VN H R T R A K A L E P Q L E I I W P N V

MDS16cMDS16aMDS16b
MDS16cMDS16aMDS16bCTCGATCGAACCAATCATACAGCCTTCTGGCGTAGACAGTGGATAAAACATGGCACTTGTCTCGATCGAACCAATCATACAGCCTTCTGGCGTAGACAGTGGATAAAACATGGCACTTGTCTCGATCGAACCAATCATACAGCCTTCTGGCGTAGACAGTGGATAAAACATGGCACTTGTL D R T N H T A F W R R Q W I K H G T C
L D R T N H T A F W R R Q W I K H G T C
L D R T N H T A F W R R Q W I K H G T C240

MDS16cMDS16aMDS16b
MDS16cMDS16aMDS16bGGGTATCCCACAATACAGAACGAGAATGATTACTTCGAAACAGTAGTCAAAATGTACATAGGGTATCCCACAATACAGAACGAGAATGATTACTTCGAAACAGTAGTCAAAATGTACATAGGGTATCCCACAATACAGAACGAGAATGATTACTTCGAAACAGTAGTCAAAATGTACATAG Y P T I Q N E N D Y F E T V V K M Y IG Y P T I Q N E N D Y F E T V V K M Y IG Y P T I Q N E N D Y F E T V V K M Y I300

MDS16cMDS16aMDS16b
MDS16cMDS16aMDS16b

ACCGAGAAACAAAACGTCTCTAAAATCCTCTCAAATGCAAAGATTGAACCGGACGGGATAACTGAGAAACAAAACGTCTCTAAAATCCTCTCAAATGCAAAGATTGAACCGGACGGGATAACCGAGAAACAAAACGTCTCGAAAATCCTCTCAAATGCAAAGATTGAACCGGACGGGATAT E K Q N V S K I L S N A K I E P D G IT E K Q N V S K I L S N A K I E P D G I
T E K Q N V S K I L S N A K I E P D G I

MDS16cMDS16aMDS16b
MDS16cMDS16aMDS16bAAGAGAACACTGGCGGACCTTGAAATTGCCATACGCAGTGGTACGGACAACAAGAAACCAAAGAGAACACTGGCGGACCTTGAAATTGCCATACGCAGTGGTACTGACAACAAGAAACCAAAGAGAACACTGGCGGACCTTGAAATTGCCATACGCAGTGGTACGGACAACAAGAAACCAK R T L A D L E I A I R S G T D N K K P
K R T L A D L E I A I R S G T D N K K P
K R T L A D L E I A I R S G T D N K K P

MDS16cMDS16aMDS16b
MDS16cMDS16aMDS16b

AAATTCAAGTGCCAAAAGAAGAGAAGGGTGACTGAATTAGTTGAGGTCACTCTTAAATTCAAGTGCCAAAAGAAGAGAAGGGTGACTGAATTAGTTGAGGTCACTCTTAAATTCAAGTGCCAAAAGAAGAGAAGGGTGACTGAATTAGTTGAGGTCACTCTTK F K C Q K K R R V T E L V E V T L
K F K C Q K K R R V T E L V E V T L
K F K C Q K K R R V T E L V E V T L

20C21204018060C380100RC4360120420140C5474158Fig．
１ Alignment of the nucleotide and deduced partial amino acid sequences of S16c(=16)-，S16a(=16=27a)-，and S16b(=22=27b)-RNase in ̀Bohnapfel', ̀Baskatong' and ̀Alkmene' apple, respectively.
８

Genomic DNA sequence of S16c(=16)-RNase in apple: re-numbering of S16(=27a)-and S22(=27b)-allele to S16a and S16b

Genomic DNA encoding S16c(=16)-RNase was amplified from S16-allele of ̀Bohnapfel' by PCR using the１３）
４９．
PCR product was directly sequenced by dideoxy chain termination
primers FTQQYQ７） and OWB２

on an ABI PRISMTM ３
１０DNA sequencer（Perkin-Elmer）using dRhodamine Terminator Cycle Sequencing Kits（Perkin-Elmer）． The conserved regions C２，C３，RC４ and C５，and hypervariable region RHV（MDS 16 c）in ̀Bohnapfel' was deposited under the DDBJ accession
are boxed. The sequence of S16c(=16)
number AB１２６３２
２． S16a(=16=27a)（MDS 16 a）and S16b(=22=27b)（MDS 16 b）were from the studies of Verdoodt et１３）
１４）
and Van Nerum et al ．，
respectively. The different sites are shown in bold. The site of the intron isal ．

shown by a triangle.
GTAATATTATTAGTAGTAAGACAGTCAATATTGTTTATTTCATTTATCTATATTTATCTG60TATATATCAAGGGAGAAGACAAAAATGATGAAACATTCATAATTTCAAAATTGCCCTCTT20CCATTAAATTTTCAAATAAAGTAAGAAATTCACAAAATTAAAAAAATTAAAATATCTCCA180TGGAGTGGTTAGCAACCAACTTTATTATTTTTTAAATGAGATTTTTGAACTTTAATCCTT240GACTATATAAATAAAGGGAGAAGACAAAAGTGGTGAAACATTCATAATTCCAAAATTGCC300

CTCTTCCATTAAATTTTCAAATAAAGTAAAAAATTCACAAAATAAAAAAAAATCTCCACG360

GAGTAGTTAGCAACTAACTTTATTATTTTTTAAATGAGATTTTTGAACTTTAATCCTTGA420AGAAGATTAAAATTTAATAAAAACTAGGTGTTAGATTGGATATTGATTTTAGTCCTTTAA480

TGAGTACTTAATTCAAATTCATATTATATTTTGTTTTAATATTTAATAGATATATTTTTT540

AATTCCATTACACTAAATTTTAAATTTATTATTATACACATTTTAATTCATTAATTTCAT600

TTAACTTCCTCTATTTAGTGTACATAAACGCCTGTATCATAATATATTTTTAGAAACCTT660

CATTTTAATCCTTGGCAAAGATGACTTTTAGATTTAAACCATGMGTAAGACCAAAATCTA

720

ATTTTAGTCATTTGATACATTAATAACCTGATTYATTAATTATATTTTGATTTTTTTTMA780

TTATTTATCTTTTTGYTCCTAATTTTTATTTTCATTTCATTTATTGTAATATATTTCGTT840GTAAACATTTAAATAAATTAATTTTTGTTATACAAYWTATTTCAATGCACTTTGTCTTCT900

TCATTGAGGTACCATTAAATTCATTATACTACATTTCAGTACATACATTTAGGTMMACAT960

ATTTTCGTACATACCATTTTGATACAAACATTTAGGTACATTAATTTTATATAATACATT1020

TTGGTACTAACATTTCGGTGCATACATTTCAATATACAAATTTAGATACATTAACTTTAA1080

TATAATACATTTCGGTGCATATATTTCGGTACATACATTTCGGTACTAACATTTAAATAC1140

ATTTGTTCAATATAATACATTTCGGTACACACATTTTGGTACATTAATTGAGTCTTTCAA1200

GTTTGAAGAATGTTAAATAAAATTAATAAATTAAAAAAAATCTTTTTTGGTAAAAAAATA

1260AATTAAAATTTGTGTAGTAATAAATTTAAATATTTTATGGGAGACATTAAATAAAATGCA

1320

CATTAATTATTTTGAATTAAGGACACATCAAGAGATTATAATCAATATGTAATCTAACAT1380

CAGGTTTATTTTAAATTTCAATCTTCTTAAAGGATTAAAGTTAAATGCTAAATTAGTGTA

1440

CTGAAGTGTCTGTTCCTAAATATATTGTAGTACCTAACTATATTGTAGGGATTTAGTGGC

1500ACATAAGAAAATATGCAAAAACGCAAGTGTACTGAAAATTTTGTACCTAAATATTTGATA1560

CTAAACTAGTGTACCGAAATGTCGGTGTCTAAATGTATTATACTAAGCCAGTGCACCGAA

1620

ATGTCCTTATCTAAACATATTATATTAACTTAGTGTACCGAAATATTTGTACCTAAATGT1680

ATTATAATGAATTAGTGGCACTTACGAAAATACGCAAAAACATAAGTGTACCGAAAAATC1740

TCTATGAAAATATTTTCTTATATAAGATACTTACCAGAATGACAATTAAAATTTATAATA

1800

TTTTCATAAAATTTAATTTATGAACACCATAAATATAAATAAAAAAGAGAGACATTGAAT1860

ACATGTGCTGCCATTAAATAGAGTTTAGGGACTAGAATCAATTTTTAATCTGGTATAAGA1920

CATTTTTCTAAACTTCATCTTTTTAAGGATTAAAATCTAGTTTTATATTATTATTTTTTT1980

TTCTATTTTTTTTTCTATTTTTTTTTCAAATAAAAAATCCAAAGAGCCAATTGCTAGTAC2040９Shogo Matsumoto and Yui Furusawa

ATTTTTTGTTTTTTTTTTCTTTCAAAAAAATAAAATCAAAAGACCCAATTGCTACACACT2100TACGCATGTAGCAAGGGGCTAGTATACTTTTGTGTGTGCGCACGGGTGTATACTTTTTAA

2160

TACAATTAATTTAAAATCTAATCATAAATGTTTTATATTGTACATATTACCTTGTCAG

2218

Fig．
２ DNA sequence of the intron in S16c(=16)-allele.
The sequence was determined as described in Fig．
１．
１７）
divided into three groups based on the length of the intron within S -RNases．
The first group consisting of S3１１，１２）
１１００bp of intron．
The second group consisting of S1=f -，Sg=20a-，Sg=20b-and S24=h-RNase
and S5-RNase had ca．
５，８，１７）
The third group consisting of S2-，S7=d-，S9=c-，S28=e- and S30=t-RNase had１１８-１８０bp
had３
１８-３
４４bp of intron．
１５，１７）
Since the introns of closely related S -alleles, such as those belonging to the second group showedof intron．

an extremely high similarity（８３．
９−９４．
４％）
，S16=27a and S22=27b may have similar size with S16 in ‘Bohnapfel’. We
grouped the alleles into a fourth group. We could not find any retrotransposon-like sequence in the intron, which is１８）
linked to the S -locus of Nicotiana alata．

Two hypervariable regions（HVa and HVb）alone are sufficient for S -allele discrimination in closely related１９）
Since rosaceous S -RNases have only one hypervariable region RHV located at a posiSolanaceae S -RNases．
２０）
it is reasonable that the RHV region is sufficient fortion corresponding to that of the solanaceous region HVa，

allele discrimination of closely related apple S -alleles. As the deduced amino acid sequence of S16 in ‘Bohnapfel’ is１４）
deidentical to S16=27a and S22=27b, these three alleles are thought to be functionally identical. Van Nerum et al ．

scribed the two identical alleles in S27 to be S27a and S27b. Afterward, Broothaerts２） re-numbered S27a and S27b to S16and S22，respectively.
We determined the S16-RNase sequence, and concluded that the S16 in ‘Bohnapfel’, S16=27a and S22=27b should be renumbered to S16c, S16a and S16b, respectively（Table１）．

Table 1. S-alleles of apple cultivars.
Proposed Former
S-allele
S-allele

Cultivar

S-alleles

Database
Accession #16a16b

BaskatongAlkmene
Delbard Jubile
Merlijin
Bohnapfel

16a52
36b

AF016919
AF327222
AF327222
AF327222
AB126322

16c

16, 27, 27a
22, 27b23, 27b
25,z 27b
16,y 27ax

z2616b16b16b916c

yS25-allele in 'Merlijin', S16-allele in 'Bohnapfel',x S27a-allele in 'Bohnapfel'

Acknowledgements
We wish to thank Ms. Jnge De Wit for supplying the plant sample. This work was supported in part by a Grantin-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan（Nos．
１４３６００１９and１５２０８００４）．

１０Genomic DNA sequence of S16c(=16)-RNase in apple: re-numbering of S16(=27a)-and S22(=27b)-allele to S16a and S16bReferences
１） Matsumoto, S., Furusawa, Y., Kitahara, K., and Soejima, J., Partial genomic sequences of S6-，
S12-，
S13-，
S14-，
S17-，
S19-，
，
２０，３２３-３
２９（２００３）．
and S21-RNases of apple and their allele designations. Plant Biotech．
２） Broothaerts, W., New findings in apple S-genotype analysis resolve previous confusion and request the re-numberingof some S-alleles. Theor. Appl. Genet．
，
１０６，７０３-７
１４（２００３）．
３） Kobel, F., Steinegger, P., and Anliker, J., Weitere Untersuchungen über die Befruchtungsverhaltnisse der Apfel-undBirnsorten. Landw. Jahrb. Schweiz，
５３，
１６０‐１９１（１９３９）．
４） Sassa, H., Nishio, T., Kowyama, Y., Hirano, H., Koba, T., and Ikehashi, H., Self-incompatibility（S ）alleles of the Ro，
２５０，
５４７-５
５７saceae encode members of a distinct class of the T２／S ribonuclease superfamily. Mol. Gen. Genet．
（１９９６）．
５） Matsumoto, S., Kitahara, K., Komori, S., and Soejima, J., A new S -allele in apple, ‘Sg’ and its similarity to the ‘Sf ’ allele from ‘Fuji’. HortSci．
，
３４，
７０８-７
１０（１９９９）．
６） Kitahara, K., Soejima, J., Komatsu, H., Fukui, H., and Matsumoto, S., Complete sequences of the S -genes, Sd-and ShRNase cDNA in apple. HortSci．
，３５，７１２-７
１５（２０００）．
７） Matsumoto, S., and Kitahara, K., Discovery of a new self-incompatibility allele in apple. HortSci．
，３５，１３２９-１
３３２（２０００）．
８） Matsumoto, S., Kitahara, K., Komatsu, H., and Soejima, J., A functional S -allele, ‘Sg’, in the wild apple possessing asingle amino acid, S-RNase ‘Sg’-RNase’, differed from ‘Sg-RNase’ in Malus x domestica cultivars. J. Hortic. Sci.
Biotechnol ．
，
７６，
１６３-１
６６（２００１）．
９） Kitahara, K., and Matsumoto, S., Sequence of the S10 cDNA from ‘McIntosh’ apple and a PCR-digestion identification
method. HortSci．
，
３７，
１８７-１
９０（２００２）．
１０） Kitahara, K., and Matsumoto, S., Cloning of the S25 cDNA from ‘McIntosh’ apple and an S25-allele identification
method. J. Hortic. Sci. Biotechnol ．
，
７７，
７２４-７
２８（２００２）．
１１） Broothaerts, W., Janssens, G.A., Proost, P., and Broekaert, W.F., cDNA cloning and molecular analysis of two selfincompatibility alleles from apple. Plant Mol. Biol ．
，
２７，
４９９-５
１１（１９９５）．
１２） Janssens, G.A., Goderis, I.J., Broekaert, W.F., and Broothaerts, W., A molecular method for S -allele identification inapple based on allele-specific PCR. Theor. Appl. Genet．
，
９１，６
９１-６
９８（１９９５）．
１３） Verdoodt, L., Van Haute, A., Goderis, I.J., De Witte, K., Keulemans, J., and Broothaerts, W., Use of the multi-allelicself-incompatibility gene in apple to assess homozygocity in shoots obtained through haploid induction. Theor. Appl.
Genet．
，９６，２
９４-３００（１９９８）．
１４） Van Nerum, I., Geerts, M., Van Haute, A., Keulemans, J., and Broothaerts, W., Re-examination of the selfincompatibility genotype of apple cultivars containing putative ‘new’ S -alleles. Theor. Appl. Genet．
，
１０３，
５８４-５
９１（２００１）．
１５） Matsumoto, S., Komori, S., Kitahara, K., Imazu, S., and Soejima, J., S -genotypes of １５ apple cultivars and selfcompatibility of ‘Megumi’. J. Japan Soc. Hort. Sci．
，６８，２３６-２４１（１９９９）．
〉′ R., and Tobutt, K. R., Correlation of stylar ribonuclease isoenzymes with incompatibility alleles in apple.
１６） Boskovic,Euphytica，
１０７，２９-４３（１９９９）．
１７） Matsumoto, S., Hayashi, S., Kitahara, K., and Soejima, J., Genomic DNA sequences encoding Malus x domestica
Borkh. “Akane”, “Delicious” and Malus transitoria S-RNases. DNA Seq．
，
１２，
３８
１-３８３（２００１）．
１８） Royo, J., Nass, N., Matton, D. P., Okamoto, S., Clarke, A. E., and Newbigin, Ed., A retrotransposon-like sequencelinked to the S-locus of Nicotiana alata is expressed in styles in response to touch. Mol. Gen. Genet．
，
２５０，
１８０-１８８（１９９６）．

１１Shogo Matsumoto and Yui Furusawa

１９） Matton, D. P., Maes, O., Laublin, G., Xike, Q., Bertrand, C., Morse, D., and Cappadocia, M., Hypervariable domainsof self-incompatibility RNases mediate allele-specific pollen recognition. Plant Cell ，９，
１７５７-１
７６６（１９９７）．
２０） Ushijima, K., Sassa, H., Tao, R., Yamane, H., Dandekar, A. M., Gradziel, T. M., and Hirano, H., Cloning and characterization of cDNAs encoding S-RNases from almond（Prunus dulcis）：primary structural features and sequence diversity of the S-RNases in Rosaceae. Mol. Gen. Genet．
，
２
６０，２
６
１-２
６８
（１９９８）．

１２