Plant Mol Biol (2010) 74:143–154
DOI 10.1007/s11103-010-9662-z

Apple S locus region represents a large cluster of related,
polymorphic and pollen-specific F-box genes
Mai Minamikawa • Hiroyuki Kakui •Sanhong Wang • Nobuhiro Kotoda •Shinji Kikuchi • Takato Koba • Hidenori Sassa

Received: 9 February 2010 / Accepted: 1 July 2010 / Published online: 15 July 2010
Ó Springer Science+Business Media B.V. 2010

Abstract Gametophytic self-incompatibility (GSI) of
Rosaceae, Solanaceae and Plantaginaceae is controlled by acomplex S locus that encodes separate proteins for pistil andpollen specificities, extracellular ribonucleases (S-RNases)
and F-box proteins SFB/SLF, respectively. SFB/SLFs of
Prunus (subfamily Prunoideae of Rosaceae), Solanaceae
and Plantaginaceae are single copy in each S haplotype,while recently identified pollen S candidates SFBBs of
subfamily Maloideae of Rosaceae, apple and Japanese pear,
are multiple; two and three related SFBBs were isolated

Sequence data from this article have been deposited with DDBJ/
EMBL/GenBank Data Libraries under accession nos. AB539844(MdFBX1), AB539845 (MdFBX2), AB539846 (MdFBX3), AB539847(MdFBX4), AB539848 (MdFBX5), AB539849 (MdFBX6), AB539850(MdFBX7), AB539851 (MdFBX8), AB539852 (MdFBX9), AB539853(MdFBX10), AB539854 (MdFBX11), AB539855 (MdFBX12),
AB539856 (MdFBX13), AB539857 (MdFBX14), AB539858(MdFBX15), AB539859 (MdFBX16), AB539860 (MdFBX17),
AB539861 (MdFBX18), AB539862 (MdFBX19), AB539863(MdFBX20).

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11103-010-9662-z) contains supplementarymaterial, which is available to authorized users.
M. Minamikawa  H. Kakui  S. Wang  S. Kikuchi  T. Koba 
H. Sassa (&)
Graduate School of Horticulture, Chiba University, Matsudo,
Chiba 271-8510, Japane-mail: sassa@faculty.chiba-u.jp

from each S haplotype of apple and Japanese pear, respectively. Here, we show that apple (Malus 9 domestica)
SFBBs constitute a gene family that is much larger thaninitially thought. Twenty additional SFBB-like genes/alleles
were isolated by screening of a BAC library derived from
S3S9 genotype, and tentatively named MdFBX1-20. All but
one MdFBX showed S haplotype-specific polymorphisms.
All the polymorphic MdFBXs were completely linked to
S-RNase in 239 segregants. In addition, FISH revealed
that the monomorphic gene MdFBX11 is also located near
S-RNase, and the S locus is located in a subtelomeric regionof a chromosome and is not close to the centromere. All
MdFBXs were specifically expressed in pollen, except for apseudogene MdFBX4 that showed no expression in any
organs analyzed. Phylogenetic analysis revealed that theclosest relatives of most MdFBXs were from a different
S haplotype, suggesting that proliferation of MdSFBB/FBXs
predates diversification of the S haplotypes.
Keywords Self-incompatibility  S locus  Rosaceae 
F-box  Pollen
Abbreviations
GSI
Gametophytic self-incompatibility
SFBB
S locus F-box brothers
S-RNase S-ribonuclease
BAC
Bacterial artificial chromosome

S. Wang
Department of Horticulture, Nanjing Agricultural University,
210095 Nanjing, China
N. Kotoda
Apple Breeding and Physiology Research Team, National
Institute of Fruit Tree Science, Shimo-Kuriyagawa, Morioka
020-0123, Japan

Introduction
Self-incompatibility (SI) is a genetic mechanism to prevent
inbreeding and to promote outcrossing found in many

123

144

flowering plants. The S-RNase-based gametophytic selfincompatibility (GSI) system has been found in the families Solanaceae, Rosaceae, and Plantaginaceae. Haplotypesof a single S locus control the specificity of self/non-self
discrimination; when an S haplotype in the haploid pollen
matches one of two S haplotypes in the pistil, then thepollen is recognized as ‘‘self’’ and rejected by the pistil (deNettancourt 2001). The S haplotype contains two tightly
linked S-determinants, pistil S and pollen S (Kao andTsukamoto 2004; Franklin-Tong 2008).
Pistil S is known as the S-RNase gene which encodes anextracellular ribonuclease (Anderson et al. 1986; McClureet al. 1989; Sassa et al. 1996, 1997; Tao et al. 1997; Xueet al. 1996). S-RNases are taken up by both compatible andincompatible pollen tubes, but thought to act as cytotoxins
only in incompatible pollen tubes to arrest growth (Goldraij
et al. 2006; Luu et al. 2000). The identification of F-box
genes as pollen S (candidates), SLF (S locus F-box)/SFB(S haplotype-specific F-box), raised the possibility thatubiquitin/proteasome pathway plays a pivotal role in GSI
because the predominant function of F-box protein issubstrate recognition as a component of SCF complex, akind of E3 ubiquitin ligase (Entani et al. 2003; Lai et al.
2002; Qiao et al. 2004a; Sijacic et al. 2004; Ushijima et al.
2003, 2004; Wang et al. 2004). It has been hypothesizedthat SLF/SFB recognizes non-self S-RNase in compatible
pollen tubes and ubiquitinates it for degradation by the 26S
proteasome (Hua and Kao 2006; Hua et al. 2008; Qiao
et al. 2004b; Ushijima et al. 2003, 2004). However,Goldraij et al. (2006) showed that S-RNase is sequesteredinto vacuoles in pollen tubes and that the amount of
S-RNase is not significantly different between compatible
and incompatible pollination. Consequently, how SLF/SFB
and S-RNase interact with each other to trigger the selfincompatibility reaction in vivo remains largely unclear.
Furthermore, genetic studies suggested divergence of pollen S function among taxa.
In Solanaceae and subfamily Maloideae of Rosaceae(e.g., apple and pear), it has been well-documented thatcoexistence of different pollen S in a pollen grain as aresult of polyploidization or translocation of the S locus
causes breakdown of SI in pollen leaving the pistil SI
function unaffected (Adachi et al. 2009; Crane and Lewis
1941; de Nettancourt 2001; Golz et al. 2001; Lewis andModlibowska 1942). This phenomenon, known as ‘competitive interaction’, was recently reported also in Plantaginaceae, Antirrhinum (Xue et al. 2009). In contrast, in
species of the subfamily Prunoideae of Rosaceae (e.g.,almond and sweet cherry), it is suggested that the pollen
S gene does not cause competitive interaction. Sour cherry(Prunus cerasus) is a naturally occurring tetraploid species
and includes both SI and self-compatible (SC) lines (Lansari and Iezzoni 1990). Hauck et al. (2006b) conducted

123

Plant Mol Biol (2010) 74:143–154

genetic analyses of sour cherry and proposed that the
breakdown of SI is caused by the accumulation of nonfunctional S haplotypes and not by competitive interactionin heteroallelic pollen. Consistent with this, different pollen-part SC mutants of Prunus contained SFB genes
encoding truncated proteins (Hauck et al. 2006a; Tsukamoto et al. 2005; Ushijima et al. 2004). Furthermore,
Sonneveld et al. (2005) showed complete deletion of SFB
in a SC haplotype of sweet cherry. This is in marked
contrast to the case of Solanaceae, in which pollen S isconsidered to be required for pollen tube growth. In
Nicotiana (Solanaceae), no pollen S deletion type mutant
but pollen S duplication types were recovered after large
scale screening of mutagenized pollen by incompatible
pollination, suggesting that pollen S is essential for pollen
tube growth (Golz et al. 2001). Pollen S deletion typemutant is not known in Maloideae either.
Consistent with the probable difference in pollen
S function between Maloideae and Prunoideae, recently
identified pollen S candidates of Maloideae show a uniquefeature, multiplicity (Sassa et al. 2010). Two related F-box
genes were identified as pollen S candidates by sequencingof the S locus region of apple (Malus 9 domestica) and
named SFBBs (S locus F-box brothers; Sassa et al. 2007).
Three SFBB genes were isolated by PCR from eachS haplotype of Japanese pear (Pyrus pyrifolia). All the
SFBBs showed S haplotype-specific polymorphisms, linkage to the S locus and pollen-specific expression. Here, weshow that many more SFBB-like genes are clustered in the
S locus region of apple. We isolated twenty SFBB-likegenes from apple with S3S9 genotype, and tentatively
named them MdFBX1-20. Linkage to the S-RNase gene,
S haplotype-specific polymorphisms and pollen-specificexpression were found in all the MdFBX genes, except fora pseudogene and a monomorphic gene. We discuss theimplications of the MdSFBB/FBX cluster at the apple
S locus for the probable divergence of the GSI mechanism
among taxa.

Materials and methods
Plant material
Eight apple cultivars, Sekai-ichi (S3S9), Natsumidori(S3S9), Kitaro (S3S9), Hatsuaki (S3S9), Golden Delicious(S2S3), Megumi (S2S9), Gala (S1S6) and Morioka64 (S3S5),
were used. 175 progenies obtained by crossing Kitaro(S3S9) and Morioka64 (S3S5) and 64 progenies obtained bycrossing Hatsuaki (S3S9) and Megumi (S2S9) were used toanalyze the linkage of F-box genes with the S-RNase gene.
Seedlings with S9 haplotype that were derived from open
pollinated Fuji (S1S9) fruits were selected by PCR (KimPlant Mol Biol (2010) 74:143–154

et al. 2009) and used for fluorescence in situ hybridization(FISH) analysis.

145

the procedure of Kim et al. (2009). PCR products weretreated with Sphl to differently digest S3-, S5- and S9-RNase
fragments (Kim et al. 2009).

Isolation of nucleic acids
FISH
Genomic DNA was isolated from leaves as described by
Sassa (2007). RNA was isolated from leaves and floralorgans as described by McClure et al. (1990).
Screening of a BAC library and cloning of SFBB-likegenes
A BAC library of an apple cultivar, Florina, covering fivegenome equivalents (Vinatzer et al. 1998) was obtained
from Texas A&M University and screened using
MdSFBB9-b (Sassa et al. 2007) as a probe. SFBB-like genes
were amplified from the selected BAC clones using theprimers FSMF1 and RSMF1(Sassa et al. 2007). A standard
PCR mixture (in 20 ll total volume) included BAC plasmid, 0.4 lM of each primer, 200 lM dNTP, 19 PCR
Buffer, and 0.5 U of ExTaq (TaKaRa). Amplification was
carried out for 35 cycles of denaturation at 94°C for 15 s,
annealing at 50°C for 15 s, and extension at 72°C for1.25 min, with a final extension at 72°C for 7 min. The PCR
products were cloned into a plasmid vector and sequenced.
For three genes that were not amplified from BAC clones
with FSMF1 and RSMF1, partial sequences were amplified
by different primer pairs: FjpFB1 (Sassa et al. 2007) andRSMF1 for MdFBX17 and 19, and aBACf6 and aBACr8 for
MdFBX20. Flanking sequences were determined by using
DNA Walking SpeedUp Premix Kit (SeeGene) or by
50 RACE. The primer sequences used for amplification of
those three genes are listed in Supplemental Tables 1 and 2.
Sequence data was analyzed with BioEdit ver.7.0 software(http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and ClustalW (Thompson et al. 1994). The identity of amino acidsequences of 20 MdFBXs was examined using GENETYXMAC program (GENETYX Co. Tokyo, Japan).

CAPS and dCAPS analyses
Genomic DNA of apple cultivars was used as the template
for amplification of MdFBX genes. The PCR products weredigested with restriction enzymes to detect specific cleaved
amplified polymorphic sequence (CAPS) bands. Derived
CAPS (dCAPS; Neff et al. 1998) analysis was conducted todetect MdFBX10, 15 and 20-specific fragments. The
primers and enzymes are listed in Supplemental Table 1.
The products were separated on 1 or 2% agarose gels andvisualized by staining with ethidium bromide. CAPS
analysis of the S-RNase genes was conducted according to

Root tips of seedlings with S9 haplotype derived from Fuji
(S1S9) were pretreated with 2 mM 8-hydroxyquinoline for
2 h at room temperature and 4 h at 4°C, and fixed in 3:1(v/v) ethanol: acetic acid for [24 h. After washing indistilled water, chromosome slides were prepared byenzymatic maceration (2% cellulase and 2% pectolyaseY-23) at 37°C for 20 min and squash method. The slides
were incubated in 100 ng/ll RNase for 1 h at 37°C in a wet
chamber, and then incubated in 5 ng/ll pepsin for 20 min
at 37°C, followed by post-fixation in 3:1 (v/v) ethanol:
acetic acid for 5 min. BAC clones were labeled with either
biotin-16-dUTP or digoxigenin-11-dUTP by nick translation and used as FISH probes. A 10 ll hybridization
solution (50% formamide, 10% dextran sulfate, 29 SSC,
20 ng of each probe, 3.0 lg of apple genomic blockingDNA) was applied to the slides. Chromosomal DNA wasdenatured in the hybridization solution for 5 min at 80°C
and then kept in the chamber at 37°C for 2 days. Afterwashing in distilled water, FITC-conjugated streptavidin
and mouse anti-digoxigenin were applied to slide andincubated for 30 min at 37°C. After washing, Alexa fluor
568-conjugated rabbit anti-mouse IgG (H ? L) wasapplied as secondary antibody and incubated for 30 min
at 37°C. Chromosomes were counterstained with 4, 6
diamidino-2- phenylindole (DAPI).
RT-PCR/CAPS
RNA samples from the leaves and the floral organs of
Sekai-ichi (S3S9) were treated with DNaseI (Nippongene).
cDNA was synthesized by ReverTra Ace (TOYOBO) andused for RT-PCR/CAPS. As the control in the RT-PCR,
ef1-a (translation elongation factor 1a, AY338249) was
amplified by the primers ef1-af1 (50 -ATTGTGGTCATT
GYCAYGT-30 ) and ef1-ar1 (50 -CCTATCTTGTAVACATCCTG-30 ).
Construction of phylogenetic trees of S locus-linked
F-box proteins
The amino acid sequences of the S locus-linked F-box
proteins of Solanaceae, Rosaceae and Plantaginaceae werealigned using ClustalW (Thompson et al. 1994). A neighbor-joining tree was constructed using the alignment(Saitou and Nei 1987). Maximum parsimony phylogenetic
tree was generated by MEGA4 (Kumar et al. 2008).

123

146

Plant Mol Biol (2010) 74:143–154

Results
Twenty new SFBB-like genes isolated from a BAC
library of apple Florina (S3S9)
Screening of a Florina (S3S9) BAC library covering
five 9 haploid-genome equivalents (Vinatzer et al. 1998)
with the MdSFBB9-b probe was conducted under low
stringent hybridization condition (50°C), and identified 39
positive clones. SFBB-like genes were amplified from thepositive BAC clones by PCR. Identical sequences wereobtained from several clones, probably because the BACs
are overlapping. The overlapping of BAC clones was
confirmed by fingerprinting the clones with restriction
digestion (data not shown). Finally, twenty new SFBB-likegenes were isolated and tentatively named MdFBX1 *20.
All 20 MdFBX genes encoded SFBB-like proteins except
for MdFBX4, which encoded a truncated protein because of
in-frame stop codons, and was considered to be a pseudogene. MdFBXs and previously identified MdSFBBs
were highly homologous with each other showing 59.6%(MdFBX14 and MdFBX10) to 98.0% (MdFBX5 and

MdFBX1MdFBX2
MdFBX3
MdFBX4
MdFBX5
MdFBX6
MdFBX7
MdFBX8
MdFBX9
MdFBX10
MdFBX11
MdFBX12
MdFBX13
MdFBX14
MdFBX15
MdFBX16
MdFBX17
MDFBX18
MdFBX19
MdFBX20
MdSFBB3-alpha
MDSFBB3-beta
MdSFBB9-alpha
MdSFBB9-beta

1 ----------------MSLVCESETPEDQLVEILSWLRPKSLMRFKCIRKSWCTIINSSSFVAKHLSNSIDNKLSSSTCILLNRCQVHDFPDRSWKQDVFWSMINLSIDSDKNNLHYDVEDL-NIPFPM-EDQDNVELHGYCNGIVCVIV
1 ----------------MSQVRECETPEDKVVEILSKLPPKSLIRFKCIRKSWCTIINSPSFVAKHLRISMDNKLSSTTCILLNRCQVHVFPDRSWKQDVFWSMTNLSIDSDEHNLHYDVEDL-NIPFPM-EDQDNVEIHGYCNGIVCVIV
1 ----------------MSQVSETETPEDRVVAIMSKLPPKSLMRFKCIRKSWCTVINSPSFVAKHLSNSVDNKFSSSTCILLNRSQVHVFPDKSWKHEVLWSMINLFNERVARTLYYDVEDL-NIPFPR-DDHQHVLIHGYCNGIVCVIS
1 ----------------MSQVRETETPEDRVVTIMSKLPPKSLM*FKCIRKSWCTLINSPTFVAKHLSNYVDNKFSSSTCIILNHS*VHVFPDGSWKHEDLWSMINLFNDRVAQTLYYGVEDL-NIPFTR-DDHQHVLIHGYCNGIVCVIS
1 ----------------MSQVHESETPEDKVVEILSRLSPKSLMRFKCVHKSWCTIINSPSFVAKHLSNTVDNKFSSFTCILFNRCQVHVFPDRSWKRDVFWSMINLSIDSDEHNLHYDVEDR-NIPFPI-EVQDNVQLYGYCNGIVCVIV
1 ----------------MSQLHETETPEDKVVEILSRLPPKCLMRFKCIRKSWCTLINSPSFVAKHLNNSMDNKLSSSTCILLNRSQAHIFPDQSWKQEVFWSTINLSIDSDEHNLHYDVEDL-IIPFPL-EDHDFVLIFGYCNGIICVDA
1 ----------------MSQVRESETPEDRVVEILSRLSPKSLLRFKCIRKSWCTLVNSPSFVAKHLSNSLENKLSSSTCILLNRSQFHIFPDQSWKREILWSMINLSIDSDVHNLHYDVKPL-NIPFPR-DDHNPVQIHGYCNGIVCLIE
1 ----------------MSQEHESEAPEDRVVEILSRLPPKSLMRFKCIRNSWCTLINSPRFVAKHINNSVDNKLSSSTCILLHRSQTPIFPYDSWKREFFWSMINFSIDSDESNLHYDVEDLTNVPLLQWEDHHEVEIHGYCNGIVCVTV
1 ----------------MSQVCESETPEDRVVETLSRLPPKSLMRFKCICKSWCTLINSLSFVAKHLNNSMDNKLSSSTCILLSRSQAHVFPDNSWKPEVFWSMINLSIDSDEHNLHYDVEDL-NIPFPL-EGHDFVQIEGYCNGIVCVIA
1 ----------------MSQVRECETPEDRMVEILSRLPPKSLMRFKCIRKSWCTLINSPCFVAKHLSDSVDNKLSSSTCILLNCSQAHVCPEESWKQEVLWSMINLSIDGDE--LHYDVEDLTNVPFLK-DDHHELEIHGYCDGIVCVTV
1 ----------------MSQVHENETLEDRVVEILSRLLPKSLMRFKCIRKSWCTLINSPSFVAKQLSNSVDNKFSSSTCILLNRSQTHVFPDNSWKQEVFWSMINLSIDSDEHNLHYDVEDL-NIPFPL-EDHDYVLILGYCNGIVCVTA
1 ----------------MSQVHDSETLEDRIVEILSRLPPKSLMRFKCIRKPWCTLINSPSFVAKYLSNSVDNKLSSSTCILLNRTQMYVFPDQSWKYETLWSMMNLSNYSDEHNLHYDFKDL-NIPFPM-EDHHPVQIHSYCNGIVCVIT
1 -------------------MLENITPEDRMIEILSKLPAKSLTRFKCIRKSWCTLINSPSFVAKHLNNSADSKLSSSTCILVNHSQPHIFPDKNWKQEVFWSMINISIDSDEHSLHYDVVDL-NIPFPL-EDHDFVQIHGYCNGIVCVIV
1 MSQVRKSETLKDRVTEMTQVRESETPEVRVAEILSRLPPKSLMRFKCIRKSWCTAINNPSFTAKHLSNSVDNKFSSSTCILLHRSHMPVFPDRSWKREYFWSMINLSHDSDEHNLYYDVEDL-NIQFPL-EDHDLVSVHGYCNGIVCLIV
1 ----------------MSQVHESETPEDKVVEILSRLSPKSLMRFKCVHKSWCTIINSPSFVAKHLSNTVDNKFSSCTRILFNRCQVHVFPDRSWKRDVFWSMINLSFDSDEHNLHYDVEDR-NIPFPI-EVQDNVQLYGYCNGIVCVIV
1 ----------------MFQVRESETPENSVVETLSRLPPKSLMRFKCIRKSWCTLINSPSFVAKHFNNSMDNKLSSSTCILLSRSQAHVFPDNSWKPEVFWSMINLSIDSDEHNLHYDVEDL-NIPFPL-EGHDFVQIEGYCNGIVCVIA
1 ----------------MSQVRESETPEDRMVGILSRLSPKSLLRFKCIRKSWCTLINSPSFVAKHLSNSLDNKLSSSTCILLNRSQFHIFPDQSWKREVLWSMINLSSDSDVHNLHYDVKPL-NIPFSR-DDHNPVQIHGYCNGIVCLIE
1 ----------------MSQVRESETPEDRMVEILSRLPPKSLMRFKCIRKSWCTLINSPCFVAKHLSDSVDNKLSSSTCILLNCSQAHIFPDQSWKQGVLWSVIYLSIDGDE--LHYDIEDLTNVPFLK-DDHHEVEIHGYCDGIVCVTV
1 ----------------MSHVRQSKTPEDRVIEILSRLPPKSLMRFKCLHKSWFSLINSLSFVDKHLSNSVDNKLPSSTCILLNRSQAHIFPDQSWKQEVFWSMINFSIDSDENNLHYDVEDL-NIPFPL-NDHDFVLIFGYCNGIVCIEA
1 ---------MLNKTVEMSQVHDSETPEDRVVEILSRLPPKSLMRFKCIHKSWCTLINSPSFVAKYLSNSVDNKLSSSTCILLNRTQMHVFPDQSWKYETLWSMMNLSNYSDEHNLQYDFKDL-NIPFPT-EDHHPVQIHSYCNGIVCVIT
1 ----------------MSHVRESETPEDRVVEILSRLPPKSLMRFKCIHKSWFSLINNLSFVAKHLSNSVDNKLSSSTCILLNRSQAHIFPDQSWKQEVFWSMINFSIDSDENNLHYDVEDL-NIPFAL-KDHDFVLIFGYCNGILCVEA
1 ----------------MSQVHESETPEDKVVEILCRLPPKSLMRFKCIRKSWCTLINRPSFVAKHLNNSVDNKLSSSTCILLNRSQAHIFPDQSWKQEVFWSTINLSIDSDEHNLHYDVEDL-IIPFPL-EDHDFVLIFGYCNGIVCVDA
1 ----------------MSQVRESETPEDQVVEILSRLPPKSLMRFKCIRKSWCTIINSSSFVAKHLSNSIDNKLSSSTGILLNRCQVHVFSDRSWKQDVFWSMINLSIDSDKNNLYYDVEDL-NIPFPM-EDQDNVELHGYCNGIVCVIV
1 ----------------MSQVRESETLEDKVVEILSKLPPKSLMRFKCIRKSWCTIINSPSFVAKHLNNSMDYKLSSTTCILLNRCQVHVFPDRSWKQDVFWSMINLSIDSDEHNLHYDVEDI-NIPFPM-EDQDNVELHGYCNGIVCVIV.... .....*.........*..*.*.................................*...**..... ..... ... ......................... ..... .... . ............

132
132
132
132
132
132
132
134
132
131
132
132
129
148
132
132
132
131
132
139
132
132
132
132

MdFBX1MdFBX2
MdFBX3
MdFBX4
MdFBX5
MdFBX6
MdFBX7
MdFBX8
MdFBX9
MdFBX10
MdFBX11
MdFBX12
MdFBX13
MdFBX14
MdFBX15
MdFBX16
MdFBX17
MDFBX18
MdFBX19
MdFBX20
MdSFBB3-alpha
MDSFBB3-beta
MdSFBB9-alpha
MdSFBB9-beta

133 GKNVL-----LCNPATGEFRQLPDSSLLLP--LPKGRFGLETVFKGLGFGYDCKAKEYKVVRIIENCDCEYSEGEESYYERILLPHTAEVYTMTTNSWKEIKIDVTSDTD-PYCIPYSCSVYLKGFCYWFAMDNGEYIFSFDLGDEIFHI
133 GKNVL-----LCNPATREFRQLPNSSLLLP--LPKGRFGLETTFKGMGFGYDCKTKEYKVVRIIENCDCEYSEDGESYYERILLPHTAEVYTTTANSWKEIKIDISIETR-WYCIPYSCSVYLKGFCYWFAYDNGEYVFSFDLGDEIFHR
133 GKNIL-----LCNPATREFRQLPDSFLLLPS-PLGGKFELETDFGGLGFGYDCRAKDYKIVRIIENC--EYSDDERTYYHRIPMPHTAEVFTMATNYWKEIKIDISSKT-----YPCSCSVYLKGFCYWFTRDGEEFILSFDLGDERFNR
133 GKYIL-----LCNPATREFRQLPDSFLLLPS-PLGGKFELETDFGGLGFGYDCRAKDYKVVRIIENC--EYSDDEGTYYHRIPLPHTAEVYTMATNSWKEIKIDISSKT-----YPCSCSVYLKGFCYWFTRDGEEFILSFDLGDERFHR
133 GENVL-----LCNPATREFKQLPDSSLLLP--LPTGKFGLETLFKGLGFGYDCKTKEYKVVRIIENCDCEYSDGKESYIERILLPYTAEVYTTVANSWKEIKIDTSSDTD-PYCIPYSCSMYLKGFCYWFANDNGEYIFSFDLGDEIFHR
133 GKNVL-----LCNPATREFRQLPHSCLLLP--PPKGKFELETTFQALGFGYDCNSKDYKVVQIIENC--EYSDDEQTFHHRIALPHTAEVYTTAANSWKEIKIEISSQT-----YHCSCSVYLKGFCYWFASDSEEYILSFYLGDETFHI
133 GDNVL-----LCNPSTREFRLLPNSCLLVP--HPEGKFELETTFHGMGFGYDCKAKEYKVVQIIENC--EYSDDGQTYQHCIAYPYTAEVYTTAANFWKEIKINISSTT-----HPYPFSVYLKGFCYWFATDGEECILSFDLGDEIFHR
135 GEYFF-----LCNPATGEFSQLPNSRLLLPLPRGKGKFGLETTVKGLGFGYDCKAKEYKVVRIIENYDCEYSDGEETYIEHTALPHTAEVYTTTANSWKEIKINISSKILSLYSYPYSCSVYLKGFCYWLSSDDEEYICSFNLGDEIFDR
133 GTSLYLINVLLCNPATGKFRQLPPSYLLLPS-RPQGKFQLESIFGGLGFGYDCKAKDYKVVQIIENC--EYSDDQQYYYHRIALPHTAEVYTMAANSWRVIKIDISSET-----YHYSSSVYLNGFFYWFAIDGEKYILSFDLGDEIFHR
132 DENFF-----LCNPATGEFRQLPDSCLLLPLPGVKEKFGMETTLKGLGFGYDCKAKEYKVVRIIDNYDCEYSDDGETYIEHIALPHTAEVYTMAANSWKEITIDILSKILSSYSEPYSYSVYLKGFCYWLSCDVEEYIFSFDLANEISDM
133 GKNIL-----LCNPTTREFMRLPSSCLLLPS-RPKGKFELETVFRALGFGYDCKAKEYKVVQIIENS--EYSDDERTYYHRIPLPHTAEVYTTAANSWREIKIDISTKT-----YSCSCQVYLKGFCYWYATDAEEYILSFDLGDEIFHR
133 GKSVR----ILCNPATREFRQLPSSCLLVPS-PPEGKFQLETIFEGLGFGYDYKAKEYKVVQIIENC--EYSDDERRYYHRIALPHTAEVYTTTANSWKEIKIEISSKT-----YQCYGSEYLKGFCYWLANDGEEYILSFDLGDEIFHI
130 GKKFL-----LCNPATREFMQLPNSCLLLP--PAEGKFELDTTFEALGFGYDCKGKEYKVVQIIENC--EYSDDEQTFNHCTTLPHTAEVYTTVANSWKEIKIDISSTT-----YSWSCSVYLKGFCYWYATDDEEYVLSFDLCDETFHR
149 GKHAV-----LYNPATRELKQLPDSCLLLPS-PPKGKFELESSFQGMGFGYDSKAKEYKVVKIIENC--EYSDYERTFSHRIALPHTAEVYVTTTNSWRVIKIEISSDT-----YNCSCSVYLKGFCYWFASDDEEYILSFDLGDEIFHR
133 GENVL-----LCNPATREFKQLPDSSLLLP--LPTGKFGLETLFKGLGFGYDCKTKEYKVVRIIENCDCEYSDGKESYIERILLPYTAEVYTTAANSWKEIKIDTSSDTD-PYCIPYSRSMYLKGFCYWFANDNGEYIFSFDLGDEMFHR
133 GTSLYLINVLLCNPATGKFRQLPPSCLLLPS-RPTGKFQLESIFGGLGFGYDCKAKEYKVVQIIENC--EYSDDQQYYYHRIALPHTAEVYTMAANSWRVIKIDISSET-----YHYSSSVYLNGYFYWFAIDGEKYILSFDLGDEIFHR
133 GDNVL-----LCNPSTREFRLLPNSCLLVP--HPEGKFELETTFHGMGFGYDCKANEYKVVQIVENC--EYSDDEQTYQHCIAYPYTAEVYTTAANFWKEIKIDISSST-----HPYPFSVYLKGFCYWFATDGEECILSFDLGDEIFHR
132 DENFF-----LCNPATGEFRQLPDSCLLLPLPGVKVKFGLETTLKGLGFGYDCKAKEYKVVRIIDNYDCEYSDDGETYIEHIALPHTAEVYTMAANSWKEITIDILSKILSSYSEPYSYSVYLKGFCYWLSCDLEEYIFSFDLANEISDM
133 GKNVL-----LCNPATREFRQLPDSCLLLPS-PPEGKFELETSFQALGFGYDSNAKEHKVVRIIENC--EYSDEERTFYHRIALPHTAELYTATANSWKEIKIDISSTT-----YSCSRSVFMKGFCYWYATDGEEYILSFDLSDDKFHI
140 GKSVR----ILCNPATREFRQLPASCLLLPS-PPEGKFQLETIFEGLGFGYDYKAKEYKVVQIIENC--EYSDDERRYYHRIALPHTAEVYTATANSWKEIKIEISSKT-----YQCYGSEYLKGFCYWLANDGEEYILSFDLGDEIFHK
133 GKNVL-----LCNPATREFKQLPDSCLLLPS-PPERKFELETNFQALGFGYDCNAKEYKVVRIIENC--EYSDDERTYYYRIALPHTAELYTTTANSWKEIKIDISSTT-----YSCSRSVFMKGFCYWYATDGEEYILSFDLGDDTFHI
133 GKNVL-----LCNPATREFRQLPDSCLLQP--PPKGKFELETTFQALGFGYDCNAKEYKVVRIVENC--EYSDDEQTFYHRIALPHTAEVYTTAANFWKEIKIDISIKT-----YHCSCSVYLKGFCYWFASDNEEYILAFYLGDETFHI
133 GKNVL-----LCNPATGEFRQLPDSSLLLP--LPKGRFGLETVFKGLGFGYDCKAKEYKVVRIIENCDCEYSEGEESYYERILLPHTAEVYTMTADSWKEIKIDVSSDTD-PYCIPYSCSVYLKGFCYWFACDNGEYIFSFDLGDEIFHI
133 GKNVL-----LCNPATGEFRQLPNSPLLLP--LPKGRFGLETTFKGMGFGYDCKSKEYKVVRIIENCDCEYSDDGESYYERILLPHTAEVYTMTANSWKEIKIDISIETR-WYCIPYSGSVYLNGFCYWFAYDNGEYVFSFDLGDEIFHK.....
............. ..*...
. ... ... . ..*................... ...... ................. ......*.*.... .
. .. ............ . ...............

274
274
269
269
274
268
268
279
274
276
269
270
265
285
274
274
268
276
269
277
269
268
274
274

MdFBX1MdFBX2
MdFBX3
MdFBX4
MdFBX5
MdFBX6
MdFBX7
MdFBX8
MdFBX9
MdFBX10
MdFBX11
MdFBX12
MdFBX13
MdFBX14
MdFBX15
MdFBX16
MdFBX17
MDFBX18
MdFBX19
MdFBX20
MdSFBB3-alpha
MDSFBB3-beta
MdSFBB9-alpha
MdSFBB9-beta

275 IELPSRREFGFKFYGIFLYNESITSYCSRYEED------CKLFEIWVMDDDDGVKSSWTKLLTVGPFKDI-DYPLTLGKCDEVLMLGSYGRAAFCNSSIGNLKYLHIPPIIN--WMID-----YVKSIVPVK----------275 IELPSRRESDFKFYGIFLYNESVTSYCYRHEED------CELFEIWVMDDYDGVKSSWTKQLTIGPLKDI-DYPLTLWKCDEILMLGSYGRAASCNSSTGNLKYLHIPPIIN--WMID-----YVKSIVPVK----------270 IQLPSRRESGLEFYYIFLCNESIASFCSRYDRS-EDS---KSCEIWVMDDYDGVKSSWTKLLVAGPFKGI-EKPLTLWKCDELFMIDTDGRVISYNSSIGYLSYLHIPPIIN--RVIDSQALIYVESIVPIK----------270 IQLPSRRESGFEFYYIFLCNESIASFCSLYNQS-EDS---KSCEIWVMDEYDGVKNSWTKLLVAGPFKGI-EKPLTLWKCDELLMLATDGRVISYNSSIGYLTYLHIPPIIN--RVTDSQALIYVESIVPVK----------275 IELPFRRESDFKFCGLFLYNESVASYCSCYEED------CKLVETWVMDDYDGVKSSWTKLLTVGPFKDI-ESPLKFWKCDEVLILSSYGKATSYNSSTGNLKYFHIPPIIN--WMID-----YVETIVPVK----------269 IQLPSRRESGFTFDYIFLLNESLASFCSPYNPS-EDS---KLFEIWVMDDYDGVKSSWTKLLTVGPFKGI-EYPLTLWKCDELLMLASDGRATSYNSSTGNLKYLHIPPILN--KVVDFEGLIYVKSIVPLN----------269 IQLPSKIESGFEFCGLFLYNESITSYCCHNDPS-EDS---KLFEIWVMDGYGGVNSSWTKILTIGPSKDI-EYPLTFWKCDELLMFASGRRVTSYNSSTGNLKDLHIPPIMH--QVTDLEALIYEESLVPIK----------280 IELPSRRESGFKLDGIFLYNESITYYCTSYEERS------RLFEIWVMDNYDGVKSSWTKHLTAGPFKGI-EFPLTLRKHDELLMIASDGRATSYNSNTGNLKYLHIPVIIYRNRVID-----YAKSIVPVKRVEGKVPFSPI275 IQLPSRRESDFEFSNIFLCNKSIASFCSCCDPSDEDS---TLCEIWVMDDYDAVKRSWTKLLTFGPLKDI-ENPFTFWKTDELLMVAAGGRATTYNSSTGNLNYLHIPPILN--EVRDFEALIYMESIVPVK----------277 IELPFRGEFGFKRDGIFLYNESLTYYCTSYEEPS------TLFEIWVMGYDDGFKSSWTKHLTAGPFKDM-EFPLTPWKRDELLMIASDGRAASYNSCTGNFKYLHIPVIINENRVVD-----YVKSIILVN----------270 IQLPSRRESGFKFYYIFLRNESLASFCSRYDRS-DKS---ESCEIWVMHNYDGVKSSWTKLLIIGPLQAI-GKPLTFWKSDELLMLASDERATSYNSSTGNLKYLHIPPILN--RVVDFQALIYVKSIVSFK----------271 MQLPSRRESGFKFYNIFLCNESIASFCCCYDPTNEDS---TLCEIWVMDDYEGVKSSWTKLLTVGPLKGINENPLAFWKSDELLMVSCDGRVTSYNCSTKNLSYLHIPPIVN--EVRDFQAVIYVESIVPVK----------266 IQFPSRGESGFTFFYIFLRNESLTSFCSRYDRS-GDS---QSCEIWVMDDYDGVKSSWTKLLTVGPLQAI-EKPLTFWKSDELLMLASDGRTTSYNSSTRNMKYIHIPPILN--TVVDFQALTYVESIVPLK----------286 IQLPRRKESGFLFYDLFLFNESIASFCSHYDKS-DNSRILEILEIWVMDDCDGVKSSWTKLQTLGPFKDN-ENLLTFWKSDELLMVTSDNRVISYNSSTGNLKYIHIPPIIN--KITGFEALIYVESIVSVKRVEGKVPFSPI275 IELPFRRESDFKFCGLFLYNESVASYCSCYEED------CKLVEIWVMDDYDGVKSSWTKLLTVGPFKDI-ESPLKFWKCDEVLILSSYGKATSYNSSTGNLKYFHIPPIIN--WMID-----YVETIVSVK----------275 IQLPSRIESDFEFSNIFLYNKSIASFCSCCDPSDADS---TLCEIWVMDDYDGVKRSWTKLLTFGPLKDI-ENPFTFWKTDELLMVAAGGRATTYNSSTGNLNYLHIPPILN--EVRDFEALIYVESIVPVK----------269 IQLPSKIESGFNFCGLFLYNESITSYCCRYDPS-EDS---KLFEIWVMDGYGGVKNSWTKLLTVGPFKGI-EYPLTFWKCNELLMVASSRRVTSYNSSTGNLKDLHIPPIIH--QVTDLQAFIYEESLIPIK----------277 IELPFRGEFGFKRDGIFLYNESLTYYCTSYEEPS------TLFEIWVMDYDDGFKSSWTKHLTVGPFKDM-EFPLTPWKRDELLMIASDGRAASYNSCTGNFKYLHIPVIINENRVVD-----YVKSIVLVN----------270 IQLPSRRESGFRFYYIFMRNESLASFCSRYDRS-EDS---ESCEIWVMDDYDGIKRSWTKLLTIGPLQGI-KKPLTFWKSDELLMLDSDGRATSYNSSTGNLNYIHIPPILN--RVVDFEVLIYVKSIVHVK----------278 IQLPSRRESGFKFCNIFLCNESIASFCCCYDPKNEDS---TLCEIWVMDDYGGVKSSWTKLVTVGPLKGINENPLAFWKSDELLMVSCDGSVTSYNSSTKNLSYLNIPPILN--EVRDFQAVIYVESIVSVK----------270 IQLPSRRESGFRFYYIFLRNESLASFCSRYDRS-EDS---ESCEIWVMDDYDGDKSSWTKLLNIGPLQGI-KKPLTFWRSDELLMLDSDGRATSYNYSTRNLKYLHIPPILN--RVVDFEVLIYVKSIVHVK----------269 IQLPSRRESGFTFDYIFLRNESLASFCSPYNPS-EDS---KLFEIWVMDDYDGVSSSWTKLLTVGPFKGV-EYPLTLWKCDELLMLASDGRATSYNSSTGNLKYLHIPPILN--KVVDFEGLIYVKSIVPLK----------275 IELPSRREFGFKFYGIFLYNESITSYCSRYEED------CKLFEIWVMDDYDGVKSSWTKLLAVGPFKDI-DYPLTLGKFDEVLMLGSYGRAAFCNSSTGNLKYLHIPPIIN--WMID-----YVKSIVPVK----------275 IDLPSRRESDFKFYGIFLYNESVTSYCYRHEED------CELFEIWVMDDYDGVKSSWTKLLTIGPLKDI-DYPLTLWKCDEVLMLGSYGRAASCNSSTGNLRYLHIPPIIK--WMMD-----YVKSIVPVQ----------........... . ... ... .. .. .
.
. .................... .. . . . ...... ..... ....................... .
. .
... ......

392
392
394
392
392
393
393
410
400
396
394
397
390
424
392
400
393
396
394
404
394
393
392
392

F-box

FBA1

Fig. 1 Amino acid sequence alignment of the MdSFBB/MdFBX
genes. Amino acid sequences of MdSFBB/MdFBX proteins werealigned using ClustalW. Conserved sites and relatively conservative
sites are marked with asterisks and dots, respectively. The 28th and123

70th asterisks of MdFBX4 are premature stop codons. A doubleheaded arrow denotes the F-box region. FBA_1 (F-box associated)
motif detected by Pfam (http://pfam.sanger.ac.uk) was denoted by aline

Plant Mol Biol (2010) 74:143–154

147

82.7%
76.1%
73.8%

FBX9

FBX20

FBX12

(

70J19, 42J3, 6N9,
( 69A4,
10D16, 27P17, 21M5, 9P16

Non-S
-specific
FBX11

96.0%

94.7%

(*pseudogene)

FBX10

FBX13

FBX4

22C6,
89I13,( 33E2,( 9J18,
68N5
92P4, 20J13(

FBX6 FBX16

FBX5

FBX7

FBX18

92.9%

(

(45M19, 78B14, 34G16, 96N6, 14P21, 90A5)

FBX19

FBX14

78.7%
96.0%

MdSFBB9-β

FBX3( 22H23,( 27M4,
73B7
69O22(25N15)

98.0%

FBX17

(72L2, 52B7, 25J12)

(9L6)

FBX15

( 24C22,
21O2

(

FBX8

94.1%

S9
MdSFBB9-α S9-RNase

(

93.4%

(

(

95.7%

92.1%

86I6, 33J19,
34H13, 29J19(

FBX1 MdSFBB3-β S3-RNase MdSFBB3-α FBX2(

68F11, 11A21,
51O8

(

(

(92E20, 49F18, 72N11, 66L6)

68I12, 7L12,30J20, 56J20

(

S3

79.4%

Fig. 2 The BAC contigs for the S locus region of apple. Black andwhite boxes denote MdSFBB/MdFBX genes and S-RNase genes,
respectively. A gray box represents a pseudogene MdFBX4. Arrows
indicate closest relatives of the MdSFBB/MdFBX genes. Solid, dashedand dotted lines denote the amino acid identities C90%, 80.0–89.9%
and 70.0–79.9%, respectively. BAC clones that constitute the contigs
were in the parentheses. Relative order and orientation of BAC
contigs are not determined. Bars are not in scale

MdFBX15) amino acid sequence identity (Figs. 1, 2;
Supplemental Table 3). All the MdSFBB/FBXs contained
FBA_1 motif (F-box associated) in addition to the N-terminal F-box region. No intron was found in all the
MdSFBB/FBX genes.

(S3S9) 9 Megumi (S2S9) were analyzed by the CAPS/dCAPS
system to examine linkage between MdFBXs and S-RNase
genes. Figure 3 shows representative results of the linkage
analysis for the Kitaro (S3S9) 9 Morioka64 (S3S5) population. Analysis of the S-RNase gene of the two populations
showed that genotypes of Kitaro (S3S9) 9 Morioka64 (S3S5)
were segregated as S3S5:S5S9 = 92:83, while Hatsuaki(S3S9) 9 Megumi (S2S9) resulted in S2S3:S2S9 = 30:34.
CAPS/dCAPS analysis of MdFBXs showed that all
S3-associated genes (MdFBX1,2,3,5,7,8,9,14,18 and 20)
were completely linked to S3-RNase in a total of 239 plants.,
i.e., the S3-associated MdFBX genes were detected in 92
S3S5 plants and 30 S2S3 plants but not in other plants with the
S5S9 or S2S9 genotype. Similarly, complete linkage between
S9-associated genes (MdFBX4,6,10,12,13,15,16,17 and 19)
and S9-RNase was observed in the 239 plants.
Because MdFBX11 was monomorphic and cannot be
subjected to genetic analysis, linkage of MdFBX11 to the
S locus was examined by FISH analysis. First, two BAC
clones derived from the S locus, BAC 45M19 containing
SFBB9a and BAC 34G16 containing S9-RNase (Sassa et al.
2007), were used as FISH probes to examine the chromosomal location of the S locus. 45M19 was clearly detectedin the subtelomeric region of a chromosome and overlappedwith 34G16 (Fig. 4a). In addition, the signals of 34G16
were observed at centromeric regions of each chromosome,probably because 34G16 contains repetitive sequence(s)that is/are abundant in the pericentromeric region. Thisfinding suggests that the S locus is not localized at the
pericentromeric region. Next, the chromosomal position ofmonomorphic MdFBX11 was analyzed. BAC 45M19 as theS haplotype-specific sequence polymorphismsof MdFBXs
Since the SFBB/FBX genes are similar with each other (Sassa
et al. 2007; Supplemental Table 3), cleaved amplified
polymorphic sequence (CAPS) and derived cleaved amplified polymorphic sequence (dCAPS) procedures were used
to determine whether the newly identified MdFBXs show
S haplotype-specific polymorphisms. CAPS and dCAPS
analyses were conducted using different S genotypes andshowed that all twenty genes were correlated with S haplotypes except for MdFBX11. Ten genes (MdFBX1,2,3,5,7,
8,9,14,18 and 20) were associated with the S3 haplotype,while nine genes (MdFBX4,6,10,12,13,15,16,17 and 19)
were specific to the S9 haplotype (Supplemental Fig. 1;
Fig. 2). S haplotype-specific sequence motif was not detected in those F-box genes. The sequences of MdFBX11
derived from two genotypes (S2S5 and S3S9) were completely
identical (data not shown), showing that MdFBX11 is amonomorphic gene.
MdFBX genes are linked to S-RNases
Two segregating populations derived from a cross between Kitaro (S3S9) 9 Morioka64 (S3S5) and Hatsuaki

123

148
Fig. 3 Linkage analysis of the
MdFBXs and the S-RNases ofapple. 239 progenies from
Kitaro (S3S9) 9 Morioka64(S3S5) and Hatsuaki(S3S9) 9 Megumi (S2S9) wereanalyzed using CAPS.
Representative results of Kitaro(S3S9) 9 Morioka64 (S3S5)
population were shown

Plant Mol Biol (2010) 74:143–154

(bp)
1659
899
408
420

S5-RNase
S3-RNase
S9-RNase
RsaI

MdFBX1 (S3)
AluI

430

MdFBX2 (S3)HphI + BfaI

341
252
582

MdFBX4 (S9)MdFBX3 (S3)
BbvI

MdFBX5 (S3)

Tru1I + PdmI
388
350

MdFBX6 (S9)SFBB3β (S3)
VspI

386

MdFBX7 (S3)
BstBI

628
843
678

MdFBX8 (S3)MspI

MdFBX16 (S9)MdFBX9 (S3)
SspI

265

MdFBX10 (S9)
RsaI

625

444
331

MdFBX12 (S9)TaqI

MdFBX13 (S9)
NspI

525
281
279

966

MdFBX14 (S3)
BstSNIMdFBX15 (S9)Tsp45IMdFBX17 (S9)HaeΙΙΙMdFBX18 (S3)TruI + XbaI415

MdFBX19 (S9)
RsaI

160

S locus probe and BAC 9L6 as the MdFBX11 probe were
hybridized to chromosomal DNA. FISH showed that
MdFBX11 was located near the S locus (Fig. 4b). The FISH
result was confirmed to be reproducible in repeated experiments (n = [50).

123

MdFBX20 (S3)MdFBX genes are specifically expressed in pollen
To examine the organ-specific expression of MdFBX
genes, RT-PCR followed by CAPS analysis (RT-PCR/
CAPS) was conducted. For MdFBX17, restriction digestion

Plant Mol Biol (2010) 74:143–154

(A)

149

(B)

Discussion
Many SFBB-class F-box genes are clusteredin the S locus region in apple

Fig. 4 FISH mapping of BAC clones including S9-RNase,
MdSFBB9-a and MdFBX11 on chromosomal DNA. Bars = 10 lm.
Arrows indicate the S locus region. a BAC 45M19 containing SFBB9a(red; Alexa fluor 568) was located in a subtelomeric region of achromosome and overlapped with 34G16 containing S9-RNase(green; FITC). b The green signal (FITC) of BAC 9L6 containing
MdFBX11 was located near the red signal (Alexa fluor 568) of 45M19of the RT-PCR product was omitted because it was foundto be specifically amplified by the RT-PCR condition. The
monomorphic gene MdFBX11 was also analyzed by
RT-PCR. Results showed that all the MdFBX genes, except
for the pseudogene MdFBX4, were specifically expressedin pollen (Fig. 5). Expression of the pseudogene MdFBX4
was not observed in any organs analyzed.
Phylogenetic analysis of MdFBXs
The full length amino acid sequences of the S locus linked
F-box proteins of Rosaceae, Solanaceae and Plantaginaceae were aligned and used to construct a phylogenetic treeusing the neighbor-joining method with Arabidopsis
thaliana S locus SLF-related F-box (NP_180975) as an
outgroup (Fig. 6). SFBBs/FBXs of Maloideae formed awell-supported (bootstrap value = 100%) compact clade.
In Prunus, F-box proteins formed three distinct clades;
SFBs, SLFL2, and SLFL1 and 3. SFBBs/FBXs were moresimilar to probable non-S F-box proteins SLFL1 and 3 ofPrunus than to Prunus pollen S proteins SFBs as previously
suggested (Matsumoto et al. 2008; Sassa et al. 2007).
Topology of maximum parsimony tree was similar to thatof neighbor-joining tree (Supplemental Fig. 2).
For most, if not all, SFBBs/FBXs, closest relatives wereof different haplotypes, e.g., MdFBX9 (S3) was closest to
MdFBX16 (S9) (Fig. 6; Supplemental Table 3). Relative
position was, however, not same in some closely related
gene pairs (Fig. 2). For example, two F-box genes SFBB3-aand SFBB3-b are proximately localized to the S3-RNase,
while their closest relatives, FBX19 and FBX6, respectively,
are not located beside the S9-RNase. Similarly, SFBB9-b is
proximately localized to the S9-RNase, while its closest
relative, FBX2, is not located in the S3-RNase-containing
BAC contig.

In different species of Rosaceae, Solanaceae and Plantaginaceae, the S locus region contains several F-box genes,
the pollen S genes SLF/SFBs, and other (probable) non-S
F-box genes SLFLs (Lai et al. 2002; Entani et al. 2003;
Ushijima et al. 2003; Yamane et al. 2003; Zhou et al. 2003;
Wang et al. 2004; Tsukamoto et al. 2005). Pollen S F-box
genes are less homologous to non-S F-box genes, and are
single copy in S haplotypes. In contrast, a unique feature of
the pollen S candidate SFBB of Maloideae, apple and pear,is that related and multiple genes are located at the S locus.
Two and three SFBBs were identified in each S haplotype
of apple and Japanese pear, respectively (Sassa et al. 2007).
In this study, we conducted a comprehensive analysis of
the SFBB genes of apple and showed that the SFBB family
is larger than initially thought. We identified twenty additional SFBB-like genes/alleles that were provisionallynamed MdFBXs, and showed that all of them are linked to
the S locus. Ten and nine MdFBXs were genetically linked
to the S3- and S9-RNase, respectively. FISH analysis
showed physical linkage of monomorphic MdFBX11 to the
S locus. Although the possibility that several S locus-linked
SFBB/FBX genes remain to be identified can not be
excluded, because we didn’t determine complete sequencesof the isolated BAC clones, this study revealed that the
S locus region of apple represents the largest F-box genecluster known, composed of more than ten highly homologous members.
CAPS/dCAPS and sequence comparison analyses
revealed that all SFBB/FBX genes showed S haplotypespecific polymorphisms, except for MdFBX 11. A monomorphic F-box gene that is highly homologous to pollen
S was also found in Petunia and named PaF1, althoughlinkage of PaF1 to the S locus is unclear (Tsukamoto et al.
2005). Phylogenetic analysis revealed that SFBB/FBX
proteins didn’t form S haplotype-specific groups, and,instead, closest relatives were from different S haplotypes
for most, if not all, proteins. This finding suggests that eachclosely related gene pair shares a common ancestor, andproliferation of the SFBB/FBX genes at the S locus predates
diversification of the S haplotypes. Relative order of thegenes was not conserved in some closely related gene pairs,e.g., for two F-box genes that are closely localized to the
S3-RNase, SFBB3-a and SFBB3-b, their closest relatives,
FBX19 and FBX6, respectively, are not located corresponding position of the S9 haplotype, but are locatedoutside the BAC contig containing the S9-RNase. In addition, highly similar partner (C90% amino acid identity)
was not detected in some FBX genes, suggesting that order

123

150
Fig. 5 RT-PCR/CAPS analysisof the expression of MdFBXs indifferent organs. MdFBX17 was
specifically amplified by the
RT-PCR condition and thus the
restriction digestion step wasomitted. Monomorphic
MdFBX11 was also analyzed by
RT-PCR. Lf leaf, Pd pedicel, Sp
sepal, Pt petal, Pg pollen grain,
Ov ovary, St style, gDNA
genomic DNA(S3S9)

Plant Mol Biol (2010) 74:143–154
RT-PCR

(bp)

+RT

-RT

Lf Pd Sp Pt Pg Ov St

Lf Pd Sp Pt Pg Ov St

CAPS
DNA

(bp)

Pg

DNA

420

599

RsaI

MdFBX1 (S3)
AluI

590
430

MdFBX2 (S3)HphI + BfaI

547

582

341
252
582

MdFBX4 (S9)MdFBX3 (S3)
BbvI

MdFBX5 (S3)
Tru1I + PdmI

570

388
350

MdFBX6 (S9)SFBB3β
VspI

602
386

MdFBX7 (S3)
BstBI

853

628

843

843
678

265

265

838

625

1023

444
331

MdFBX8 (S3)MspI

MdFBX16 (S9)MdFBX9 (S3)SspI

MdFBX10 (S9)
RsaI

595

MdFBX12 (S9)TaqI

MdFBX13 (S9)
NspI

525

MdFBX14 (S3)
BstSNI

281

281

966

966

825

415

MdFBX15 (S9)HaeΙΙΙMdFBX18 (S3)TruI + XbaIMdFBX19 (S9)

RsaI

188
160

532

MdFBX11279

MdFBX17 (S9)MdFBX20 (S3)ef1-α

and repertoire of SFBB/FBX genes were rearranged during
the course of evolution. The relationship between themolecular mechanism of the GSI of apple and selective

123pressure that favored proliferation of the SFBB/FBX genes
at the S locus region would be an intriguing issue to beaddressed.

Plant Mol Biol (2010) 74:143–154

55

96

0.1

PiSLF3PiSLF1
Pa17
88
PiSLF2100
Pa19
97
97 PhSLFv
PaF1
NaDD3
PiSLFL1c
98
NaDD4
57
NaDD1
NaDD2
57
PiSLF3A134
100
NaDD10
NaDD9
NaDD8
100
NaDD5
99
NaDD6
PiSLFL2b
99
PiSLFL2d
NaDD7AhSLF2CAhSLF4
51
100
AhSLF5AhSLF2
100
AmSLFLAhSLF1E
95 MdFBX14
PpSFBB4-beta
100
PpSFBB5-beta
MdFBX9100 MdFBX16
MdFBX3100 MdFBX7
MdFBX17
65
MdFBX12100 MdFBX20
MdFBX11
MdFBX13
99
MdFBX676
MdSFBB3-beta
MdFBX1972
100 MdSFBB3-alpha99
MdFBX1
MdSFBB9-alpha97 MdFBX2
100 91
MdSFBB9-beta
59 97 MdFBX5
MdFBX15
99 PpSFBB4-alpha57 PpSFBB5-alpha
MdFBX8
MdFBX18
85
MdFBX10100
PpSFBB6-gamma
61 PpSFBB9-gamma
PpSFBB8-gamma
97 PpSFBB4-gamma
PpSFBBk-gamma
PpSFBB1-gamma
90
PpSFBB2-gamma
PpSFBB7-gamma
PpSFBB3-gamma
PpSFBB5-gamma
77 PmSLFL3-S7
PmSLFL3-Sf
100
PavSLFL3-S4
PavSLFL1-S1
100
PavSLFL1-S2
100
PmSLFL1-Sf
90 PavSLFL1-S4
50 PavSLFL1-S6
PavSLFL2-S4
PmSLFL2-S7
100
93 PmSLFL2-S1
66 PmSLFL2-Sf
PdSFBaParSFBc
100
PavSFB571
PsSFBcPmSLF7
100 PdSFB1
PdSFBdPmSFB1
PavSFB2
100 PcSFB9
PavSFB9PmSFB3
PdSFBf
52
PavSFB6
AT2G34280

151b Fig. 6 Neighbor-joining tree of the S locus-linked F-box proteins.
Abbreviations for the F-box proteins are Md, apple (Malus 9 domestica); Pp, Japanese pear (Pyrus pyrifolia); Pd, almond (Prunus dulcis);
Pm, Japanese apricot (Prunus mume); Pav, sweet cherry (Prunus
avium); Pc, sour cherry (Prunus cerasus); Ps, Japanese plum (Prunus
salicina); Par, apricot (Prunus armeniaca); Pi, Petunia inflata; Ph,
Petunia hybrida; Pa, Petunia axillaris; Na, Nicotiana alata; Ah,
Solanaceae
Antirrhinum hispanicum; Am, Antirrhinum majus; At, Arabidopsis
thaliana. Numbers in the phylogenetic tree indicate the percentage of
100 bootstrap replicates in which a group was found (values \50%
not shown). MdSFBBs/FBXs from the S3 haplotype are boxed while
MdSFBBs/FBXs from the S9 haplotype are shaded in black. The
alignment used to generate the phylogenetic tree is shown in
Supplemental Fig. 3. Accession numbers: MdSFBB3-alpha(AB270795), MdSFBB3-beta(AB270796), MdSFBB9-alpha(AB270793),
MdSFBB9-beta(AB270794), PpSFBB1-gamma(AB297933), PpSFB
Plantaginaceae
B2-gamma(AB297934), PpSFBB3-gamma(AB297935), PpSFBB4alpha(AB270797), PpSFBB4-beta(AB270798), PpSFBB4-gamma(AB270799), PpSFBB5-alpha(AB270800), PpSFBB5-beta(AB270801), PpSFBB5-gamma(AB270802), PpSFBB6-gamma(AB297936),
PpSFBB7-gamma(AB297937), PpSFBB8-gamma(AB297938), PpSF
BB9-gamma(AB297939), PpSFBBk-gamma(AB297940), PdSFB1(FJ514938), PdSFBa(AB092966), PdSFBd(AB081648), PdSFBf(AB361036), PcSFB9(DQ827717), PsSFBc(DQ849084), ParSFBc(DQ422946),
PavSFB2(AB111519),
PavSFB5(AB111520),
PavSFB6(AB096858), PavSFB9(DQ422809), PavSLFL1-S1(AB360339), PavSLFL1-S2(AB360340), PavSLFL1-S4(AB280953), PavSLFL1-S6(AB360342), PavSLFL2-S4(AB280954), PavSLFL3S4(AB280955), PmSFB1(AB101440), PmSFB3(AB376968), PmSLF7(AB092622), PmSLFL1-Sf(AB280956), PmSLFL2-S1(AB092625), PmSLFL2-S7(AB092626), PmSLFL2-Sf(AB280957), PmSL
FL3-S7(AB092627), PmSLFL3-Sf(AB280958), PhSLFv(FN429077),
PiSLF1(AY500390), PiSLF2(AY500391), PiSLF3(AY500392), PiSMaloideae
LF3A134(AY363975), PiSLFL1c(EF614191), PiSLFL2b(EF614188), PiSLFL2d(EF614187), Pa17(AY766153), Pa19(AY766154),
PaF1(AY766155), NaDD1(EF420251), NaDD2(EF420252), NaDD3(EF420253), NaDD4(EF420254), NaDD5(EF420255), NaDD6(EF420256), NaDD7(EF420257), NaDD8(EF420258), NaDD9(EF420259), NaDD10(EF420260), AhSLF1E(AJ515535), AhSLF2(CAC33022),
AhSLF2C(DQ462204),
AhSLF4(AJ515534),
AhSLF5(AJ515536), AmSLFL(DQ862124)
Rosaceae

Multiple, related, polymorphic and pollen-specific
SFBB/FBX genes and the GSI mechanism

Prunoideae

Sequence analyses have revealed that the S loci of Prunus,
Petunia, and Antirrhinum contain several F-box genes in
addition to the pollen determinant SFB/SLF (Lai et al.
2002; Entani et al. 2003; Ushijima et al. 2003; Yamane
et al. 2003; Zhou et al. 2003; Wang et al. 2004; Tsukamoto
et al. 2005). However, those non-S F-box genes, SLFLs, areless homologous to the pollen S genes SLF/SFBs and someof them are also expressed in other organs. Hua et al.
(2007) showed that SLFLs can not substitute the pollen
S function of SLF in transgenic Petunia. Although a recent
study on Nicotiana identified many S locus-linked SLF-likegenes named DDs, the DD group is composed of highly
divergent genes and some of them are located outside thefunctional S locus, expressed in non-S specific manner,

123

152

and/or expressed not only in pollen but also in other organs(Wheeler and Newbigin 2007), suggesting that many of theDDs correspond to SLFL genes of Nicotiana. In contrast, aunique feature of the S locus of Maloideae is that it contains related and multiple pollen S candidates, SFBB/FBX
genes. The feature of SFBB/FBXs might be related to theprobable difference in pollen S function between two
subfamilies of Rosaceae, Maloideae (Malus and Pyrus) andPrunoideae (Prunus).
Loss of SI in heteroallelic pollen has been known as theresult of competitive interaction of pollen S alleles in
Maloideae (Adachi et al. 2009; Crane and Lewis 1941;
Lewis and Modlibowska 1942), and also in Solanaceae andPlantaginaceae (de Nettancourt 2001; Xue et al. 2009),
while genetic studies suggested that it doesn’t occur in
Prunus (Hauck et al. 2006b; Yamane and Tao 2009).
Furthermore, pollen S is considered to be essential forpollen tube growth in Nicotiana (Solanaceae; Golz et al.
2001). Deletion-type pollen-part SC mutant is also not
known in Maloideae. In contrast, in Prunus, the mutation,even complete deletion, of SFB is not lethal to pollen, but,instead, confers pollen-part SC (Hauck et al. 2006a; Sonneveld et al. 2005; Tsukamoto et al. 2006; Ushijima et al.
2004). The difference of pollen S function between Prunoideae and Maloideae is consistent with the difference inthe features of the pollen S (candidate) of them, i.e., theSFB of Prunoideae is single copy in each S haplotype,while the SFBB/FBXs of Maloideae are multiple, and morehomologous to SLFL than to SFB of Prunoideae (Sassa
et al. 2010).
Given that the SFBB/FBX genes show S haplotypespecific polymorphisms, linkage to the S locus and pollenspecific expression, they are candidates for pollen S of
Maloideae. In the 317 kb S locus region of apple, no othergood candidate was identified (Sassa et al. 2007). However,it seems unlikely that all the SFBB/FBX genes, more thanten in an S haplotype, are involved in pollen S specificity.
The tentative nomenclature of SFBB/FBX genes should be
revised in future based on the function of each member.
It has been postulated that pollen S protein degrades orinhibits the non-self S-RNases, possibly through ubiquitination, with leaving self S-RNase unaffected (Hua et al.
2008). In this scenario, heteroallelic pollen loses SI,
because both two S-RNases are non-self for one of twopollen S proteins and thus are degraded/inhibited. If one orsome of the SFBB/FBX genes is/are pollen S determinant(s), some mechanism may differentiate pollen S andnon-S SFBB/FBX genes. Without such mechanism, nonS SFBB/FBX are likely to degrade/inhibit S-RNases irrespective of their S haplotypes, leading to SC. Pollen
S SFBB/FBX proteins only are possible to be accumulated
in pollen. It is also possible that pollen S SFBB/FBX but
non-S SFBB/FBX can interact with S-RNase, although we

123

Plant Mol Biol (2010) 74:143–154

detected no sequence motif that characterizes probablepollen S proteins among the SFBB/FBX.
Among the three families with the S-RNase based-GSI,
Rosaceae is distantly related to the other two families
Solanaceae and Plantaginaceae; the former belongs to
subclass Rosidae while the latter two are in Asteridae. In
addition, in Rosaceae, pollen-part SI function seems todiffer among taxa, Maloideae and Prunoideae. Functional
characterization of pollen S genes of Maloideae and comparisons to those of other species will provide insight into
the evolutionary history and divergence of the S-RNase
based-GSI.
Chromosomal location of the apple S locus
In this study, we physically mapped the apple S locus to thesubtelomeric region of a chromosome using FISH. This isconsistent with previous genetic linkage analyses thatmapped the S locus to the bottom of linkage group 17 ofapple (Igarashi et al. 2008; Maliepaard et al. 1998). FISH
analysis also revealed that the apple S locus is not locatednear the centromeric region. Suppression of recombination
at the S locus region is required to maintain the pair of thepistil- S and pollen-S alleles, the S haplotype, for SI
function (de Nettancourt 2001; Franklin-Tong 2008). In
Solanaceae, pericentromeric localization of the S locus has
been considered to be an important factor in the suppression of recombination (Entani et al. 1999), while in Brassica and Ipomoea, the S loci were mapped to thesubtelomeric regions and were not close to the centromeres(Iwano et al. 1998; Suzuki et al. 2004). In Brassica andIpomoea, sequence heterogeneity among S haplotypes has
been implicated for the suppression of recombination(Casselman et al. 2000; Iwano et al. 1998; Rahman et al.
2007; Suzuki et al. 2004). Given that the distance between
S-RNase and SFB of Prunus is 380–40 kb (Ikeda et al.
2005), if one or more of the newly identified MdFBXs is
involved in pollen S specificity, the physical size of theapple S locus would be remarkably larger than that ofPrunus, because the MdFBXs are located outside the previously analyzed 317 kb S locus BAC contig (Sassa et al.
2007). A large-scale comparative sequence analysis ofdifferent apple S haplotypes would be useful to estimate theextent of the physical size and elucidate mechanism ofrecombination suppression of the S locus.
Acknowledgments We thank Dr. K. Abe of National Institute ofFruit Tree Science for plant materials, Dr. K. Ushijima of Okayama
University for comments, and M. Miyamoto for technical help. This
work was supported by the Grants-in-Aid for Scientific Research (B,20380003) from the Ministry of Education, Science, Sports andCulture of Japan. H.K and S.W. were supported by JSPS Research
Fellowship for Young Scientists and JSPS Postdoctoral Fellowshipfor Foreign Researchers, respectively.

Plant Mol Biol (2010) 74:143–154

References
Adachi Y, Komori S, Hoshikawa Y, Tanaka N, Abe K, Bessho H,
Watanabe M, Suzuki A (2009) Characteristics of fruiting andpollen tube growth of apple autotetraploid cultivars showing
self-compatibility. J Jpn Soc Hort Sci 78:402–409
Anderson MA, Cornish EC, Mau S-L, Williams EG, Hoggart R,
Atkinson A, Bonig I, Grego B, Simpson R, Roche PJ, Haley JD,
Penschow JD, Niall HD, Tregear GW, Coghlan JP, Crawford RJ,
Clarke AE (1986) Cloning of cDNA for a stylar glycoprotein
associated with expression of self-incompatibility in Nicotiana
alata. Nature 321:38–44
Casselman AL, Vrebalov J, Conner JA, Singhal A, Giovannoni J,
Nasrallah ME, Nasrallah JB (2000) Determining the physicallimits of the Brassica S locus by recombinational analysis. Plant
Cell 12:23–33
Crane MB, Lewis D (1941) Genetical studies in pears. III. Incompatibility and sterility. J Genet 43:31–44
de Nettancourt D (2001) Incompatibility and incongruity in wild andcultivated plants. Springer, Berlin
Entani T, Iwano M, Shiba H, Takayama S, Fukui K, Isogai A (1999)
Centromeric localization of an S-RNase gene in Petunia hybrida
Vilm. Theor Appl Genet 99:391–397
Entani T, Iwano M, Shiba H, Che F-S, Isogai A, Takayama S (2003)
Comparative analysis of the self-incompatibility (S-) locus
region of Prunus mume: identification of a pollen expressed
F-box gene with allelic diversity. Genes Cells 8:203–213
Franklin-Tong VE (2008) Self-incompatibility in flowering plants—
evolution, diversity, and mechanisms. Springer, Berlin
Goldraij A, Kondo K, Lee CB, Hancock CN, Sivaguru M, VazquezSantana S, Kim S, Phillips TE, Cruz-Garcia F, McClure B (2006)
Compartmentalization of S-RNase and HT-B degradation in selfincompatible Nicotiana. Nature 439:805–810
Golz JF, Su V, Oh H-Y, Kusaba M, Newbigin E (2001) Genetic
analysis of Nicotiana pollen-part mutants is consistent with thepresence of an S-ribonuclease inhibitor at the S locus. Proc Natl
Acad Sci USA 98:15372–15376
Hauck NR, Ikeda K, Tao R, Iezzoni AF (2006a) The mutatedS1-haplotype in sour cherry has an altered S haplotype-specific
F-box protein gene. J Hered 97:514–520
Hauck NR, Yamane H, Tao R, Iezzoni AF (2006b) Accumulation of
nonfunctional S-haplotypes results in the breakdown of gametophytic self-incompatibility in tetraploid Prunus. Genetics
172:1191–1198
Hua Z, Kao T-H (2006) Identification and characterization ofcomponents of a putative Petunia S-locus F-box-containing E3ligase complex involved in S-RNase-based self-incompatibility.
Plant Cell 18:2531–2553
Hua Z, Meng X, Kao T-H (2007) Comparison of Petunia inflata
S-Locus F-box protein (Pi SLF) with Pi SLF like proteins reveals
its unique function in S-RNase based self-incompatibility. Plant
Cell 19:3593–3609
Hua Z-H, Fields A, Kao T-H (2008) Biochemical models for
S-RNase-based self-incompatibility. Mol Plant 1:575–585
Igarashi M, Abe Y, Hatsuyama Y, Ueda T, Fukasawa-Akada T,
Kon T, Kudo T, Sato T, Suzuki M (2008) Linkage maps of theapple (Malus 9 domestica Borkh.) cvs ‘Ralls Janet’ and‘Delicious’ include newly developed EST markers. Mol Breed
22:95–118
Ikeda K, Ushijima K, Yamane H, Tao R, Hauck NR, Sebolt AM,
Iezzoni AF (2005) Linkage and physical distances between the
S-haplotype S-RNase and SFB genes in sweet cherry. Sex Plant
Reprod 17:289–296
Iwano M, Sakamoto K, Suzuki G, Watanabe M, Takayama S, Fukui
K, Hinata K, Isogai A (1998) Visualization of a self-

153
incompatibility gene in Brassica campestris L. by multicolor
FISH. Theor Appl Genet 96:751–757
Kao T-H, Tsukamoto T (2004) Molecular and genetic bases of
S-RNase-based self-incompatibility. Plant Cell 16:S72–S83
Kim H, Kakui H, Kotoda N, Hirata Y, Koba T, Sassa H (2009)
Determination of partial genomic sequences and development ofa CAPS system of the S-RNase gene for the identification of 22
S haplotypes of apple (Malus 9 domestica Borkh.). Mol Breed
23:463–472
Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: a biologistcentric software for evolutionary analysis of DNA and protein
sequences. Brief Bioinform 9:299–306
Lai Z, Ma W, Han B, Liang L, Zhang Y, Hong G, Xue Y (2002) An
F-box gene linked to the self-incompatibility (S) locus of
Antirrhinum is expressed specifically in pollen and tapetum.
Plant Mol Biol 50:29–42
Lansari A, Iezzoni A (1990) A preliminary analysis of selfincompatibility in sour cherry. Hort Sci 25:1636–1638
Lewis D, Modlibowska I (1942) Genetical studies in pears IV. Pollentube growth and incompatibility. J Genet 43:211–222
Luu DT, Qin X, Morse D, Cappadocia M (2000) S-RNase uptake bycompatible pollen tubes in gametophytic self-incompatibility.
Nature 407:649–651
Maliepaard C, Alston FH, van Arkel G, Brown LM, Chevreau E,
Dunemann F, Evans KM, Gardiner S, Guilford P, van Heusden
AW, Janse J, Laurens F, Lynn JR, Manganaris AG, den Nijs
APM, Periam N, Rikkerink E, Roche P, Ryder C, Sansavini S,
Schmidt H, Tartarini S, Verhaegh JJ, Vrielink-van Ginkel M,
King GJ (1998) Aligning male and female linkage maps of apple(Malus pumila Mill.) using multi-allelic markers. Theor Appl
Genet 97:60–73
Matsumoto D, Yamane H, Tao R (2008) Characterization of SLFL1, a
pollen-expressed F-box gene located in the Prunus S locus. Sex
Plant Reprod 21:113–121
McClure BA, Haring V, Ebert PR, Anderson MA, Simpson RJ, Sakiyama
F, Clarke AE (1989) Style self-incompatibility gene products ofNicotiana alata are ribonucleases. Nature 342:955–957
McClure BA, Gray JE, Anderson MA, Clarke AE (1990) Selfincompatibility in Nicotiana alata involves degradation of pollen
rRNA. Nature 347:757–760
Neff MM, Neff JD, Chory J, Pepper AE (1998) dCAPS, a simple
technique for the genetic analysis of single nucleotide polymorphisms experimental applications in Arabidopsis thaliana genetics. Plant J 14:387–392
Qiao H, Wang F, Zhao L, Zhou J, Lai Z, Zhang Y, Robbins TP, Xue
Y (2004a) The F-box protein AhLF-S2 controls the pollen
function of S-RNase-based self-incompatibility. Plant Cell
16:2307–2322
Qiao H, Wang H, Zhao L, Zhou J, Huang J, Zhang Y, Xue Y (2004b)
The F-box protein AhSLF-S2 physically interacts with S-RNases
that may be inhibited by the ubiquitin/26S proteasome pathwayof protein degradation during compatible pollination in Antirrhinum. Plant Cell 16:582–595
Rahman MH, Tsuchiya T, Suwabe K, Kohori J, Tomita R, Kagaya Y,
Kobayashi I, Kakeda K, Kowyama Y (2007) Physical size of the
S locus region defined by genetic recombination and genome
sequencing in Ipomoea trifida, Convolvulaceae. Sex Plant
Reprod 20:63–72
Saitou N, Nei M (1987) The neighbor-joining method: a new method
for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Sassa H (2007) A technique to isolate DNA from woody andherbaceous plants by using a silica-based plasmid extraction
column. Anal Biochem 363:166–167
Sassa H, Nishio T, Kowyama Y, Hirano H, Koba T, Ikehashi H(1996) Self-incompatibility (S) alleles of the Rosaceae encode

123

154
members of a distinct class of the T2/S-ribonuclease superfamily. Mol Gen Genet 250:547–557
Sassa H, Hirano H, Nishino T, Koba T (1997) Style-specific selfcompatible mutation caused by deletion of the S-RNase gene inJapanese pear (Pyrus serotina). Plant J 12:223–227
Sassa H, Kakui H, Miyamoto M, Suzuki Y, Hanada T, Ushijima K,
Kusaba M, Hirano H, Koba T (2007) S locus F-box brothers:
multiple and pollen-specific F-box genes with S haplotypespecific polymorphisms in apple and Japanese pear. Genetics
175:1869–1881
Sassa H, Kakui H, Minamikawa M (2010) Pollen-expressed F-box
gene family and mechanism of S-RNase-based gametophytic
self-incompatibility (GSI) in Rosaceae. Sex Plant Reprod
23:39–43
Sijacic P, Wang X, Skirpan AL, Wang Y, Dowd PE, McCubbin AG,
Huang S, Kao T-H (2004) Identification of the pollen determinant of S-RNase-mediated self-incompatibility. Nature
429:302–305
Sonneveld T, Tobutt KR, Vaughan SP, Robbins T (2005) Loss ofpollen-S function in two self-compatible selections of Prunus
avium is associated with deletion/mutation of an S haplotypespecific F-box gene. Plant Cell 17:37–51
Suzuki G, Tanaka S, Yamamoto M, Tomita RN, Kowyama Y, Mukai
Y (2004) Visualization of the S-locus region in Ipomoea trifida:toward positional cloning of self-incompatibility genes. Chromosome Res 12:475–481
Tao R, Yamane H, Sassa H, Mori H, Gradziel TM, Dandekar AM,
Sugiura A (1997) Identification of stylar RNases associated withgametophytic self-incompatibility in almond (Prunus dulcis).
Plant Cell Physiol 38:304–311
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W:
improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucl Acids Res
22:4673–4680
Tsukamoto T, Ando T, Watanabe H, Marchesi E, Kao T-H (2005)
Duplication of the S-locus F-box gene is associated withbreakdown of pollen function in an S-haplotype identified in anatural population of self-incompatible Petunia axillaris. Plant
Mol Biol 57:141–153

123

Plant Mol Biol (2010) 74:143–154
Tsukamoto T, Hauck N, Tao R, Jiang N, Iezzoni A (2006) Molecularcharacterization of three non-functional S-haplotypes in sourcherry (Prunus cerasus). Plant Mol Biol 62:371–383
Ushijima K, Sassa H, Dandekar AM, Gradziel TM, Tao R, Hirano H(2003) Structural and transcriptional analysis of the self-incompatibility locus of almond: identification of a pollen-expressed Fbox gene with haplotype-specific polymorphism. Plant Cell
15:771–781
Ushijima K, Yamane H, Watari A, Kakehi E, Ikeda K, Hauck NR,
Iezzoni AF, Tao R (2004) The S haplotype-specific F-box
protein gene, SFB, is defective in self-compatible haplotypes of
Prunus avium and P. mume. Plant J 39:573–586
Vinatzer BA, Zhang H-B, Sansavini S (1998) Construction andcharacterization of a bacterial artificial chromosome library ofapple. Theor Appl Genet 97:1183–1190
Wang Y, Tsukamoto T, Yi K-W, Wang X, Huang S, McCubbin AG,
Kao TH (2004) Chromosome walking in the Petunia inflata selfincompatibility (S-) locus and gene identification in an 881-kb
contig containing S2-RNase. Plant Mol Biol 54:727–742
Wheeler D, Newbigin E (2007) Expression of 10 S-class SLF-likegenes in Nicotiana alata pollen and its implications forunderstanding the pollen factor of the S locus. Genetics
177:2171–2180
Xue Y, Carpenter R, Dickinson HG, Coen ES (1996) Origin of allelicdiversity in Antirrhinum S locus RNases. Plant Cell 8:805–814
Xue Y, Zhang Y, Yang Q, Li Q, Cheng Z, Dickinson HG (2009)
Genetic features of a pollen-part mutation suggest an inhibitoryrole for the Antirrhinum pollen self-incompatibility determinant.
Plant Mol Biol 70:499–509
Yamane H, Tao R (2009) Molecular basis of self-(in)compatibility
and current status of S-genotyping in rosaceous fruit trees. J Jpn
Soc Hort Sci 78:137–157
Yamane H, Ikeda K, Ushijima K, Sassa H, Tao R (2003) A pollenexpressed gene for a novel protein with an F-box motif that isvery tightly linked to a gene for S-RNase in two species ofcherry, Prunus cerasus and P. avium. Plant Cell Physiol
44:764–769
Zhou J, Wang F, Ma W, Zhang Y, Han B, Xue Y (2003) Structuraland transcriptional analysis of S-locus F-box genes in Antirrhinum. Sex Plant Reprod 16:165–177