Journal of Heredity 2006:97(5):514–520
doi:10.1093/jhered/esl029
Advance Access publication September 19, 2006
ª The American Genetic Association. 2006. All rights reserved.
For permissions, please email: [email protected].
The Mutated S1-Haplotype in Sour
Cherry Has an Altered S-Haplotype–
Specific F-Box Protein Gene
NATHANAEL R. HAUCK, KAZUO IKEDA, RYUTARO TAO,
AND
AMY F. IEZZONI
From the Department of Horticulture, Michigan State University, East Lansing, MI 48824 (Hauck and Iezzoni); and the
Laboratory of Pomology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan (Ikeda and Tao).
Address correspondence to A. F. Iezzoni at the address above, e-mail: [email protected].
Abstract
Gametophytic self-incompatibility (GSI) is an outcrossing mechanism in flowering plants that is genetically controlled by
2 separate genes located at the highly polymorphic S-locus, termed S-haplotype. This study characterizes a pollen part mutant
of the S1-haplotype present in sour cherry (Rosaceae, Prunus cerasus L.) that contributes to the loss of GSI. Inheritance of
S-haplotypes from reciprocal interspecific crosses between the self-compatible sour cherry cultivar Újfehértói Fürtös carrying
the mutated S1-haplotype (S1#S4 Sd Snull) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type
S1-haplotype revealed that the mutated S1-haplotype confers unilateral incompatibility with a functional pistil component and
a nonfunctional pollen component. The altered sour cherry S1-haplotype pollen part mutant, termed S1#, contains a 615-bp
Ds-like element within the S1-haplotype–specific F-box protein gene (SFB1 #). This insertion generates a premature in-frame
stop codon that would result in a putative truncated SFB1 containing only 75 of the 375 amino acids present in the wild-type
SFB1. S1# along with 2 other previously characterized Prunus S-haplotype mutants, Sf and S6m, illustrate that mobile element
insertion is an evolutionary force contributing to the breakdown of GSI.
Sour cherry (Prunus cerasus L.) is a segmental allotetraploid produced by hybridization of the diploid sweet cherry (Prunus
avium L.) and the tetraploid ground cherry (Prunus fruticosa
Pall.) (Olden and Nybom 1968; Beaver and Iezzoni 1993).
Like sweet cherry, sour cherry exhibits an S-RNase–based gametophytic self-incompatibility (GSI) system (Yamane et al.
2001). However, in contrast to sweet cherry where natural
types are self-incompatible (SI), sour cherry includes both
SI and self-compatible (SC) types (Lansari and Iezzoni 1990).
In sweet cherry, as in other diploid GSI systems, matching S-haplotypes in the pollen and style will result in an incompatible reaction, and the growth of this ‘‘self ’’-pollen
tube will be inhibited (de Nettancourt 2001). In sour cherry,
the genetic control of self-pollen recognition is more complicated because a pollen grain contains 2 S-haplotypes. Sour
cherry pollen is incompatible if 1 or 2 of the S-haplotypes in
the pollen matches an S-haplotype in the style (Hauck et al.
2006). In contrast, SC sour cherry pollen must contain 2 Shaplotypes that have lost pollen-S and/or pistil-S function,
termed nonfunctional S-haplotypes. Therefore, the
genotype-dependent loss of SI in sour cherry is due to the
accumulation of at least 2 nonfunctional S-haplotypes (Hauck
et al. 2006).
514
In sour cherry, 4 common sweet cherry S-haplotypes, S1,
S4, S6, and S9, have been identified and determined to retain
their SI function. In addition, 7 nonfunctional S-haplotypes
have been identified in sour cherry based on genetic inheritance studies (Hauck et al. 2006). This includes a nonfunctional S-haplotype, termed Snull, that is hypothesized to be a
deletion containing the S-haplotype because no restriction
fragment length polymorphism (RFLP) fragment associated
with Snull was visualized with either an S-RNase or an SFB
probe (Hauck et al. 2006). To date, the molecular changes
resulting in nonfunctional S-haplotypes have been reported
for 4 of the 6 other mutants, including the stylar part mutant,
S6m (Yamane et al. 2003; Tsukamoto et al. 2006). This mutant
has a 2.6-kbp insert approximately 800 bp upstream of
the S6-RNase. S6m was originally identified as a novel
S-haplotype as it exhibited a unique RFLP fragment that resulted from an altered HindIII cut site compared with the
wild-type S6-haplotype (Yamane et al. 2001).
The S-haplotype mutant described in this work was also
initially identified due to a unique RFLP fragment (Yamane
et al. 2001). In a survey of the S-RNases in sour cherry, only
one selection, Újfehértói Fürtös (UF), had a unique HindIII
S-RNase fragment of 9.6 kb (Yamane et al. 2001). This
Hauck et al. The Mutated S1-Haplotype in Sour Cherry
Table 1. Segregation of pollen-derived S-haplotypes in interspecific crosses between UF and sweet cherry
Segregation of paternal S-haplotypes
Parents (S-genotype)
a
UF (S1#S 4 Sd Snull ) EF (S3S4)
UF (S1#S 4 Sd Snull ) Regina (S1S3)
Population size
Observed ratio
Expected ratiob
v2 (P)
45
88
45:0 (S3:S4)
0:88 (S1:S3)
1:1
1:1
45.0 (,0.0001)
88.0 (,0.0001)
a
The S-haplotypes being tested are indicated in bold.
b
Observed ratios were tested for fit to the ratio expected if the shared S-haplotype is nonfunctional (1:1). If the shared S-haplotype were functional, it would not
be inherited from the paternal parent.
fragment was tentatively named the Se-haplotype, and the
UF S-haplotype phenotype was determined to be S4SdSe.
Segregation analysis subsequently determined that the
fourth S-haplotype in UF was Snull (Hauck et al. 2006). In
self-pollinated progeny of UF (S4SdSeSnull), the S4-haplotype
segregated 1:1 (presence:absence) as would be expected for
a fully functional S-haplotype. In contrast, both the Sd - and
Se-haplotypes were present in all the self-pollinated progeny,
indicating that the Sd - and Se-haplotypes have impaired
functions (Hauck et al. 2006).
UF is a SC selection from the SI sour cherry landrace variety Pándy (syn. Crisxana, Köröser) that is native to Hungary
and Romania (Iezzoni et al. 1990). The S-haplotype phenotype for Pándy was determined to be S1S4Sd, and the functionality of the S1- and S4-haplotypes were confirmed using
reciprocal crosses with sweet cherry (Rainier, S1S4) (Hauck
et al. 2002). The origin of UF as an SC selection from the
SI Pándy landrace, and the similar S-haplotype phenotypes
between these selections, led to the hypothesis that Se is a mutated nonfunctional S1-haplotype that contributes to the SC
of UF. Herein, we confirm this hypothesis and report that
UF has an altered S1-haplotype, termed S1#, in which the
S-haplotype–specific F-box protein gene (SFB) (Ushijima
et al. 2003; SLF, Entani et al. 2003) is nonfunctional due
to the insertion of a Ds-like element.
Materials and Methods
Plant Material and DNA Extraction
Three sour cherry cultivars, UF, Pándy 114 (P114), and
Montmorency (Mont; S-phenotype S6S13mSa) (Hauck et al.
2006; Tsukamoto et al. 2006), and 3 sweet cherry cultivars,
Emperor Francis (EF), Regina, and Summit, were used.
S-alleles have been previously reported for the sour cherry
cultivars (Yamane et al. 2001; Hauck et al. 2006) and sweet
cherry cultivars (Schmidt and Timmann 1997; Boskǒvić and
Tobutt 2001). The cultivars were grown at the Michigan
State University Experimental Station, Clarksville, MI. Triploid progenies were generated from fully fertile reciprocal
interspecific crosses between UF and Regina, EF, and Summit. Progeny genotyping was done using DNA extracted
from mature seed from which the testa was removed using the procedure of Hauck et al. (2006). DNA extraction
from leaves was done as previously described (Hauck et al.
2001).
S-RNase and SFB Genotyping
The S-RNase gene–specific primer set, Pru-C2 and PCE-R
(Tao et al. 1999; Yamane et al. 2001), was used for S-haplotype
determination of the progeny/seed from the interspecific
crosses. This primer pair can differentiate between most
S-RNase alleles based on polymorphisms in the length of
the second intron in the Prunus S-RNase. However, the S2RNase allele could not be reliably amplified using this primer
pair. Instead, the S2-allele–specific primer pair PaS2-Fnew/
PaS2-R (Sonneveld et al. 2003) was used. The SFB gene–
specific primer set, SFB-C1F and FB3R (Ushijima et al.
2003), was used to amplify all SFB alleles and for the initial
discovery of an insertion in SFB1 #: In addition, the SFB1 allele
was amplified with the allele-specific primer set PaSFB1-F
and PaSFB1-R (Ikeda et al. 2005). A newly designed primer
PcSFB1#-R (5#-GTTCGGTCCGGTTCTGAC-3#) was used
in conjunction with PaSFB1-F (Ikeda et al. 2005) for differentiating between the S1- and S1#-haplotypes. Polymerase chain
reaction (PCR) conditions were identical to Ikeda et al. (2005)
but with an annealing temperature of 62 C.
S-RNase and SFB Reverse Transcriptase–PCR
Total RNA was isolated from styles with stigma or from
pollen grains at the balloon stage of flower development
as described elsewhere (Tao et al. 1999). One microgram
of DNase (Roche Applied Science, Indianapolis, IN)-treated
RNA was used for first strand cDNA synthesis by SuperScript II reverse transcriptase (RT) (Invitrogen, Carlsbad,
CA) following the manufacturer’s protocol. For S1-RNase
expression, PaS1-F and PaS1-R (Sonneveld et al. 2001)
were used on stylar cDNA. For SFB1 and SFB1 # expression,
PaSFB1-F and PcSFB1#-R were used on pollen cDNA. Expression of the actin gene was used as a reference with the
primers ActF1 and ActR1 (Yamane et al. 2003). PCR conditions were identical to those described by Yamane et al.
(2003).
Genomic Library Construction and Screening
DNA from UF and P114 was used to create fosmid libraries
using the CopyControl Fosmid Library Production Kit (Epicentre Technologies, Madison, WI). Clones containing either
SFB1 # or SFB1 were obtained using a digoxigenin–labeled
SFB1 probe as previously described (Ushijima et al. 2001).
515
Journal of Heredity 2006:97(5)
Table 2. Punnett square indicating the number of progeny that resulted from each gamete combination between Summit or Regina
and UF. All 4 gamete types from UF resulted from homologous pairing (S1#S4/SdSnull)
#
Summit (S1S2)
or
Regina (S1S3)
Expected ratio
v2
P
UF
Expected
S4Snull
S1#Sd
S1#Snull
S4Sd
Total
Ratio
v2
P
S1
2
5
4
3
14
1:1
0.360
0.5485
S2/S3
5
3
1
2
11
Total
7
8
5
5
25
$
1:1:1:1
1.08
0.7819
Plasmid DNA was prepared using Wizard Plus Minipreps
DNA Purification Kit (Promega, Madison, WI).
SFB1 and SFB1# Sequencing
Two micrograms of plasmid DNA was used as templates to
sequence the SFB1 # and SFB1 by primer walking using the primers SFB-C1F, SFB-C2R, SFB-C5F, and FB3R (Ushijima et al.
2003). In addition, PcSFB1#-R was used to obtain the sequence
of the 5# end of the SFB1 # gene. Sequencing was done by
the Michigan State University Research Technology Support
Facility using an ABI PRISM 3100 Genetic Analyzer.
The S1 #-haplotype was also present in a progeny from
the cross UF Mont. The S-genotype of this individual
was S1 #S4 S6 Sa , and the individual was determined to be SC
(Hauck et al. 2006). Because S4 and S6 are fully functional
S-haplotypes, both Sa and S1 # would need to be nonfunctional
for this individual to be SC.
To determine the molecular basis of the nonfunctional
SFB1 #, 2 fosmid clones each from UF and P114 that contained the S1-RNase were identified. PCR analysis using S-RNase primers amplified an S1-RNase fragment of identical
size from the fosmid clones, UF and P114 (Figure 1A).
As expected, S-RNase fragments representing the S4- and
Sd -haplotypes were also amplified from UF and P114 (Hauck
Results
The ability of UF (S1 #S4 Sd Snull ) styles to arrest S4 and S1 pollen
was tested by pollinating UF with pollen from the sweet
cherry cultivars EF (S3S4) and Regina (S1S3). All the progenies
from these 2 crosses contained the S3-haplotype (Table 1).
This indicates that EF and Regina S3-pollen were compatible
in the UF styles, whereas Regina S1-pollen and the EF S4pollen were selectively arrested by the presence of functional
S1- and S4-RNases. Therefore, the UF S1- and S4-haplotypes
exhibit functional stylar-S components. Because the UF styles
were able to carry out this SI reaction in an allele-specific
manner, the genetic changes causing self-compatibility in
UF must disrupt specific GSI interactions through disruption
of pollen-S function.
Previously we demonstrated that UF has a functional S4SFB as UF S4-containing pollen was unsuccessful when
placed on EF (S3S4) pistils (Hauck et al. 2006). We tested
the compatibility of S1#-containing UF (S1 #S4 Sd Snull ) pollen
by placing UF pollen on pistils of Summit (S1S2) and Regina
(S1S3). The presence of progeny resulting from the UF gametes S1# Sd and S1 #Snull indicates that the UF S1#-haplotype has
a nonfunctional pollen component (Table 2). If the S1 pollen
component had been functional, S1Sd and S1Snull pollen would
have been incompatible because the match of one-functional
S-haplotype between the pollen and style results in incompatibility in Prunus (Hauck et al. 2006). This mutated SFB1
S-haplotype is termed S1# according to the Prunus nomenclature for a pollen part mutant (Lewis and Crowe 1954).
516
Figure 1. PCR amplification of S-RNase and SFB alleles.
(A) The S1-RNase amplification products are identical in size
using the Pru-C2/PCE-R primer set. (B) The SFB1 # is
approximately 600 bp longer than the SFB1 using primers
SFB-C1F/FB3R.
Hauck et al. The Mutated S1-Haplotype in Sour Cherry
SFB1'
SFB1
ATGGCATTTACAATACGTAAGAAAGAAATCTTAATCGACATCCTGGTGAGACTACCTACA
ATGGCATTTACAATACGTAAGAAAGAAATCTTAATCGACATCCTGGTGAGACTACCTACA
************************************************************
60
60
SFB1'
SFB1
AAGTCCCTCGTTCGATTTCTGTGTACATGCAAGTCATGGAGTGATTTTATTGGCAGCTCG
AAGTCCCTCGTTCGATTTCTGTGTACATGCAAGTCATGGAGTGATTTTATTGGCAGCTCG
************************************************************
120
120
SFB1'
SFB1
AGTTTTGTTAGCACACACCTTGATAGGAATGTCACAAAACATGCCCATGTCTATCTACTC
AGTTTTGTTAGCACACACCTTGATAGGAATGTCACAAAACATGCCCATGTCTATCTACTC
************************************************************
180
180
SFB1'
SFB1
TGCCTCCACCACCCAAATTTTGAATGTCACGTCGACCCTGATGACTAGGGGTGGGCATCG
TGCCTCCACCACCCAAATTTTGAATGTCACGTCGACCCTGATGAC--------------*********************************************
240
225
SFB1'
SFB1
GGACCGGAAAACCGGAACACCGAACCGAATCGGTGAAAAAAAACCGGAAAAAAAACCGGT
------------------------------------------------------------
300
SFB1'
SFB1
TGACCAAAAAAGTCAACAAACCGGACCGGACCGGACCGAACCGGTTCCAACCGGTTCCGG
------------------------------------------------------------
360
SFB1'
SFB1
TTCCGGTTTTACACTACACCGCACCGGACCGGACCGAACCGGACCGGTCATATTTTTTTA
------------------------------------------------------------
420
SFB1'
SFB1
TTTTTTTTTTATTTTTTTTATTTTTTATTTACAAATTTTTAAAATATAATCTCATATTTT
------------------------------------------------------------
480
SFB1'
SFB1
AAATGTTATAATCACTATAGTTCAACTTCAAAAAATTTCTAAATGTATGAATTGGATTAT
------------------------------------------------------------
540
SFB1'
SFB1
TTGTTAATTTTTAAGCCAAAAAAATAAGTTATTTATTATAAATTTTTAATTAAAAAAATA
------------------------------------------------------------
600
SFB1'
SFB1
AAAAAAAAAAAAATTTTTAAAAAAATAAAAAAATAATTAAAAAATAAAAAATTAATAAAC
------------------------------------------------------------
660
SFB1'
SFB1
CGGTTCAAAACCGGAACCGGTCAGAACCGGACCGGAACCGGTCAGAACCGAACCGGCCGG
------------------------------------------------------------
720
SFB1'
SFB1
TTTTTGAATTTTTTTTTACTTAAACCGGACCGAACCGGACCGGTTAAATAGTACCGATTT
------------------------------------------------------------
780
SFB1'
SFB1
CGGTTCCGGTTTAAGGTCAGAACCGGACCGAACCGGACCGCGCCCACCCCTACTGATGAC
------------------------------------------------------------
840
SFB1'
SFB1
CCATATGTTAAAAAAGAATTTCAATGGTCTCTTTTTCCCAATCAAACATGTGAGGTGTTC
CCATATGTTAAAAAAGAATTTCAATGGTCTCTTTTTCCCAATCAAACATGTGAGGTGTTC
************************************************************
900
285
Figure 2. Partial nucleotide alignment of the SFB1 and SFB1 # alleles. SFB1 # has a 615-bp insert located 225 bp downstream from
the start codon of the SFB1 # gene. The first 3 nucleotides of the insert encode for an in-frame stop codon (bold). The insert is
bordered by two 11-bp complementary inverted repeats (underlined) that are flanked by identical 8-bp direct repeats (shaded).
et al. 2006). The nucleotide and amino acid sequences for
280 bp of the Pru-C2/PCE-R S1 fragment from the UF fosmid clone were identical to those of the reported S1-RNase
(GenBank number AB028153, data not presented).
The UF and P114 fosmid clones containing the S1-RNase
also contained SFB1 # and SFB1, respectively. This was
expected as the intergenic distance between the S-RNase
and SFB in the S1-haplotype is only approximately 600 bp
(Ikeda et al. 2005). The SFB1 # fragment amplified with the
universal SFB primers SFB-C1F and FB3R was approximately 600 bp longer than expected for wild-type SFB alleles,
including the wild-type SFB1 from P114, SFB26, and SFB13
(Figure 1B). The increased length of SFB1 # compared with
SFB1 indicates the presence of an insertion.
The nucleotide alignment of SFB1 from P114 and sweet
cherry from which it was originally sequenced was identical
(GenBank number AB111518, data not presented). In contrast, SFB1 # from UF contained a 615-bp insertion located
þ225 bp from the translation start site (Figure 2). The first
3 nucleotides of the insert encode an in-frame stop codon.
The insert has 11-bp inverted repeats at the termini that are
flanked by a 8-bp direct repeat. Both the terminal inverted
517
Journal of Heredity 2006:97(5)
PcSFB1
PcSFB1'
PaSFB4
PaSFB6
PcSFB9
PcSFB26
MAFTIRKKEILIDILVRLPTKSIVRFLWTCKSWSDFIGSSSFVSTHLDRNVTKHAHVYLL
MAFTIRKKEILIDILVRLPTKSLVRFLCTCKSWSDFIGSSSFVSTHLDRNVTKHAHVYLL
MTFTLRKKEILIDILARLPAKSLIRFLCTCKSWSDLIGSSSFVSTHLHRNVTKHDHVYLL
MTFTLRKKDILIDILVRLPAKSLVRFLCTCKSWIDLIGSSSFISTHLHRNATKHTHVYLL
MTFTLRKNEILIDILARLPTKSLVRFLCTCKSWSDLIGSSSFVSTHLHWNVTKHARGYLL
MAFTLRKKEIVIDILVRLPAKSLLRFLSTCKSWSDLIGSSSFVSTHLHRNVTKHTHVYLL
* ** ** * **** *** ** *** ***** * *** ** **** * ***
***
60
60
60
60
60
60
PcSFB1
PcSFB1'
PaSFB4
PaSFB6
PcSFB9
PcSFB26
CLHHPNFECHVDPDDPYVKKEFQWSLFPNQTCEVFYKLSHPLGNTEHYGIYGSSNGLVCI
CLHHPNFECHVDPDD--------------------------------------------CLHHPNFERLVDPDNPYFKKEFQWSLFSNETFKQCYKLNHPLGSTEHYVIYGSSNGLVCI
CLHHPNFERNDDPDDPYVEQEFQWSLFSNETFEECSKLSHPSGSTKHYVIYGSSNGLVCI
CLHHPDVERQADPDDPYVKQELQWSLFSNETFEECSKLSHPLGSTEHYMIYGSSNGLVCI
CLHHPNFERNEDPDDPYVEQEFQWSLFSNETFEECSKLSHPLGSTEHYVIYGSSNGLVCI
***
*
*** *
* ***** * *
** ** * * ** ******* **
PcSFB1
PcSFB1'
PaSFB4
PaSFB6
PcSFB9
PcSFB26
SDEILNFDSPIHIWNPSVRKLRTTPISTNINMKFSLVSLQFGFHPVVNDYKAVRMMRTNK
-----------------------------------------------------------SDEILNFDSPIHIWNPSVRKFRTPPMSTSINIKFNYIALQFGFHPRVNDYKAVRMMRTNK
SEEILNFDSPIHIWNPSVKKFRTPPMSTNINIKFSHVALQFGFHPGVNDYKAVRMMRTNK
SDEILNFDSPIHIWNPSVRKLRTTSMSTNINIKFSHVALQFGFHPGVNDYKAVRMMRTNK
SDEILNFDSPIHIWNPSVRKLRSTPISTNINIKFSHIALQFGFHPGVDDYKAVRMMRTNK
* **************** * *
** ** **
******* * * **** *****
180
PcSFB1
PcSFB1'
PaSFB4
PaSFB6
PcSFB9
PcSFB26
GALAVEVYSLRTDSWKMIQAIPPWLKCTWQHHKGTFSNGVAYHIIEKGHIISIMSFNSGS
-----------------------------------------------------------DALAVEVYSLRTDSWKMIEAIPPWLKCTWQHHKGTFFNGISYHIIEKCPIFSIMSFDSGS
NALAVEVYSLKTDSWKMIEAIPPWLKCTWQHHKGTFFNGVAYHIIEKGPICSIMSFDSGS
NALVVEVYSLRTDSWKMIKAIPPWLKCTWQPFKGTFFNGVAYHIILKGPIFSIMSFDSDS
NAFAVEVYSLKTDCWKMIEAIPPWLKCTWQHHKGTFFNGIAYHIIEKGPIFSIVSFDSGS
****** ** **** *********** *** ** **** * * ** ** * *
240
PcSFB1
PcSFB1'
PaSFB4
PaSFB6
PcSFB9
PcSFB26
EEFEEFIAPDAVCSSWRSCIEVYKEQICCFLTLS-CEEEGMEKFDLWVLQEKRWKQLCPF
-----------------------------------------------------------EEFEEFIAPDVICSSWGLFIDLYKEQICLLSSFYSCEEKGMRKIDFWVLQEKRWKQLCPF
DEFEEFIAPDAICSPSELCIDIYKERVCLLFSFYSCDEEGMVPNDLWVLQEKRWKQLCPF
EEFEEFIAPDAICHSWELCIDVYKEQICLLFSCYSCEEEDMEKVDLWVLQEKRWKLLCPF
EEFEEFIAPDAICTSVGLWIDVYKDQICLLFKCYGCEEEGMDKVDLWVLQDKRWKQLCPF
********* *
* **
*
* * *
* **** **** ****
299
PcSFB1
PcSFB1'
PaSFB4
PaSFB6
PcSFB9
PcSFB26
IYSSDYCYRTIGISIDNELLTARIGFDKGVADLCLCNYETNQLLETGIKLAIMRYGEIEF
-----------------------------------------------------------IYPSHY-YGTLGISKDNELLMEKRDFSRGIGDLHLCNYESKQVLETGIDLATIKYGEIEF
IYPAGS-YGTIGITIDNKLLMVRRDFLRGAGDLCLCNYESKQVFETGIELAVMKYGEIEL
IYPLGYDYRPIGISIDNKLSMQRKDYTWGIADLHLCDYESKQVQETGIKLAIMKYDEIEF
ISSFDC-CGPVGISVDNELLIEIRDFTSGVGHLHLCNYESRQVLDTRIKLAVMNYGEIEF
*
** ** *
*
* ** ** *
* * **
* ***
PcSFB1
PcSFB1'
PaSFB4
PaSFB6
PcSFB9
PcSFB26
LFAITYIESSGLLNNH
---------------LFAITYIESLVLLNNT
LFAITYIESLALLNKY
LFSITYIKSLVLLNND
LFSITYIESLFLLNNY
** **** * ***
F-Box
120
75
120
120
120
120
V1
180
180
180
180
240
240
240
240
300
300
300
300
V2
359
359
359
360
359
HVb
HVa
375
375
375
376
375
Figure 3. Amino acid alignment of 6 cherry SFB alleles. SFB1 #, but not SFB1, contains a stop codon in the V1 region. Conserved
residues are indicated by asterisks. The location of the F-box motif, V1, V2, HVa, and HVb are boxed (Ushijima et al. 2003; Ikeda et al.
2004). PaSFB4 (GenBank accession number AB111521) and PaSFB6 (AB096858) are previously reported sweet cherry SFB
sequences that have also been identified in sour cherry (Yamane et al. 2001). PcSFB1, PcSFB1#, PcSFB9, and PcSFB26 are newly
sequenced sour cherry SFB alleles.
repeat and the direct repeat are characteristic of Ds transposable elements (Kunze and Weil 2002).
The premature in-frame stop codon in SFB1 # is located in
the V1 region of SFB, upstream from the 3 other SFB variable
regions, V2, HVa, and HVb (Figure 3) (Ikeda et al. 2004).
Therefore, SFB1 # lacks 3 of the 4 variable regions and has
only 75 amino acids from the N-terminus of the protein compared with wild-type SFB1 protein that has 375 amino acids.
RT–PCR using the SFB1 forward primer, PaSFB1-F,
and a newly designed reverse primer using sequence from
518
the SFB1 # insert, named PcSFB1#-R, amplified a fragment
from UF pollen cDNA, indicating that SFB1 # is transcribed
(Figure 4). Therefore, it is possible that a truncated variant
of the SFB1 protein is encoded from the S1#-haplotype.
Discussion
S1 # is the fourth pollen part mutant identified in Prunus
for which the molecular basis of the mutation has been
Hauck et al. The Mutated S1-Haplotype in Sour Cherry
Figure 4. RT–PCR of the SFB1 # from pollen cDNA of
UF (S-phenotype: S1#S4Sd), P114 (S1S4Sd), and Mont (S6S13mSa)
using (A) SFB1 #-specific primers, PaSFB1-F and PcSFB1#-R,
and (B) actin control primers, ActF1 and ActR1 (Yamane et al.
2003). Genomic DNA of UF and non-RT RNA (RT) from
UF pollen were used as quality controls.
characterized. The other 3 pollen part mutants are the naturally occurring Sf in Prunus mume and the irradiation-induced
pollen part mutants in sweet cherry, S3# and S4#. All 4 of these
pollen part mutants contain structural alternations in SFB
(Ushijima et al. 2004; Sonneveld et al. 2005). The most severe
structural change is present in the sweet cherry irradiationinduced S3# (Lewis and Crowe 1954), as the SFB3-coding
region is completely deleted (Sonneveld et al. 2005). In contrast, the other 3 altered SFBs, Sf, S1#, and S4#, are at least
partially transcribed, suggesting that they encode putative
truncated proteins (Ushijima et al. 2004; Sonneveld et al.
2005). The F-box domain and the N-terminus of these proteins are unaffected; yet, the variable and hypervariable
domains at the C-terminus, which are thought to be involved
in specific interactions (Ikeda et al. 2004), would be changed
or lost. The altered putative proteins are therefore unlikely to
perform their normal specificity function. Compared with Sf
and S4 #; S1 # is noteworthy in that it would encode the shortest protein, completely lacking the variable region V2 and the
hypervariable regions HVa and HVb.
The finding that alterations disrupting SFB function lead
to pollen part mutants suggests that in Prunus, S-allele–
specific S-RNase degradation follows the modified inhibitor
model (Luu et al. 2001; see review by Wang et al. 2003). In
this model, the pollen-S determines specificity and a separate
inhibitor prevents the cytotoxic activity of the S-RNases.
Therefore, SFB would act to block the binding of a general inhibitor to its cognate S-RNase, and the absence of
SFB would result in the binding of a general inhibitor to
the S-RNases and compatibility. This mechanistic model is
also consistent with the ‘‘one-allele match’’ genetic model
for the control of SC and SI in sour cherry (Hauck et al.
2006). If 1 or 2 of the SFBs in the 2 sour cherry pollen
match a stylar S-RNase, the pollen would be incompatible
because the SFBs would protect their cognate S-RNases from
general inhibitor binding. Therefore, these S-RNases would
maintain their cytotoxic activity.
SFB1 # is the third nonfunctional S-haplotype mutation
that is caused by the insertion of a transposable element.
The sour cherry stylar part mutant S6m-haplotype has a
2.7-kb Mutator-like element insertion in the putative promoter region of the S6-RNase (Yamane et al. 2003). The
S6-RNase failed to express in the mutant plant. SFBf has
a 6.8-kb insertion that is a nonautonomous long terminal
repeat element (Ushijima et al. 2004). Like the Ds insertion
in SFB1 #; this insertion also results in a premature stop
codon. The above examples illustrate that mobile element
insertion is an evolutionary force contributing to the breakdown of SI in Prunus, either by suppressing the expression
or by interrupting the coding region of the relevant genes.
For centuries, the SI sour cherry landrace variety Pándy
was grown in backyards and lined village streets in its native Hungary and Romania due to its excellent fruit quality
(Iezzoni et al. 1990). In the village of Újfehértó, Hungary,
the local residents identified and vegetatively propagated
a particular selection of Pándy that was SC and therefore
more productive. This selection, named Újfehértói Fürtös
(translated ‘‘Bunched of Újfehértó’’) subsequently became
the most widely planted sour cherry cultivar grown in Hungary. Pándy has the S-haplotype phenotype S1S4Sd. This lineage suggests that the SC in UF is at least partially due to the
loss of function of the S1#-haplotype in the Újfehértó selection from the Pándy landrace. Therefore, SFB1 # represents a
novel allele resulting from Ds-element insertion that was selected and maintained in the population by local people due
to the increased yield accompanying self-compatibility.
Acknowledgments
We thank Audrey Sebolt for technical assistance and Ning Jiang for her
critical review of the manuscript. This project was supported by the USDA
Cooperative State Research, Education and Extension Service, National
Research Initiative—Plant Genome Program.
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Received January 16, 2006
Accepted July 16, 2006
Corresponding Editor: Reid Palmer