Download An S Receptor Kinase Gene in Self-Compatible

The Plant Cell, Vol. 5, 531-539, May 1993 O 1993 American Society of Plant Physiologists
An S Receptor Kinase Gene in Self-Compatible
Brassica napus Has a l-bp Deletion
Daphne R. Goring, Tracy L. Glavin, Ulrike Schafer, and Steven J. Rothstein'
Department of Molecular Biology and Genetics, Univers/ty of Guelph, Guelph, Ontario, Canada N1G 2W1
S locus glycoprotein (SLG) and S locus receptor kinase (SRK) cDNAs were isolated from an S allele present in a number
of self-compatible Brassica napus lines. This Aí0 allele did not segregate with self-incompatibility in crosses involving
other self-incompatible B. napus lines. The SLG-Aí0 cDNA was found to contain an intact open reading frame and was
predicted to encode an SLG protein with sequence similarities to those previously associated with phenotypically strong
self-incompatibility reactions. SLG-Aí0 transcripts were detected in the developing stigma at steady state levels even
higher than those detected for SLG alleles linked with self-incompatibility. Analysis of the corresponding SRK-Aí0 cDNA
showed that it was very similar to other S locus receptor kinase genes and was expressed predominantly in the stigma.
However, a l-bp deletion was detected in the SRK gene toward the 3' end of the SLG homology domain. This deletion
would lead to premature termination of translation and'the production of a truncated SRK protein. The A10 allele was
determined to represent a R oleracea S allele based on its segregation pattern with the R oleracea SZ4allele when both
these alleles were present in the same B. napus background. These results suggest that a functional SRK gene is required for Brassica self-incompatibility.
INTRODUCTION
In the Brassica family, fertilization can be controlled by a selfincompatibilitysystem that is inheritedas a dominant genetic
locus called the S locus (for reviews, see Nasrallahet al., 1991;
Dzelzkalns et al., 1992). Although the diploid species Brassim oleracea and B. campesfris are typically self-incompatible,
B. napus, an allotetraploidcomposed of both of these genomes,
generally occurs as a self-compatible plant (Downey and
Rakow, 1987). Some naturally occurring self-incompatible B.
napus lines have been isolated (Olsson, 1960; Gowers, 1974),
and in addition, 8. napus can be made self-incompatible by
the introgression of S alleles from one of its progenitor species (Mackay, 1977). The presence of a functional S allele
results in a barrier to fertilization when the pollen grain originates from a plant carrying the same S allele as the pistil. The
pollen phenotype, which is determined by the diploid parenta1
genotype and not by the haploid pollen, is thought to be
propagated by putative S allele gene products deposited on
the outside wall of the pollen grain by the surrounding tapetum during pollen development (de Nettancourt, 1977). Thus,
during pollen-pistil interactions, there is a recognition of self
versus nonself, leadingto either a block in pollen germination
when both parents carry the same Sallele or successful fertilization when different S alleles are present.
There are multiple alleles at the S locus, and in B. oleracea,
up to 50 different alleles have been identified (Ockendon, 1974,
1982). In addition, two different genes cosegregate with the
1
To whom correspondence should be addressed.
self-incompatibility phenotype: the S locus glycoprotein (SLG)
gene and the S locus receptor kinase (SRK)gene. At a single
locus, there is considerable DNA sequence homology between
the SLG and SRK genes, with the 5'end of the SRK gene showing m90% DNA homology with the SLG gene (Stein et al., 1991;
Goring and Rothstein, 1992). By analogy to other receptor kinases, the protein domain coded for by this part of the gene
would be the receptor that recognizes self-pollen (Hanks et
al., 1988). The remainder of the SRK gene encodes a putative
transmembranedomain and a cytoplasmic kinase domain that
has been shown to encode a functional serinelthreonine kinase (Goring and Rothstein, 1992).
The DNA sequences of SLG and SRK genes from different
S alleles are somewhat variable and have been associated
with different self-incompatibility phenotypes. The majority of
characterized SLG genes fall into one group with DNA homologies greater than 80% (Dwyer et al., 1991; Goring et al., 1992a,
1992b). Some of these alleles were isolated from lines with
phenotypically strong self-incompatibility reactions and showed,
in the presence of a second allele from this group, a nonhierarchical pattern of dominance in the pollen (Thompson and
Taylor, 1966). A second group of alleles was found to be always recessive to the first group in the pollen and tended to
display a weaker self-incompatibility reaction (Thompson and
Taylor, 1966). The two characterized alleles from this group,
S2 and S5, share high levels of homology, but only show .u7Oo/o
DNA homology with the first group (Chen and Nasrallah, 1990;
Scutt and Croy, 1992).
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The Plant Cell
Transformation experiments have shown that the SLG gene
is not sufficient to confer self-incompatibility to a self-compatible
B. napus (Nishio et al., 1992). However, there is evidence that
SLG genes are required for self-incompatibility. A naturally occurring variant of B. campestris with normal SRK expression,
but low levels of SLG expression, has been associated with
self-compatibility (Nasrallah et al., 1992). In this study, we have
examined an S locus present in a number of self-compatible
8. napus lines to determine possible causes for selfcompatibility in these lines. Whereas the SLG gene appears
to be normal, the SRK gene has acquired a mutation that is
predicted to lead to a truncated gene product. This suggests
that the SRK gene is also required for self-incompatibility.
RESULTS
Occurrence of the SLG-A10 Allele
The SLG-A10 cDNA was isolated during a search for an SLG
allele associated with self-incompatibility in the selfincompatible B. napus ssp oleifera (canola) lines R2 and T2
(Goring et al., 1992a). While the SLG-AW cDNA was one of
the more predominant SLG cDNAs isolated from the stigma
cDNA library, it did not segregate with self-incompatibility in
these lines. A survey of different canola cultivars revealed that
the A10 allele was present in several of these lines. As shown
in Figure 1, the SLG-A10 cDNA hybridizes to two Hindlll fragments at 8.4 and 9.2 kb, and further analysis has revealed that
the SLG-A10 gene is represented by the lower 8.4-kb Hindlll
band, while the upper 9.2-kb band results from crosshybridization of the SLG-A10 cDNA to the SRK-A10 gene (see
below).
In the self-compatible canola cultivars Ceres (Figure 1, lane
1), Regent (Figure 1, lane 2), and Westar (Figure 1, lane 5),
the/170 allele is present in a homozygous form as determined
by the segregation of this allele into progeny. This locus is also
occasionally found in the self-compatible Topas line, as is
shown for the two Topas plants tested, where one is heterozygous for the AW allele because not all self-progeny carry this
allele (Figure 1, lane 4) and the other does not carry the A10
allele (Figure 1, lane 3). In the self-incompatible line W1, which
was developed from a cross between a self-incompatible B.
campestris and the self-compatible canola cultivar Westar
(Goring et al., 1992b), the AW allele is again present in a
homozygous state. A cross between W1 and a self-compatible
winter canola line, which does not carry the AW allele, shows
the segregation pattern of the AW allele relative to selfincompatibility. Of the five genomic DMA samples from selfincompatible plants, only two (Figure 1, lanes 9 and 11) contain
the/170 allele in their genomes, while genomic DMA from four
self-compatible plants (Figure 1, lanes 12 to 15) carry this allele. The allele responsible for self-incompatibility in this line
has been determined to be the 970 allele (Goring et al., 1992b),
J__2__3_jL-5-_L_i__3.J»
9.4-
,-SRK-AIO
(
V
6.6-
SLG-A10
—SLG-910
6.64.4-
—SRK-910
1
2
3
4
5
6
7
8
9
10
11 12 13 14 15
Figure 1. Genomic DMA Gel Blot Analysis of the A10 Locus in SelfCompatible and Self-Incompatible B. napus.
Genomic DNA samples were digested with Hindlll and hybridized to
the SLG-AW cDNA in the top panel and the SLG-910 cDNA in the bottom panel. Ceres, Regent, Topas, and Westar (lanes 1 to 5) represent
different self-compatible canola cultivars. W1 (lane 6) is a selfincompatible line derived from Westar (see Methods for details). Lanes
7 to 15 represent segregating progeny from a cross involving selfincompatible W1 and a self-compatible winter canola line. The plants
from lanes 6 to 11 are self-incompatible W1 and a self-compatible winter canola line. The plants from lanes 6 to 11 are self-incompatible
(SI); and from lanes 1 to 5 and 12 to 15 are self-compatible (SC). The
910 locus represents the S locus associated with self-incompatibility
in W1 (Goring et al., 1992b). Bands, other than those marked, represent cross-hybridization of the SLG probes to related sequences in
the Brassica genome. There was some variability in the amount of
DNA loaded in each lane.
which is only present in the W1 plant (Figure 1, lane 6) and
in the resulting self-incompatible plants from the cross involving W1 (Figure 1, lanes 6 to 11). Thus, in theW1 line, the A10
allele does not segregate with self-incompatibility.
Sequence Analysis of the SLG-A10 cDNA
The sequence of the SLG-A10 cDNA was analyzed to determine if some alteration was responsible for the inability of this
allele to elicit a self-incompatibility reaction. It was found that
the SLG-AW sequence could be translated into a full-length
SLG protein, as shown in Figure 2. The predicted amino acid
sequence shows that the SLG-A10 protein contains several
characteristic features of SLG proteins, such as the signal peptide at the 5' end, several potential sites for N-glycosylation,
and the 12 conserved cysteine residues at the carboxyl end
of the sequence (Figure 2) (Nasrallah et al., 1987; Dwyer et
al., 1991). The SLG-AW sequence shows high levels of DNA
homology to a majority of characterized SLG genes (Trick and
Flavell, 1989; Dwyer et al., 1991; Goring et al., 1992a, 1992b),
ranging from 84 to 91% for DNA and 79 to 86% similarity for
the predicted amino acid sequences. Some of these alleles
have been associated with phenotypically strong selfincompatibility reactions (Thompson and Taylor, 1966; Goring
A Nonfunctional S Locus
et al., 1992a, 1992b). The amino acid homology is much lower
(67%) for the pollen recessive S2 allele (Chen and Nasrallah,
1990). Thus, the sequence predictions suggest that the SLGA10 allele should be able to promote a strong self-incompatibility reaction.
533
Plasmid (pg)
SLG-A10
3.4-
<£
1.8-
Expression of the SLG-A10 Gene
To determine if the lack of a self-incompatibility reaction was
due to an altered expression pattern of the SLG-AW allele, RNA
levels in different tissues from the W1 line were examined. The
SLG-A10 steady state mRNA levels were then compared to that
of the SLG-910 allele, which is associated with W1 selfincompatibility. As shown in Figure 3, both genes were found
to be predominantly expressed in the stigma tissue, with mRNA
transcripts detected in samples before and after anthesis
(Figures 3A and 3B). When the levels were compared to plasmid controls, the steady state levels of SLG-A10 mRNA were
found to be approximately four to eight times higher than the
SLG-9W transcripts (Figures 3A and 3B). Similar results were
also detected in the R2 line when the SLG-A10 steady state
mRNA levels were compared to that of the SLG-A14 gene, the
allele associated with self-incompatibility in the R2 line (data
not shown). When the developmental profile of the SLG-A10
mRNA levels was examined in stigma samples from developing W1 buds (Figure 3C), it was determined to be very similar
to that observed for the SLG-970 allele (Goring et al., 1992b).
Finally, loss of self-incompatibility was not associated with an
absence of SLG-A10 expression because high levels of SLGAW transcripts were also detected in stigma RNA samples from
developing buds in the self-compatible Westar line (data not
shown). Thus, there are no indications that the absence of selfincompatibility is due to an altered expression profile, unless
the higher steady state levels of SLG-AW RNA somehow exert a negative effect.
MKGIRKNYENSYTLSFLLVFFVLILLPPAFSINTLSSEESLTISSNRTLV
50
SRGDVFELGFFKTTSSSRWYLGIWYKKFPYRTYVWVANRDNPLPNSIGTL
100
KISNMNLVLLDHSNKSVWSTNLTRRNERTPVMAELLANGNFVMRDSNNND
150
ASEFLWQSFDYPTDTLLPEMKLGYNLKKGLNRFLISWRSSDDPSSGDYSY
200
KLEPRRLPEFYLLQGDVREHRSGPWNGIRFSGILEDQKLSYMEYNFTETS
250
EEVAYTFRMTNNSFYSRLTLSSTGYFERLTWAPSSWWNVFWSSPNHQCD
300
MYKICGPYSYCDVTTSPVCNCIQGFRPKNRQQWDLRISLRGCIRRTRLSC
350
SGDGFARMKYMKLPETTMAIVDRSIGVKECEKRCLSDCNCTAFANADVRN
400
GGTGCVIWTGRLDDMRNYVPDHGQDLYVRLAAADLV
436
Figure 2. The Predicted Amino Acid Sequence of the SLG-A10 cDNA.
The underlined section represents a putative signal peptide. Conserved
cysteine residues are marked by stars above the amino acid residues.
Potential N-glycosylation sites are represented by double underlines.
SLG-910
Plasmid (pg)
B
*3
v
<y <T
SLG-A10
3.4-
1.8-
SLG-910
Stigma RNA
3.4-
Figure 3. RNA Analysis of the SLG-AW Gene.
(A) Total RNA from different W1 tissues was hybridized with the SLG910 cDNA, which represents the active S locus in the W1 line. Samples were taken from 1 day before anthesis (1) and at anthesis (2). On
the right are plasmid controls to show that hybridization was specific
and to determine the mRNA abundance.
(B) Hybridization of the SLG-A 10 cDNA to total RNA from different W1
tissues. On the right are plasmid controls to show that hybridization
was specific and to determine the mRNA abundance. Samples were
taken from 1 day before anthesis (1) and at anthesis (2).
(C) Total stigma RNA from different bud sizes increasing from ~1 to
6 mm in length (lanes 1 to 7) and open flowers (lane A) was hybridized
to the SLG-A10 cDNA.
The positions of the 18S (1.8-kb) and the 25S (3.4-kb) rRNAs are marked
on the left.
Isolation and Expression of the SRK-10 cDNA
Because the sequence and expression pattern of SLG-A10 was
similar to a functional SLG gene, the SRK gene associated with
the AW allele was then analyzed. Using the polymerase chain
reaction (PCR) with primers to conserved regions in SLG genes,
an 800-bp internal fragment from the SRK-AW gene (Figure
534
The Plant Cell
1, upper band) was first isolated and sequenced. The corresponding 2.7-kb cDNA was then amplified using the rapid
amplification of cDNA ends (RACE) technique with specific
primers derived from the genomic fragment (Frohman et al.,
1988). To determine in which tissues the SRK-A10 gene was
expressed, W1 and Westar RNA samples were analyzed by
RNA PCR. As shown in Figure 4, the SRK-A10 gene is predominantly expressed in the pistils throughout bud
development in both W1 and Westar RNA samples. This is
the primary site of expression seen for other SRK genes in
Brassica (Stein etal., 1991; Goring and Rothstein, 1992). However, while very weak expression of other SRK genes has also
been found in the anthers, SRK-A10 transcripts were not detected in this tissue.
Sequence Analysis of the SRK-A10 Gene
The SRK genes from different S alleles encode proteins with
similar features, namely a region at the N-terminal end very
similar in sequence to SLGs, a transmembrane domain, and
a C-terminal kinase domain. Alignment of the SRK-A10 cDNA
sequence to other SRK genes revealed that it was very similar to the SRK-910 (Goring and Rothstein, 1992) and SRK6
(Stein et al., 1991) genes showing 86 and 87% DMA homology, respectively. However, as determined for other SLG-SRK
pairs, the SRK-A10 sequence was most similar to the SLG-A10
sequence in the SLG domain (92%). An unusual feature of
this homology is the presence of a 590-bp region with 100%
sequence identity between the two genes, suggesting that a
gene conversion event has occurred, as shown in Figure 5A.
Whereas SLG-SRK pairs at a locus are very similar to each
other, the three S loci characterized to date do not have an
analogous region of identical sequence. As shown in Figure
5B, a comparison of the SLG-A10 and SRK-A10 sequences
revealed a few deletions or insertions occurring in multiples
i———— Westar ————i
anthers
J_ .2- 3.
pistils
anthers
_ 2. 3_
pistils
J_ _J_ _3
321-
Figure 4. RNA PCR Analysis of the SRK-A10 Gene.
First strand cDNAs from different W1 and Westar RNA samples were
amplified with SflK-A JO-specific primers spanning the kinase domain.
The anther and pistil samples were extracted from different bud size
lengths: (lanes 1) 2 to 3 mm; (lanes 2) 4 to 5 mm; (lanes 3) 6 to 7 mm.
Amplification was performed for 30 cycles for the pistil samples and
35 cycles for the remaining samples. PCR products were hybridized
to the SRK-A10 cDNA. Only the top hybridizing band, which represents
the expected length of 1.1 kb, was visible after staining with ethidium
bromide. The smaller bands may have been derived from incorrectly
spliced mRNAs, which have been detected during the cloning of another SRK gene, the SRK-910 cDNA (O.R. Goring, unpublished results).
of 3 bp, which would result in the removal or addition of amino
acids in the SLG domain. This also has been detected in the
SLG-SRK pairs at the S2 and S6 loci. However, just downstream
of the region of 100% homology at position 948 in the SRKA10 sequence, there is a 1-bp deletion (Figure 5, arrow) leading to a shift in the reading frame. Translation of the DMA
sequence revealed that premature termination would occur
at nucleotide 978 and a truncated protein would be produced
(Figure 5B, double underlined). Except for this 1-bp deletion,
this gene codes for the predicted structures of a receptor kinase, including the transmembrane and kinase domains. The
predicted kinase domain contains all of the conserved amino
acids found in kinases, and similar to the other S receptor
kinase genes, it shows greatest sequence similarity to serine/threonine kinases (Hanks et al., 1988). Thus, due to the
frameshift mutation, this gene no longer codes for a functional
S receptor kinase.
Segregation of the A10 Allele with a Functional S
Allele
Because the A10 allele is not associated with self-incompatibility, it was not known whether this allele was genetically linked
to the S locus or if it was present in another part of the Brassica genome. There are potentially two S loci in B. napus
because it is composed of the genomes of both B. campestris
and B. oleracea. The location of the A10 allele can be determined by studying the segregation patterns in crosses between
the self-compatible Westar line, which is homozygous for the
AW allele, and a self-incompatible B. napus line that is produced by the introgression of a B. campestris or B. oleracea
S allele. In the W1 line, both the B. campestris 910 allele and
the A10 allele are homozygous, suggesting that the A10 allele
is not at the S locus position in the B. campestris component
of the B. napus genome. Furthermore, in lines in which both
the 970 and A10 alleles are present as heterozygotes, these
alleles segregate randomly in the self-progeny (data not shown).
The segregation pattern of the AW alleles was next compared to a B. oleracea allele (S24) present in the canola
cultivar Karat. The self-incompatible 824 Karat homozygote
(negative for the A10 allele) was crossed to the self-compatible
Westar (homozygous for the A10 allele), and the resulting F1
progeny were self-pollinated (using salt to break down selfincompatibility) to produce a segregating F2 population. The
AW allele was detected in seedlings both by PCR amplification using primers specific to the SLG-A10 gene and by DMA
blot hybridization. Surprisingly, all 30 F2 progeny tested carried the AW allele. Because a DMA probe for the S24 allele
was not available, 19 of these plants were analyzed for the presence of this allele by testing for self-incompatibility, and the
S24SLG gene was then identified by cross-hybridization to the
SLG-A10 cDNA. Approximately two-thirds of the plants tested
(13 of 19) were found to be self-incompatible. Furthermore, all
the self-incompatible F2 plants, as shown in Figure 6 (lanes
7 to 10), were found to carry a cross-hybridizing Hindlll band
A Nonfunctional S.Locus-
A
535
de
SLG-A1O
SRK-A1O
B
91
66
197
160
297
260
397
360
497
460
591
560
691
660
191
760
89J
860
996
951
SRK-AIO
SLG-AlO
TMCTGTATACMGGGTTCGATCCCAGGRRTTTGCAGCAGTGGGCTCTGAGRRTCTCATTMGGGGGTGT~GGAGGACGCTGCTGA~TGCMTGGA 1096
---------C---------AGA-.--A---CCG-.---------A--------------------------T---A-------G-----------G---1057
SRK-AIO
SLG-AlO
GATGGTTTTACCAGGATGRGMTATGGAGTTGCCAG~CTACGATGG~TATTGT~GACCGCAGTATA~TGAGA~AGMTGTMGAAGAGGT~CTTA
---------G-----------T-----A----.---.--------------------.----------T----T----------G---------------
SRK-AIO
SLG-AIO
CCGATTGTMTTGTACCGCGTTTGCAMTGCGGATATCCGGMTGGT~GACGGGTTGTGTGATTT~ACTGGAMTCTCGCTGATATGC~MTTACGT 1296
G---C-----------A------------------G----------------------------------C---CGG--T-AC-----------------
1257
SRK-A10
SLG-AIO
TGCTG
ACGGTCMGATCTTTATGTCAGATTGGCTGCGGCTGATCTCGTTMGMGAGTMCGCG M T G G G W T C A T M G T T T G A T TGTTGG
-C---ATC---------------------------C--T-----C--T---TAGCTCTT-CT-TTAA----RRA-C-CGGA-CCG-C-A--T-A-CGA-A
1389
1357
SRK-A10
SLG-AlO
AGTTRGTGT TCTGCTTCTTCTGATCATGTTTTG CCTCTGGAAAAGGAMCRCGAGMAAATCMGTGCAGCATCTATTGCAMTCGACAGAGAA 1 4 8 1
---CC-M-A-G--ACG-RR----T-M-A--T-G-TA-C----M---A----A-TCTT---T--CG-C-CA-A-AT-T--TGA-TC1452
SRK-AIO
SLG-AIO
A C C W T T T G C C T A T G M C G G G A T A G T A C G A T C M G C M G A G A C A G T T G T C T G G A G A G M C ~ T T G A G G A ~ T G G A A C T T C C A T T G A T A G A G T T1587
G~
-TA--G--A-A--G--CGTATCC
1475
SRK-AIO
AGCTATTGTC~GCCACCGAAAATTTCTCCMTTCTMC~TTGGACMGGTGGTTTTGGTATTGTTTACMGGGGATATTACTTGAC~MGM
SRK-A10
ATCGCGGT~GGCTATCAMGACGTCAGTTCAAGGGGTTGATGAGTTTATGMTGAGGTGACATTMTCGCGAGGCTTCMCATGTAMTCTTGTGC
1187
SRK-AIO
A M T T C T T G G C T G T T G C A T T G A C G C A G A T G A G M G A T G C T G A T A T A T G A G T A T T T G G ~ T T T ~ G C C T C G A T T C T T A T C T C T T C ~ ~ C T C G M1887
G
1196
1157
1687
SRK-A10
GTCTMGCTAhATTGGMGGAGAGATTCGACATTACCRRTGGTGTTGCTCGAGGGCTTTTATATCTTCATCMGACTCCCGCTTTAGGATAATCCACAGA
SRK-AIO
GATTTG~GTMGTMCATTTTGC~TGATAGRRATATGGTCCCAMGATCTCGGATTTTGGRRTGGCCA~ATATTTGRRAGAGACGA~AA~~A 2087
1987
SRK-AIO
ACACAATGAAGGTGGTCGGACTTACGGCTACATGTCCCCAGAGTACGCMTGGGTGGGATATTCTCGG~TCAGATGTTTTCAGTTTTGGAGTCAT
2181
SRK-A10
GGTTCTTGRAATTATTACTGGGMGAG~CAGAGGATTCGACGMGACMTCTTCTM~TGTGCATGGAGRRATTGGMGGMGGMGAGCGCTAGM2281
SRK-AIO
ATAGTAGATCCAGTCATCGTAAATTCATTTTCACCACTGTCATCACCATTTCMCTACAAGMGTCCT~TGCATACAMTTGGTCTCTTGTGTGTTC
2387
SRK-AIO
MGMCTTGCAGAGMCAGACCMCCATGTCGTCTGTGGTTTGGATGCTTGGCMTGMGCMCAGAGATTCCTCAGCCTAMTCGCCAGGTT~GTCAG
2481
SRK-AIO
MGMGTCCTTACGAACTTGATCCTTCATCMGTAGGCAGCGCGACGATGATGMTCT~~~ACGGTGAACCAGTACACCTGCTCAGTAATCGAT~CC~~~T
2581
SRK-AIO
R R T A T G A A A G C T G T G A G M T G T T C A T T T M T T ~ T T A C T R R A T ~ ~ ~ T G T G A C T C M T A C C A T A T G T G ~ G M G T ~ T A A A T T C T C A A T 2685
AG~
Figure 5. Alignment of the SLGA70 and SRK-A70 cDNA Sequences.
(A) The coding region of the SLGA70 cDNA was aligned with the SRK-A70 cDNA sequence. A region of 1000/0 homology between the two genes
is marked. The 1-bp deletion in the SRK-AIO gene leading to premature termination is marked by the arrow. Underneath the SRK-AlO cDNA is
a representation of the S receptor kinase domains that would be translated if the deletion was not present.
(B) Sequence alignment of the SLGAlO and SRK470 cDNAs. ldentical nucleotides are marked by dashes, and gaps are represented by blank
spaces. The predicted start and stop codons for both genes are undarlined. The 1-bp deletion in the SRK-A70,gene is marked by an arrow. The
nucleotide sequences for SLG-AIO and SRK-AIO have been submitted to GenBank as accession numbers L08608 and LO8607, respectively.
The Plant Cell
536
SM Karat/Westar F2
#•—— SI
——SC ——
—S M
—-SRK-A10
9.46.61
2
3
4
5
6
7
8
9
10
11 12
13
^
Figure 6. Segregation of the AW Locus with a 6. oleracea 5 Allele
in the 8. napus Genome.
Genomic DMA samples were digested with Hindlll and hybridized to
the SLG-A10 cDNA under cross-hybridizing conditions (washes at 50°C
in 0.1 x SSC, 0.1% SDS). Sources of genomic DMA are the following:
self-incompatible S24 Karat line (lanes 1 and 2); F, progeny from a
cross between S24 Karat and Westar (lanes 3 and 4); self-compatible
Westar canola line (lanes 5 and 6); and F2 progeny from the selfpollination of a S24 Karat/Westar F, plant (lanes 7 to 13). The plants
from lanes 1 to 4 and 7 to 10 are self-incompatible (SI); and from lanes
5 and 6, and 11 to 13 are self-compatible (SC).
at ~12 kb; this band was present in the S24 Karat parental line
(Figure 6, lanes 1 and 2) and in the S24 Karat/Westar F, plants
(Figure 6, lanes 3 and 4), but absent in the Westar parental
line (Figure 6, lanes 5 and 6) and in the self-compatible F2
plants (Figure 6, lanes 11 to 13).
The AW allele (represented by the two Hindlll bands at 8.4
and 9.2 kb) was present in the S24 Karat/Westar F! plants (Figure 6, lanes 3 and 4), the Westar parental line (Figure 6, lanes
5 and 6), and all of the F2 plants (Figure 6, lanes 7 to 13). The
signal intensity of the A10 allele in the F, and F2 populations
suggested that there is one copy of the A10 allele in the selfincompatible FT and F2 plants and two copies in the selfcompatible F2 plants and the Westar parental line. Therefore,
all 30 progeny plants tested had the AW allele. Of the 19 tested
further, 13 of 19 were self-incompatible, and all appeared to
be heterozygous for the A10 allele, with the six self-compatible
plants being homozygous for \r\eAW allele. Although the S24
and AW alleles appear to be segregating in a 0:2:1 ratio into
the progeny with no S24 homozygotes, there is a statistically
significant probability that the actual ratio is 0:1:1.
DISCUSSION
Self-incompatibility is one of several mechanisms that promote
outbreeding. In self-incompatible species like B. campestris
and B. oleracea, progeny of self-fertilized plants show severe
inbreeding depression and are less capable of competing with
their cross-bred cousins. It is generally thought that selfcompatible species have arisen via mutation of the genes
important for self-incompatibility (Stebbins, 1957). There are
a variety of circumstances where self-compatibility and the
resultant capability for inbreeding might prove advantageous.
These include cross-pollination difficulties due to the low density of a species under some environment conditions, selfing
as an isolating mechanism due to polyploidy, and fixing of a
chance variant having local adaptive advantage (for a more
complete discussion, see Jain, 1976). 1. napus is an amphidiploid species derived from a combinati' n of the B. campestris
and 8. oleracea genomes (Downey and Rakow, 1987). Presumably the selection for self-compatibility in the original B. napus
lines was very strong for a variety of reasons, including an initially low population density and a lack of severe inbreeding
depression due to polyploidy. It is tempting to conclude that
the mutation in the AW allele occurred during the process of
B. napus speciation. However, this species has been domesticated for centuries (Downey and Rakow, 1987), and it should
be recognized that the cultivars that are presently utilized have
a complex breeding history. This makes it difficult to determine when a particular mutation in the self-incompatibility
genes occurred.
The AW allele is present in a number of B. napus cultivars
and yet all of these are self-compatible lines. The SLG-AW gene
is expressed at high levels in the stigma, and sequence analysis suggested that it should encode a functional SLG protein.
It has recently been shown that a self-compatible B. oleracea
line also carries a normal SLG gene and that its gene product
is expressed in a manner similar to that for SLGs from selfincompatible lines (Gaude et al., 1993). Analysis of the SRKAW gene showed that it also is expressed in stigma tissues;
however, the coding region contains a 1-bp deletion in the
receptor domain when compared to other SLG and SRK genes.
This deletion creates a shift in the reading frame that leads
to premature translation termination toward the end of the
receptor domain and to the production of a truncated receptor. Receptor kinases have been implicated in a variety of
systems to play important roles in signal transduction during
cell-cell communication (Cantley et al., 1991; Karin, 1992).
Thus, the absence of a functional receptor kinase would presumably lead to a block in the signal pathway that would
normally be activated during the self-incompatible response.
Close to the frameshift mutation in the SRK-AW gene is a
region of identical sequence between the SLG-AW and SRKAW genes, which is a characteristic product of gene conversion. One explanation for these results is that the SRK-AW gene
suffered DNA damage followed by recombinational repair. During the repair process, if the SLG-AW gene was used to repair
the SRK-AW gene, the result would be gene conversion. This
type of homologous recombination between duplicated sequences present on the same chromosome has been shown
to be induced by mutagens in mammalian cells (Wang et al.,
1988). The SLG gene and the SLG domain of the SRK gene
from a single allele are more similar in sequence to each other
than they are to these genes isolated from other alleles (Stein
et al., 1991; Goring and Rothstein, 1992). Occasional gene
A NonfunctionalS Locus
conversion between the SLG and SRKgenes ata single allele
would explain why these sequences have remained so similar.
Through crosses to self-incompatible 6. napus lines carrying known 6. campestris and 6, oleracea S alleles, the A70
allele was shown to be closely linked to the B. oleracea S allele. The evidence of linkage between the A70 allele and the
B. oleracea $4 allele is twofold. First, when the S24 allele is
present, the A70 allele does not segregate randgmly and is
in fact present in all progeny plants. This is in contrast to what
occurs when the A70 allele is present in the same plant as
a 6. campestris S allele or in self-compatible lines. Second,
the A70 allele appears to be heterozygous to lhe S24 allele in
all self-incompatible progeny, while being homozygous in the
self-compatible Fp plants. At this point, i1 is not possible to determine whether the A70 allele is present right at the S locus,
because not enough progeny were analyzed to detect rare
recombination events.
The segregation of A10 and B. oleracea S2, alleles into the
F2 progeny was not as expected. For a sporophytic selfincompatibilitysystem such as that found in the Brassicaspecies, the parenta1 genotypes determine whether a particular
pollinationwill be rejected. Therefore, if one breaks down the
self-incompatibility system by treating the stigmas with salt prior
to pollination,the mechqnisms preventing pollen germination
on the stigma surface would be overcome for all pollen. Therefore, self-pollination of the A7O/SZ4heterozygotes should yield
progeny at a normal Mendelian ratio of 1:2:1.There is evidence
that there also may be a gametophytic component in Brassica self-incompatibility (Zuberi and Lewis, 1988).Furthermore,
Sato et al. (1991)have demonstratedthat an SLG-6 promoter8-glucuronidase (GUS) fusion gene introduced into Brassica
is expressed not only in the stigma papillae cells (sporophytic)
but also in the transmitting tissue of the stigma, style, and ovary
(gametophytic). This also may indicate a gametophytic component to Brassica self-incompatibility.
If a gametophytic component does exist and is not affected
by salt treatment, then one would expect the growth of the pollen tubes from gametes carrying the Sp4 allele to be hindered,
allowing the pollen tubes carrying the A70 allele to successfully fertilize the ovules. In this case, one would expect a
progeny ratio in the F2generation of 1 A101S24heterozygote
to 1 A10 homozygote. The segregation ratio of the AIO and
S24 alleles into progeny was aberrant, with all progeny tested
having the A70 allele and concomitantly none being Sp4
homozygotes. Although the ratio of 13 heterozygotes to 6
homozygotesfits a segregation ratio of 0:2:1,not enough samples were tested to statistically eliminate the possibility that
the real ratio is 0:l:l.Therefore, it is not possibleto demonstrate
that there is a gametophytic self-incompatibility component
in this case or whether some other mechanism is at work. It
should be noted that one does not always fail to detect progeny homozygous for self-incompatibility at the expected frequency when heterozygous plants having one functional Sallele are self-pollinated. The actual ratio varies considerably
depending on which S allele is present (D.R. Goring, unpub-
537
lished results). It is unclear whether this is due to the strength
of a certain allele or the result of another closely linked locus
(Zuberi and Lewis, 1988).
Because having a single functional S allele is sufficient to
make a plant self-incompatible, both the 6. campesfris and
B, oleracea S loci must be mutated to produce self-compatible
8, napus. Whereas the A70 allele represents a B. oleracea S
allele in severa1B. napus lines, it is unclear what has happened
to the 6. campestris S locus in these lines. Under crosshybridizing conditionswith SLG probes, bands that may represent a nonfunctional6. campesfris S allele were not detected
(for example, see Figure 6). Therefore, these genes may be
absent due to a deletion removing these genes. However, the
B. campestris locus would not be detected if the SLG and SRK
genes have been masked by other cross-hybridizing bands
or have divergedsufficiently not to be detected by hybridization.
Whereas direct transformation of self-compatible plants with
the SLG and SRK genes would be the most conclusive evidente demonstrating that these genes are involved in the
self-incompatibility reaction, the results in this study add to
the growing evidence that these genes are responsiblefor selfincompatibility. We have shown that the A70 locus.does segregate with an S allele linked to self-incompatibility and that
the A70 locus itself is not associated with self-incompatibility.
The presence of a frameshift mutation in ths SRK-AjO gene
suggests that loss of receptor kinase activity is responsible
for the loss of self-incompatibility at tliis locus, althougll we
cannot rule out the possibilitythat there are other unidentified
genes at the S locus that are also affected. Nasrallah'et al.
(1992)have shown that decreased SLG expression is associated with loss of self-incompatibility. Thus, from these
studies, it appears that both the SLG and SRK genes are required for the self-incompatibility response. However, it is
certainly possible that an additional gene product, such as
a ligand recognized by the receptor kinase, is required to confer self-incompatibility.
METHODS
Plant Material and Crosses
The S allele in the W1 line originated from a self-incompatible6rassica cahpestrisplant and was introgressedinto the 6.napus ssp olefera
'cv Westar line as described by Goring et'al., (1992b).The S allele in
a 6, oleracea broccoli F, hybrid was identified as the S24allele based
'on crosses to a 6. oleracea tester set (supplied by Dr. D. J. Ockendon,
Horticultural Research Institute, Wellsbourne, U.K.) and then introgressed into the 6. napus ssp oleifera cv Karat line (V. Ripley and
W. Beversdorf, manuscript in preparation). Plants were grown either
in a greenhouse or in agrowth room under lchr-daylightand 8-hr-dark
conditions.Followinganthesis under pollination bags'(toenforce selfpollination), plants were tested for self-incompatibility by measuring
seed setor by staining pistils for pollen tube growth with an'iline blue
and examining therh Únder a fluorbscence microscope (Kho and Baer,
1968). Forced self-pollinationof the self-incompatibleplantshvolved
538
The Plant Cell
the use of 5% NaCl to block expression of self-incompatibility temporarily and permit self-fertilization.
lsolatlon of the SLG-A10 and SRKaA10 cDNAs
The S locus glycoprotein (SLG)-A70cDNA was isolated from a cDNA
library constructed from stigmas collected 1 to 2 days before anthesis
and hybridized with the BS29-2 SLG probe (Trick and Flavell, 1989)
under cross-hybridizing conditions (Goring et al., 1992a).
The S receptor kinase (SRK)-A70cDNA was isolated using polymerase chain reaction(PCR) techniques described by Goring et al. (1992b).
W1 genomic DNA, digested with Hindlll and fractionated on an agarose
gel, was used to amplify an 800-bp region using PCR primers to conserved regions in publishedSLG sequences. The PCR products were
cloned into pBluescript KS+ (Stratagene) and identified by sequencing and genomic blot hybridization patterns. Specific primers derived
from the genomic PCR clone were then used to amplify the 5' and
3'cDNAends using the RACE (rapid amplification of cDNA ends) procedure (Frohman et al., 1988)with modifications described by Goring
et al. (1992b).
DNA Sequencing
PCR clones were partially sequenced using dideoxy sequencing and
the sequence enzyme (U.S. Biochemical Corp.) to confirm that they
were derived from the SRK-AIO gene. To completely sequence the fulllength SLG-A70 cDNA clone and the SRK-A70 PCR clones, deletions
were made using exonuclease 111 and mung bean nuclease according to the procedure in the Stratagene kit. Overlapping deletions were
sequenced for both strands. To avoid errors that may have been introduced during PCR, several cDNA ends derived from separate PCR
amplificationswere sequenced. The 1-bpdeletion in the SRK-A70 gene
was detected in both the genomic PCR clones and in the 5' RACE
products. All DNA and protein sequence analyses were performed on
the DNASIS and PROSIS software (HitachiAmerica Ltd., San Bruno,
CA).
Genomic DNA Blots
Genomic DNA was extracted from leaves as described previously
(Goring et al., 1992b). Approximately 5 to 10 pg of genomic DNA was
digested with Hindlll, fractionated through a 0.7% agarose gel, and
transferred to a Zetabind membrane (CUNO, Meriden, CT).The membranes were prehybridized and hybridized as described previously
(Goring et al., 1992b). Washing conditions involvedtwo 30-min washes
in 0.1 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate), 0.1%
SDS at 50 to 53OC for cross-hybridization, and at 65 to 68OC for specific hybridization. For the probes, the full-length cDNAs were gel
purified from vector sequences and labeled by random priming
(Feinberg and Vogelstein, 1983).
RNA Analysis
Total RNA was extracted f,rom W1 tissues using the method of Jones
et al. (1985). Poly(A)+ FINA samples were extracted from Westar tissue using the Micro-FastTrack mRNA isolation bits (Invitrogen, San
Diego, CA). For the RNA blots, 10 pg of W1 total RNA was fractionated
through a 1.2% formaldehyde gel (Sambrook et al., 1989) and transferred to Zetabind membranes. Hybridization conditionswere the same
as used for the genomic blots, and washing conditions were followed
for specific hybridization.
To examine the expression of the SRK-A70 gene using PCR, W1 total RNA samples and Westar poly(A)+ RNA samples were amplified
with specific SRK-A70 primers that span the kinase domain, which contains several introns. Twenty micrograms of total RNA and -1 pg of
poly(A)+ RNA were first treated with DNase I to remove any contaminating genomic DNA; these samples were then used to synthesize
first strand cDNA accordingto the methodof Harvey and Darlison (1991).
Controls without reverse transcriptase were also set up for each sample. One-quarter of each cDNA sample was amplified for 30 cycles
for the pistil samples and 35 cycles for the remaining samples and
then subjected to gel electrophoresis and DNA gel blot analysis.
ACKNOWLEDGMENTS
Daphne R. Goring and Tracy L. Glavin have contributed equally to this
paper. We are very grateful to Van Ripley for supplying the S2,Karat
and S,,Karat/Westar
plants. We would also like to thank Rajeev
Rastogi, Art Hilliker, and David Evans for their comments on the manuscript. This work was supported by agrant from the Natural Sciences
and Engineering Research Council (NSERC) to S.J.R. T.L.G. and D.R.G.
were recipients of an NSERC postgraduate fellowshipandan NSERC
postdoctoral fellowship, respectively.
Received February 2, 1993; accepted March 23, 1993
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Plant Cell 1993;5;531-539
DOI 10.1105/tpc.5.5.531
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