Journal of Experimental Botany, Vol. 54, No. 380, pp. 131-139,
January 1, 2003
© 2003 Oxford University Press
Genomic organization of the Papaver rhoeas self-incompatibility S1 locus
Received 12 June 2002; Accepted 26 July 2002
Wolfson Laboratory for Plant Molecular Biology, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
1 To whom correspondence should be addressed. Fax: +44 (0)121 414 5925. E-mail: m.wheeler{at}bham.ac.uk
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Abstract |
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 Top Abstract IntroductionSequence of the Papaver...Fluorescence in situ...DiscussionReferences |
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The self-incompatibility (SI) response in Papaver rhoeas depends upon the cognate interaction between a pollen-expressed receptor and a stigmatically expressed ligand. The genes encoding these components are situated within the S-locus. In order for SI to be maintained, the genes encoded by the S-locus must be co-inherited with no recombination between them. Several hypotheses, including sequence heterogeneity and chromosomal position, have been put forward to explain the maintenance of the S-locus in the SI systems of the Brassicaceae and the Solanaceae. A region of the Papaver rhoeas genome encompassing part of the self-incompatibility S1 locus has been cloned and sequenced. The clone contains the gene encoding the stigmatic component of the response, but does not contain a putative pollen S-gene. The sequence surrounding the S1 gene contains several diverse repetitive DNA elements. As such, the P. rhoeas S-locus bears similarities to the S-loci of other SI systems. An attempt to localize the P. rhoeas S-locus using fluorescence in situ hybridization (FISH) has also been made. The potential relevance of the findings to mechanisms of recombination suppression is discussed.
Key words: FISH, Papaver rhoeas, recombination, retro transposon, self-incompatibility, S-locus.
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Introduction |
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TopAbstractIntroductionSequence of the Papaver...Fluorescence in situ...DiscussionReferences |
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Self-incompatibility (SI) is a recognition mechanism that enables flowering plants to distinguish between self and non-self pollen. SI systems in most flowering plants are controlled by a single multi-allelic locus termed the S-locus. Self-pollination is prevented when pollen carrying an S-allele genetically identical to that expressed in the pistil on which it alights is recognized and inhibited, whereas pollen carrying an S-allele different from that of the pistil is allowed to grow. The classical model of SI proposes that genes situated at an S-locus encode a pollen component and a corresponding pistil component, the interaction of which leads to the SI response, and that these two components are physically linked and invariably co-inherited (de Nettancourt, 1977).
SI in the field poppy, Papaver rhoeas is under gametophytic control (GSI) where the S-phenotype of pollen is determined by its haploid S-genotype. However, the response in the Papaveraceae is very different from GSI found in the Solanaceae and Rosaceae. In these families the SI response takes place as pollen tubes grow through the stylar transmitting tract and depends upon the action of stylar S-ribonucleases degrading rRNA within incompatible pollen (McClure et al., 1989). By contrast, the SI response in the Papaveraceae takes place at the stigma surface and is mediated by a complex signalling cascade in pollen initiated following interaction between S-proteins secreted by the stigma and their cognate receptors in the pollen tube. Growth inhibition of incompatible pollen in P. rhoeas is rapid, acting on a time scale of minutes rather than hours (Wheeler et al., 2001).
Considerable insight into the Papaver SI system has been obtained following the characterization of several stigmatic S-alleles (S1, S3, S8 from P. rhoeas and Sn1 from P. nudicaule) (Foote et al., 1994; Walker et al., 1996; Kurup et al., 1998). The stigmatic S-proteins of P. rhoeas are small (
15 kDa), extracellular signalling molecules. It is postulated that S-proteins interact with the pollen S-gene product, believed to be a plasma membrane receptor. Inhibition of incompatible pollen in P. rhoeas is mediated by the activation of a calcium-based signal transduction pathway in the pollen. Evidence for this comes from calcium imaging studies demonstrating that increases in cytosolic Ca2+ are stimulated within a few seconds of an incompatible interaction and precede the inhibition of pollen tube growth (Franklin-Tong et al., 1993, 1995, 1997). Downstream of the initial Ca2+ signals, induction of SI results in the phosphorylation of several proteins in an S-specific manner. One of these proteins, p26, has since been cloned and found to be an inorganic pyrophosphatase whose activity is inhibited by phosphorylation (JJ Rudd et al., unpublished results). One possible result of this is that synthesis of biopolymers essential to pollen tube growth is reduced. There is also evidence that a mitogen activated protein kinase (MAPK) is activated during the SI response in incompatible pollen (JJ Rudd, FCH Franklin, VE Franklin-Tong, unpublished data). As well as the initiation of a phosphorylation cascade, dramatic re arrangements to the actin cytoskeleton follow the SI response in P. rhoeas (Snowman et al., 2000). Further downstream of these events, S-specific nuclear DNA fragmentation, a hallmark feature of programmed cell death (PCD), has been detected 4–12 h after induction of the SI response (Jordan et al., 2000). The Papaver SI system is thus characterized by a complex network of events acting downstream of the interaction between products of the S-locus. As such it has provided an excellent model system for examining cell signalling in plants.
The nature of the pollen component of the SI response is unclear at present. One candidate protein, SBP (S-protein binding protein), a pollen plasma membrane glycoprotein of 70–120 kDa, has been identified by Western ligand blotting (Hearn et al., 1996). Binding of SBP to S-proteins is at least partly dependent upon the presence of its glycan moieties. Whilst SBP specifically binds S-proteins in vitro, studies suggest that this binding is not S-allele specific. Hence, SBP may be an accessory receptor rather than the pollen S-receptor itself. However, mutagenesis studies of S-proteins reveals that all mutants that exhibit reduced ability to inhibit incompatible pollen also have reduced SBP binding activity (Jordan et al., 1999). The fact that binding to SBP and loss of biological function of S-proteins cannot be separated means that it is conceivable that SBP is itself the pollen S-receptor. An explanation for the binding of S-proteins to SBP in a non-S-specific manner may be accounted for by the presence of both low affinity binding to all S-proteins via the glycan moieties on SBP and high affinity binding to ligands of the same S-genotype via specific, protein binding domains. The presence of multiple binding sites of differing affinities within one receptor has been observed in mammalian systems (Shraga-Levine and Sokolovsky, 1998). This question will remain unresolved until either SBP and/or the pollen S-receptor is isolated.
As both stigma and pollen components of the SI response are encoded at the S-locus, a potential route to the identification of the gene encoding the pollen S-receptor is the examination of sequence surrounding the stigma S-gene. Indeed, the eventual identification of the pollen component of the SI response in Brassica followed sequencing around the stigmatic component of the response, SRK (Schopfer et al., 1999; Suzuki et al., 1999). Ultimately, the work discussed in this paper is aimed towards identifying the Papaver pollen S-gene.
As previously mentioned, one feature of all functioning SI systems is the maintenance of an intact S-locus. Recombination between the two components of the S-locus would inevitably result in the breakdown of SI, as progeny are no longer able to recognize their own pollen as ‘self’. Certainly, breakdown of SI in this way has never been observed in studies of SI in P. rhoeas. It is commonly thought that recombination suppression is important in the maintenance of the S-locus. However, Casselman et al. (2000) found relatively uniform recombination frequencies across a region surrounding the S-locus in B. oleracea. Nevertheless, this study does not rule out recombination suppression as a mechanism for maintenance of the integrity of S-haplotypes as in the same study no recombination events between SRK (S-receptor kinase) and SLG (S-locus glycoprotein) (two components of the Brassica S-locus expressed in the stigma) were detected.
Several hypotheses have been proposed to explain how suppression of recombination may occur. Firstly, the small physical size of some S-haplotypes in Brassica spp. may account for lack of recombination between the components. This explanation is problematic when it is considered that some of the Brassica S-haplotypes are large (up to 400 kb; Boyes and Nasrallah, 1993), yet no evidence for recombination within the S-locus has been forthcoming. Secondly, recombination may occur at a low level, but progeny resulting from these recombinations may be less fit due to increased homozygosity of deleterious recessive alleles (Casselman et al., 2000). A problem with this explanation is the lack of observed breakdown of SI. A third hypothesis maintains structural rearrangements due to deletions and chromosomal rearrangements may play a role (Cui et al., 1999; Boyes et al., 1997). Fourth, sequence divergence between S-loci may act to stop recombination. In the case of both structural heterogeneity and sequence divergence it is possible that repetitive elements may play a role, indeed studies on S-haplotypes of B. napus (Yu et al., 1996; Cui et al., 1999) have identified various repetitive elements in the immediate surroundings of SRK and SLG. Similarly, repetitive elements have been detected in the S-loci of Solanaceae (Bernatzky et al., 1989; Coleman and Kao, 1992; Matton et al., 1995). Finally, the location of the S-locus may play a role. It is known that recombination does not occur uniformly across chromosomes. The S-locus may, therefore, be situated in a region of the chromosome with intrinsically low recombination. It is known that recombination frequencies in most species are noticeably higher distally to the centromere (John, 1990). The possibility that the proximity of the S-locus to the centromere affects intra-locus recombination frequency has been cited in the case of Petunia hybrida (ten Hoopen et al., 1998).
In this study a start has been made to characterize the structure of the S1 locus of P. rhoeas. It is believed that obtaining sequence from around the stigma S-gene may lead to the identification of the pollen-S-gene in this species. It is also hoped that examination of the structure of the S-locus may be enlightening as to the mechanisms involved in preventing recombination at the S-locus. Studies using fluorescence in situ hybridization (FISH) in order to locate the S-locus within the P. rhoeas genome have also been initiated to examine whether the position of the S-locus in Papaver may play a role in maintaining its integrity.
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Sequence of the Papaver rhoeas S1 locus |
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TopAbstractIntroductionSequence of the Papaver...Fluorescence in situ...DiscussionReferences |
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Genomic DNA was extracted from leaf tissue of plants of genotype S1S3, partially digested with Sau3A and cloned into
EMBL3 previously digested with BamH1. The resultant library was screened using probes corresponding to cDNA from the stigma S-genes, S1 and S3. A 13.5 kb clone containing the S1 gene was isolated. Fragments of the S1 clone were subsequently subcloned into pBS-SK+ (Stratagene). Sequencing of subclones was then carried out using primers to vector sequence and, by walking into each subclone, primers designed to novel sequence. Sequence was then analysed using BLAST software provided by NCBI. Northern analysis was carried out to detect any transcripts from genes contained within the S1 clone.
The translation start of S1 was located within the clone and the sequence confirmed that the S-gene lacked introns. Analysis of sequence suggested that there were no parts of the clone that exhibited homology with known plant receptor genes (the likely identity of pollen S in P. rhoeas). However, several areas of the clone had significant homology to repetitive DNA elements previously found in plants. Extending over 720 bases and terminating 1400 bases downstream of S1 lies an area with significant homology (21% identity, 38% similarity in amino acid sequence over 728 bp) to the reverse transcriptase of a non-LTR retrotransposon found in Arabidopsis thaliana (AAC18922) (Fig. 1). Also, approximately 4 kb downstream of S1 lies a retro-element with homology to gag/pol polyprotein of Ty1/copia-type transposons. The deletion of large parts of both of these repetitive DNA elements and the fact that their open reading frames contain numerous stop codons and frameshifts indicates that both probably derive from ancient transposition events. Meanwhile, 3 kb upstream from S1, lying at the border of the S1 clone lies a small region of sequence (300 bp) that shows significant homology (including 96% identity over 100 bases) to tRNA genes, encoding an apparently functional tRNASER transcript. Interestingly, the homology is closest to trn genes encoded in the chloroplast genome. The most likely explanation for its occurrence within the S-locus is a transposition event. As such, the element shows similarities to short interspersed nuclear elements (SINEs) that have been identified in other plant species (Deragon et al., 1994). These findings provide evidence of transposition events in P. rhoeas between organellar and nuclear genomes.
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In order to assess the repetitive nature of the elements found within the
S1 clone, reverse Southern analysis was carried out (Fig. 2a). P. rhoeas S1,3 genomic DNA was digested with HindIII, labelled with 
32P-dCTP and hybridized to the S1 clone cut with a variety of enzyme combinations. This confirmed the highly repetitive nature of the sequence surrounding the S1 gene. Reverse Southern analysis also identified another region, bordering the opposite side of the clone to the tRNA gene, upstream of the Ty1/copia-like element, containing highly repetitive DNA. This region did not contain sequence of known homology to any previously identified repetitive elements. The distribution of repetitive sequence within the S1 clone is shown in Fig. 3. Finally, northern analysis identified hybridization of the border of the clone to a leaf-specific transcript (Fig. 2b). Sequence analysis of this part of the clone has failed to identify homology to any known genes or indeed an open reading frame, however, it is possible that only non-coding sequence of this transcript is present on the S1 clone. The fact that this transcript appears to be leaf-specific is of interest. Studies of the S-locus in Brassica spp. suggest that the two central components of the S-locus, SCR (S-locus cysteine rich) (SP11 in B. campestris) and SRK (S-receptor kinase) lie adjacent to each other with no intervening open reading frames (Suzuki et al., 1999; Schopfer et al., 1999). If this situation is repeated in the P. rhoeas S-locus then it is likely that the gene encoding the pollen component of the SI response lies beyond the tRNA gene at the end of the S1 clone. Chromosome walking beyond this border may thus lead to identification of pollen S. A map of the S1 clone is shown in Fig. 3.
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In order to ‘walk’ to the next clone in the library, screening was attempted using the ends of the clone as probe DNA. Unfortunately, due to the repetitive nature of the sequence several hundred positive clones were identified. To circumvent this problem inverse PCR (IPCR) was used to amplify flanking sequence using a method described by Ochman et al. (1988). Nested primers were designed between a HindIII site and the tRNA gene at the end of the clone in an area of sequence identified, by reverse Southern analysis, as likely to contain little repetitive sequence. Using the Expand Long Template PCR system (Roche), one set of 30 amplification cycles was carried out using each of the nested primer sets on ligated, HindIII cut genomic DNA. Amplification produced a band of
5.5 kb. The amplification product was cloned into pCR2.1 (Invitrogen) and sequencing of the ends of the clone confirmed it to be contiguous with the S1 clone. As well as 2 kb of sequence from the S1 clone the fragment contained 3.5 kb of adjacent sequence. Analysis of this sequence again failed to detect any potential receptor genes, however, a portion of the sequence again showed homology to reverse transcriptases of non-LTR retrotransposons and is similar to the repetitive element found downstream of S1. |
Fluorescence in situ hybridization (FISH) analysis of the P. rhoeas S-locus |
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TopAbstractIntroductionSequence of the Papaver...Fluorescence in situ...DiscussionReferences |
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FISH analysis of P. rhoeas chromosomes (2n=14) was attempted using metaphase chromosomes from root tip preparations and, in order to facilitate greater resolution of potential signals along P. rhoeas chromosomes, extended pachytene chromosomes. Metaphase chromosomes were prepared from root tips in which mitosis was interrupted by treatment with colchicine. Pachytene chromosome preparations were produced from anther meiocytes.
Anther preparations were examined microscopically to determine the presence of cells in pachytene. A drop spreading technique developed for the use of FISH on Arabidopsis thaliana chromosomes was employed (Armstrong et al., 1998). Briefly, anthers were washed in 0.01 M citrate buffer (0.01 M citric acid, 0.01 M sodium citrate) and then incubated at 37 °C in 0.3% pectolyase, 0.3% cytohelicase and 0.3% cellulase for 2 h. The anther was then tapped out with a fine needle on a glass slide. Three 15 µl aliquots of 60% acetic acid were then added and the slide heated at 45 °C for 20 s. Slides were then fixed in 100 µl 3:1 ethanol/acetic acid and dried. FISH was carried out using nick-translated DNA probes labelled with biotin or digoxygenin. Hybridization was accomplished by the addition of probe DNA to 71% deionized formamide, 3x SSC, 14% dextran sulphate. Results were detected by UV-microscopy using avidin-FITC or avidin-Cy3 for biotin-labelled probes or anti-digoxygenin-FITC or anti-rhodamine. Chromosomes were counter-stained with 4,6-diamidine-2-phenylindole (DAPI). Analysis was carried out using a Nikon T600 fluorescence microscope and software provided by Applied Imaging International.
Initial experiments used the entire S1 clone as a probe. This showed hybridization to all parts of the P. rhoeas genome other than centromeres and 18.26 S rDNA repeats, thus confirming the highly repetitive nature of the repeat sequences seen on reverse Southern analysis. The experiment was refined to use a 4 kb fragment containing S1 and the non-LTR retroposon-like sequence. Again, hybridization was noted at multiple loci spread uniformly across all chromosomes (Fig. 4a). However, the level of hybridization was significantly reduced compared to that found with the whole S1 clone. In order to reduce the background signals associated with hybridization to repetitive elements, two modifications were used. First, a 1.5 kb section of the S1 clone thought to contain little repetitive sequence was amplified by PCR, cloned into pCR2.1 (Invitrogen) and used to probe chromosome preps. Secondly, in order to block hybridization of probe to repetitive DNA, C0t-1 DNA from P. rhoeas (containing highly repeated sequence) was added. This was isolated according to the method of Zwick et al. (1997). On a hybridization experiment using this probe with metaphase spreads, one chromosome containing a telomeric 18.26 S rDNA repeat did give a reproducible signal. On this chromosome a signal proximal to the centromere was noted on three different chromosome preparations (Fig. 4b). However, other fluorescent loci were visible on the same chromosome and other chromosomes although their position was not repeated between different spreads. As such the locus proximal to the centromere appears at this stage to be the best candidate for the position of the S-locus in P. rhoeas. Hybridizations with this probe against pachytene chromosomes failed to localize the S-locus although the length of P. rhoeas chromosomes inevitably leads to overlying of chromosomes and thus makes analysis of signal position difficult. The potential significance of a possible centromeric location for the S-locus is discussed below.
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Discussion |
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TopAbstractIntroductionSequence of the Papaver...Fluorescence in situ...DiscussionReferences |
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Analysis of the sequence of the
S1 clone has shown no evidence of genes transcribed in pollen and thus no candidate for pollen-S in P. rhoeas is to be found in this clone. Findings in Brassica place the pollen and pistil components of the SI response adjacent to each other within the S-locus. The fact that there appears to be a leaf-specific transcript at one end of the S1 clone suggests that the S-locus would extend over the other end of the S1 clone if this situation was repeated in P. rhoeas. Due to the repetitive nature of the sequence of the IPCR contig fragment, screening of the library for overlapping clones is difficult at present. However, the findings do offer the possibility that, by a combination of isolating larger clones containing segments of the S-locus and chromosome walking into adjacent clones, the gene encoding pollen-S can be isolated. A cosmid library is presently being constructed to this end.
Repeated screening of the EMBL3 library failed to produce a clone containing part of the S3 locus. In order to compare the structure of S-loci and to determine whether heterogeneity of structure is important in maintenance of S-locus by recombination suppression it is essential that the sequences of other S-loci are deduced. The S1S3 cosmid library that is currently being constructed is envisaged to help resolve this. The availability of cosmid clones will enable both the analysis of larger fragments of the S-locus and a comparison of the structure of the S1 and S3 locus. However, insight into the structure of the locus in the immediate surroundings of the stigmatic S1 gene has been gained. Multiple repetitive elements are now known to surround this gene and the relevance of these findings to the functioning of the S-locus is discussed here.
The finding of repetitive elements surrounding the S1 gene in P. rhoeas reflects previous findings in the S-locus of both the Brassicaceae and Solanaceae. Cui et al. (1999) compared the sequence of a 65 kb contig from the 910 S-haplotype of Brassica napus with an 88 kb contig from S-haplotype A14. Although the region downstream of SLG was found to be highly colinear, the intervening region between SLG and SRK was found to vary greatly in size. It appears that this intergenic region has abundant retro-elements and S-haplotype specific genes. At least some of the repetitive elements had homology to Ty1/copia-like retro-elements, a finding that has been replicated in P. rhoeas. The region also contained an Athila-like retro-element. The region downstream of SRK was also found to be highly heterogeneous. Coleman and Kao (1992) examined genomic clones isolated from the S1 and S3 alleles of Petunia inflata. In both cases the sequences were found to contain repetitive elements although these were of unknown type. Again, the sequences were found to be highly heterogeneous. It is clear from analysis of the P. rhoeas S1 locus that within 8 kb either side of the stigmatic gene there are at least four elements of repetitive DNA. Either side of S1 lie truncated non-LTR retrotransposon-like elements. Downstream lies an element with homology to Ty1/copia gag/pol genes. The presence of frameshifts and stop codons indicates the likelihood of these sequences being remnants of ancient transposition events. Stop codons within these sequences are indeed a common occurrence (Brandes et al., 1997), for instance of 27 Ty1/copia elements analysed in Vicia faba 80% contained stop codons (Navarro-Quezada and Schoen, 2002). Upstream of S1 lies a trnS gene, probably derived from the insertion of a chloroplast tRNA transcript. Interestingly, regarding this last finding, Bernatzky et al. (1989) identified the presence of a mitochondrial-derived DNA in the S2 locus of Nicotiana alata. Whether or not the presence of these diverse elements within the P. rhoeas S-locus is significant is discussed below.
There appears to be at least some structural commonality between S-loci of the Solanaceae, Brassicaceae and the Papaveraceae. Both Cui et al. (1999) and Coleman and Kao (1992) hypothesize that the heterogeneity found between the S-loci they examined could be a factor in suppression of recombination within these loci. Heterogeneity between loci has been cited as a reason for a lack of recombination between alleles in other systems notably in the Adh1 locus of Zea mays (Sachs et al., 1986) and in the mating-type locus of Chlamydomonas reinhardtii (Ferris and Goodenough, 1994). More recent research suggests that the presence of repetitive DNA elements per se may be a factor in lowering recombination levels. Studies of the bronze locus in maize, found that the level of recombination varied by two orders of magnitude between adjacent genic and retrotransposon regions (Fu et al., 2002). This confirms previous findings that intragenic recombination rates are higher than the average for the genome (Brown and Sundaresan, 1991). The presence of higher levels of recombination within genes is proposed as an explanation for the similarities in genetic length between organisms where there are gross disparities in physical length. Thus the presence of retro-elements around the S1 gene of P. rhoeas may explain at least part of the mechanism behind suppression of recombination.
Does the presence of retro-elements in the S-loci of three independently evolved systems, imply function, or is it merely a reflection of the large amount of repetitive DNA sequence now known to be present in plant genomes? It is known that plant genomes contain multiple repetitive DNA sequences. Retro-elements have been found to represent up to 50% of nuclear DNA in some species (Bennetzen, 1996; San Miguel and Bennetzen, 1998). It is widely thought that the large differences in genome size are at least partially explained by differing amounts of repetitive elements (Flavell et al., 1974). Also, it has been observed that retro-elements preferentially insert into the 5' control regions of existing plant genes (White et al., 1994). Thus, the presence of repetitive elements around the P. rhoeas S-locus may be entirely expected. Whether the S-loci of the Papaveraceae are heterogeneous as in the Brassicaceae and Solanaceae remains to be determined when the sequences of other S-loci are examined. The heterogeneity found between S-loci is an interesting result and may well play a role in recombination suppression. However, it must be borne out that, quite apart from heterogeneity being important in preventing recombination, heterogeneity will almost certainly be seen simply as a result of a lack of recombination between S-loci. In any case, the findings of Fu et al. (2002) suggest that heterogeneity may not be required for recombination suppression given the presence of retrotransposons at a locus.
Attempts have been made to use FISH to locate the position of the S-locus in the Solanaceae (ten Hoopen et al., 1998; Entani et al., 1999). ten Hoopen et al. (1998) used lines of Petunia hybrida that had been shown to carry T-DNA inserts genetically linked to the S-locus (ten Hoopen et al., 1996). The T-DNA inserts most tightly linked to the S-locus were found to localize to a subcentromeric position. The position of the S-locus at this point concurs with the phenomenon, previously observed by Brewbaker and Natarajan (1960), that mutants carrying centric fragments have an additional S-specificity. Entani et al. (1999), using FISH with a probe derived from the region around SB1, confirmed the position of the S-locus near to the centromere of P. hybrida chromosome III. In fact, the presence of a centromere-specific sequence within a larger clone indicates that the S-locus may be as little as 12 kb from the centromere itself.
An attempt to localize the S-locus in P. rhoeas using FISH was made. The presence of repetitive elements adjacent to the S-locus makes this approach challenging. The 450 bp stigmatic S-gene is single copy but the size of the gene makes its use as a probe untenable. To circumvent this problem studies localising the S-locus in the Solanaceae have used T-DNA tagged lines where linkage between the tag and S-genotype has been shown (ten Hoopen et al., 1998), or larger fragments around the S-RNase gene (Entani et al., 1999). The first of these methods in P. rhoeas is not viable due to an absence of tagged lines. Using the second of these approaches, the lack of information in the databases regarding repetitive elements in the Papaveraceae means there are difficulties in predicting where the borders of some elements lie and thus probes designed around the S-locus are liable to contain at least some repetitive DNA. This inevitably means that resolution of single signals is difficult. This problem is exemplified by the finding on reverse Southern analysis of a potential repetitive element at the border of the S1 clone even though the sequence at this point shows no evidence of homology to known transposable elements. To circumvent this problem a method was used that combined both using the area solely around the S-gene together with the use of non-labelled highly repeated DNA as a block.
The image in Fig. 4a vividly illustrates the repetitive nature of the elements found at the P. rhoeas S-locus. A reduction in the amount of background annealing was obtained both by using C0t-1 DNA as a block and by designing the probe outside the obvious repetitive regions of the S1 clone. Unfortunately, the hybridizations failed to resolve into single signals consistently, although a reproducible signal was noted on one of the chromosomes containing a telomeric 18.26 S rDNA repeat. This position of the signal must as yet be taken as provisional. Resolution of this issue will only take place by co-localizing signals derived from further sequence around the S1 locus. The observation of consistent signals in a chromosome containing 18.26 S rDNA repeats is also interesting in view of work accomplished by Copenhaver et al. (1998). In this work it was found that chromosomes carrying ribosomal DNA arrays exhibited considerably reduced levels of recombination. To summarize then, these results at least do not rule out that the S-locus in P. rhoeas is situated near to a centromere. Thus it remains possible that chromosomal position may play a role in the maintenance of the S-locus of the Papaveraceae.
The conclusions to this work are that there do appear to be some similarities between the P. rhoeas S-locus and those of the Brassicaceae and Solanaceae. The presence of transposable elements reiterates the findings of research into the other SI systems. There is also some evidence to support the findings in the Solanaceae, which point to a positional arrangement of the S-locus affecting recombination. Further investigation should give information on structural differences between S-loci in the Papaveraceae. As well as this sequencing around the S-locus in P. rhoeas may lead to the identification of the pollen component of the response.
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References |
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Armstrong SA, Fransz P, Marshall DM, Jones GH. 1998. Physical mapping of DNA repetitive sequences to mitotic and meiotic chromosomes of Brassica oleracea var. alboglatra by fluorescence in situ hybridization. Heredity 81, 666–673.[CrossRef]
Bennetzen JL. 1996. The contribution of retro-elements to plant genome organization, function and evolution. Trends in Microbiology 4, 347–353.[CrossRef][Web of Science][Medline]
Bernatzky R, Mau S-L, Clarke AE. 1989. A nuclear sequence associated with self-incompatibility in Nicotiana alata has homology with mitochondrial DNA. Theoretical and Applied Genetics 77, 320–324.[CrossRef]
Boyes DC, Nasrallah JB. 1993. Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea. Molecular and General Genetics 236, 369–373.
Boyes DC, Nasrallah ME, Vrebalov J, Nasrallah JB. 1997. The self-incompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin. The Plant Cell 9, 237–247.[Abstract]
Brandes A, Heslop-Harrison JS, Kamm A, Kubis S, Doudrick RL, Schmidt T. 1997. Comparative analysis of the chromosomal and genomic organization of Ty1-copia-like retrotransposons in pteridophytes, gymnosperms and angiosperms. Plant Molecular Biology 33, 11–21.[CrossRef][Web of Science][Medline]
Brewbaker JL, Natarajan AT. 1960. Centric fragments and pollen-part mutations of incompatibility alleles in Petunia. Genetics 45, 699–704.
Brown J, Sundaresan V. 1991. A recombination hotspot in the maize A1 intragenic region. Theoretical and Applied Genetics 81, 185–188.[CrossRef]
Casselman AL, Vrebalov J, Conner JA, Singhal A, Giovannoni J, Nasrallah ME, Nasrallah JB. 2000. Determining the physical limits of the Brassica S locus by recombinational analysis. The Plant Cell 12, 23–33.
Coleman CE, Kao T-H. 1992. The flanking regions of two Petunia inflata S-alleles are heterogeneous and contain repetitive sequences. Plant Molecular Biology 18, 725–737.[CrossRef][Web of Science][Medline]
Copenhaver GP, Browne WE, Preuss D. 1998. Assaying genome-wide recombination and centromere functions with Arabidopsis tetrads. Proceedings of the National Academy of Sciences, USA 95, 247–252.
Cui Y, Brugière N, Jackman L, Bi Y, Rothstein SJ. 1999. Structural and transcriptional comparative analysis of the S-locus regions in two self-incompatible Brassica napus lines. The Plant Cell 11, 2217–2231.
de Nettancourt D. 1977. Incompatibility in angiosperms. New York: Springer-Verlag.
Deragon J-M, Landry BS, Pelissier T, Tutois S, Tourmente S, Picard G. 1994. An analysis of retroposition in plants based on a family of SINEs from Brassica napus. Journal of Molecular Evolution 39, 378–386.[CrossRef][Web of Science][Medline]
Entani T, Iwano M, Shiba H, Takayama S, Fukui K, Isogai A. 1999. Centromeric localization of an S-RNase gene in Petunia hybrida Vilm. Theoretical and Applied Genetics 99, 391–397.[CrossRef]
Ferris PJ, Goodenough UW. 1994. The mating type locus of Chlamydomonas reinhardtii contains highly rearranged DNA sequences. Cell 76, 1135–1145.[CrossRef][Web of Science][Medline]
Flavell RB, Bennett MD, Smith JB, Smith DB. 1974. Genome size and proportion of repeated nucleotide sequence DNA in plants. Biochemical Genetics 12, 257–269.[CrossRef][Web of Science][Medline]
Foote HCC, Ride JP, Franklin-Tong VE, Walker EA, Lawrence MJ, Franklin FCH. 1994. Cloning and expression of a distinctive class of self-incompatibility (S-) gene from Papaver rhoeas L. Proceedings of the National Academy of Sciences, USA 91, 2265–2269.
Franklin-Tong VE, Hackett G, Hepler PK. 1997. Ratio-imaging of Ca2+i in the self-incompatibility response in pollen tubes of Papaver rhoeas. The Plant Journal 12, 1375–1386.[CrossRef]
Franklin-Tong VE, Ride JP, Franklin FCH. 1995. Recombinant stigmatic self-incompatibility (S-) protein elicits a Ca2+ transient in pollen of Papaver rhoeas. The Plant Journal 8, 299–307.[CrossRef]
Franklin-Tong VE, Ride JP, Read ND, Trewawas AJ, Franklin FCH. 1993. The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium. The Plant Journal 4, 163–177.
Fu H, Zheng Z, Dooner HK. 2002. Recombination rates between adjacent genic and retrotransposon regions in maize vary by two orders of magnitude. Proceedings of the National Academy of Sciences, USA 99, 1082–1087.
Hearn MJ, Franklin FCH, Ride JP. 1996. Identification of a membrane glycoprotein in pollen of Papaver rhoeas which binds stigmatic self-incompatibility (S-) proteins. The Plant Journal 9, 467–475.[CrossRef]
ten Hoopen R, Harbord RM, Maes T, Nanninga N, Robbins TP. 1998. The self-incompatibility (S) locus in Petunia hybrida is located on chromosome III in a region, syntenic for the Solanaceae. The Plant Journal 16, 729–734.[CrossRef]
ten Hoopen R, Robbins TP, Fransz PF, Montijn BM, Oud JL, Gerats AGM, Nanninga N. 1996. Localization of T-DNA insertions in Petunia hybrida by fluorescent in situ hybridization: physical evidence for a suppression of recombination. The Plant Cell 8, 823 830.
John B. 1990. Meiosis. Cambridge: Cambridge University Press.
Jordan ND, Kakeda K, Conner A, Ride JP, Franklin-Tong VE, Franklin FCH. 1999. S-protein mutants indicate a functional role for SBP in the self-incompatibility response of Papaver rhoeas. The Plant Journal 20, 119–125.[CrossRef][Web of Science][Medline]
Jordan ND, Franklin FCH, Franklin-Tong VE. 2000. Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas. The Plant Journal 23, 471–479.[CrossRef][Web of Science][Medline]
Kurup S, Ride JP, Jordan N, Fletcher G, Franklin-Tong VE, Franklin FCH. 1998. Identification and cloning of related self-incompatibility S-genes in Papaver rhoeas and Papaver nudicaule. Sexual Plant Reproduction 11, 192–198.[CrossRef]
Matton DP, Mau S-L, Okamoto S, Clarke AE, Newbigin E. 1995. The S-locus of Nicotiana alata: genomic organization and sequence analysis of two S-RNase alleles. Plant Molecular Biology 28, 847–858.[CrossRef][Web of Science][Medline]
McClure BA, Haring V, Ebert PR, Anderson MA, Simpson RJ, Sakiyama F, Clarke AE. 1989. Style self-incompatibility gene products of Nicotiana alata are ribonucleases. Nature 342, 955–957.[CrossRef][Medline]
Navarro-Quezada A, Schoen DJ. 2002. Sequence evolution and copy number of Ty1-copia retrotransposons in diverse plant genomes. Proceedings of the National Academy of Sciences, USA 99, 268–273.
Ochman H, Gerber AS, Hartl DL. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623.
Sachs MM, Dennis ES, Gerlach WL, Peacock WJ. 1986. Two alleles of maize alcohol dehydrogenase 1 have 3' structural and poly(A) addition polymorphisms. Genetics 113, 449–467.
San Miguel P, Bennetzen JL. 1998. Evidence that a recent increase in maize genome size was caused by the massive amplification of intergene retrotransposons. Annals of Botany 82, 37–44.
Schopfer CR, Nasrallah ME, Nasrallah JB. 1999. The male determinant of self-incompatibility in Brassica. Science 286, 1697–1700.
Shraga-Levine Z, Sokolovsky M. 1998. Functional role for glycosylated subtypes of rat endothelial receptors. Biochemical and Biophysical Research Communications 246, 495–500.[CrossRef][Web of Science][Medline]
Snowman BN, Geitmann A, Clarke SR, Staiger CJ, Franklin FCH, Emons AMC, Franklin-Tong VE. 2000. Signalling and the cytoskeleton in pollen tubes of Papaver rhoeas. Annals of Botany 85A, 49–57.
Suzuki G, Kai N, Hirose T, Fukui K, Nihio T, Takayama S, Isogai A, Watanabe M, Hinata K. 1999. Genomic organization of the S-locus: identification and characterization of genes in the SLG/SRK region of S9 haplotype of Brassica campestris (syn.) rapa. Genetics 153, 391–400.
Walker EA, Ride JP, Kurup S, Franklin-Tong VE, Lawrence MJ, Franklin FCH. 1996. Molecular analysis of two functional homologues of the S3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations. Plant Molecular Biology 30, 983–994.[CrossRef][Web of Science][Medline]
Wheeler MJ, Franklin-Tong VE, Franklin FCH. 2001. The molecular and genetic basis of pollen–pistil interactions. New Phytologist 151, 565–584.[CrossRef]
White SE, Habera LF, Wessler SR. 1994. Retrotransposons in the flanking regions of normal plant genes: a role for copia-like elements in the evolution of gene structure and expression. Proceedings of the National Academy of Sciences, USA 91, 11792–11796.
Yu KF, Schafer U, Glavin TL, Goring DR, Rothstein SJ. 1996. Molecular characterization of the S-locus in two self-incompatible Brassica napus lines. The Plant Cell 8, 2369–2380.[Abstract]
Zwick MS, Hanson RE, McKnight TD, Islam-Faridi MN, Stelly DA, Wing RA, Price HJ. 1997. A rapid procedure for the isolation of C0t-1 DNA from plants. Genome 40, 138–142.
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