Annals of Botany 85 (Supplement A): 105-112, 2000 doi: 10.1006/anbo. 1999.1062, available online at http://www.idealibrary.com on ID E Lo

The Molecular Genetics of Self-incompatibility in Petunia hybrid

T. P. ROBBINS*, R. M. HARBORD, T. SONNEVELD and K. CLARKE Plant Science Division, School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK Downloaded from https://academic.oup.com/aob/article/85/suppl_1/105/102776 by guest on 29 September 2021 Received: 21 July 1999 Accepted: 20 October 1999

The cultivated petunia (Petunia hybrida) has been a popular system in which to study genetic, physiological and biochemical aspects of gametophytic self-incompatibility. As with other members of the Solanaceae a number of S-RNase genes have been isolated for functional S-alleles. We have identified S-RNase sequences for two additional functional S-alleles, S and S3. These alleles are more similar to alleles from other families of the Solanaceae (Nicotiana and Solanum) than to any petunia alleles reported previously. The total number of S-alleles in P. hybrida is at least ten in spite of its cultivated origin. However, most cultivars of P. hybrida are in fact self-compatible and this appears to arise from the prominence of a single previously described allele SO. The implications of this observation for the origin of self-compatibility in P. hyhrida are discussed. The S-locus of P. hybrida has recently been mapped using an indirect method involving T-DNA insertions. Seven T-DNA insertions that were previously shown to be closely linked to the S-locus were physically mapped on the long arm of chromosome III using fluorescent in-situ hybridization. The most tightly linked T-DNA insertions are in a sub-centromeric position. This is consistent with the centric fragments of P. inflata obtained by irradiation mutagenesis that carry additional S-loci and confer a pollen- part mutant phenotype. An S-linked restriction fragment length polymorphism (RFLP) marker, CP100 was used to confirm this chromosomal assignment and has provided evidence for S-locus synteny in the Solanaceae. © 2000 Annals of Botany Company

Key words: Petunia hybrida, petunia, self-incompatibility, self-compatibility, S-RNase, PCR, T-DNA.

INTRODUCTION (Linskens and Straub, 1978; Ascher, 1984; this paper) the predominant condition is self-compatibility with abundant Over the past 30 years the cultivated petunia, Petunia seed set on self-pollination. hybrida, has contributed to many aspects of self-incompati- As with all other solanaceous species studied to date, the bility research. The large flowers are amenable to controlled self-incompatibility in P. hybrida is gametophytic with a pollinations and provide sufficient anther and pistil material single multiallelic S-locus. Polymorphic pistil proteins were for biochemical analyses. The contributions of P. hybrida to found to be associated with the S-alleles originally described both genetic and biochemical studies have been reviewed by Linskens and Straub (Kamboj and Jackson, 1986). These previously (Ascher, 1984). In this review, recent molecular were subsequently purified and N-terminal sequences were genetic studies will be discussed in relation to the earlier obtained for three alleles Sl, S2 and S3 (Broothaerts et al., findings. The contribution of P. hybrida will be placed in the 1989). These sequences showed homology with the Cl context of the major advances in our understanding of conserved domain of pistil S-proteins previously identified S-ribonuclease (S-RNase) based incompatibility systems in in Nicotiana alata and Lycopersicon peruvianum (Ioerger other Solanaceae, and in particular the wild relative et al., 1991). These proteins were subsequently shown to P. inflata. have ribonuclease activity (Broothaerts et al., 1991) similar P. hybrida is believed to have been derived in the early to that reported for the pistil S-proteins of N. alata part of the last century from hybridization of two or more (McClure et al., 1989). These pistil S-proteins therefore wild petunia species (Sink, 1984). Molecular fingerprinting show all the properties anticipated of S-ribonucleases (Peltier et al., 1994; Cerny et al., 1996) has supported the (S-RNases) that have now been extensively characterized view that major contributions were from a purple flowered in the Solanaceae (reviewed in Kao and McCubbin, 1996). species (probably P. integrifolia) and a white flowered The first S-RNase cDNA sequences reported in P. hybrida species (P. axillaris or P. parodii). The former species are were obtained by screening cDNA libraries with an invariably self-incompatible outbreeders whereas the latter oligonucleotide probe based on a conserved region (Clark species can be either self-incompatible or self-compatible et al., 1990). They reported S-RNase sequences for three S , from an (Tsukamoto et al., 1999). Although a number of self- alleles also named, S,, S2 and 3 but obtained source (Ascher, 1984). A comparison of the incompatible cultivars of P. hybrida have been identified independent putative mature S-RNase sequences encoded by these

* For correspondence. Fax +44 (0) 115-951-6334, e-mail tim. alleles confirms that they are distinct from the Linskens robbins(a'nottingham.ac.uk alleles previously characterized by Broothaerts (Fig. 1). In 0305-7364/00/0A0105 + 08 $35.00/00 © 2000 Annals of Botany Company 106 Robbins et al.-Self-incompatibility in Petunia Allele Source N-terminal sequence approaches (Lee, Huang and Kao, 1994) is consistent with the view that the pollen determinant of self-incompati- * ******* bility (pollen-S) is controlled by a separate, as yet S1 Ascher SFDHWQLVLTWPAGYCKVKG unidentified, gene at the S-locus. S2 Ascher NFDYFQLVLTWPASFCYPKN S3 Ascher NFDYFQLVLTWPASFCYPKN IDENTIFICATION OF TWO NOVEL S1 (SiL) Linskens DFDYMQLVLTWPASFXYRPR S-RNASES IN SELF-INCOMPATIBLE P. HYBRIDA Linskens S2 (S2L) YFEYMQLVLTWPPAFXHIKX We have studied two self-incompatible petunia cultivars, S3 (S3L) Linskens EFELLQLVLTWPASFXYANH one (T2U) homozygous for the S3 allele (Linskens and Ai at al. DFDYMQLVLTWPASFCYRPR Straub, 1978), the other cultivar (V13) homozygous for a Downloaded from https://academic.oup.com/aob/article/85/suppl_1/105/102776 by guest on 29 September 2021 S.5. Ai et al. AFDHWQLVLTWPAGYCKIKG previously uncharacterized allele provisionally called Sv (Harbord, Napoli and Robbins, 2000). To further charac- Broothaerts AFDHWQLVLTWPAGYXKIKG terize these alleles a degenerate PCR cloning procedure was this paper YFEYMQLVLTWPPAFCHIKR adopted to identify S-RNase sequences expressed in pistils. FIG. 1. A comparison of N-terminal sequences reported for S-RNase A degenerate primer based on the C2 conserved domain of genes of P. hybrida. The N-terminal sequences of the Linskens alleles S-RNases from the Solanaceae (loerger et al., 1991) was reported by Broothaerts et al. (1989) are aligned with those of the used to amplify partial S-RNase sequences by RT-PCR of Ascher alleles derived from cDNA sequences (Clark et al., 1990). The amino acids conserved between the Cl domain of all the Solanaceae pistil RNA (Xue et al., 1996). Several identical cloned PCR S-RNases (loerger et al., 1991) are indicated with asterisks. Also products with homology to other S-RNases were obtained included are the S. and SO alleles reported by Ai et al. (1992), the Sb for each allele. allele reported by Broothaerts et al. (1991) and the S sequence Confirmation that these cDNA sequences corresponded reported here. Allele names in parentheses are those proposed to avoid future confusion. to the Sv and S3 RNases was obtained by designing allele specific primers based on the hypervariable domain and the 3' untranslated region. These were used for genomic PCR addition they are all distinct from the three S-RNase analysis of a small family that segregated for both alleles sequences reported for the wild species P. inflata (Ai et al., (Fig. 2). Allele specific PCR products were found to 1990). Two additional S-RNase sequences have been cosegregate perfectly with the S-genotypes determined by reported in P. hybrida, S and S, the former of which test pollination. In a more extensive analysis of 90 progeny appears to be non-functional (Ai, Kron and Kao, 1991; Ai, from the same cross, the two allele specific PCR products Tsai and Kao, 1992). There may be as many as ten distinct segregated as alleles of the same locus (Clarke and Robbins, S-alleles in P. hybrida cultivars suggesting a fairly complex data not shown). These findings suggest that the cloned breeding history involving more than two parent indivi- sequences represent functional alleles and not S-like duals. RNases that are unlinked to the S-locus such as X2 in Expression analysis in P. hybrida at the protein level P. inflata (Lee, Singh and Kao, 1992). Additional allele showed that the Linskens S2 glycoprotein accumulates in specific primers were subsequently used to isolate full the top half of the pistil during the latter stages of floral bud coding region sequences by 5' rapid amplification of expansion (Broothaerts et al., 1989). The accumulation of cDNA ends-polymerase chain reaction (5'RACE-PCR) as corresponding mRNAs was studied using the Ascher SI-S3 described by Xue et al. (1996). alleles by Clark et al. (1990). They showed that all three The full amino acid sequences predicted for the S3 and Sv alleles were highly expressed in styles but only weakly in S-RNase precursors are aligned in Fig. 3. Both S-RNase ovaries. The accumulation of stylar mRNA is consistent sequences contain the conserved domains Cl-C5 typical with the onset of self-incompatibility as revealed previously for S-RNase sequences from the Solanaceae (loerger et al., in Nicotiana alata (Cornish et al., 1987). As in other 1991). The S sequence is most closely related to S7 from Solanaceae, it is possible to overcome self-incompatibility Nicotiana alata (67% identity) and the most similar allele in by pollinating immature pistils, before the S-RNase has P. hybrida is S (51% identity; Ai et al., 1992). The S3 accumulated to sufficient levels, and this allows the genera- sequence is most closely related to S from Solanum tion of homozygous S-allele stocks. chacoense (66% identity) and the most similar allele in In addition to the anticipated expression in the pistil, P. hybrida is again S, (58% identity). The higher level very low levels of expression were detected using reverse of interspecific rather than intraspecific similarity is an transcriptase-polymerase chain reaction (RT-PCR) in established feature of many S-RNase sequences in the immature anthers for the S but not the S3 RNase (Clark Solanaceae (Ioerger, Clark and Kao, 1990). and Sims, 1994). These findings are similar to those in The predicted amino acid sequence of S3 can be compared N. alata (Dodds et al., 1993), and recent studies in Lycoper- with the N-terminal sequence for the purified S3 protein sicon peruvianum call into question the relevance of these published previously (Fig. 1). There is full agreement low levels of expression for self-incompatibility (Dodds between the cDNA and protein sequence for all 19 amino et al., 1999). The pollen-part mutations described in acid positions that were determined unambiguously P. infiata (Brewbaker and Natarajan, 1960) and the inability (Broothaerts et al., 1989). The predicted molecular weight to alter pollen phenotype using S-RNase transgenic (22.1 kD) is in approximate agreement with that reported Robbins et al.-Self-incompatibility in Petunia 107 Downloaded from https://academic.oup.com/aob/article/85/suppl_1/105/102776 by guest on 29 September 2021

FIG. 2. S-RNase allele specific PCR products cosegregate with S-phenotypes in P. hybrida. A small family of plants segregating for the S3 and Sv alleles were generated by crossing an SS, stock with pollen from an S3S stock. The progeny were genotyped by test pollination with the S3Sb stock and found to segregate 1: 1 for SSb and SVSb as anticipated. Progeny determined to carry the S3 or Sv allele are indicated together with the results obtained from two different allele specific genomic PCR reactions. A 500 bp product from Sv allele specific PCR was found to cosegregate with the Sv phenotype (upper panel) and a 450 bp product from the S allele specific PCR with the S phenotype (lower panel). Methods: Genomic DNA was prepared from 11 genotyped plants as described elsewhere (Harbord et al., 2000) and approx. 30-50 ng was used for genomic PCR. Genomic PCR was carried out in a manufacturer's buffer with 3 mM MgCI2, 02 mM dNTPs and 0.5 units Taq polymerase (Bioline UK, Ltd) for 35 cycles of: 94°C 30 secs, specified annealing temperature 30 secs, 72°C 60 secs. The S allele specific primers (V13-F1 5' GGACGAAGCTGATTGTAAGGG and V13-R1 5' CGATTTTCATATATTGGC) were used at 0-1 M and 45°C annealing temperature. The S3 allele specific primers (PhS3-F1 5' CACTACATTCGCGGGTAAGATGCTC and PhS3-RI 5' CGCATGTATCACTTTGACGACAGG) were used at 0.2 IM and 60°C annealing temperature.

previously after deglycosylation (25.1 kD; Broothaerts et al., in this report although they are presumably derived from 1991). The Sv allele was known to be functionally distinct distinct cultivars. from S3 and Sb in test pollinations but it was not clear how it was related to other previously described P. hybrida S-alleles. Based on the predicted sequence of the Cl region MOLECULAR BASIS OF SELF-COMPATIBILITY SUGGESTS A it is possible that Sv is equivalent to the S2 allele from Linskens allowing for some ambiguous amino acid COMMON ORIGIN sequences. Unfortunately we have not been able to obtain In spite of its contribution to self-incompatibility research, any S2 stocks and we prefer to identify S as a novel most cultivars of P. hybrida are in fact self-compatible. Self- S-RNase until shown otherwise. It is worth pointing out compatibility in P. hybrida has been studied by Ascher and that the S2 and S 3 alleles of Ascher also appear identical in colleagues and has been reviewed previously (Ascher, 1984). this region yet they are known to be functionally distinct Ascher uses the term pseudo-self-compatibility (PSC) to (Dana and Ascher, 1986) and have different cDNA describe both physiological and genetic causes for a break- sequences (Clark et al., 1990). down in self-incompatibility. In some cases there is complex To avoid future confusion between the S-S 3 alleles polygenic control and intermediate levels of seed set are described by Linskens and Straub (1978) and the S-S 3 observed. In at least one case it has been shown that PSC is alleles from Ascher that were sequenced by Clark et al. not a result of reduced expression of an S-RNase (Clark (1990) it seems desirable to introduce a suffix, L, as shown et al., 1990). Ascher also describes lines with 100% PSC or in Fig. 1. Since this report contains the first full length rather full self-compatibility and in one case this arises from sequence for one of Linsken's alleles we propose that this a loss of pollen function (Dana and Ascher, 1986). allele should subsequently be called S3L. It is also Interestingly, this line seems to lack a centric fragment important in any sequence comparisons to distinguish the which is characteristic of the pollen-part mutants arising P. hybrida alleles from the structurally distinct alleles of from duplications described in Petunia inflata (Brewbaker P. inflata that are also called SI-S3 (Ai et al., 1990). During and Natarajan, 1960). The mutation appears to be tightly, the preparation of this manuscript two additional P. hybrida but not absolutely, linked to the S-locus. S-RNase sequences were submitted to the European Mole- The numerous self-compatible cultivars of P. hybrida cular Biology Laboratory (EMBL) database (accession developed for commercial and research purposes offer a numbers AB016522, AB016523). These amino acid diverse source of self-compatible stocks. In a previous study sequences are identical to the S,v and S3L alleles described a commercial F hybrid was crossed to self-incompatible 108 Robbins et al.-Self-incompatibility in Petunia ----C1----- C C - Sv MFRSQLTSVIFVLFFSLPPIYGYFEYMQLVLTWPPAFCHIKRCRR-TPNN 50 11 IIIII II I11 11 1111111 I11 III S3L MFKSQLTSALFVVLFFLSPTYGEFELLQLVLTWPASFCYANHCERIAPNN

---C2--- C------HVa------HVb Sv FTIHGLWPDNYSTMLNFCTDD-EFVKFTDDDKKDKLDKRWPDLITDEADC 100 I1111111111I I III I I II S3L FTIHGLWPDNVTIRLQYCKPKPTYTTFAGK-MLNDLDKHWIQLKYKEAYA

--C3-- ---C4--- Sv KGTQDFWKREYEKHGTCCLSSYNQEQYFELAMVLKDRFDLVKSFRNHGII 150 Downloaded from https://academic.oup.com/aob/article/85/suppl_1/105/102776 by guest on 29 September 2021 I 11 I I I I 11 I I 11 11 1 I I I I I I 1 S3L RREQPTWKYQYQKHGSCCQTKYKQIPYFSLALRLKDRFDLLTTLRTHHIV

C --C5-- Sv, PGTAGHTVQKINNTVKAITQGFPNLACTK ----ALELKEIGICFDRTGKN 200 I I 11 I I I I II 11111 S3L PGSS-YTFDDIFDAVKTVTQMNPDLKCTEVTKGTQELDEIGI CFTPKADK

C C S VINCPHPRTCKQTRTGIKFP 214 aa I 11 I1 S3L MFPCRQSDTCEKTRKILFRG 218 aa

FIG. 3. Full amino acid sequences of two previously unreported P. hybrida S-RNase sequences. The full precursor protein sequences of the Sv and S3L S-RNases are shown based on cDNA sequences. The amino terminus of the mature S3 protein is underlined (see Fig. I). The five conserved domains for S-RNases from the Solanaceae (CI-C5), the two major hypervariable domains (HVa and HVb) and several conserved cysteine (C) residues are indicated (loerger et al., 1991). Vertical lines show positions that are identical between the two P. hybrid S-RNase sequences. Methods: Total pistil RNA was prepared from S-allele homozygotes maintained by bud selfing and used for 3'RACE-PCR using the method and C2 degenerate primer described by Xue et al. (1996). PCR products were cloned into the TA cloning vector (Invitrogen) and clones with homology to S-RNases identified. To obtain full length sequences the 5'RACE-PCR procedure was used with a commerical kit (Bethesda Research Laboratories) and allele specific primers (sequences available on request). All DNA sequences are based on at least three independent clones and analysed using the DNAstar software and the Clustal alignment method (Madison, USA). The DNA sequences have been submitted to the 2 EMBL database under the accession numbers AJ271062(Sv) and AJ 71065(S3 L).

P. inflata to test the genetic basis of the self-compatibility were self-compatible and the remainder were either fully (Ai et al., 1991). Two distinct S-alleles derived from the self-incompatible (27.7%) or partially incompatible P. hybrida parent, S, and So, were identified in progeny by (20.0%) setting intermediate levels of seed (Harbord et al., SDS-PAGE analysis. All progeny carrying the S allele 2000). The latter class may be equivalent to the pseudo-self- were self-compatible whereas progeny carrying the S, allele compatible types described previously by Ascher (1984). were either self-compatible or self-incompatible. This Intermediate levels of seed set were also observed in the suggested that the S allele is non-functional and the Sx progeny of P. hybrida x P. inflata crosses that carry the S, allele is conditionally functional perhaps due to the allele (Ai et al., 1990). segregation of modifier genes. Subsequently cDNA clones To analyse the segregation of S-alleles in this backcross were obtained for both S-RNases (Fig. 1) and both alleles two approaches were utilized. The first approach was were shown to have ribonuclease activity (Ai et al., 1992). indirect, using an S-linked RFLP marker CP100 which We have obtained similar results in crosses between the suggested that all self-incompatible progeny were homo- self-incompatible cultivar V13 (SvSv) and a self-compatible zygous for the S-linked allele of CPI00 (Harbord et al., inbred cultivar V26 (Harbord, Napoli and Robbins, 2000). 2000). This suggested that the S allele may be non- The self-compatible stock was assumed to be homozygous functional and this was tested further by the cloning of an for a non-functional S-allele, termed Sc = self-compatible. S-RNase from the V26 line (Robbins, unpubl. res.). Using An F hybrid was generated and found to be self- the same approach described above for the functional compatible but incompatible with pollen from the V13 alleles S and S3, 3'RACE-PCR was used to amplify parent. This indicated that the Sv allele is functional in the sequences that contain the conserved C2 domain from pistil pistil of these F plants. Using the early bud pollination RNA of the self-compatible V26 cultivar. Three indepen- method this F hybrid was backcrossed to the self- dent clones with S-RNase homology were identical in incompatible parent V13 and 65 backcross individuals sequence to the previously published SO allele (Ai et al., were tested for self-compatibility. It was anticipated that 1992). Allele specific primers were designed based on the half of these backcross progeny would be homozygous SvS v full length published S sequence and found to amplify and half heterozygous SvS c . Approximately half (52.3%) identical size bands in So and S (data not shown). These Robbins et al.-Self-incompatibility in Petunia 109 Downloaded from https://academic.oup.com/aob/article/85/suppl_1/105/102776 by guest on 29 September 2021

FIG. 4. An SO allele-specific PCR product cosegregates with self-compatibility in the backcross ScSv x SvS v . A family of 65 plants was generated from a (V13 x V26) x V13 backcross and tested for self-incompatibility (see Harbord et al., 2000). This family should segregate for the S. allele of V26 and a selection of 12 progeny that were unambiguously self-incompatible (SI) or self-compatible (SC) were tested for the presence of the So(So) allele by genomic PCR. The expected 800bp product was detected in a V26 control and all SC progeny. Methods: Genomic PCR was carried out as decribed in Fig. 3 except that the primers were used at 0-2 gM and an annealing temperature of 57°C. The primers used (SoF2: 5'

GGTTATTGCAAAATTAAGGG and V26-R5: 5' ATGTTCTCTCTTCGAAGTTCGCG) were based on the published 5'UTR sequence of SO (Ai et al., 1992) and the 3'UTR of So(S) from V26 (Robbins, unpubl. res.). primers were used to monitor the segregation of the S, allele of the parental species, is frequently self-compatible in the in the backcross progeny described above. All the fully self- wild (Tsukamoto et al., 1999). This is in contrast to incompatible progeny were Sv/S v whereas the fully self- P. integritblia which has been proposed as the other major compatible progeny were Sv/S o (Fig. 4). The partially species contributing to P. hybrida (Peltier et al., 1994; Cerny self-incompatible progeny were Sv/S v (data not shown) et al., 1996), which is an obligate outbreeder. It will be which was consistent with the genotyping using the CP100 interesting to see whether the SO allele is present in any of marker (Harbord et al., 2000). This confirms that the SO these natural populations of P. axillaris. An alternative allele is non-functional as reported by Ai et al. (1990), and possibility is that the self-compatibility arose as a result of suggests that unlinked modifiers are also segregating that hybridization. Some evidence to support this view is that weaken the self-incompatibility in the Sv/S v homozygotes. the SO allele is expressed at high levels, has all the structural The finding that the same SO allele is present in two features of an S-RNase, and most significantly has been apparently unrelated commercial stocks prompted us to shown to have ribonuclease activity (Ai et al., 1992). These conduct a survey of commercial cultivars and research findings are consistent with the view that the S-RNase of stocks. Using the allele specific primers designed for SO we the SO allele is fully functional, and whilst RNase function obtained genomic PCR products of the expected size for is not the sole prerequisite for stylar expressed S-gene 12 out of 15 commercial F i hybrids tested and 13 out of function in the Solanaceae, the self-compatibility is most 16 research stocks obtained from the Free University of likely to result from a mutation affecting pollen-S function. Amsterdam (data not shown). This suggests that the SO A similar situation may apply in Nicotiana sylvestris allele is present in approx. 80% of existing research stocks which is exceptional amongst self-compatible Nicotiana and commercial cultivars and is probably a major species in having an apparently functional S-RNase (Golz contributor to the self-compatibility observed in many P. hybrida varieties propagated today. However, it cannot be excluded that modifier loci are also present and prelimin- Petunia axillaris X Petunia integrifolia? ary data suggest that at least two inbred lines other than Sl or SC? Sl V26 (W80 and W1 15) show backcross segregations consistent with modifiers and polygenic control (Robbins and Harbord, unpubl. res.). The self-compatibility of V26 and other cultivars may therefore represent a compound First Petunia hybrids effect of the SO allele and other unlinked compatibility Sl or SC? factors. It is also possible that self-compatible cultivars carry functional S-alleles that are rendered inactive due to | Breeders select for SC such modifiers as proposed for the original source of the S, allele (Ai et al., 1991). Modern day Petunia hybrida It is interesting to speculate on why the SO allele should predominate in self-compatible varieties (Fig. 5). One SC (and Sl) possibility is that the self-compatible allele was present in FIG. 5. Origin of self-compatibility in Petunia hybrida. The possible the original crosses between wild species that gave rise to origin of self-compatibility (SC) and self-incompatibility (SI) in P. hybrida. Indeed, P. axillaris, which is believed to be one modern cultivated P. hybrida is shown schematically. 110 Robbins et al.-Self incompatibility in Petunia et al., 1998). Phylogenetic analysis places the N. sylvestris family of 48 individuals (Harbord et al., 2000). In a S-RNase with other functional S-RNases and the authors different mapping population the CPI00 marker was found proposed the term 'relic S-RNase'. The self-compatibility of to cosegregate with a peroxidase isozyme locus (PrxA) N. sylvestris may also result from a mutation in pollen-S. In previously mapped to chromosome III (ten Hoopen et al., contrast to this, a rare self-compatible accession in 1998). Consistent with this localization was the observation Lycopersicon peruvianum revealed an active site mutation that S-linked T-DNAs were loosely linked to a leaf visual that abolishes ribonuclease activity (Royo et al., 1994). This marker, yg3 previously assigned to chromosome 111. These is consistent with the observation that S-RNases with a observations are not in agreement with the reported linkage modified active site are also inactive in transgenic Petunia between the S-locus and the Undulata mutation on infiata (Huang et al., 1994). It will be interesting to examine chromosome V. However, this was based on segregation whether pollen-part mutations are a frequent cause of distortions that were subsequently called into question by

self-compatibility in other cultivated species such as Reimann-Phillip (de Nettancourt, 1977). Downloaded from https://academic.oup.com/aob/article/85/suppl_1/105/102776 by guest on 29 September 2021 L. esculentunm. In other members of the Solanaceae, the S-locus has been assigned to a chromosome genetically. Thus the S-locus of Lycopersicon peruvianum was assigned to chromosome I by CYTOGENETIC AND GENETIC MAPPING segregation distortion (Tanksley and Loaiza-Figueroa, OF THE S-LOCUS IN P. HYBRIDA 1985) and the S-RNase of Solanum tuberosum was placed Until relatively recently the cytological position of the S- on the homeologous chromosome by RFLP analysis locus in P. hybrida was uncertain. There was a single report (Gebhardt et al., 1991). There is evidence of synteny of linkage between the Grandiflora/Undulata locus on around the S-locus in the Solanaceae, the CP100 marker is chromosome V and the S-locus based on segregation S-linked in petunia (Harbord et al., 2000) and potato distortion (de Nettancourt, 1977). The seven chromosomes (Gebhardt et al., 1991), and an anodal peroxidase is linked of P. hybrida can be divided into three classes based on to the S-locus in both petunia (ten Hoopen et al., 1998) and centromere position (Smith, Oud and de Jong, 1973). The tomato (Bernatzky and Tanksley, 1986). Evidence is thus chromosomes are amenable to direct gene localisation by gathering in support of the view that no major rearrange- fluorescent in-situ hybridization (FISH) as demonstrated by ment of the S-locus region has occurred during speciation ten Hoopen et al. (1996). However, the ability to map of the Solanaceae although local rearrangements sufficient S-RNase genes directly by FISH has been hampered by the to inhibit recombination between alleles cannot be small target size (ten Hoopen and Robbins, unpubl. res.). A excluded. much larger target is provided by a T-DNA integrated into During the preparation of this manuscript it was reported the genome and several transgenes have been localized that a P. hybrida S-RNase gene has been localized directly previously on chromosome II (ten Hoopen et al., 1996). by FISH at, or very near to, the centromere of chromosome Recently, several T-DNA insertions have been mapped III (Entani et al., 1999). Genomic clones of the S-RNase close to the S-locus of P. hybrida using a segregation revealed that the gene was embedded in repetitive sequences distortion assay (Harbord et al., 2000). These T-DNA present at the centromere of all petunia chromosomes. A insertions offered an opportunity to map the S-locus by an centromeric localization for the S-locus opens up the indirect method. possibility that recombination around the S-locus may be In collaboration with colleagues at the University of suppressed by centromeric heterochromatin. Recombina- Amsterdam we have localized seven of these T-DNA tion is likely to be suppressed at the S-locus to maintain insertions by fluorescent in-situ hybridization (ten Hoopen linkage between a coevolved gene complex including the et al., 1998). All seven S-linked T-DNA insertions were pollen recognition gene and the S-RNase gene. Comparison localized to the long arm of chromosome III. The two of S-RNase sequences in the Solanaceae lends support to T-DNA insertions most tightly linked to the S-locus (within this view (Clark and Kao, 1991). 2 cM) were shown to be within 20% of total chromosome If it is a selective advantage for the S-locus to be located length of the centromere whereas more loosely linked within a centromere then this should be observed in other T-DNAs had a more distal location. This suggests that the members of the Solanaceae. There is some support for this S-locus is located in the vicinity of the centromere, a finding in tomato based on S-linked RFLP markers (Bernatzky and consistent with a class of self-compatible mutants generated Miller, 1994) showing linkage to centromeric repeats by irradiation in P. inflata (Brewbaker and Natarajan, (Broun and Tanksley, 1996). However, a centromeric 1960). These mutants carry 'centric fragments', small location for the S-locus in other members of the Solanaceae centromere-bearing chromosome fragments that confer an may only be a reflection of synteny (ten Hoopen et al., additional S-specificity to otherwise normal diploid plants 1998). For this reason it will be particularly interesting to (see Golz et al., 2000). establish the cytological position of S-RNase loci in other An independent confirmation that the S-locus of more distantly related families such as the Scrophulariaceae P. hybrida maps to chromosome III was obtained using and Rosaceae (Richman, Broothaerts and Kohn, 1997). an anonymous potato RFLP marker, CP100. This RFLP marker had previously been shown to map genetically to FUTURE PERSPECTIVES the same position as a potato S-RNase gene (Gebhardt et al., 1991). Similarly, in P. hybrida this RFLP marker was The total number of S-alleles in P. hybrida currently stands shown to cosegregate with S-phenotypes in a testcross at ten, and since the same alleles are now being recovered Robbins et al.-Self-incompatibility in Petunia 111 independently in unrelated cultivars it may not rise signifi- Ai Y, Tsai D-S, Kao T-h. 1992. Cloning and sequencing of cDNAs cantly in future surveys. This remains a surprisingly high encoding two S proteins of a self-compatible cultivar of Petunia hybrida. Plant Molecular Biology 19: 532-528. level of diversity considering that plant breeders probably Ai Y, Singh A, Coleman CE, loerger TR, Kheyr-Pour A, Kao T-h. 1990. selected for self-compatibility at an early stage. This has Self-incompatibility in Petunia inflata: isolation and characteris- almost certainly contributed to the prevalence of the non- ation of cDNAs encoding three S-allele associated proteins. functional S allele in current stocks of P. hybrida. The Sexual Plant Reproduction 3: 130 138. molecular basis of self-compatibility of the S allele is Ascher PD. 1984. Self-incompatibility. In: Sink KC, ed. Petunia: Monographs on theoretical and applied genetics Vol. 9. Berlin: uncertain, but the available data are consistent with a Springer-Verlag, 92-109. functional S-RNase. One possibility is that it is a pollen- Bernatzky R, Miller D. 1994. Self-incompatibility is codominant in part mutation of a previously functional allele, perhaps as a intraspecific hybrids of self-compatible and self-incompatible result of a duplication. It will be interesting to compare the Lycopersicon peruvianum and L. hirsutum based on protein and DNA marker analysis. Sexual Plant Reproduction 7: 297-302. situation in P. hybrida with other cultivated Solanaceae that Bernatzky R, Tanksley SD. 1986. Toward a saturated linkage map in Downloaded from https://academic.oup.com/aob/article/85/suppl_1/105/102776 by guest on 29 September 2021 were derived from self-incompatible species. Understanding tomato based on isozymes and random cDNA sequences. Genetics the mechanisms that have led to self-compatibility during 112: 887-898. cultivation may facilitate the genetic engineering of self- Brewbaker JL, Natarajan AT. 1960. Centric fragments and pollen-part compatibility into self-incompatible species of both the mutation of incompatibility alleles in Petunia. Genetics 45: 699-704. Solanaceae and the Rosaceae which share the same S- Broothaerts WJ, van Laere A, Witters R, Preaux G, Decock B, RNase mechanisms (Richman et al., 1997). van Damme J, Vendrig JC. 1989. Purification and N-terminal Self-compatibility can also arise from the presence of sequencing of style glycoproteins associated with self-incompat- modifier genes as demonstrated in P. hybrida. The identity ibility in Petunia hybrida. Plant Molecular Biology 14: 93-102. of such genes is difficult to predict in view of the Broothaerts WJ, Vanvinckenroye P, Decock B, van Damme J, Vendrig simple JC. 1991. Pentunia-hybridaS-proteins: Ribonuclease-activity and models of self-incompatibility currently proposed (reviewed the role of their glycan side-chains in self-incompatibility. Sexual by Kao and McCubbin, 1996). However, P. hybrida offers Plant Reproduction 4: 258-266. several examples of such modifier genes (Ascher, 1984; Ai Broun P, Tanksley SD. 1996. Characterization and genetic mapping of et al., 1991; Harbord et al., 2000) that could be genetically simple repeat sequences in the tomato genome. Molecular and General Genetics 250: 39-49. characterised. Such modifier genes may not show allele Cerny TA, Caetano-Anolles G, Trigiano RN, Starman TW. 1996. specificity and therefore offer a more general method of Molecular phylogeny and DNA amplification fingerprinting of inactivating self-incompatibility systems. Petunia taxa. Theoretical and Applied Genetics 92: 1009-1016. Finally, as in all of the self-incompatibility systems Clark KR, Okuley JJ, Collins PD, Sims TL. 1990. Sequence variability discussed in this volume, a major goal remains the identifi- and developmental expression of S-alleles in self-incompatible and pseudo-self-compatible petunia. The Plant Cell 2: 815-826. cation of the pollen determinant of S-locus specificity. The Clark AG, Kao T-h. 1991. Excess nonsynonymous substitution at pollen-S gene must reside at the S-locus and high resolution shared polymorphic sites among self-incompatibility alleles of the genetic maps, including tightly linked flanking markers Solanaceae. Proceedings of the National Academy of Sciences such as CP100, should allow the limits of the S-locus to be (USA) 88: 9823-9827. defined. The Clark KR, Sims TL. 1994. The S-ribonuclease gene of Petunia hybrida centromeric location and the suppression of is expressed in nonstylar tissue, including immature anthers. Plant recombination anticipated would predict that this may Physiology 106: 25-36. correspond to a physically large region. The application of Cornish EC, Pettitt JM, Bonig , Clarke AE. 1987. Developmentally S-linked transgenes to preselect for rare recombination controlled expression of a gene associated with self-incompat- events around the S-locus (Harbord et al., 2000) should ibility in Nicotiana alata. Nature 326: 99-102. Dana MN, Ascher PD. 1986. Sexually localized expression of pseudo- facilitate this approach. In addition, these S-linked T- self compatibility (PSC) in Petunia hybrida Hort. I. Pollen DNAs carry maize transposable elements which opens up inactivation. Theoretical and Applied Genetics 71: 573 577. the possibility of genetic screens to identify pollen-S. Dodds PN, Bonig I, Du H, Rodin J, Anderson MA, Newbigin E, Clarke AE. 1993. S-RNase gene of Nicotiana alata is expressed in developing pollen. The Plant Cell 5: 1771-1782. Dodds PN, Ferguson C, Clarke AE, Newbigin E. 1999. Pollen-expressed ACKNOWLEDGEMENTS S-RNases are not involved in self-incompatibility in Lycopersicon peruvianum. Sexual Plant Reproduction 12: 76-87. The authors would like to thank Dr Wim Broothaerts for Entani T, Iwano M, Shiba H, Takayama S, Fukui K, Isogai A. 1999. supplying seed carrying the S3L allele originally character- Centromeric localisation of an S-RNase in Petuniahybrida Vilm. ized by Prof. H. F. Linskens. Also Dr Ronald Koes (Free Theoretical and Applied Genetics 99: 391-397. University of Amsterdam) for providing V13, V26 and Gebhardt C and 11 others. 1991. RFLP maps of potato and their alignment with the homoeologous tomato genome. Theoretical other inbred lines of P. hybrida and Dr Christiane Gebhardt and Applied Genetics 83: 49-57. (Max Planck Institute, Cologne) for the potato RFLP Golz JF, Clarke AE, Newbigin E. 2000. Mutational approaches to the marker, CP100. This work was supported by the UK study of self-incompatibility: Revisiting pollen-part mutants. Biotechnology and Biological Sciences Research Council. Annals of Botany 85: 95-103. Golz JF, Clarke AE, Newbigin E, Anderson MA. 1998. A relic S-RNase is expressed in the styles of self-compatible Nicotiana sylvestris. The Plant Journal 16: 591-600. LITERATURE CITED Harbord RM, Napoli CA, Robbins TP. 2000. Segregation distortion of T-DNA markers linked to the self-incompatibility (S) locus in Ai Y, Kron E, Kao T-h. 1991. S-alleles are retained and expressed in a Petunia hybrida. Genetics (in press). self-compatible cultivar of Petunia hybrida. Molecular and General ten Hoopen R, Harbord RM, Maes T, Nanninga N, Robbins TP. 1998. Genetics 230: 353-358. 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