Sex Plant Reprod (2005) 17: 211–217 DOI 10.1007/s00497-004-0235-7
O R I GI N A L A R T IC L E
Yoshinobu Takada Æ Takayuki Nakanowatari Æ Jun Sato Katsunori Hatakeyama Æ Tomohiro Kakizaki Æ Akiko Ito Go Suzuki Æ Hiroshi Shiba Æ Seiji Takayama Akira Isogai Æ Masao Watanabe
Genetic analysis of novel intra-species unilateral incompatibility in Brassica rapa (syn. campestris) L. Received: 21 May 2004 / Accepted: 13 October 2004 / Published online: 4 December 2004 Springer-Verlag 2004
Abstract Plants have evolved many systems to prevent inappropriate fertilization. Among them, incompatibility is a well-organized system in which pollen germination or pollen-tube growth is inhibited in pistils. Self-incompatibility (SI), rejecting self-pollen, promotes outbreeding in flowering plants. On the other hand, inter-species incompatibility, preventing gene flow among species to restrict outbreeding, usually occurs unilaterally, and is known as unilateral incompatibility (UI). In Brassicaceae, little is known about the molecular mechanism of UI, although S-locus genes involved in recognition of self-pollen have been characterized in the SI system. In the present study, we characterized novel UI observed between members of the same species, Brassica rapa; pollen of Turkish SI lines was specifically rejected by pistils of the Japanese commercial SI variety
The revised version was published online in December 2004 with corrections to figure 1. Y. Takada Æ T. Nakanowatari Æ J. Sato Æ T. Kakizaki Æ A. Ito M. Watanabe (&) Laboratory of Plant Breeding, Faculty of Agriculture, Iwate University, Iwate Morioka, 020-8550, Japan E-mail:
[email protected] Tel.: +81-19-6216152 Fax: +81-19-6216107 K. Hatakeyama Department of Leaf and Root Vegetables, National Institute of Vegetable and Tea Science, Mie Ano, 514-2392, Japan G. Suzuki Division of Natural Science, Osaka Kyoiku University, Osaka Kashiwara, 582-8582, Japan H. Shiba Æ S. Takayama Æ A. Isogai Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara Ikoma, 630-0101, Japan Present address: A. Ito Iwate Biotechnology Research Center, Kitakami, 024-0003, Japan
‘Osome’. The incompatible phenotype of this intra-species UI closely resembled that of SI. Segregation analysis revealed that the pollen factor of this UI was not linked to the S-locus. Keywords Brassica rapa (syn. campestris) L. Æ Intra-species incompatibility Æ Pollen-stigma interaction Æ Self-incompatibility Æ Unilateral incompatibility
Introduction Sexual reproduction plays an important role in the evolution of living organisms. This process provides a unique opportunity to exchange genetic information between individuals. However, in the case of the plants, most gametes are not useful resources for fertilization and evolution, because many kinds of plant species are grown in a field. To avoid such inappropriate pollination events, plants have evolved various sophisticated mechanisms. One such system is known as inter-species incompatibility, which rejects pollen from other species. On the other hand, self-incompatibility (SI) prevents self-fertilization and generates genetic diversity within a species. Inter-species incompatibility prevents gene flow, and is thus thought to underlie generation of new species. Inter-species incompatibility operates between closely related species belonging to the same family, or cluster of families, hosting the same SI system (de Nettancourt 2001). Harrison and Darby (1955) first revealed the relationship between the self and inter-species pre-zygotic barrier with the description of unilateral incompatibility (UI). In the case of inter-species crosses, UI is defined as the following phenomenon: pollen from one species is rejected on the stigma of another species, whereas crosses in the reverse direction are compatible. UI has been also observed as an intra-species unilateral barrier in crosses between different populations of the
212
green-fruited Lycopersicon hirsutum (Martin 1963). As for the relationship between SI and UI, UI has been frequently observed in reciprocal crosses between selfincompatible (SI) and self-compatible (SC) species, and incompatible pollen rejection occurs only when the pistillate parent is SI and the staminate parent is SC (Harrison and Darby 1955; Lewis and Crewe 1958; Hogenboom 1972). This phenomenon was defined as the ‘‘SI · SC rule’’, although it cannot be applied in all cases. In species possessing gametophytic SI, e.g., Nicotiana, an S-gene product was shown to be involved in inter-species UI (Murfett et al. 1996). In Lycopersicon, factors playing an essential role in inter-species UI were mapped not only to the S-locus but also to two other loci by restriction fragment length polymorphism (RFLP) analysis based on classical UI segregation studies (Martin 1963, 1964, 1967; Murfett et al. 1996; Hardon 1967; Chetelat and De Verna 1991). In Brassica species, the SI system is sporophytically controlled by a highly divergent S-locus (Bateman 1955; Nou et al. 1993b; Ockendon 2000), and three major studies (Lewis and Crowe 1958; Sampson 1962; Hiscock and Dickinson 1993) have shown the occurrence of inter-species UI with the SI · SC rule. At the Brassica S locus, there are two tightly linked polymorphic genes encoding SP11/SCR (S locus protein 11 or S locus cysteine-rich protein) and SRK (S receptor kinase) (reviewed in Watanabe et al. 2001, 2003). SP11/ SCR is a small cysteine-rich pollen coat protein and functions as sole determinant of the pollen SI phenotype (Suzuki et al. 1999; Schopfer et al. 1999; Takayama et al. 2000b; Shiba et al. 2001). SRK is a membrane-spanning serine/threonine receptor kinase and functions as sole determinant of the stigma SI phenotype (Stein et al. 1991; Takasaki et al. 2000). A haplotype-specific physical interaction between SP11/SCR and SRK has been observed, and is thought to cause a signal cascade that leads to rejection of self-pollen on the stigma (Kachroo et al. 2001; Takayama et al. 2001). To date, we have isolated and characterized 30 different S haplotypes from two different populations of B. rapa, in Japan and Turkey (Nou et al. 1991, 1993a, b; Hatakeyama et al. 1998). Furthermore, two additional S haplotypes, S52 and S60, were also isolated from a Japanese commercial hybrid variety (cv. Osome) for Agrobacterium-mediated transformation (Takasaki et al. 1999, 2000). However, detailed pollination analysis of these two S haplotypes has not yet been performed. In the present study, we found that the stigma of several plants in the progeny of S52 and S60 haplotypes specifically rejected pollen derived from Turkish S haplotypes, but accepted pollen derived from Japanese S haplotypes. In crosses in the reverse direction, the stigmas of both Turkish and Japanese S haplotypes were able to accept pollen derived from the progeny of S52 and S60 haplotypes, indicating that this phenomenon was a novel intra-species UI between self-incompatible plants of B. rapa. From linkage analysis with F2 progeny of S29 (Japanese line) and S40 (Turkish line), the locus regulating this UI phenotype of pollen was found not to be
linked to the S locus. We discuss the relationship between SI and UI in B. rapa.
Materials and methods Plant materials The 11 S-homozygous lines of B. rapa used in the present experiment were selected from those produced by Nou et al. (1991, 1993a, b) containing two populations, one from Oguni in Japan, and the other from Balcesme in Turkey (Table 1). In addition to these 11 lines, 2 S homozygous lines, S60 and S52, which were derived from a Japanese commercial hybrid variety (cv. Osome; Takasaki et al. 1999), were also used in this experiment. For determination of linkage to the S locus, the F2 progeny of a cross between S29 (7-5j) and S40 (83-5t) was raised. Test pollinations In B. rapa, anthesis takes place from the lower to the upper part of an inflorescence. In the test-cross pollinations, a flower bud was numbered from the lowest bud based on an inflorescence as described in Gonai and Hinata (1971). Because the average daily flowering number is about three, the flower buds at stages -1¢, -2¢, and -3¢ should be expected to bloom on the following day, and stigmas of these three buds have a potential to exhibit a normal SI reaction (Gonai and Hinata 1971). Non-pollinated flower buds were cut at the peduncle. After pollination, they were placed on 1% solid agar for about 24 h under room conditions. Thereafter, pistils of the pollinated flowers were softened in 1 N NaOH for 1 h at 60C, and stained with basic aniline blue (100 mM K3PO4, 0.1% aniline blue). Samples were mounted in 50% glycerol (fluorescence microscope grade) on slides and observed by UV fluorescence microscopy (Kho and Bear 1968). At least three flowers were used in each cross combination, and observations were generally replicated Table 1 The origin of the S alleles used in this investigation of Brassica rapa. Code numbers in parenthesis indicate strain numbers in Nou et al. (1991, 1993a, b; j S-alleles isolated from Oguni, Japan, tS-alleles isolated from Balcesme, Turkey). The cv. Osome lines (S52 and S60) were isolated by Takasaki et al. (1999) Japanese lines S24 S28 S29 S43
(S-12j) (S-9j) (7-5j) (S-8j)
cv. Osome line S52 S60
Turkish lines S24 S35 S37 S38 S40 S47 S49
(27-1t) (7-12t) (79-1t) (22-7t) (83-5t) (26-6t) (82-3t)
213
two or three times for each cross combination on different dates. The degree of compatibility in each testpollination was scored on a five-point scale based on pollen tube penetration as follows: (1) penetration of more than 20 pollen tubes into the style; (2) penetration of 1–20 pollen tubes into the style; (3) penetration of pollen tubes into papilla cells but not into the style; (4) germinated pollen but no pollen-tube penetration; (5) no germination of pollen. We calculated the average point of the degree of compatibility from the replicated data in each bud position. Scores under three were defined as incompatible, and scores over three as compatible. Linkage analysis For linkage analysis, the S genotype of each F2 plant was determined by DNA gel blot analysis with SP11 probes (Shiba et al. 2002). Total DNA was extracted from young leaf tissue of B. rapa by the procedure of Murray and Thompson (1980). DNA (1 lg) was digested with HindIII, loaded on 1% agarose gels, and transferred to nylon membranes (Roche, Mannheim, Germany). Hybridization and detection of the hybridized probe were carried out as described in Takada et al. (2001) and Matsuda et al. (1996). The UI phenotype of the same F2 progeny was determined by test pollination with S29 (7-5j) and S40 (83-5t) as described above.
Results Pollination test of S52 and S60 haplotypes In order to characterize S52 and S60 haplotypes derived from Japanese cv. Osome, the self-pollinated progeny of S52 and S60 homozygous plants was propagated, and 15 plants in each S haplotype were used in the pollination analysis. As representative tester lines, we selected four S haplotypes; S24 (S-12j) and S28 (S-9j) derived from a Japanese population, and S24 (27-1t) and S40 (83-5t) Fig. 1 Results of test-pollination between four S homozygotes and selfed progeny of S52 and S60 homozygotes. The selfed progeny of S52 and S60 homozygotes were classified into two groups (first, second ) according to the test-pollination to Turkish lines [S24 (27-1t) and S40 (83-5t)]. The stigma of the second group rejected the pollen derived from two Turkish lines. The other combinations except self-pollination were compatible. C Compatible combination between left and upper plants, I (shaded box) incompatible combination between left and upper plants, * results of test pollination were dependent on S haplotype
derived from a Turkish population (Table 1). When the four S tester lines were used as a pollen parent, and the S52 and S60 haplotypes were used as a pistil partner, we were able to classify the S52 and S60 homozygous individuals into two different groups based on their phenotypes in pollination. For the first group, which consisted of seven S52 homozygous plants (S52-1, -3, -4, -5, -6, -14, -18) and nine S60 homozygous plants (S60-2, -3, -4, -8, -10, -13, -16, -18, -19), pollen tubes of the four tester lines could penetrate into the stigma as a compatible phenotype (Figs. 1, 2a). Interestingly, for the second group, which consisted of eight S52 homozygous plants (S52-2, -7, -8, -10, -12, -13, -15, -17) and six S60 homozygous plants (S60-1, -7, -9, -12, -14, -17), pollen tubes of the two Turkish tester lines of S24 (27-1t) and S40 (83-5t) could not penetrate into the stigma as an incompatible phenotype, whereas those of the two Japanese tester lines S24 (S-12j) and S28 (S-9j) could penetrate into the stigma as a compatible phenotype (Figs. 1, 2b, c). This incompatible phenotype in the combination of the second-group stigma and Turkish pollen could not be distinguished from that in the self-pollination of SI plants by using fluorescence microscopic analysis (Fig. 2b, d). As reverse combinations of pollination, when the S52 and S60 haplotypes and the four S tester lines were used as pollen and pistil parents, respectively, penetration of pollen tubes was observed in all combinations (Fig. 1). In this experiment, we found no difference in the compatible phenotype between the S52 and S60 homozygous individuals. Thus, this one-way incompatible phenotype observed in the stigma of the second-group S52 and S60 plants against the pollen of Turkish strains is a novel intra-species UI between self-incompatible Brassica plants. Characterization of a novel intra-species UI To confirm the Turkish-pollen-specific incompatibility, we further characterized the S52-12 and S60-9 plants as the representative second-group plants showing UI phenotype. Pollen-tube behavior was carefully determined by reciprocal crosses between the 2 plants and 11 tester lines (4 Japanese and 7 Turkish lines; Table 1). In the case of reciprocal crosses between the two secondgroup plants (S52-12 and S60-9) and Japanese tester lines [S24 (S-12j), S28 (S-9j), S29 (7-5j), and S43 (S-8j)], pollentube penetration was observed in all combinations, indicating that the UI phenotype was not observed in combinations between the second-group plants and
214
Fig. 2a–d Representative results of test-pollination. Photographs taken under UV fluorescence microscopy. a Cross-pollination of an S52-1 stigma with S40 (83-5t) pollen. Pollen tubes penetrated into the stigma papilla cells. b Cross-pollination of an S52-12 stigma with S40 (83-5t) pollen. Pollen tubes could not penetrate into the stigma papilla cells of S52-12. c Cross-pollination of an S52-12 stigma with S24 (S-12j) pollen. Pollen tubes penetrated into the stigma papilla cells. d Self-pollination of an S52-12. Pollen tubes could not penetrate into the stigma papilla cells of S52-12
Fig. 3a–d Results of test-pollination between 11 S homozygotes and S52-12 and S60-9 lines. The developmental stages of flower buds (-1¢, -2¢, -3¢, -4¢) are given in Materials and methods. a Testpollination with S52-12 pollen. As stigma parents, four representative lines are shown; all combinations were compatible. b Testpollination with S60-9 pollen. As stigma parents, four representative lines were used; all combinations were compatible. c Reciprocal test-pollination to a. On S52-12 stigma, pollen grains derived from four Japanese lines except ‘Osome’ (S52-12) were compatible. In contrast, pollen grains derived from four Turkish lines were incompatible. d Reciprocal test-pollination to b. A trend similar to that seen in c was observed. Numbers indicate the degree of compatibility in each test-pollination (see Materials and methods). Shaded boxes Incompatible combinations, other values compatible combinations, not determined
Japanese lines (Fig. 3a–d). In contrast, when Turkish lines [S24 (27-1t), S35 (7-12t), S37 (79-1t), S38 (22-7t), S40 (83-5t), S47 (26-6t), and S49 (82-3t)] were used for reciprocal crosses, the UI phenotype was clearly observed as rejection of the pollen of Turkish lines on the stigma of the S52-12 and S60-9 plants. In some Turkish lines, an unstable UI phenotype at flower bud position -2¢ to -4¢ was also observed, indicating that the difference between the stable and unstable UI phenotype was due to the genetic background between lines (Fig. 3c, d). Because the pollen rejection observed in these UI plants was physiologically similar to that in SI plants, we attempted bud pollination (pollination using immature stigmas) of the second-group plants in the UI combinations. In the case of bud pollination of the SI combinations, mature stigmas are usually incompatible to self-pollen, and immature stigmas are usually compatible. The Turkish pollen was rejected by mature stigmas, whose flower bud position in the fluorescence was -1¢ to -4¢, as described above (Fig. 3c, d). In the case of immature stigmas, whose flower bud position was under -5¢, the score of degree of compatibility was gradually larger according to the inflorescence. Furthermore, pollen tube penetration into the style, and seed set, were observed. These results indicate that the immature stigmas were compatible (data not shown). In contrast, mature and immature stigmas were fully compatible to Japanese lines, and seed sets were also observed (data not shown). Interestingly, S24 (27-1t) from Turkey and S24 (S-12j) from Japan were incompatible with each other, indicating that they share the same S haplotype (Nou et al. 1993b; Matsushita et al. 1996; Fig. 1). As for the SI genes, the nucleotide sequences of SLG24 and S24-SP11 are completely identical (Matsushita et al. 1996; this work). In addition, the partial nucleotide sequence of SRK24 is also identical to the latter two genes (M. Matsushita, K Hinata, and M. Watanabe, unpublished data). However, S52-12 and S60-9 were compatible to Japanese S24 (S-12j) in reciprocal crosses, and were incompatible to the pollen of Turkish S24 (27-1t), suggesting that regulation of this pollen-side UI phenotype was not related to the S locus (Fig. 3c, d).
215
Fig. 4 Restriction fragment length polymorphism (RFLP) linkage analysis of an F2 population segregating for S29 (7-5j), S40 (83-5t) and their pollen phenotype to S60-9 and S52-12 stigma. Genomic DNA isolated from parental (P) plants homozygous for either the S29- or S40-homozygote, their F1 heterozygote (F1), and 18 F2 progeny plants (F2), was digested with HindIII, and hybridized with a mixed-SP11 probe (a mixture of S29-SP11 and S40-SP11 digoxigenin-labeled probes). The unilateral incompatibility (UI) phenotype of pollen of each plant to S52-12 and S60-9 stigma was determined by pollination tests. The S genotype and UI phenotype are shown below in each lane: 29 S29-homozygote, 40 S40homozygote, H S29 S40-heterozygote, C compatible to S52-12 and S60-9 stigma (non-UI); I incompatible to S52-12 and S60-9 stigma (UI)
Linkage analysis to the S locus In order to determine whether the pollen-side UI phenotype is linked to the S-locus, we performed RFLP analysis by using S29 (Japanese)/S40 (Turkish) F1 heterozygote and its F2 progeny. The pollen UI phenotype of F1 and F2 progeny plants was determined by test cross pollination using S52-12, and the S60-9 secondgroup UI plants as female partners. The pollen phenotype of five S29/S40 F1 plants was compatible to the stigma of the second-group plants (Fig. 2). The pollen phenotype of the F2 progeny was clearly segregated as incompatible (UI) or compatible (non-UI). The ratio of segregation of non-UI:UI of 148:51 corresponded closely to the single locus segregation ration, 3:1 (v2=0.041, P=0.838, df=1), suggesting that the pollen UI phenotype is controlled genetically by a single recessive gene. DNA blot analysis using HindIII-digested genomic DNA with S29-SP11 and S40-SP11 probes showed distinct hybridization patterns discriminating the S-genotype of each F2 plant (Fig. 4). From comparison of the S-genotype with the UI phenotype of the F2 progeny, it appeared that the pollen-side UI was not linked to the S locus (v2=5.556, P=0.351, df=5).
Discussion We characterized unilateral pollen rejection in B. rapa, which occurred between the cultivated SI line ‘Osome’ and the Turkish SI line. Of 30 Osome plants, 14 individuals showed unilateral rejection of pollen derived from all Turkish plants on their stigmas, whereas they showed reciprocal compatibility to the Japanese line. Importantly, all plants used represented a distinct SI phenotype, suggesting that they have an active self/non-self-recognition system. The second group, including S52-12 and S60-9, possessed not only a barrier to self-pollen, but also to pollen from the Turkish strain. Thus, the phenomenon
observed in this study is a novel UI system between SI plants, to which the SI · SC rule does not apply. The phenotype of pollen rejection in the UI described here resembles those in SI, in which incompatible pollen often fail to germinate or the pollen tube is not able to penetrate into the stigmatic cuticle with concomitant deposition of callose in the stigmatic papilla cells. Furthermore, like SI, UI could also be overcome by bud pollinations. A common downstream shared by UI and SI signal transduction might have resulted from the same incompatible events. In the case of UI in grasses, HeslopHarrison (1982) suggested that the mechanisms of pollen rejection in SI and UI are the same, but that the recognition and subsequent signaling events that trigger the rejection process are different. If this hypothesis is correct, the SI · SC rule in general UI is possibly due to the absence of the incompatible signal pathway in SC plants. In the present UI between SI plants, the pathway downstream of SI is active, indicating that Turkish pollen would be recognized and rejected on ‘Osome’ stigma as a novel pollen-stigma interaction. The phenotype of pollen rejection in the intra-species UI described here is also similar to those observed in the inter-species UI of Brassica species (Hiscock and Dickinson 1993). In the incompatible combination of both UI events, pollen cannot germinate, or the pollen tube cannot penetrate into the stigmatic papilla cells. In the case of UI in Brassica, Hiscock and Dickinson (1993) have proposed a ‘lock and key’ model in which the incompatibility of inter-species crosses might be attributed to the presence of a specific ‘key’ molecule in pollen that always matches a ‘lock’ molecule in the pistil within the species, but does not match between species as a prezygotic barrier. These ‘key’ and ‘lock’ differences can be regarded as functional differences between the pollen molecules that overcome the stigmatic barrier. It could be that the present intra-species UI is also caused by differences in the pollen ‘key’ molecules in the pollenstigma interaction. To identify the molecular players in this novel pollenstigma interaction, and clarify the reason for unilateral pollen rejection, we performed a genetic analysis, revealing that the pollen factor of the UI was not linked to the S locus. Therefore, it is clear that SP11 does not play an active role in the UI process. As a candidate for the pollen factor, the possible involvement of SP11-like pollen coat proteins (PCPs; Doughty et al. 1998, 2000) in UI is worth investigating in the future. Over ten different small basic PCPs, with eight cysteine residues, have been identified in Brassica pollen coats (Takayama et al. 2000a). As for the stigma factor, we found that almost
216
half of the ‘Osome’ progeny showed the UI phenotype, and that the S genotype (S52 or S60) and the UI phenotype were not correlated. We infer from this that the stigma factor of the UI was not also related to the S-locus products. Fine genetic mapping of the stigma factor, as well as the pollen factor, is necessary to determine the genetic regulation of UI. In the interspecies UI of genera possessing gametophytic SI, there have been reports showing a clear link between the S-locus gene product and UI (Murfett et al. 1996), and several loci not linked to the S locus might also play a role in UI in Solanaceae (Martin 1967; Murfett et al. 1996; Hardon 1967; Chetelat and De Verna 1991). However, a molecular system regulating UI recognition has not yet been characterized. The present study is the first step towards characterizing pollen and stigma factors of the novel UI system observed in Brassica, which will provide new insights into pollen-stigma interactions. Interestingly, both pollen and stigma parents belong to the same species, B. rapa, in the present UI system, although almost all other UI systems reported to date have been observed in the crossing of inter-species combinations. To the best of our knowledge, this is the first report showing intra-species UI in Brassica. Only a geographic difference in the origin of the lines—Japan vs. Turkey—affected the pollen UI phenotype; all the Turkish pollen grains were rejected, but all the Japanese pollen grains were accepted on the stigmas of S52-12 and S60-9. This suggests that the two populations (Japanese ‘Osome’ and Turkish lines) were isolated geographically, and acquired their respective genetic variations independent of diversification of the S alleles, before they diversified their respective reproductive mechanisms. In a transition status of speciation, UI between ‘Osome’ and Turkish lines might function as a reproductive barrier within species, which could be related to the creation of new species in B. rapa. Molecular dissection of the intra-species UI mechanism will contribute to our knowledge of the evolutionary history of speciation as well as to our understanding of the molecular mechanisms of the pollen-stigma interaction. Acknowledgements This work was supported in part by Grants-inAid for Special Research on priority Areas (Nos. 11238201 and 14360002) and Grants-in-Aid for the 21st Century Center of Excellence Program to Nara Institute of Science and Technology and Iwate University from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and a Grant-in-Aid for Creative Scientific Research (No. 16G3016) from Japan Society for Promotion of Science (JSPS). Y.T. and T.K. are the recipients of a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. The authors are grateful to Ayako Chiba, Yukiko Ohyama, and Hiroyuki Ishikawa (Iwate University) for technical assistance. We also thank Dr. Kokichi Hinata (Iwate Biotechnology Research Center) for his helpful discussion.
References Bateman AJ (1955) Self-incompatibility systems in angiosperms. III. Cruciferae. Heredity 9:52–68
Chetelat CB, De Verna JW (1991) Expression of unilateral incompatibility in pollen of Lycopersicon pennellii is determined by major loci on chromosomes 1, 6 and 10. Theor Appl Genet 82:704–712 Doughty J, Dixon S, Hiscock SJ, Willis AC, Parkin LA, Dickinson HG (1998) PCP-A1, a defensin-like Brassica pollen coat protein that binds the S-locus glycoproteins, is the product of gametophytic gene expression. Plant Cell 10:1333–1347 Doughty J, Wong HY, Dickinson HG (2000) Cysteine-rich pollen coat proteins (PCPs) and their interactions with stigmatic S (incompatibility) and S-related proteins in Brassica; putative roles in SI and pollination. Ann Bot 85:161–169 Gonai H, Hinata K (1971) Growth of pistils in relation to phenotypic expression of self-incompatibility in Brassica. Jpn J Breed 21:137–142 Hardon JJ (1967) Unilateral incompatibility between Solanum pennellii and Lycoperisicon esculentum. Genetics 57:795–808 Harrison BJ, Darby LA (1955) Unilateral hybridization. Nature 176:952 Hatakeyama K, Watanabe M, Takasaki T, Ojima K, Hinata K (1998) Dominance relationships between S-alleles in selfincompatible Brassica campestris L. Heredity 80:241–247 Heslop-Harrison J (1982) Pollen stigma interaction and crossincompatibility in the grasses. Science 215:1358–1364 Hiscock SJ, Dickinson HG (1993) Unilateral incompatibility within the Brassicaceae: further evidence for the involvement of the self-incompatibility (S)-locus. Theor Appl Genet 86:744–753 Hogenboom NG (1972) Breaking breeding barriers in Lycoperisicon 5. The inheritance of unilateral incompatibility between L. peruvianum (L.) MILL. and L. esculentum MILL. and the genetics of its breakdown. Euphytica 21:405–414 Kachroo A, Schopfer CR, Nasrallah ME, Nasrallah JB (2001) Allele-specific receptor-ligand interactions in Brassica selfincompatibility. Science 293:1824–1826 Kho YO, Bear J (1968) Observing pollen tubes by means of fluorescence. Euphytica 17:298–302 Lewis D, Crowe LK (1958) Unilateral incompatibility in flowering plants. Heredity 12:233–256 Martin FW (1963) Distribution and interrelationship of incompatibility barriers in the Lycopersicon hirsutum Humb. and Bonpl. complex. Evolution 17:519–528 Martin FW (1964) The inheritance of unilateral incompatibility in Lycopersicon hirsutum. Genetics 50:459-469 Martin FW (1967) The genetic control of unilateral incompatibility between two tomato species. Genetics 56:391–398 Matsuda N, Tsuchiya T, Kishitani S, Tanaka Y, Toriyama K (1996) Partial male sterility in transgenic tobacco carrying antisense and sense PAL cDNA under the control of a tapetumspecific promoter. Plant Cell Physiol 37:215–222 Matsushita M, Watanabe M, Yamakawa S, Takayama S, Isogai A, Hinata K (1996) The SLGs corresponding to the same S24haplotype are perfectly conserved in three different selfincompatible Brassica campestris L. Genes Genet Syst 71:255– 258 Murfett J, Strabala TJ, Zurek DM, Mou BQ, Beecher B, McClure BA (1996) S RNase and inter-species pollen rejection in the genus Nicotiana: multiple pollen-rejection pathways contribute to unilateral incompatibility between self-incompatible and selfcompatible species. Plant Cell 8:943–958 Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325 Nettancourt D de (2001) Incompatibility and incongruity in wild and cultivated plants, 2nd edn. Springer, Berlin Heidelberg New York Nou IS, Watanabe M, Isogai A, Shinozawa H, Suzuki A, Hinata K (1991) Variation of S-alleles and S-glycoproteins in a naturalized population of self-incompatible Brassica campestris L. Jpn J Genet 66:227–239 Nou IS, Watanabe M, Isuzugawa K, Isogai A, Hinata K (1993a) Isolation of S-alleles from a wild population of Brassica campestris L. at Balcesme, Turkey and their characterization by S-glycoproteins. Sex Plant Reprod 6:71–78
217 Nou IS, Watanabe M, Isogai A, Hinata K (1993b) Comparison of S-alleles and S-glycoproteins between two wild populations of Brassica campestris in Turkey and Japan. Sex Plant Reprod 6:79–86 Ockendon DJ (2000) The S-allele collection of Brassica oleracea. Acta Hortic 539:25–30 Sampson DR (1962) Intergeneric pollen-stigma incompatibility in Cruciferae. Can J Genet Cytol 4:38–49 Schopfer CR, Nasrallah ME, Nasrallah JB (1999) The male determinant of self-incompatibility in Brassica. Science 286:1697–1700 Shiba H, Takayama S, Iwano M, Shimosato H, Funato M, Nakagawa T, Suzuki G, Watanabe M, Hinata K, Isogai A (2001) An anther-expressed gene SP11 determines the pollen S-specificity in the self-incompatibility of Brassica rapa. Plant Physiol 125:2095–2103 Shiba H, Iwano M, Entani T, Ishimoto K, Shimosato H, Che F-S, Satta Y, Ito A, Takada Y, Watanabe M, Isogai A, Takayama S (2002) The dominance of alleles controlling self-incompatibility in Brassica pollen is regulated at the RNA level. Plant Cell 14:491–504 Stein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB (1991) Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc Natl Acad Sci USA 88:8816–8820 Suzuki G, Kai N, Hirose T, Fukui K, Nishio T, Takayama S, Isogai A, Watanabe M, Hinata K (1999) Genomic organization of the S locus: identification and characterization of genes in SLG/SRK region of S9 haplotype of Brassica campestris (syn. rapa). Genetics 153:391–400 Takada Y, Ito A, Ninomiya C, Kakizaki T, Takahata Y, Suzuki G, Hatakeyama K, Hinata K, Shiba H, Takayama S, Isogai A,
Watanabe M (2001) Characterization of expressed genes in the SLL2 region of self-compatible Arabidopsis thaliana. DNA Res 8:215–219 Takasaki T, Hatakeyama K, Watanabe M, Toriyama K, Isogai A, Hinata K (1999) Introduction of SLG (S-locus glycoproteins) alters the phenotype of endogenous S-haplotype, but confers no new S-haplotype specificity in Brassica rapa L. Plant Mol Biol 40:659–668 Takasaki T, Hatakeyama K, Suzuki G, Watanabe M, Isogai A, Hinata K (2000) The S receptor kinase determines self-incompatibility in Brassica stigma. Nature 403:913–916 Takayama S, Shiba H, Iwano M, Asano K, Hara M, Che FS, Watanabe M, Hinata K, Isogai A (2000a) Isolation and characterization of pollen coat proteins of Brassica campestris that interact with S locus-related glycoproteins 1 involved in pollenstigma adhesion. Proc Natl Acad Sci USA 97:3765–3770 Takayama S, Shiba H, Iwano M, Shimosato H, Che FS, Kai N, Watanabe M, Suzuki G, Hinata K, Isogai A (2000b) The pollen determinant of self-incompatibility in Brassica campestris. Proc Natl Acad Sci USA 97:1920–1925 Takayama S, Shimosato H, Shiba H, Funato M, Che FS, Watanabe M, Iwano M, Isogai A (2001) Direct ligand-receptor complex interaction controls Brassica self-incompatibility. Nature 413:524–538 Watanabe M, Hatakeyama K, Takada Y, Hinata K (2001) Molecular aspects of self-incompatibility in Brassica species. Plant Cell Physiol 42:560–565 Watanabe M, Takayama S, Isogai A, Hinata K (2003) Recent progresses on self-incompatibility research in Brassica species. Breed Sci 53:199–208
