A Gene Block Causing Cross-Incompatibility Hidden in Wild and Cultivated Rice

Corresponding author: Yoshio Sano, Kita 9, Nishi 9, Department of Applied Biotechnology, Hokkaido University, Sapporo, 060-8589 Japan., rysano{at}abs.agr.hokudai.ac.jp (E-mail)

Communicating editor: A. PATERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Unidirectional cross-incompatibility was detected in advanced generations of backcrossing between wild (Oryza rufipogon) and cultivated (O. sativa) rice strains. The near-isogenic line (NIL) of T65wx (Japonica type) carrying an alien segment of chromosome 6 from a wild strain gave a reduced seed setting only when crossed with T65wx as the male. Cytological observations showed that abortion of hybrid seeds occurred as a consequence of a failure of early endosperm development followed by abnormalities in embryo development. The genetic basis of cross-incompatibility reactions in the female and male was investigated by testcrosses using recombinant inbred lines (RILs) that were established through dissecting the introgressed segments of wild and cultivated (Indica type) strains. The results revealed that the cross-incompatibility reaction was controlled by Cif in the female and by cim in the male. When the female plant with Cif was crossed with the male plant with cim, a failure of early endosperm development was observed in the hybrid zygotes. Among cultivars of O. sativa, cim was distributed predominantly in the Japonica type but not in the Indica type. In addition, a dominant suppressor, Su-Cif, which changes the reaction in the female from incompatible to compatible was proposed to present near the centromere of chromosome 6 of the Indica type. Further, the death of young F₁ zygotes was controlled by the parental genotypes rather than by the genotype of the hybrid zygote itself since all three genes acted sporophytically, which strongly suggests an involvement of parent-of-origin effects. We discuss the results in relation to the origin of a crossing barrier as well as their maintenance within the primary gene pool.

RECENT evolutionary studies have focused on how genes that cause isolating barriers can be fixed in a species during speciation in spite of the fact that such genes reduce fitness when they coexist within a population (ORR 1996 ; LYNCH and FORCE 2000 ). Reproductive isolation is considered to result from a disharmonious interaction of genes from the parents, and complementary genes are frequently reported in various plant species (STEBBINS 1958 ; GRANT 1981 ). If one of the complementary genes were selectively neutral to another within a population, then it could become fixed with a minimal reduction in fitness. Cross-incompatibility is one of the most effective isolating barriers that restricts gene flow between diverged populations. Cross-incompatibility after pollination is classified into pre- and postfertilization barriers in plants (STEBBINS 1958 ; MACNAIR 1989 ), with the former resulting mainly from pollen-pistil interactions and the latter from an arrest of the development of young zygotes. Sexual affinity or cross-compatibility has been widely surveyed in crops and their wild relatives since knowledge about the primary gene pool is a prerequisite for hybridization breeding (HARLAN and DE WET 1971 ), but our present understanding of the genes involved in these phenomena and their distribution within the primary gene pool is limited.

The present study was carried out to examine the genetic basis of the unidirectional cross-incompatibility observed in hybrid derivatives between cultivated (Oryza sativa) and wild (O. rufipogon) rice strains. A domesticated plant and its progenitor generally belong to the same biological species, which consists of groups of potentially interbreeding populations, and the corresponding cultivated and wild forms of rice are regarded to be the O. sativa-O. rufipogon complex (HARLAN 1975 ; OKA 1988 ). The unidirectional cross-incompatibility was detected after introducing an alien chromosomal segment of O. rufipogon into O. sativa and observing that in plants with the introduced (or introgressed) segment, abortion of hybrid zygotes occurred only when the plants were pollinated with pollen grains of the recurrent parent (SANO 1992 ). This provides a unique example in which genes for crossing barriers were present within the primary gene pool and a distinct isolating barrier resulted from hybridization and recombination, although no distinct crossing barrier has been reported within the rice species complex (CHU et al. 1969 ; SITCH et al. 1989 ). To examine the unidirectional cross-incompatibility, it was assumed that, as reported for maize (RASHID and PETERSON 1992 ), the cross-incompatibility reactions in the female and male are determined by the parental genotypes and that a disharmonious interaction between parental reactions causes a crossing barrier. Cross-incompatibility between distantly related species frequently results from an arrest of endosperm development, which may be explained by a genomic imbalance showing parent-of-origin effects (JOHNSTON et al. 1980 ; SCOTT et al. 1998 ), although few genetic studies have aimed at testing this. The present phenomenon found in closely related taxa should give us an opportunity to examine the genetic basis and the chromosomal localization of the genes responsible for the cross-incompatibility observed within species. We report here that three putative genes responsible for the cross-incompatibility are located on the introduced segments, and we propose a model for the genic interactions responsible for the cross-incompatibility reactions in the female and male. The distribution of these three genes in the genome is also discussed in relation to the origin of cross-incompatibility.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant materials:
The materials used were three near-isogenic lines (NILs): T65wx (OKA 1974 ), W593A (SANO 1992 ), and 868A (DUNG et al. 1998 ). T65wx is a NIL of Taichung 65 (designated T65, Japonica type of O. sativa from Taiwan), in which the wx gene on chromosome 6 from Kinoshita-mochi (Japonica type from Japan) has been introduced by successive backcrosses (from BC14). The other two NILs, W593A and 868A, were established by successive backcrosses (from BC8) using T65wx as the recurrent parent. Those NILs were made originally to study the gene expression of Wx alleles responsible for grain quality (SANO 1984 ). The NILs grow normally, showing a high fertility like the recurrent parent; however, late-heading plants were detected in the segregating lines (SANO 1992 ; DUNG et al. 1998 ). The late-heading plants carried most parts of the short arm of chromosome 6 and the homozygotes were used in the present study. W593A carries a segment that contains Wx, Cif (Cross-incompatibility in the female, formerly designated Lcr), C (Chromogen for anthocyanin), Se1 (Hd1, photoperiod sensitivity; YANO et al. 2000 ), and S6 (hybrid sterility) on chromosome 6 from W593 (O. rufipogon from Malaysia), and 868A carries a segment from Wx to Se1 from Patpaku (designated 868, an Indica type of O. sativa from Taiwan). The S6 gene caused F₁ hybrid sterility but showed no effect on cross-incompatibility (SANO 1992 ). All three NILs carry the cytoplasm of T65.

To examine the cross-incompatibility or crossability with W593A carrying Cif, 12 cultivated strains (7 of Japonica type, 1 of Javanica type, and 4 of Indica type; Table 1) were used. Although Asian cultivated rice strains are often treated differently in the literature, the three varietal groups, Indica (continental), Javanica (tropical insular), and Japonica (temperate insular) were used in the present study, according to OKA 1953 and CHANG 1976 . The parental seeds were obtained from the National Institute of Genetics, Mishima, Japan.

Cultivation and hybridization:
Seeds were germinated in petri dishes at 30° in late April, and each of the 4-week-old seedlings was transplanted in a plastic pot in a greenhouse. The plants were grown in a short-day field (10.5 hr) after 8 weeks from sowing due to photoperiod sensitivity. For genetic analysis of the unidirectional cross-incompatibility, the incompatible reactions in the female and male were investigated through hybridization with different lines. The female reaction of a plant is testable by pollination with the pollen grains of T65wx, while the male reaction is testable by pollination to the pistils of W593A. For crossing, the female parent was emasculated before anthesis in hot water at 42° for 7 min and then used for cross-pollination. At maturity, the numbers of plump and aborted seeds were counted for each cross, and crossability was assessed as the rate of seed setting (100 x the number of plump seeds obtained/the number of florets pollinated). Plants grown in a short-day field were used for hybridization to avoid low temperatures. All the tested plants had a high seed setting on selfing (>80%). In the present experiments, incompatible crosses always gave a high frequency of aborted or shriveled seeds (Fig 1), which was regarded as a good indication of seed arrest after fertilization.

View larger version (147K): In this window In a new window Download PPT slide   Figure 1. Processes of pollination and seed development in the compatible and incompatible crosses. Plump (A) and shrunken (B) seeds resulted from compatible and incompatible crosses, respectively. Pollen tubes of T65wx reaching the micropyle in the incompatible cross (C) showed no arrest of pollen-tube growth. The development of embryos and endosperms was observed in the compatible (D, E, H) and incompatible (F, G, I) crosses. At 2 DAP, globular embryos with normal and abnormal endosperms in the compatible (D) and the incompatible (F) crosses, respectively, were observed. At 4 DAP, a coleoptilar-stage embryo in a compatible cross (E) and the overgrowth of an embryo with a degenerated endosperm in an incompatible cross (G) were observed. A first-leaf-stage embryo with a developing endosperm from a compatible cross (H) and a giant embryo without endosperm from an incompatible cross (I) at 8 DAP are shown. Arrows indicate degenerated endosperms (F and G). Bars: 1 mm for A and B; 0.5 mm for C; 50 µm for D, E, F, and G; and 100 µm for H and I.

Cytological observations:
For cytological observations of the growth of pollen tubes, florets were sampled 4 hr after pollination. The samples were fixed in a solution (3:1 ethanol:acetic acid) and were stored at 4° in 70% ethanol until use. The dissected pistils and ovaries were washed twice with distilled water and then incubated in a solution of 8 N NaOH for 1 hr. Then the samples were stained in 0.1% aniline blue in K₃PO₄ buffer and examined under UV illumination to visualize the callose of pollen tubes. To examine the development of seeds after fertilization, florets were collected and fixed in FAA (1:1:18 formalin:glacial acetic acid:70% ethanol) at 2, 4, and 8 days after pollination (DAP). After overnight incubation, the samples were stored at 4° in a solution of 70% ethanol until use. For paraplast sections, dissected pistils were dehydrated in a graded n-butanol series, embedded in paraplast, and then sectioned at 10 µm. The sections were stained with safranin and counterstained with fast green.

Fragmentation of alien chromosomal segments:
The introduced chromosomal segment was dissected by repeated self-pollination after hybridization to examine the location of the genes of interest. Recombinant inbred lines (RILs) were made thereby from crosses of T65wx x W593A (R lines), T65wx x 868A (P lines), and W593A x 868A (RP lines). Since W593A and 868A were NILs of T65wx, the resultant RILs were expected to have the same genetic background except for the region of chromosome 6 noted above. Of the three sets of NILs, 16, 16, and 5 lines were used after genotyping the alien segments in the R, P, and RP lines, respectively.

Genotyping of alien segments in the NILs:
Genomic DNA was isolated from 2-month-old seedlings by the cethyltrimethyl-ammonium bromide method according to MURRAY and THOMPSON 1980 . The length of the chromosome segment introduced from alien strains was surveyed using 20 molecular markers on chromosome 6. Thirteen of these markers (RZ516, RZ398, RZ588, RG264, RZ192, C764, S1520, G200, C235, R538, R111, R32, and G2028) were restriction fragment length polymorphism (RFLP) markers provided by S. McCouch, Cornell University, and T. Sasaki, Rice Genome Research Program, National Institute of Agrobiological Resources, Tsukuba, Japan. For RFLP analysis, after digestion with restriction enzymes the DNAs were subjected to electrophoresis on 1% agarose gels and transferred to BIODYNE B membranes (Pall). Southern blotting was performed by using ECL direct nucleic acid labeling and detection systems (Amersham). The remaining markers were PCR-based markers from the Wx gene (MIKAMI et al. 2000A ), the OsC1 gene (MIKAMI et al. 2000B ), and three microsatellite markers RM204, RM253 (CHEN et al. 1997 ), and RM136 (TEMNYKH et al. 2000 ). In addition, the following gene-specific primers were designed from the database (DDBJ accession nos. AB041837 and AP000399): 5'-gtcagtgcttacacagattccatc-3' and 5'-ccttcttcttctccctgtacctgag-3' for Hd1 and 5'-atggggatgctgaatctgatg-3' and 5'-gacagaagagagcatgggaaat-3' for E12. To detect polymorphisms for E12, the amplified products were digested with HinfI.

A linkage map of the markers was made using 165 F₂ plants of T65wx x IR36 and 99 F₂ plants of T65wx x W593A. Recombination values were calculated by the maximum-likelihood method (ALLARD 1956 ) and converted to centimorgans using the Kosambi function (KOSAMBI 1944 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cytological observations:
Unidirectional cross-incompatibility was observed between a specific type of female (W593A) and a specific type of male (T65wx) parent (Table 1). W593A showed a low seed setting (12.1%) when crossed with pollen grains of T65wx in spite of the fact that the reciprocal cross gave a high seed setting (66.7%). A previous study revealed that the dominant gene Cif caused abortion of hybrid zygotes, although it showed low expressivity (SANO 1992 ). Cytoplasmic differences were ruled out as the causal factor because both parental lines had the cytoplasm of T65. The incompatible crosses frequently gave aborted seeds, suggesting that it was a postfertilization barrier (Fig 1A). To examine when and how the unidirectional cross-incompatibility took place, histological observations were carried out. The pollen tubes from the incompatible male parent (T65wx) penetrated immediately after pollination into the styles of the incompatible female parent (W593A) and reached the region around the micropyle 4 hr after pollination (Fig 1C). Double fertilization seemed to be accomplished normally since each of two sperm cells fused with the egg cell and the binucleated central cell.

The development of the embryos proceeded normally morphologically until 2 DAP in the incompatible cross, as in the compatible cross (Fig 1D and Fig F). However, at 4 DAP the embryos in the incompatible cross began to overgrow in comparison with those in the compatible cross (Fig 1G), and at 8 DAP giant embryos were formed with defective formation of differentiated tissues (Fig 1I). On the other hand, the triploid endosperm began to deteriorate within a few days after pollination in the incompatible cross (Fig 1F). This suggests that abortion of seeds occurred as a consequence of the failure of early endosperm development followed by abnormalities in embryo development.

Responses of cultivated strains to W593A carrying Cif:
To examine whether or not T65wx responds uniquely to W593A, 12 strains belonging to three varietal groups of O. sativa were crossed reciprocally with W593A (Table 1). All of the eight strains of the Japonica and Javanica types gave a high seed setting (41.3–83.3%) when pollinated with the female W593A; however, the reciprocal cross gave a low seed setting (2.4–26.1%). In addition, the reciprocal cross frequently produced aborted seeds, suggesting that the seed abortion was caused by the Cif gene. Thus, the distinct difference in the reciprocal crosses revealed that the eight strains responded to W593A in a similar manner to T65wx. On the other hand, all four strains of Indica type always showed a high seed setting (47.8–73.3%), without differences between the reciprocal crossings, suggesting that the response to W593A was different among the varietal groups of rice.

Mapping of Cif by using RILs:
In a previous study, it was concluded that Cif was loosely linked to Wx. To locate Cif more precisely, 16 RILs (R lines) derived from T65wx x W593A were further investigated. On the basis of the 16 molecular markers on chromosome 6, these RILs were grouped into 11 types. Each of the lines was crossed with the male parent of T65wx to determine whether the Cif gene was present or absent. Different lines with the same graphical genotype showed similar rates of seed setting, and therefore their data were pooled (Fig 2). A low seed setting was always associated with the production of aborted seeds. The crossing experiments suggested that Cif is located between RZ516 and RM204, because only the lines carrying the region between these two markers showed a distinct reduction in seed settings (10.5–18.6%). The results also revealed that no other region on the short arm of chromosome 6 was associated with a reduced seed setting.

View larger version (34K): In this window In a new window Download PPT slide   Figure 2. Testcrosses to determine the location of Cif by using RILs. T and R indicate the markers from T65wx (Japonica) and W593 (O. rufipogon), respectively. The possible range of the alien segment is shown by boxes. The location of Cif is shown by a solid bar. The number of plump seeds obtained/number of florets pollinated is shown in parentheses.

Since W593A itself was self-fertile, the difference in the reciprocal crosses could not be explained only by the Cif gene, and thus it was considered likely that additional gene(s) on the introduced segment were involved. The cross-incompatibility reaction in the female could be investigated by pollinating T65wx. When T65wx was used as the male parent (Table 2), the W593A x T65wx F₁ plant (Cif/cif) showed crossability as low (8.1%) as that of the homozygote (Cif/Cif). This suggests that Cif acts sporophytically, which is consistent with the previously reported finding of 3:1 Mendelian inheritance (SANO 1992 ).

Unidirectional cross-incompatibility reaction in the male:
W593A had a high seed setting upon selfing in spite of the presence of the Cif gene as mentioned. One possible explanation is that an additional gene(s) suppressing the effect of Cif is present on the introduced segment. To examine this possibility, three different RILs (R-1, R-5, and R-6) were used to pollinate W593A (Fig 3). All crosses gave a high seed setting, showing that R-5 and R-6 without the Cif gene were compatible with the Cif female. This suggests the presence of a gene(s) that modified the male reaction of T65wx and was located near the centromere. The above observation led us to consider the possibility that the unidirectional cross-incompatibility might be regulated not only by the female reaction but also by the male reaction.

View larger version (36K): In this window In a new window Download PPT slide   Figure 3. Testcrosses to determine the location of cim by using recombinants. T, R, and P indicate the markers from T65wx (Japonica), W593 (O. rufipogon), and 868 (Indica), respectively. The possible range of the alien segment is shown by boxes. The location of cim is shown by a solid bar. The number of plump seeds obtained/number of florets pollinated is shown in parentheses.

As mentioned before, all four strains of Indica type were compatible with W593A, whereas the eight strains of Japonica and Javanica types were not (Table 1), suggesting that the Indica strains might carry the same gene for the male reaction as W593A. To confirm this, 868A was used as the male parent to pollinate W593A, since 868A carried the short arm of 868 (Patpaku) in the genetic background of T65wx (Fig 3). The cross gave a high seed setting (64.9%), showing that a gene(s) that modified the male reaction of T65wx was present on the introduced segment. The segment introduced from 868 was then segmented by repeated selfing of T65wx x 868A F₂ plants. Sixteen of the resultant RILs (P lines) were used in the present experiments and were divided into 10 groups by genotyping with 14 molecular markers. When the lines were used to pollinate the female W593A, they were clearly classifiable into compatible and incompatible lines (Fig 3). R-6, P-4, and P-10 gave high seed settings (76.0, 63.6, and 55.0%), indicating that a gene(s) modifying the male reaction of T65wx was present between R111 and G2028. On the basis of the estimated position in W593A and 868A, it was suggested that both of these lines had the same gene for the male reaction. Accordingly, the causal gene near the centromere was tentatively designated cross-incompatibility in the male reaction (cim).

When the F₁ hybrid of T65wx x 868A was crossed to the female W593A, the seed setting was as high as 55.9%, and few aborted seeds were produced (Table 2). This indicates that the gene carried by 868A is dominant and that it acts sporophytically since the crossability would be reduced by half, owing to the generation of aborted seeds if it acted gametophytically in the heterozygote. Therefore, 868 and T65wx were assumed to carry Cim and cim, respectively. Cim seemed to have no effect on the cross-incompatibility reaction in the female, because W593A carried Cim as well as Cif.

Suppressor of Cif in the female reaction:
Regarding the female reaction, the following unexpected result was obtained when the F₁ hybrid of W593A x P-4 was pollinated with the male T65wx (Table 2). The genotype of the F₁ hybrid was expected to be Cif Cim/cif Cim on the basis of the graphical genotype of P-4 (Fig 3). The cross gave a high seed setting (61.9%) in spite of the fact that it should have given a reduced seed setting since Cif was dominant. This revealed that the segment introgressed from 868 carried a dominant gene(s) that changed the incompatible reaction in the female. No such segments modifying the female reaction were detected in RILs from W593A and T65wx. The high seed setting with few aborted seeds indicated that the dominant gene acted sporophytically, too. The suppressor of Cif was tentatively designated Suppressor of Cif (Su-Cif).

To determine the location of the suppressor more precisely, the segment introgressed from 868A was dissected in the derivatives of W593A x 868A hybrids. In the F₄ generation, five different RILs (RP lines) were selected using 12 markers, and each was pollinated with the male T65wx to determine the female reaction (Fig 4). Four of the five lines, i.e., all except RP-1, carried Cif from W593A together with varying lengths of segments from 868A. All lines tested gave a high seed setting (50.0–92.7%), as high as that of the F₁ of W593A x P-4. Although R32 and G2028 were monomorphic between 868A and W593A, the present results demonstrated that the suppressor is located between Hd1 and G2028.

View larger version (24K): In this window In a new window Download PPT slide   Figure 4. Testcrosses to determine the location of Su-Cif by using recombinant inbred lines. T, R, and P indicate the markers from T65wx (Japonica), W593 (O. rufipogon), and 868 (Indica), respectively. A dash indicates monomorphism between W593 and 868. The possible range of the alien segment is shown by boxes. To determine whether the alien segment carried Su-Cif, all the lines except for P-4 were crossed with T65wx. In the case of P-4 (not carrying Cif), the F1 of W593A x P-4 was crossed with the male T65wx (see Table 2). The location of Su-Cif is shown by a solid bar. The number of plump seeds obtained/number of florets pollinated is shown in parentheses.

The assumed location of Su-Cif suggested that NILs with a segment from 868A would not have Su-Cif (P-1 and P-2; Fig 3). Since the two lines were highly crossable to the male T65wx (57.2 and 64.7%), 868A seemed not to have Cif. Thus, the haploid genotypes of T65wx, 868A, and W593A were assumed to be cif su-Cif cim, cif Su-Cif Cim, and Cif su-Cif Cim, respectively, with those genes forming a gene block on chromosome 6.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genic interactions among three genes that appear to account for the unidirectional cross-incompatibility in rice:
Cross-incompatibility after fertilization is manifested as F₁ inviability and is caused by the failure of young F₁ zygotes to develop, especially by failure of endosperm development in plants (GRANT 1981 ; BERGER 1999 ). It is well documented in plants that hybrid inviability is often controlled by a set of complementary genes as well as by nucleo-cytoplasmic interactions and the occurrence of abnormalities in F₁ and F₂ depends on the degree of dominance of the responsible genes (OKA 1988 ; MACNAIR 1989 ). Thus, cross-incompatibility after fertilization is genetically different from internal barriers that appear in the later stages of development such as F₁ sterility and F₂ breakdown. In most cases, it is assumed that the death of a young F₁ zygote depends on the genotype of itself and is caused by a set of complementary dominant genes derived from the parents. This assumption suggests that differing gene dosages in triploid endosperm could give rise to differences between the reciprocals (CHU and OKA 1970 ). Cross-incompatibility was frequently observed in wide hybridization but not within the primary gene pool (CHU et al. 1969 ; SITCH et al. 1989 ), although numerous genes for isolating barriers except for crossability were reconfirmed to appear in crosses between cultivars of O. sativa (LI et al. 1997 ; HARUSHIMA et al. 2002 ). The present results showed that the unidirectional cross-incompatibility was detected in the O. sativa-O. rufipogon complex and that this cross-incompatibility was controlled by at least three genes, as shown in Fig 5, although further studies are needed since two of these genes (cim and Su-Cif) were not recombined in the present study. The simple inheritance observed for each of these three presumptively responsible genes ruled out nucleo-cytoplasmic interactions as the causal factor. These three genes control whether the incompatible or compatible reactions occur in the females or males. The Cif and cim genes are responsible for the incompatible reactions (Cif and cim) in the female and male, respectively. In addition, the Su-Cif gene that was detected only from an Indica strain was proposed to change the incompatible reaction (Cif) to the compatible reaction (+) in the female. All three genes act sporophytically, so that, for example, the heterozygote of Cim/cim produces only male gametes with the compatible reaction (+) even if half of the gametes carry cim. The deterioration of endosperm took place only when female gametes with the incompatible reaction (Cif) were fertilized with male gametes with the incompatible reaction (cim). On the basis of the molecular mapping in rice, the locations of the three genes differ from those of S5 (YANAGIHARA et al. 1995 ) and esa1 (LIU et al. 2001 ), which cause abortion of female gametes. The introduced segment carries S6 near the centromere and their interactions on cross-incompatibility are under investigation.

View larger version (17K): In this window In a new window Download PPT slide   Figure 5. Putative genetic model by which the three genes Cif, cim, and Su-Cif are involved in unidirectional cross-incompatibility. Cross-incompatibility occurs when gametes with the cross-incompatibility reactions in the female (Cif) and the male (cim) are used for fertilization. "+" indicates the cross-compatibility reaction.

Furthermore, W593A is self-fertile because it carries Cif and Cim; however, it undergoes seed abortion when crossed with male T65wx, indicating that maternally inherited Cim has no effect on rescuing the F₁ zygote and paternally inherited Cif has no effect on the deterioration of endosperm. Whether tissue-specific expression of Su-Cif occurs is uncertain, since no recombinant with Su-Cif and cim was obtained here, as mentioned. On the basis of the genic interactions proposed, if Cim were replaced by cim in W593A, the plant would be expected to be self-incompatible. This means that cross- and self-incompatibility could be convertible, depending on the combination of genes participating.

Sex-specific expression:
All three genes for cross-incompatibility found in the present experiments acted sporophytically in spite of the fact that the degeneration took place after fertilization. The paternally derived cim from the heterozygote (Cim/cim) had no effect in seed abortion, suggesting that seed abortion is determined not by the genotype of the zygote but by the genotypes of the parents. Therefore, it is not due to zygotic lethality. Sporophytic expression means that the reactions are determined before meiosis in the parent. Our cytological observations suggested that the degradation results from an arrest in early endosperm development, as is frequently observed in interspecific hybrids of plants. Numerous genes regulate the formation of gametes and the seed development in plants (FREELING and WALBOT 1993 ; GOLDBERG et al. 1994 ) and maternally and paternally derived factors also play significant roles in early seed development (EVANS and KERMICLE 2001B ; BERGER 2003 ).

The sporophytic expression detected in this study might possibly be explained by mechanisms such as a transmission of some products and signals from gametes into hybrid zygotes or their epigenetic modifications depending on the parental genotypes. In one known example of the first case, a paternally contributed factor is actually transmitted to the fertilized egg and takes part in the early development of the embryo in Caenorhabditis elegans (HILL et al. 1989 ; BROWNING and STROME 1996 ). In an example of the second case, the paternal copy of the gene is silenced via a mechanism with the features of imprinting in the Arabidopsis mutant medea (GROSSNIKLAUS et al. 1998 ; KINOSHITA et al. 1999 ).

Genetic comparisons between pre- and postfertilization barriers:
Regarding prefertilization barriers, in intergeneric hybridizations, including those among bread wheat, rye, and Hordeum bulbosum, cross-incompatibility or crossability is regulated by three Kr genes that cause an arrest of pollen-tube growth at the base of the stigma, thereby preventing the subsequent penetration of the ovary wall (SNAPE et al. 1980 ). The recessive alleles promote crossability and act additively. Genetic mechanisms for prefertilization barriers have also been reported in maize and its relatives. Within maize, the phenotype of a spontaneous mutant showing unidirectional cross-incompatibility was explained by three recessive genes (RASHID and PETERSON 1992 ). One of them controls the cross-incompatibility reaction in the female and the others control the cross-incompatibility reaction in the male, suggesting that these reactions reflect altered affinities in the style and pollen, respectively. If these recessive genes were accumulated in an individual, the plant would be expected to be self-incompatible. It was also reported that between maize and teosinte, cross-incompatibility was controlled by a series of alleles of the Ga1 gene, which was originally detected as a gametophyte gene causing a distorted segregation through certation or pollen competition within maize (KERMICLE and ALLEN 1990 ; EVANS and KERMICLE 2001a). Regarding pollen-pistil interactions, the breakdown of pollen-tube growth occurs between different genotypes in cross-incompatibility whereas it occurs between the same genotypes in self-incompatibility. Recent studies have revealed that an S allele is formed in a gene complex in which different elements are responsible for the incompatible reactions in the stigma or pollen, suggesting that a gene complex might play a role in self-incompatibility (SCHOPFER et al. 1999 ; TAKAYAMA et al. 2000 ). The complex nature of the genetic organization around the Ga1 alleles was also shown in maize and teosinte (KERMICLE and ALLEN 1990 ; EVANS and KERMICLE 2001A ); however, it is not known to what extent this complex organization is associated with the preservation of the Ga1 alleles within the primary gene pool.

In contrast to prefertilization barriers, postfertilization endosperm developmental arrest has been reported for a number of interspecific hybrids, and the present case was one such example. To explain failures of endosperm formation, it was proposed that normal development requires a proper balance of the female and male genome sets (NISHIYAMA and YABUNO 1979 ; JOHNSTON et al. 1980 ). The importance of the ratio of the parental genomes was proved by using an indeterminate gametophyte (ig) mutant of maize. A ratio within the endosperm of two chromosome sets of maternal origin to one of paternal origin is required for normal development in maize (LIN 1984 ). The failure of interspecific crosses can be well explained by assuming that the effective ploidy levels are determined in a species-specific manner (JOHNSTON et al. 1980 ; SCOTT et al. 1998 ). Although the genetic basis thereof is unknown, it has been suggested that the two parental genomes are not equivalent and that genomic imprinting might be related to the mechanism of parental conflict (HAIG and WESTOBY 1989 ; KONDOH and HIGASHI 2000 ).

Assuming that genomic imprinting is a possible mechanism for cross-incompatibility does not negate the possibility of zygotic lethality due to complementary genes. If imprinting is involved in the present case, all three genes might be modifiers for imprinting because the imprinted gene should behave in an allele-specific manner, but they all act sporophytically. Whatever the causal factor, the present results have confirmed that the two parental genomes are not equivalent for normal seed development and that sex-specific regulation is used for recognizing an appropriate partner through the cross-incompatibility in rice.

Origin of cross-incompatibility barriers:
Deleterious genes reducing fitness are eliminated due to segregational loads within populations. A simple genetic mechanism for the origin of reproductive isolation was proposed by Bateson, Dobzhansky, and Muller (DOBZHANSKY 1970 ; ORR 1996 ). Assuming two loci for simplicity, one daughter species becomes fixed for an allele at one locus, whereas the other daughter species becomes fixed for a second allele at another locus. Hybrid inviability would be established without reducing fitness if both these mutations were neutral (or advantageous) within the population in which they arose; however, they cause hybrid inviability when expressed together in the hybrid. Varietal groups of rice such as Indica and Japonica as well as wild rice tend to be more or less isolated from each other geographically and artificially (OKA 1988 ; HARUSHIMA et al. 2002 ). The wild relatives of rice are predominantly cross-pollinated; therefore, it is possible that the unidirectional cross-incompatibility might be a partial remnant phenomenon. However, this was ruled out by the fact that only prefertilization barriers were responsible for self-incompatibility minimizing the reduction of fitness. It is possible the cim found in the Japonica type is a recently derived recessive mutation that disturbs normal seed development when expressed together with Cif. Although cim is recessive, it affects the F₁ zygote through parent-of-origin effects, as mentioned above. Since Japonica-type rice carries cif, cim has no adverse effects among Japonica-type rices, supporting the above idea that fixation of neutral genes could give rise to isolating barriers.

The question of why genes causing crossing barriers are present within the primary gene pool then arises. Although the dominant suppressor (Su-Cif) seems to be carried often by the Indica type, no crossing barrier appears in hybridization between the Indica type and wild strains. Furthermore, during the procedure of backcrossing between W593 and T65wx, the cross-incompatibility became more marked in later generations, suggesting that another suppressor(s) has to be involved in the wild strain. Perhaps such suppressors could maintain genes for crossing barriers as a hidden variation in closely related taxa, which suggests that the use of alien genes might change their sexual affinities during wide-hybridization breeding. Thus, the present results show that a variety of crossing barriers could be established by the combination of genes that determined the sexual reactions for cross-incompatibility. The gene block detected here on chromosome 6 might maintain the established sexual reactions against a breakdown due to recombination, or the recombined genes might generate the diversified sexual affinities actually observed in nature.

	ACKNOWLEDGMENTS

We thank S. R. McCouch and T. Sasaki for molecular markers and Y. Kishima, H. Nagano, S. Kobayashi, N. Sawamura, and R. Suzuki for their comments and assistance.

Manuscript received February 24, 2003; Accepted for publication May 2, 2003.

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