In intraspecific crosses between cultivated rice (Oryza sativa) subspecies indica and japonica, the hybrid male sterility gene S24 causes the selective abortion of male gametes carrying the japonica allele (S24-j) via an allelic interaction in the heterozygous hybrids. In this study, we first examined whether male sterility is due solely to the single locus S24. An analysis of near-isogenic lines (NIL-F1) showed different phenotypes for S24 in different genetic backgrounds. The S24 heterozygote with the japonica genetic background showed male semisterility, but no sterility was found in heterozygotes with the indica background. This result indicates that S24 is regulated epistatically. A QTL analysis of a BC2F1 population revealed a novel sterility locus that interacts with S24 and is found on rice chromosome 2. The locus was named Epistatic Factor for S24 (EFS). Further genetic analyses revealed that S24 causes male sterility when in combination with the homozygous japonica EFS allele (efs-j). The results suggest that efs-j is a recessive sporophytic allele, while the indica allele (EFS-i) can dominantly counteract the pollen sterility caused by S24 heterozygosity. In summary, our results demonstrate that an additional epistatic locus is an essential element in the hybrid sterility caused by allelic interaction at a single locus in rice. This finding provides a significant contribution to our understanding of the complex molecular mechanisms underlying hybrid sterility and microsporogenesis.
HYBRIDS between genetically divergent species often show abnormal phenotypes that reduce fitness, such as sterility, weakness, and inviability. Such hybrid characteristics, collectively called hybrid incompatibility, are assumed to play important roles in speciation by acting as postzygotic reproductive barriers. The genetic mechanisms of reproductive barriers and their implications in evolution have been studied using a variety of plant species including Helianthus (Rieseberg et al. 1996), Mimulus (Sweigart et al. 2006), Iris (Taylor et al. 2009), and rice (Sano 1990; Koide et al. 2008). These efforts have demonstrated that diverse mechanisms of hybrid incompatibility exist and have shed some light on the roles of these mechanisms in plant evolution. However, aside from a few cases, the molecules and networks that control hybrid incompatibility remain largely unknown. It is widely accepted that interactions between genetic loci (called epistasis) contribute to reproductive isolation mechanisms. Such genetic interactions most likely reflect the existence of molecular networks that control reproductive isolation. For example, an immunity system was found to be involved in hybrid incompatibilities resulting from intraspecific crosses in Arabidopsis thaliana (Bomblies et al. 2007; Alcazar et al. 2010).
Because of its wide genetic diversity and well-characterized genetic base, cultivated rice (Oryza sativa L. 2n = 24) is a useful model for the study of hybrid sterility in plants. A number of hybrid sterility genes/QTL have been reported in hybrids between O. sativa ssp. japonica and ssp. indica. So far, two major genetic models for F1 hybrid sterility have been proposed, one involving interlocus epistasis and the other involving allelic interactions at a single locus. The interlocus epistasis model is also called the Bateson–Dobzhansky–Muller (BDM) model (Bateson 1909; Dobzhansky 1937; Muller 1942). Recent reports by Yamagata et al. (2010) and Mizuta et al. (2010) have provided experimental evidence for the BDM model, by demonstrating the reciprocal loss-of-function of duplicated genes that were involved in male gamete development. On the other hand, the interaction of alleles at a single locus model is illustrated by the hybrid sterility gene (S/Sa), which causes the selective abortion of Sa gametes in F1 heterozygotes, resulting in the predominant transmission of S alleles to their progeny. This model was first proposed for tomato by Rick (1966) and has been supported by numerous studies of gamete eliminator, pollen, and egg killer genes (Kitamura 1962; Ikehashi and Araki 1986; Sano 1990). In this model, it is assumed that stepwise mutations at a single locus underlie the development of reproductive barriers without a reduction in fitness, and consequently multiple alleles, including a neutral allele, are generated at the causal locus (Nei et al. 1983). The triallelic system of the rice S5 locus is an example of this model. S5-i (the indica allele) and S5-j (the japonica allele), cause sterility when present as a heterozygous pair, while S5-n is a neutral allele that confers fertility when combined with either S5-j or S5-i. Positional cloning revealed that the S5 gene encodes an aspartic protease (Chen et al. 2008). In another case, SaM and SaF are adjacent rice genes that encode a small ubiquitin-like modifier E3 ligase-like protein and an F-box protein, respectively. These two loci were originally thought to comprise one multiallelic locus, but it is now clear that they interact epistatically to cause hybrid male sterility (Long et al. 2008). Despite the identification of some of the gene products involved in hybrid sterility, the molecular mechanisms by which these proteins cause the sterility phenotype remain obscure. Furthermore, there have been very few investigations to discover whether the genetic background or epistasis have any effects on hybrid sterility involving allelic interactions at single loci.
The hybrid male sterility gene S24, which acts as a pollen killer gene, has a strong effect on male sterility and segregation distortion in hybrid progeny between indica and japonica (Kubo et al. 2008). Male gametes carrying the japonica allele for S24 (S24-j) are selectively aborted during the mitotic stage after meiosis in S24-i/S24-j heterozygous plants, and the indica allele for S24 (S24-i) is transmitted to about 90–100% of progeny through pollen (Kubo et al. 2008). Therefore selfing of the S24 heterozygotes produce S24-j/S24-j (pollen fertile), S24-i/S24-j (semisterile) and S24-i/S24-i (fertile) plants in an ∼0.1:1:1 ratio distorted from the expected 1:2:1 ratio. The parental homozygotes (S24-i/S24-i and S24-j/S24-j) exhibit no phenotypic abnormalities in either the pollen or other tissues. Thus, it was thought that S24 specifically affects the male gamete via an allelic interaction in heterozygous hybrids. In addition it has been found that another locus, S35 on chromosome 1, enhances pollen sterility through an interaction with S24 (Kubo et al. 2008). Using different cross combinations of indica and japonica varieties, other researchers have reported on hybrid male sterility genes named f5-Du (Wang et al. 2006) and Sb (Li et al. 2006), which map to the S24 region on chromosome 5. S24, f5-Du, and Sb occur within a single 90-kb region and are therefore likely to be the same gene (Zhao et al. 2010). Zhao et al. (2010) used a fine mapping strategy to identify two candidate genes for S24 (f5-Du and Sb), one encoding an Ankyrin domain protein and the other encoding an uncharacterized protein. Thus, S24 (f5-Du and Sb) appears to be an important factor controlling hybrid male sterility in the indica/japonica hybrids. This locus should be a useful resource for exploring the following important questions: (1) What is the molecular mechanism of the allelic interaction at the hybrid male sterility locus? (2) How does the hybrid sterility gene effect rice evolution? (3) Would it be possible to overcome reduced fitness in the rice breeding program by using a neutral allele or an epistatic restorer gene for hybrid sterility?
Our study aims to reveal the genetic and molecular bases of hybrid male sterility in rice. Our previous genetic study of S24 was performed using backcross progeny with the japonica background (Kubo et al. 2008). On the basis of its mode of inheritance, it appears that negative allelic interactions occur at the S24 locus, as in other pollen killer systems. However, there has been no previous study to determine whether the hybrid male sterility caused by allelic interaction at S24 is really controlled by a single genetic locus or whether epistasis is also involved. In this study, we addressed this question in a genetic analysis of S24 using reciprocal near-isogenic lines (NILs). By this approach we unveiled a complex genetic mechanism in which interlocus epistasis is a necessary component of the hybrid male sterility system involving negative allelic interactions at S24. On the basis of these results we propose that epistasis plays a large and essential role in most cases of postzygotic reproductive barriers in rice.
Materials and Methods
Reciprocal chromosome segment substitution lines carrying S24 segments were derived from a cross between the japonica variety Asominori and the indica variety IR24 (Kubo et al. 2002). These were backcrossed (as males) with their respective parents, and NILs for S24 were obtained by marker-assisted selection (Figure 1). All the reciprocal NILs had the Asominori cytoplasm. The populations used for further genetic analyses of hybrid male sterility were developed from a cross between the japonica variety Nipponbare and the indica variety 93-11. The backcross populations BC2F1 (N = 47), BC2F2 (N = 153), BC3F2 (N = 101), BC4F1 (N = 44), and BC4F2 (N = 189) were derived from crosses with 93-11 as the donor parent and Nipponbare as the recurrent male parent (Supporting Information, Figure S1). These populations were cultivated during 2008−2010 and plants uniformly headed by early September.
To examine the pollen phenotypes, preflowering panicles from each individual in the population were collected and fixed in a formalin/acetic acid/alcohol solution. The fixed samples were stored in a 70% ethanol solution. Three to 6 anthers collected 1 day before anthesis were stained with 1% iodine-potassium iodide and 1% acetocarmine solutions. More than 300 pollen grains were scored for each individual. Stained pollen grains with a normal size were considered to be fertile. Faintly stained small pollen grains and empty pollen grains were considered to be sterile. In this study, plants showing higher than 90% pollen fertility were classified as pollen fertile, and two classes of pollen sterility were identified: partial sterility (with 70–90% fertile pollen) and semisterility (30–70% fertile pollen).
DNA analysis and linkage map
DNA samples used for PCR analyses were crudely extracted using a 0.25 M NaOH solution, followed by neutralization with 0.1 M Tris-HCl. The crude extracts were diluted fourfold and 4.0-µl aliquots of the diluted extracts were used as DNA templates in the PCR reactions. TIGR and BLAST analyses of the genomic DNA sequences of Nipponbare and 93-11 were used to design the indel and SSR markers used in this study. The primer sequences for all markers are listed in Table S1. The PCR reactions were performed using the GO-Taq Green Master mix (Promega) in 10-µl final volumes according to the manufacturer’s instructions. The amplification reactions were generally carried out as follows: 30 cycles of denaturation at 94° for 20 s, annealing at 56° for 20 s, and elongation at 72° for 30–60 s. The PCR products were resolved on 2.0% agarose gels and visualized by ethidium bromide staining. Linkage maps of marker loci were constructed with Map Manager QTX version 0.30 (Manly et al. 2001). The recombination frequencies (%) were converted into genetic distances (in cM) using the Kosambi function (Kosambi 1944).
QTL analysis and genetic dissection of the EFS locus
The QTL and epistatic interaction analyses were carried out using a marker regression analysis with Map Manager QTX version 0.30 (Manly et al. 2001). A likelihood ratio statistic (LRS) score of 12.0 was used as the threshold to declare the presence of a putative QTL. This corresponds to an LOD score of 2.6 (LOD = LRS/4.6). To detect epistatic interactions between QTL, a genome-wide scan for all pairs of marker loci located on substituted segments was performed with Map Manager QTX, with the assumption that a QTL is right on a marker locus. The interaction effect and the magnitude were calculated using a two-way ANOVA. The EFS locus was mapped using the BC2F2, BC3F2, and BC4F1 populations derived from the cross between Nipponbare and 93-11. The fertile homozygotes for S24 did not provide any recombination information for EFS mapping. Therefore, 140 S24-heterozygous plants, identified by marker-assisted selection with the mS1 and mS2 markers, were used for mapping EFS (Figure 3D).
The S24 sterility is dependent on genetic background
S24 was first identified in backcross populations for introduction of an indica chromosome segment into a japonica cultivar of rice (Kubo et al. 2008). The S24 locus causes pollen semisterility owing to selective abortion of male gametes carrying S24-j (japonica allele for S24) in the heterozygote, leading to severe segregation distortion at the S24 locus in favor of the indica alleles in the segregating population (Kubo et al. 2008). This pollen sterility appeared to be mainly caused by an allelic interaction at the S24 locus. If this allelic interaction is sufficient to cause the phenotype, the same phenotype should be seen in any genetic background. To address the question we developed NILs carrying a single heterozygous segment harboring the S24 locus in reciprocal backgrounds and examined their pollen phenotypes. A heterozygous NIL with Asominori (japonica) genetic backgound (AI-NIL-F1), showed pollen semisterility (41.8% fertility) (Figure 1), as was demonstrated previously (Kubo et al. 2008). Its selfed progeny (AI-NIL-F2) exhibited significant segregation distortion representing preferential transmission of the indica allele at S24 (jp/jp: in/jp: in/in = 6:69:67, χ2 = 52.52, P < 0.001) (Table 1). However, a heterozygous NIL with the IR24 (indica) background (IA-NIL-F1), produced fertile pollen grains (92.9% fertility) (Figure 1). Selfed progeny of the IA-NIL-F1 (IA-NIL-F2) did not show reduced frequency of japonica allele at S24 (the frequency of japonica homozygote was 33.3%), even though the segregation ratios among the IA-NIL-F2 also deviated from 1:2:1 (jp/jp: in/jp: in/in = 23:21:25, χ2 = 10.68, P = 0.004) (Table 1). This result indicated that the S24-j male gametes in the S24 heterozygotes with the indica background were fertile and transmitted normally to their progeny. Therefore, we concluded that the S24 heterozygous alleles can induce male sterility only in the japonica background, and that epistasis must be involved in the genetic mechanisms of the pollen sterility caused by S24.
Detection of the epistatic factor for pollen sterility
Since the Nipponbare (japonica) and 93-11 (indica) genomes have been fully sequenced, we decided to carry out further analyses with these varieties. First we needed to confirm that the same S24 locus occurred in these lines and then identify the epistatic gene hidden in the japonica genetic background. To these ends we developed another set of backcross populations using Nipponbare as the recurrent parent and 93-11 as the donor parent (Figure S1). Both parents have >90.0% pollen fertility, while their reciprocal F1 hybrids, Nipponbare/93-11 and 93-11/Nipponbare, showed 37.8 ± 2.8% (N = 3) and 42.9 ± 7.6% (N = 3) pollen fertility, respectively. Among eight plants from the BC1F1 generation (Nipponbare/93-11//Nipponbare), we identified a fertile segregant (90.8% pollen fertility) that carried heterozygous segments harboring the S24 locus. To identify the other genetic factors that affect pollen sterility, the fertile BC1F1 plant was backcrossed with Nipponbare, and the segregating BC2F1 population (N = 47) was analyzed. The BC2F1 population showed a wide distribution for pollen fertility (37.7−100%) with two classes of sterility phenotypes: semisterility (37.7−70.0%) and partial sterility (70.0−90.0%) (Figure 2A). We performed a QTL analysis using the BC2F1 population with 76 PCR markers that were evenly distributed throughout the 12 rice chromosomes (Table S1). A marker regression analysis detected two putative QTL for pollen sterility, designated qPS2 and qPS5, which were located on chromosomes 2 and 5, respectively (Table 2). The qPS5 QTL was linked to markers chr05-109 and mS3 and reduced pollen fertility in plants with the heterozygous genotype. On the other hand, the qPS2 QTL at the marker locus mE4 increased pollen fertility in the heterozygous plants. Since the position and phenotypic effect of the qPS5 QTL coincided with those of the S24 locus (Kubo et al. 2008), qPS5 was considered to be S24. Next we performed a genome-wide interaction analysis to identify marker pairs that interacted epistatically to significantly affect pollen fertility. As a result, we identified two pairs of interactions, one involving the pair qPS5 (S24) and qPS2 and the other involving loci on chromosomes 4 (chr4-3173) and 9 (chr9-0755) (P < 0.001). A two-way ANOVA revealed a significant interaction effect between the marker loci mS3 (linked to S24) and mE4 (linked to qPS2) (Figure 2B, Table S2). The combination of heterozygous alleles for mS3 (S24) and homozygous Nipponbare (japonica) alleles for mE4 (qPS2) significantly reduced pollen fertility (63.7%) compared with the other genotype combinations (>95.0% fertility). This result suggests that qPS2 may be associated with S24 in its effects on pollen fertility. The qPS2 QTL has not previously been characterized and no gene/QTL for pollen sterility has previously been reported for this region of chromosome 2. Therefore we named this new locus EFS (Epistatic Factor for S24). The other pair of interacting loci, chr4-3173 and chr9-0755, significantly reduced pollen fertility (65.5%) (Table S2). However, further analyses did not show any interactive effects on sterility between these two loci (data not shown).
Genetic dissection of the EFS locus
To verify the effect and position of the EFS locus, we developed the advanced backcross populations BC3F2 and BC4F1 by making additional backcrosses between a fertile BC2F1 segregant and Nipponbare. Specific S24 and EFS alleles were identified by marker-assisted selection. The BC3F1-8-4 plant, which was used as the progenitor of the BC3F2 and BC4F1 populations, carried only small segments of chromosomes 2, 4, and 5 from 93-11 in an otherwise uniform Nipponbare background (Figure 3A). We evaluated the phenotypes of all nine genotype classes generated by different combinations of S24-linked and EFS-linked marker alleles, in both the BC2F2 and BC3F2 populations (Table S3, Figure S2). We found that the S24-i/S24-j heterozyogtes showed pollen sterility (average 70.1% in the BC3F2 population) only when they were combined with the homozygous Nipponbare EFS alleles (efs-j/efs-j). All other genotype combinations showed no abnormalities in their pollen phenotypes (Table S3). This result provided conclusive evidence for the existence of EFS and its effect of inducing male sterility in S24 heterozygotes when the recessive efs-j allele is present in the homozygous condition. The BC4F1 population was also analyzed to confirm the EFS effect on phenotype. Measurements of mean pollen fertilities, along with microscopic examinations of pollen grains, indicated that pollen sterility occurred only in plants that were heterozygous at the S24 locus and homozygous for the Nipponbare allele at the EFS locus (Figure 3, B and C). As in the BC2F1 and BC3F2 populations, the S24-i/S24-j efs-j/efs-j genotype in the BC4F1 generation showed two types of pollen sterility: semisterility (30.3–59.1% fertile) and partial sterility (74.0–82.3% fertile) (Figure S2).
We next performed a genetic dissection of the EFS locus to determine its position using the BC2F2, BC3F2, and BC4F1 populations. Since the fertile S24 homozygotes did not provide any recombination information for EFS mapping, a total of 140 plants with the S24 heterozygous genotype were examined. Of the 140 informative plants, we found 2 with recombinations between EFS and the marker loci. One recombinant, BC4F1-8-4-39, had a recombination breakpoint between mE3 and mE4. The other, BC3F2-8-4-37, was recombined between mE4 and mE5. Both recombinants showed the fertile phenotype, and we therefore concluded that the fertile EFS allele (EFS-i) resided on the substituted regions that overlapped in the two recombinants. Therefore, the EFS gene was mapped to a 817-kb region between the marker loci mE3 and mE5 on chromosome 2 (Figure 3D).
Selective transmission of the S24-j gamete is counteracted by EFS-i in the sporophyte
The transmission rates of individual S24 alleles in male gametes were examined to determine whether the EFS effect on S24 occurs in the gametophyte or the sporophyte. The transmission rates were evaluated using reciprocal crosses between the double heterozygote for S24 and EFS (NILS24+EFS) and Nipponbare. If EFS acts gametophytically, pollen with the S24-j efs-j genotype would be selectively eliminated. In contrast, if EFS acts sporophytically then no biased transmission would be observed. The results revealed that the transmission ratio of each genotype fitted a theoretical ratio of 1:1:1:1. Male gametes carrying the S24-j efs-j alleles were transmitted with a frequency of 14.3% when the double heterozygote was used as the male parent (Table 3). This was slightly lower than the expected frequency of 25.0% but higher than that observed when a plant that was heterozygous at the S24 locus and homozygous for the efs-j allele was used as the male parent [0% in our previous study (Kubo et al. 2008)]. In two different generations, the double heterozygotes for S24 and EFS produced S24-j/S24-j progeny at frequencies of 17.8% (BC3F2-8-4) and 21.7% (BC4F2-8-4-1), on the basis of the transmission frequencies of a linked marker (Table 1). These frequencies were distinctly higher than the 5.7% observed in the BC3F2-8-6 population, which was a sister line of BC3F2-8-4 that was homozygous for the Nipponbare EFS allele (genotype: S24-i/S24-j efs-j/efs-j). These results indicate that the S24-j male gametes were able to develop normally in the EFS-i/efs-j heterozygotes and be transmitted to the progeny. The results also suggest that the EFS indica allele (EFS-i) acts dominantly to counteract the sterility of the S24-j male gametes in the S24 heterozygotes. Thus, our results consistently indicate that the allelic interaction at the S24 locus requires the presence of the efs-j homozygous genotype to induce the antagonistic allelic interactions causing male sterility.
Multiple epistatic interactions affect the S24 gene
Here we present evidence that allelic interactions at the S24 locus induce the selective abortion of male gametes carrying the S24-japonica allele (S24-j) via genetic interactions with the unlinked locus EFS, which is located on chromosome 2. The recessive japonica allele for EFS (efs-j) causes male sterility in collaboration with S24. Conversely, the dominant indica allele for EFS (EFS-i) counteracts the pollen sterility by S24. Generally, pollen killer or gamete eliminator systems induce sterility only when the causal loci are heterozygous. Therefore it has been widely documented that allelic interactions are the major causes of these phenomena. Few epistatic factors regulating pollen/egg killer or gamete eliminator systems have been reported in any plant species, despite a long history of studies for nearly half a century. The reason why epistasis in such hybrid sterility systems stubbornly remains obscure might be because the allelic interaction model appears to sufficiently explain these phenomena. In fact, the allelic interaction at the S24 heterozygous locus remains an important causal factor in this male sterility system. Our study demonstrated that the allelic interaction at S24 becomes active only in the sporophyte homozygous for recessive allele of EFS (efs-j). The S24−EFS interaction can be interpreted as a sporogametophytic interaction by two independent loci as shown by model III in Figure 4A. This genetic model fits into neither of the previously recognized genetic models for hybrid sterility systems (models I and II in Figure 4A).
Another interactor, S35, has been shown to associate with S24 and enhance pollen sterility in S24 heterozygotes (Kubo et al. 2008). The S35 gene requires the presence of the S24-i allele to cause pollen sterility, but S24 can induce semisterility independently of the S35 genotype, and thus this interaction is unidirectional (Figure 4B). The relationship between EFS and S35 remains to be elucidated. However, since both S35 and EFS interact with S24 and affect its activity, we expect that S35 participates in the S24–EFS network in some way. In another case, female sterility due to epistasis among three unlinked loci has been found in one cross combination in rice. In this case, interactions among one sporophytic and two gametophytic genes induced the selective abortion of female gametes carrying a specific pair of alleles (Kubo and Yoshimura 2005). These previous findings and those presented here emphasize a large contribution of epistasis to the genetic mechanisms of hybrid sterility. They further suggest that hybrid sterility may be controlled by complicated networks composed of a variety of epistatic genes.
Molecular and functional aspects of S24 and EFS
Our studies demonstrated that hybrid sterility is strictly controlled by the S24 and EFS genotypes. In further studies we will identify and characterize the products of the S24 and EFS genes, in an effort to understand how they cause the selective abortion of the S24-j male gametes. Since the genome sequence data for both the S24 and EFS regions did not show any major structural differences such as large inversions, insertions, or deletions between Nipponbare and 93-11 (MSU Rice Genome Annotation Project ver. 6.0), the hybrid male sterility phenotype appears to be caused by negative interactions between proteins generated from polymorphic alleles at each locus. Zhao et al. (2010) identified the Ankyrin-3 (ANK3) protein as a primary candidate for the S24 product, on the basis of a map-based cloning approach. We also performed fine mapping of S24, and the candidate region of S24 was narrowed down to the region between mS1 and mS2 containing ANK3 (122–kb region) (Figure 3D). ANK is an adapter protein that exclusively mediates protein–protein interactions and is involved in various biological activities including signal transduction and the maintenance of cytoskeleton integrity. Therefore, it is possible that EFS may encode a protein capable of binding with ANK3. Because the EFS candidate region contains ∼80 predicted genes excluding transposable elements, it remains to be determined whether the EFS effects on pollen sterility involve protein interactions with ANK3. Since neither Nipponbare nor 93-11 have gene encoding ANK-domain protein at the EFS candidate region, the genetic interaction between S24 and EFS should not be due to duplication of ANK3 at these loci. Meanwhile tandem array of ANK3 (three copies) were found at the 122-kb region of Nipponbare and 93-11 alleles, suggesting that allelic diversity at these ANK3 loci may be a cause of the pollen sterility. Histological analyses revealed that the S24 genotype did not affect pollen development at the male meiotic stage, but that mitotic cell cycle arrest was observed at the mononuclear or bicellular pollen stage in affected genotypes (Kubo et al. 2008; Zhao et al. 2010). The (sporophytic) tapetal cells degenerate at this developmental stage, and this degeneration is essential for providing a supply of nutrients needed for normal pollen development. Since the EFS and S24 genes function in a sporophytic manner, it seems likely that they function in diploid tissues of reproductive organs, such as tapetal cells, which have a strong influence on pollen development. A number of mutant analyses relating to microsporogenesis have been reported in plants (Borg et al. 2009). However, the molecular networks that control microsporogenesis remain largely unknown. Further studies of the S24–EFS network should aid our understanding of the molecular mechanisms involved in hybrid male sterility as well as the molecular networks underlying microsporogenesis.
Putative supergene complex
A few issues remain unresolved by this study. One is the large variation in the pollen sterile phenotypes due to S24 (see Figure S2). Another is the incomplete elimination of the skewed transmission frequencies of male gametes in progeny of the double heterozygous plants (Tables 1 and 3). Previous studies showed that S24 (f5-Du) consistently and drastically reduced pollen fertility without the partial sterility (i.e., 70.0–90.0% pollen fertility) found in this study, even in different cross-combinations and under different environmental conditions (Wang et al. 2006; Kubo et al. 2008). In the present study, we developed a BC4F1 population carrying only a few chromosomal segments spanning the S24 and EFS loci, to evaluate the exact phenotype of the EFS gene (see Figure S1 and Figure 3). However, segregants carrying the S24-i/S24j efs-j/efs-j genotype showed large phenotypic variations (ranging from 30.3 to 82.3% pollen fertility), which were carried over from the BC2F1 and BC3F1 generations (Figure S2). Presumably, the causes of this variation are undetermined genetic factors that may or may not be linked with S24.
The incomplete elimination of the skewed transmission frequencies (mentioned above) may be due in part to an unidentified segregation distorter linked to S24. The lengths of the substituted segments in the populations analyzed in this study were different from those used in our previous study (Kubo et al. 2008). This allowed for different allele combinations via recombination within the linked loci. There are many cases in which the chromosomal region causing hybrid sterility contains two linked genetic loci (Sano 1990; Long et al. 2008). Intriguingly, a QTL analysis by Li et al. (1997) identified putative “supergenes” relating to hybrid sterility on rice chromosomes 5 and 2, in regions adjacent to the S24 and EFS loci, respectively. Supergenes are defined as groups of tightly linked genes within which recombination will cause reduced fitness (Darlington and Mather 1949). In light of these observations, the genetic and genomic features of S24 may closely resemble the segregation distorter (SD) system of Drosophila. SD is a multigene selfish gene complex (supergene cluster), in which different allele combinations regulate segregation distortion and determine phenotypic degrees (Presgraves 2007). Sd, one of the gene loci in the SD complex, encodes a truncated RanGAP that possesses enzyme activity but lacks intracellular localization domains, leading to mislocalization to the nucleus and causing segregation distortion. Unlinked suppressors have also been characterized in the SD system, suggesting that complex interactions between cis and trans components play roles in this reproductive isolation mechanism. If S24 is part of an SD-like system in rice, there may be numerous epistatic factors at both linked and unlinked loci that have yet to be identified, and these may form a complicated mechanism regulating the hybrid male sterility.
Evolutionary aspects of S24 and EFS
On the basis of the present results, the dominant EFS-i allele allows the abortive S24-j allele to be transmitted to the progeny by counteracting the pollen sterility by S24. Therefore, the S24-i allele has no strong advantage in early segregating generations of hybrid progeny between indica and japonica. Actually, no segregation distortion in the chromosomal region harboring S24 has been detected in an F7 population of recombinant inbred lines of Asominori/IR24 (Tsunematsu et al. 1996), and the same is true for the F11 generation of a set of Nipponbare/93-11 recombinant inbred lines (Huang et al. 2009). The segregation distortion observed in this study was elicited by backcrossing with japonica parents. Once the efs-j allele becomes fixed as homozygous in a population, the abundance of the S24-i allele should drastically increase due to the selective elimination of the S24-j allele. To understand the role of the EFS locus in the evolution of the allelic S24-sterility system, it would be useful to determine the timing of the appearance of mutations at both the S24 and EFS loci. It is not yet clear whether the sterility-associated S24 mutations arose before the epistatic EFS mutations in the ancestral species. For example, if the S24-j allele arose before the efs-j allele, EFS might have played an important role in allowing the deleterious S24-j mutation to spread and become fixed in the initial population without a reduction in fitness. More importantly, a sterility-neutral allele of S24 (S24-n), conferring wide compatibility with both the S24-i and S24-j alleles, has been found in an analysis of the aus variety “Dular” (Wang et al. 1998; Zhao et al. 2010). This multiple allelism could also account for the development of a sterility barrier without a reduction of fitness during the process of evolution (Nei et al. 1983). On the basis of our findings and those of others, it is also possible that the sterility barrier became established through the gradual accumulation of mutations at multiple loci showing both epistatic and allelic interactions, without any fatal phenotypes developing during population divergence. Further molecular studies to elucidate gene structures and functions, along with phylogenetic and evolutionary analyses, will provide insights into the evolutionary history of the hybrid sterility system controlled by S24 and EFS.
We thank T. Makino and Y. Gonohe (National Institute of Genetics, Mishima, Japan) for technical assistance and Y. Harushima for providing indel marker information and BC1F1 seeds derived from the cross between Nipponbare and 93-11. We thank Y. Yamagata and M. Shenton for a critical review of this manuscript. We also thank two anonymous reviewers for useful comments. This study was partly supported by a grant-in-aid for special research on priority areas from the Japanese Ministry of Education, Science, Culture, and Sports (no. 18075009 to N.K.) and partly by a grant-in-aid for young scientists (B) from the Japanese Society for the Promotion of Science and the Japanese Ministry of Education, Culture, Sports, Science, and Technology (no. 21780008 to T.K.).
Supporting information is available online at http://www.genetics.org/content/suppl/2011/08/25/genetics.111.132035.DC1.
- Received June 24, 2011.
- Accepted August 18, 2011.
- Copyright © 2011 by the Genetics Society of America