Expressivity of the fon2 mutation is reduced in F₂ plants of a cross between fon2-1 (japonica) and Kasalath (indica)

In wild-type rice, a single pistil derived from congenitally fused carpels develops into a floret, and a single ovule is formed in the pistil. After fertilization, a single seed is formed within the husks, which are derived from the palea and lemma in a floret (Figure 1A). In fon1 and fon2 mutants, by contrast, the number of floral organs such as pistils increases due to an enlargement of the FM (Figure 2B) [8],[19]. Therefore, “twin seeds” are formed within the husks in these fon mutants (Figure 1B) when two or more pistils are produced in a floret (the third and fourth seeds cannot develop to maturity). Here we used the twin seed phenotype as an indication of fon2 mutation.

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Figure 1. The fon2 mutation is suppressed in F₂ Progenies from a cross between fon2 mutant (japonica) and Kasalath (indica).

(A,B) Seed phenotype of wild type (A) and fon2-1 mutant (B) of japonica. Arrowheads indicate embryos. (C) Reduction in expressivity in F₂ plants from a cross between fon2-1 and Kasalath. Blue bars indicate the distribution of F₂ plants showing a fon2 phenotype (n = 154). Red bars indicate the distribution of the fon2-1 mutant (n = 20). Arrows indicate the median frequency in the appearance of the twin-seed phenotype per panicle. (D,E) Frequencies of genotypes of the molecular markers on chromosome 2 in F₂ plants showing a fon2 phenotype with high suppressing activity (twin seed frequency, <5%; n = 28) (D) or low suppressing activity (twin seed frequency, >45%; n = 20) (E). Arrowheads in (D,E) indicate the chromosomal position where high or low suppressing activity is closely associated with the genotypes homozygous for indica or japonica, respectively.

https://doi.org/10.1371/journal.pgen.1000693.g001

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Figure 2. Indica FOS1 suppresses the floral phenotypes caused by the fon2 or fon1 mutation.

(A) A wild-type flower with one pistil in japonica (T65). (B) A fon2-1 flower with three pistils. (C) A flower in fon2-1 that is suppressed by Agrobacterium-mediated transformation of a genomic fragment containing the FOS1 locus derived from indica (Kasalath). (D) A flower in a F₂ plant from a cross between fon2-1 and CSSL#9 that is homozygous for fon2-1 and for the FOS1 gene derived from Kasalath. The floral phenotype of fon2-1 is suppressed by indica FOS1. (E) A fon1-5 flower with two pistils. (F) A flower in fon1-5 that is suppressed by Agrobacterium-mediated transformation of a genomic fragment containing the FOS1 locus derived from indica (Kasalath). Arrows in (A–F) indicate pistils. (G) Number of stamens. (H) Number of pistils. 1, Wild type (T65); 2, fon2-1; 3, fon1-5; 4, fon2-1 plant that is transformed with an indica (Kasalath) genomic fragment containing FOS1. 5, F₂ plant from a cross between fon2-1 and CSSL#9 that is homozygous for fon2-1 and for indica FOS1 derived from Kasalath. 6, fon1-5 plant that is transformed with an indica (Kasalath) genomic fragment containing FOS1. For each strain, 100 flowers were examined.

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In a previous screen of the fon2 phenotype for positional cloning [8], we found that expressivity of the fon2 mutation was reduced in F₂ plants from a cross between the fon2-1 mutant (japonica) and Kasalath (indica). To estimate quantitatively the frequency of the appearance of the fon2 phenotype, we assessed the numbers of twin-seed phenotypes in this study. First, we counted the number of plants producing the twin-seed phenotype among F₂ plants from the fon2-1 and Kasalath cross. As a result, we found that the number of F₂ plants showing a fon2 phenotype was reduced markedly in the F₂ plants: only 4.5% of F₂ plants showed a fon2 phenotype (Table 1). Second, we found that the number of the twin-seed phenotypes per panicle was also reduced markedly (Figure 1C). The median frequency of the appearance of the twin-seed phenotype per panicle was 71% in the fon2-1 mutant (japonica); by contrast, it was reduced to 17% in the F₂ plants showing a fon2 phenotype. These results suggest that the indica (Kasalath) genome causes a reduction in the expressivity of the fon2 mutation in the F₂ progenies. In other words, it is likely that the indica genome has one or more genes that suppress the phenotype caused by the fon2 mutation.

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Table 1. Segregation ratio of the fon2 phenotype in F₂ plants from a fon2-1 and Kasalath cross.

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To address this possibility, we performed quantitative trait locus (QTL) analysis (see Materials and Methods). For QTL analysis, we checked the genotypes of the F₂ plants showing the fon2 phenotype to confirm that the fon2 locus was homozygous for the mutation. As a result, a major QTL that suppressed the fon2 mutation was detected in the region between 40 and 80 cM on chromosome 2. In a group of F₂ plants showing high suppressor activity, the frequency of genotypes homozygous for the indica genome was very high, whereas that of genotypes homozygous for the japonica genome was very low (Figure 1D). In a group of F₂ plants showing low suppressor activity, by contrast, the opposite result was obtained (Figure 1E). Thus, in this region of chromosome 2, high or low suppressor activity was closely associated with a genotype homozygous for indica or japonica, respectively.

Identification of a gene that suppresses the fon2 mutation in the indica genome

The above results indicated that a putative gene that suppresses the fon2 mutation is located in the 40–80 cM region of chromosome 2 in the indica genome. We assumed that if indica has a gene that is functionally redundant to FON2 in this region, this gene should behave as a suppressor-like gene function in genetic analyses. A strong candidate for such a gene would be a CLE gene, like FON2. A survey of rice genomic sequences identified a candidate CLE gene located at about 54 cM on chromosome 2.

Next, we examined whether the CLE gene on chromosome 2 in indica (tentatively named CLE-C2) could suppress the fon2 mutation by conducting the following two experiments. First, we introduced a 3.3-kb genomic fragment from Kasalath containing the CLE gene into the fon2-1 mutant by Agrobacterium-mediated transformation. We found that the defect in the fon2 flowers was completely rescued in the transgenic plants, suggesting that the CLE-C2 gene suppressed the fon2 mutation (Figure 2C, 2G, and 2H). Second, we applied a genetic approach using a Nipponbare/Kasalath chromosomal segment substitution line (N/K CSSL#9), in which the chromosomal segment of Nipponbare (japonica) encompassing the CLE-C2 gene was replaced by that of the Kasalath (indica) genome. We crossed fon2-1 (japonica) with N/K CSSL#9, and then screened for F₂ plants that were homozygous for both fon2-1 and the indica CLE-C2 allele by determining the genotype with molecular markers. The results indicated that the flower phenotype of the plants screened was identical to that of wild type, suggesting that the fon2 mutation was also suppressed (Figure 2D, 2G, and 2H). Taken together, these results clearly indicate that the CLE-C2 gene located at about 54 cM on chromosome 2 functions as a fon2 suppressor. Thus, we designated this CLE gene as FON2 SPARE1 (FOS1) because this gene can substitute for FON2.

Molecular characterization of FOS1

Sequence analysis revealed that FOS1 consists of one exon with a single open reading frame and encodes a putative small protein of 131 amino acids (Figure S1). FOS1 has a signal peptide that is rich in hydrophobic amino acids at its N-terminus and a CLE domain at its C-terminus (Figure 3A). Among the CLE proteins in rice and Arabidopsis, the CLE domain of FOS1 is more similar to those of CLE8 and CLE13 than to those of FON2, FCP1 and CLV3 (Figure 3B).

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Figure 3. Characteristics of FOS1 protein.

(A) Schematic representation of FOS1. The putative cleavage site of the signal peptide was predicted by signalP (http://www.cbs.dtu.dk/services/SignalP; Fletcher et al. 1999). Partial amino acid sequences of japonica and indica FOS1 are shown. (B) Alignment of amino acids from putative CLE peptides from related CLE proteins in rice (FOS1, FON2 and FCP1) and Arabidopsis (CLV3, CLE8 and CLE13).

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Notably, the nucleotide sequence of FOS1 in the fon2-1 mutant (background, Fukei71) was identical to that of standard japonica wild-type strains such as Nipponbare and Taichung65 (T65). By contrast, we found an amino acid difference at the putative cleavage site of the signal peptide between japonica (all three stains; AB455109) and indica (Kasalath; AB455108) (Figure 3A; Figure S1). It is possible that this amino acid substitution in japonica FOS1 causes a defect in the processing of FOS1 and a reduction in the amount of active CLE peptide in japonica. Except for this mutation, no nucleotide change causing indels of amino acids was detected in the FOS1 gene between indica and japonica.

FOS1 seems to be involved in meristem maintenance

We analyzed the spatial and temporal expression patterns of FOS1 by in situ hybridization. In indica, FOS1 was expressed in all aerial apical meristems, not only in the FM and the IM in the reproductive phase, but also in the SAM in the vegetative phase (Figure 4A–4C). In japonica, FOS1 transcripts were also detected in all apical meristems in a spatial distribution pattern similar to that observed in indica (Figure 4E–4G). These spatial expression patterns suggest that FOS1 may be involved in meristem maintenance in rice. In addition to the meristems, FOS1 transcripts were also detected in the primordia of lateral organs such as the leaf and the floral organs. No significant differences were observed at the transcriptional level in the expression patterns of FOS1 between indica and japonica, suggesting that the functional difference between indica FOS1 and japonica FOS1 is not due to differences at the transcriptional level. No signals were detected in the SAM and FM when sense probes were used (Figure 4D and 4H).

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Figure 4. Spatial expression pattern and constitutive expression of FOS1.

(A–H) In situ localization of FOS1 transcripts in indica (A–D) and japonica (E–H): SAM (A,E,H), IM (B,F), and FM (C,D,G). Antisense probe (A–C,E–G), sense probe (D,H). (I, J) Shoots overexpressing indica FOS1 in wild-type japonica (I) and fon1-5 (J). (K,L) Longitudinal sections of the shoot apex in a shoot expressing control vector (K) and Act1:indica-FOS1 (L). (M,N) In situ localization of OSH1 transcripts in a shoot expressing a control vector (M) and Actin1:indica-FOS1 (N). (O) A shoot overexpressing japonica FOS1 in wild-type japonica. (P) A shoot transformed with a control vector. fm, floral meristem; im, inflorescence meristem; lp, leaf primordia; sam, shoot apical meristem. Scale bars: 100 µm in (A–H,K–N); 5 mm in (I,J,O,P).

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Next, we expressed constitutively indica FOS1 by using the rice Actin1 promoter. Unlike shoots transformed with a control vector, Actin1:indica-FOS1 shoots stopped growing at the seedling stage after a few abnormal and malformed leaves were produced (Figure 4I and 4P). A longitudinal section of the shoot apex revealed that a dome-shaped shoot apical meristem (SAM) was strongly compromised in Actin1:indica-FOS1 plants, as compared with transgenic seedlings carrying a control vector (Figure 4K and 4L). Next, we examined the expression pattern of rice OSH1, an ortholog of Arabidopsis SHOOT MERISTEMLESS (STM) and maize knotted1 (kn1), which marks undifferentiated cells in the meristem [25]. OSH1 was expressed uniformly in the meristem except for the site of leaf initiation (P0) (Figure 4M). By contrast, OSH1 expression was not observed in the meristem of Actin1:indica-FOS1 shoots (Figure 4N). These results indicate that constitutive expression of indica-FOS1 terminated meristem function, suggesting that FOS1 is involved in the maintenance of stem cells in the vegetative SAM as well as in the FM.

FOS1 is likely to act independently of FON1

To address whether, FON1, a putative receptor of FON2, is required for FOS1 function, we introduced an indica genomic fragment containing FOS1 into the fon1-5 mutant, which has a severe mutation and is thought to be a null allele of fon1. The numbers of floral organs such as stamens and pistils in fon1-5 transformed with the indica FOS1 gene were identical to those in wild type, suggesting that FOS1 functions normally in this fon1 mutant (Figure 2F–2H). Next, we expressed indica-FOS1 constitutively in fon1-5. The resulting shoot showed a phenotype identical to that of wild type constitutively expressing indica FOS1 (Figure 4J). These results suggest that the FON1 receptor is not required for the function of FOS1 and that FOS1 is likely to function in an independent signaling pathway.

Origin of the japonica FOS1 allele

To elucidate when the mutation observed in japonica FOS1 occurred during rice evolution, we compared the FOS1 sequence of a number of varieties/species of domesticated rice and wild rice species such as O. rufipogon. To encompass genetic diversity, we examined a core collection of domesticated rice from around the world (WRC, 67 accessions) and from Japan (JRC, 50 accessions) (Table S1; Table S2) [26],[27].

As described above, the one-base change that causes an amino acid substitution at the putative cleavage site of the signal peptide in FOS1 and is associated with its function was found in three japonica strains (fon2-1, Nipponbare and T65). Hereafter, we call this mutation a functional nucleotide polymorphism (FNP) without reference to the japonica or indica type. Sequence analysis showed that 66 out of 68 accessions of japonica had the FNP (Haplotype B, see below), whereas 59 out of 60 accessions of indica did not (Haplotype A) (Table S1; Table S2; Figure S2). Thus, the FNP was closely associated with japonica except for three accessions (Calotoc, Pinulupot 1, Padi Perak). Although the accessions in the WRC have been designated indica or japonica by phenotypic analysis, it seemed likely that the genome of two subspecies might have been introgressed into each other during recent breeding programs. Thus, we examined the type of genome around the FOS1 locus in the three exceptional accessions by using molecular markers. The results clearly indicated that the FNP is consistent with the japonica genome, but not with the indica genome (Table 2).

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Table 2. Genotyping of the chromosomal regions around the FOS1 locus.

https://doi.org/10.1371/journal.pgen.1000693.t002

Next, we compared the FOS1 sequence from five wild rice species (22 accessions) and the African domesticated rice O. glaberrima (2 accessions), all of which have an AA genome (Table S3). Nucleotide polymorphisms were found in FOS1 among the wild and domesticated rice accessions (Figure S2). We classified the FOS1 sequences into 13 haplotypes, and generated a network of these haplotypes (Figure 5). The network indicated that the prototype of FOS1 is haplotype C, which is shared by two wild rice species, O. rufipogon and O. glumaepatula. The FOS1 sequence in wild rice species and domesticated rice may have been derived from this haplotype. None of the accessions of wild rice species showed the FNP at the processing site. Therefore, the FNP in FOS1 is specific to the genome of O. sativa japonica. Because Asian domesticated rice species, namely japonica and indica, are thought to have derived independently from O. rufipogon [28]–[30], this FNP may have occurred during the diversification of japonica from O. rufipogon.

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Figure 5. Network of FOS1 haplotypes.

The sequences of the haplotypes (A–M) are given in Figure S2. Colors indicate a species or subspecies with the AA genome in the Oryza genus. Blue, O. sativa indica; light blue, O. sativa japonica; yellow, O. rufipogon; magenta, O. glumaepatula; dark green, O. barthii; light green O. glaberrima; purple, O. meridionalis; orange, O. longistaminata. FOS1 has the FNP in the pink region but does not in the light violet region. The triangle and inverted triangle indicates insertions and deletions, respectively (Figure S2). The hatched line corresponds to a single nucleotide substitution. The number of accessions with each haplotype is roughly indicated by the size of the circle. Accession numbers for the sequences of each haplotype are as follows: FOS1 haplotype A (AB455110), B (AB455111), C (AB455112), D (AB455113), E (AB455114), F (AB455115), G (AB455116), H (AB455117), I (AB455118), J (AB455119), K (AB455120), L (AB455121), and M (AB455122).

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Identification of FOS1

The presence of a factor that suppresses fon mutations in indica was initially assumed from the low expressivity of the fon phenotype in F₂ plants from a cross between japonica and indica. Although there are two possible explanations for this low expressivity – namely, differences in the genetic background of japonica and indica, or the presence of a major gene in the genome of indica – QTL analysis provided evidence in support of the latter possibility.

Several lines of evidence suggest that the function of FOS1 is likely to be compromised in japonica. As a result, mutations at the FON2 locus result in enlargement of the FM and an increase in the floral organ number in japonica [8]. In indica, by contrast, functional FOS1 probably masks fon2 mutations by substituting for FON2 function in regulating maintenance of the FM (Figure S3). Likewise, in F₂ plants from a cross between japonica and indica, FOS1 derived from indica is likely to mask the fon2 mutation. The frequency (4.5%) of the appearance of the fon2 phenotype, which is also confirmed by the genotype, in those F₂ plants is roughly consistent with that expected for the appearance of double mutants. Overexpression of japonica FOS1 produced an abnormal shoot, as did overexpression of indica FOS1, suggesting that japonica FOS1 is not a complete loss-of-function mutant. In wild-type japonica, however, FOS1 CLE peptide, even if produced in part, would be insufficient to restrict stem cells in the FM.

Because our rice research is principally based on japonica, indica FOS1 appears to behave as though it is a suppressor of the fon2 mutation. A more likely interpretation is, however, that FOS1 regulates maintenance of the FM redundantly with FON2 in a wide range of species in the genus Oryza (see below) and that japonica is a mutant for the FOS1 locus.

In plant development, it is well known that genes that encode closely related proteins have redundant functions. APETALA1 (AP1) and CAULIFLOWER (CAL), which that encode MADS-box transcription factors, regulate floral meristem identity together with LEAFY [31],[32]. The ap1 cal double mutant has a striking phenotype, showing excessive proliferation of the inflorescence meristem, which resembles a cauliflower. This phenotype differs from that of the ap1 single mutant. Because CAL has less effect on floral meristem identity, its single mutation shows no phenotype. CAL was identified as an enhancer of the ap1 phenotype in F₂ plants from a cross between the ap1 mutant on a Landsberg electa background and wild-type Wassilewskija [31]. Thus, the identification of FOS1 in this study resembles the discovery of CAL in Arabidopsis, although FOS1 has the opposite effect; that is, it appears to be a suppressor of fon2. In the case of AP1 and CAL, functional redundancy is due to the factors themselves; by contrast, signaling pathways comprising a different signaling molecule and its receptor might be redundant in the case of meristem maintenance in rice, as discussed below.

FOS1 and FON2 function together in stem cell maintenance in the FM in rice

Our previous study demonstrated that FON2 is a negative regulator of stem cell maintenance in the FM [8]. In this study, introduction of indica FOS1 into fon2-1 by genetic methods using a chromosomal segment substitution line or by Agrobacterium-mediated transformation completely suppressed the fon2 mutation. This finding suggests that FOS1 can substitute for the function of FON2. Thus, FOS1 is likely to play an important role in maintenance of the FM in indica and either one of FOS1 and FON2 appears to be sufficient to restrict stem cell proliferation in the FM.

There are two possible explanations for the redundancy of FOS1 and FON2. Both CLE peptides may be involved in the same pathway and may share their receptors. Alternatively, there may exist two independent pathways: one involving FOS1 and one involving FON2 as signaling molecules. Two experiments in this study supported the latter possibility. First, a genomic fragment containing indica FOS1 was able to rescue a severe mutant of fon1, in which the putative receptor of FON2 is defective [7]. Second, constitutive expression of indica FOS1 in fon1 mutant showed abnormal shoots, a phenotype that is similar to that of wild type overexpressing indica FOS1. These results suggest that FON1 is not required for FOS1 function and that the signaling pathways involving FON2 and FOS1 are independent of each other. Because wild species in the Oryza genus have no mutation in the functional region of FOS1, these two pathways may function in the FM in all Oryza species except for japonica (Figure 6).

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Figure 6. Model of meristem maintenance in rice.

Rice is likely to have three negative regulators that restrict stem cell population in the three types of aerial meristem. Each pathway contributes differently depending on the type of meristem. FM, floral meristem; IM inflorescence meristem; Vegetative SAM, shoot apical meristem in the vegetative phase. The pathway indicated by the red thick line has been demonstrated by both loss-of-function and gain-of-function analyses. The pathways indicated by red or blue thin lines have been demonstrated by loss-of-function or gain-of-function analysis, respectively. Dashed lines indicate pathways to be analyzed in future studies. The line marked with X indicates a pathway with no function. See text and our two previous reports for details [8],[24].

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We found that FOS1 is expressed in the vegetative phase, and constitutive expression of FOS1 generates abnormal shoots with malformed leaves. It is, therefore, likely that FOS1 may be involved in maintenance of the vegetative SAM. Constitutive expression of FON2, by contrast, does not cause abnormalities in the shoot [24]. In this respect, FOS1 and FON2 may have diversified functionally (Figure 6).

In contrast to the FM, the vegetative SAM seems to be unaffected by loss of both FOS1 and FON2 because shoot morphology is normal in fon2 mutants in japonica [8]. Therefore, stem cell maintenance in the vegetative SAM may be regulated by an as yet unidentified negative pathway. FCP1 is likely to be involved in this pathway because its constitutive expression consumes stem cells in the vegetative SAM [24]. Thus, stem cell maintenance may be regulated by at least three negative pathways in rice, and each pathway may contribute differently to this regulation depending on the type of the meristem (Figure 6). This situation contrasts with that in Arabidopsis, where CLV signaling is the major single pathway in all meristems.

Distribution of the FNP in FOS1 in the Oryza genus

Recent genetic and phylogenetic analyses have revealed that indica and japonica arose independently from a genetically distinct population in a wild ancestor, O. rufipogon [28]–[30],[33]. Our haplotype network of FOS1 is also consistent with an independent origin of the two subspecies. In our network, haplotype C would have been the prototype of FOS1 for all domesticated and wild rice species. Haplotype A, associated with indica, and haplotype B, associated with japonica, would have been produced by the occurrence of a single nucleotide change in haplotype C during rice evolution. Indica may have been derived from an O. rufipogon species with haplotype A. The FNP at the cleavage site of the signal peptide is responsible for the generation of haplotype B. Although there is no O. rufipogon accession with haplotype B, it is possible that japonica might have been domesticated from an unidentified ancestor with this FNP.

Many types of mutation are found in FOS1 of wild rice species, including not only amino acid substitutions but also insertions or deletions (Figure S2). There are, however, no mutations that affect the function of FOS1 in the coding region, such as an amino acid change in the CLE domain or a frameshift mutation. This observation suggests that defects in FOS1 may not be neutral and that FOS1 may be essential for the growth and survival of wild rice species under natural conditions. In line with this hypothesis, it is unlikely that an O. rufipogon species that has the FNP in FOS1 will be found in the natural population at present.

Plant materials

Taichung 65 (T65) and Kasalath were used as representative strains of wild-type japonica and indica, respectively, in molecular genetic and histochemical analyses. Nipponbare/Kasalath chromosomal segment substitution line #9 (N/K CSSL#9) was obtained from the Rice Genome Resource Center, Japan. Core collections of O. sativa (World Rice Collection (WRC) and Japanese Rice Collection (JRC)) were obtained from the Genebank of National Institute of Agrobiological Sciences, Japan (Table S1; Table S2) [26],[27]. Wild rice species were obtained from the National Institute of Genetics, Japan (Table S3).

QTL analysis and identification of FOS1

F₂ plants from a cross between fon2-1 and Kasalath were used to search for a gene that suppresses the fon2 mutation. We obtained 154 F₂ plants showing a fon2 phenotype from about 2,000 F₂ plants and checked their genotypes to confirm that the fon2 locus has the mutant allele. For QTL analysis, the suppressor activity in each F₂ plant that had the fon2 mutation was estimated by calculating the frequency of the twin seeds, an indication of the fon2 mutation. Next, the genotypes of about 90 loci in the 89 F₂ plants were determined by using molecular markers [34]. As a result, a major QTL was found at around 40–80 cM on chromosome 2 (LOD score: 6.3). A gene (FOS1) encoding a protein with a CLE domain was then identified at around 40 and 80 cM on chromosome 2 by searching the rice genomic sequence database using the amino acid sequence of the FON2 CLE domain as a query.

FOS1 cDNA was amplified with the appropriate primers (Table S4) from total RNA isolated from young panicles of T65 (japonica) and Kasalath (indica). After sequencing of the RT-PCR product, the open reading frame was predicted.

Transformation of rice

To introduce indica FOS1 into the fon2 mutant, a 3.3-kb FOS1 genomic fragment, including 2.6 kb of sequence directly upstream of the initiation codon of FOS1, from the Kasalath genome was used. For constitutive expression of FOS1, a FOS1 cDNA derived from T65 or Kasalath was placed under the rice Actin1 promoter [35]. The resulting plasmids, designated Actin1:japonica-FOS1 (T65) and Actin1:indica-FOS1 (Kasalath), were introduced into Agrobacterium tumefaciens strain EHA101 and transformed into rice as described previously [36].

In situ hybridization

For the in situ hybridization probe for FOS1, a 646-bp fragment consisting of the entire coding region, the 5′ UTR (137 bp) and the 3′ UTR (113 bp) was amplified with the appropriate primers (Table S4). The fragment was cloned into a T-vector by TA-cloning (Novagen, Madison). The OSH1 probe was prepared as described in the original paper [25]. Probe synthesis, preparation of sections, in situ hybridization, and microscopic observation were performed as described previously [7],[24].

Analysis of FOS1 haplotype

The genomic region of FOS1 was amplified with the appropriate primers (Table S4). The amplified fragments were purified with Montage PCR Filter Units (Millipore, Billerica) and sequenced with the same primers used for amplification. The haplotype network was constructed by using the program TCS1 [37].