Photosensitivity plays an essential role in the response of plants to their changing environments throughout their life cycle. In soybean [Glycine max (L.) Merrill], several associations between photosensitivity and maturity loci are known, but only limited information at the molecular level is available. The FT3 locus is one of the quantitative trait loci (QTL) for flowering time that corresponds to the maturity locus E3. To identify the gene responsible for this QTL, a map-based cloning strategy was undertaken. One phytochrome A gene (GmPhyA3) was considered a strong candidate for the FT3 locus. Allelism tests and gene sequence comparisons showed that alleles of Misuzudaizu (FT3/FT3; JP28856) and Harosoy (E3/E3; PI548573) were identical. The GmPhyA3 alleles of Moshidou Gong 503 (ft3/ft3; JP27603) and L62-667 (e3/e3; PI547716) showed weak or complete loss of function, respectively. High red/far-red (R/FR) long-day conditions enhanced the effects of the E3/FT3 alleles in various genetic backgrounds. Moreover, a mutant line harboring the nonfunctional GmPhyA3 flowered earlier than the original Bay (E3/E3; PI553043) under similar conditions. These results suggest that the variation in phytochrome A may contribute to the complex systems of soybean flowering response and geographic adaptation.

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FLOWERING represents the transition from the vegetative to the reproductive phase in plants. Various external cues, such as photoperiod and temperature, are known to initiate plant flowering under the appropriate seasonal conditions. Of these cues, light is the most important, being received by several photoreceptors, including the red light (R) and the far-red light (FR)-absorbing phytochromes and the blue/UV-A absorbing cryptochromes and phototorpins (CHEN et al. 2004).

Phytochrome is the best characterized of these photoreceptors. All higher plant phytochromes are thought to exist as specific dimer combinations (SHARROCK and CLACK 2004), with each monomer being attached to a light-absorbing linear tetrapyrrole, phytochromobilin. The phytochrome apoproteins are synthesized within the cytosol and assemble autocatalytically with a chromophore to form the phytochrome holoproteins. The R-absorbing form (Pr) is thought to be inactive but is then converted to the active FR-absorbing form (Pfr) by R absorption. The absorption of light triggers the transfer of the phytochrome to the nucleus, where it regulates gene expression. In most plant species, the phytochrome apoproteins are encoded by a small gene family. Type I phytochrome is degraded in the light and is abundant in dark-grown seedlings, whereas type II phytochrome is relatively stable in the light (reviewed by BAE and CHOI 2008). In Arabidopsis, five phytochromes (PhyA–E) have been characterized (CLACK et al. 1994; QUAIL et al. 1995). PhyA is type I and is responsible for the very low fluence response and high irradiance response, whereas the other phytochromes are type II and are responsible for red-far/red reversible low fluence response (reviewed by WHITELAM et al. 1998).

It is well known that mutations in the phytochrome A gene affect the photoperiodic control of flowering. In Arabidopsis, a phyA mutant flowered later in either long-day or short-day conditions with a night break (JOHNSON et al. 1994; REED et al. 1994). In rice, combinations of mutant alleles of phytochrome genes conferred various effects on the flowering phenotype. For example, the phyA phyB and phyA phyC double mutants grown under natural-day-length conditions showed earlier flowering phenotypes than wild-type plants (TAKANO et al. 2005). In pea, a long-day plant, loss- or gain-of-function phyA mutants displayed late or early flowering phenotypes, respectively (WELLER et al. 1997, 2001). It is likely that photoperiodic response via phyA signaling is important for crop adaptation to a wide range of growing conditions.

In soybean [Glycine max (L.) Merrill], several maturity loci, designated as E loci (COBER et al. 1996a), have been characterized by classical methods. These are E1 and E2 (BERNARD 1971), E3 (BUZZELL 1971), E4 (BUZZELL and VOLDENG 1980), E5 (MCBLAIN and BERNARD 1987), E6 (BONATO and VELLO 1999), and E7 (COBER and VOLDENG 2001). Of these, the E1, E3, and E4 loci have been suggested to be related to photoperiod sensitivity under various light conditions (SAIDON et al. 1989; COBER et al. 1996b; ABE et al. 2003). In previous studies, using the same populations as in this study, three flowering-time quantitative trait loci (QTL)—FT1, FT2, and FT3 loci—were identified and considered to be identical with the maturity loci E1, E2, and E3, respectively (YAMANAKA et al. 2001; WATANABE et al. 2004). Although many loci related to soybean flowering and maturity have been identified, and some candidate genes were recognized using near isogenic lines (NILs) (TASMA and SHOEMAKER 2003), most of the genes responsible for these loci have not yet been isolated except for the E4 gene. LIU et al. (2008) reported an association between phytochrome A and photoperiod sensitivity. A retrotransposon sequence inserted into the exon of the e4 allele conferred an early flowering phenotype under long-day conditions extended by incandescent lighting.

A relationship between the E3 gene and some photoreceptor genes was suggested from different photosensitivity responses of various soybean NILs (COBER et al. 1996a). COBER and VOLDENG (1996) also reported a linkage relationship between the E3 and Dt1 loci, which is related to a determinate or indeterminate growth habit phenotype. Additionally, MOLNAR et al. (2003) reported that Satt229, on linkage group (LG) L, was a proximal simple sequence repeat (SSR) marker to the E3 loci. According to the Soybean Genome Database (SHULTZ et al. 2006a,b, 2007; http://soybeangenome.siu.edu/) and the Legume Information System (LIS; http://www.comparative-legumes.org/), there are numerous QTL and >60 loci associated with various agronomic traits in the region between Dt1 and Satt373 (30–40 cM). This extremely large number of QTL may be the result of linkage between the Dt1 and E3 loci because both loci can affect many aspects of plant morphology. Among these QTL, several associations with the E3 gene have been reported (MANSUR et al. 1996; ORF et al. 1999; FUNATSUKI et al. 2005; KAHN et al. 2008).

To identify the genes responsible for the target QTL, fine mapping and map-based cloning strategies are necessary (SALVI and TUBEROSA 2005). QTL analysis using intercross-derived populations, such as F₂ and recombinant inbred lines (RILs), have some limitations in genome resolution (10–30 cM) because of the simultaneous segregation of several loci affecting the same trait (KEARSEY and FARQUHAR 1998). Additional strategies are therefore required to locate QTL more precisely. The use of NILs that differ at a single QTL is an effective approach for fine mapping and characterization of an individual locus (SALVI and TUBEROSA 2005). However, the development of NILs through repeated backcrossing is time-consuming and laborious (TUINSTRA et al. 1997). The use of a residual heterozygous line (RHL), as proposed by YAMANAKA et al. (2004), and which is derived from RIL, is a powerful tool for precisely evaluating QTL (HALEY et al. 1994). An RHL harbors a heterozygous region where the target QTL is located and a homozygous background in most other regions of the genome. TUINSTRA et al. (1997) used a similar term, heterogeneous inbred family, for a selfed RHL population to identify the QTL associated with seed weight in sorghum.

This RHL strategy has already been used to identify loci underlying resistance to pathogens in soybean (NJITI et al. 1998; MEKSEM et al. 1999; TRIWITAYAKORN et al. 2005). After identification of the target loci, novel DNA markers tightly linked to the loci were developed using the amplified fragment length polymorphism (AFLP) method (MEKSEM et al. 2001a,b). Physical contigs, screened by sequence-characterized amplified region (SCAR) markers converted from these AFLP fragments, are ideal sources for identifying candidate genes for the target traits (RUBEN et al. 2006).

The aim of this study is to characterize the FT3 locus using a map-based cloning strategy and to confirm the gene responsible for the E3/FT3 locus by allelism tests through comparisons of gene sequences and photosensitivity of several alleles.

Plant materials and phenotypic investigation of flowering time:

A population of 156 RILs (F_8:10), derived from a cross between the two varieties Misuzudaizu and Moshidou Gong 503 was used in this study. This population had been used previously for linkage map construction and QTL analysis of agronomic traits (WATANABE et al. 2004). Three QTL for flowering time, FT1, FT2 and FT3, were identified at LG C2 (chromosome 6), LG O (chromosome 10) and LG L (chromosome 19), with the late-flowering alleles FT1, FT2, and FT3 being partially dominant over the early flowering alleles ft1, ft2, and ft3, respectively. Misuzudaizu harbored the late-flowering allele of the FT1 and FT3 loci, whereas Moshidou Gong 503 carried the late-flowering allele of the FT2 locus.

Several lines with large phenotypic variance for flowering time compared to the parents were screened. Approximately 15–60 plants of these RILs were sown on May 27, 2002, at Chiba University, Matsudo, Japan (35°78' N, 139°90' E) and grown under natural conditions. The line RIL1-146 was found to be heterozygous for the FT3 locus. One other line, RIL6-22, showed segregation for growth habit related to the determinate or indeterminate phenotype at the shoot apex after flowering. This trait is controlled by the Dt1 locus and is linked to the FT3 locus at a distance of 25 cM. The segregating region of RIL6-22 included both the Dt1 and the FT3 loci. A single plant with a genotype of dt1dt1 FT1FT1 ft2ft2 FT3ft3 was selected from RIL1-146, and 5 plants with a genotype of Dt1dt1 ft1ft1 ft2ft2 FT3ft3 were selected from RIL6-22 and designated RHL1-146 and RHL6-22, respectively. The seeds of these RHLs were sown on May 21, 2003, and seedlings were grown under natural conditions at the same location as described above. From both progenies of these RHLs, two NILs, 1-146-FT3 and -ft3 and 6-22-FT3 and -ft3 were selected. Additionally, 7 plants heterozygous for the FT3 locus were screened from the progeny of RHL1-146 using DNA markers. All seeds obtained from these plants were bulked to develop a large population for fine mapping. This segregating population consisted of 897 plants; the NILs1-146 were sown on May 18, 2004, at the Japan International Research Center for Agricultural Sciences, Hachimandai, Tsukuba (36°03' N, 140°04' E), and the seedlings were transplanted to the field on May 31 and grown under natural conditions. From this population, 14 recombinants were screened using new DNA markers tightly linked to the FT3 locus. The progenies, consisting of 24–96 plants derived from these recombinants and the NILs1-146, were sown on May 24, 2005, at Hachimandai, and seedlings were transplanted on June 7 in the field and grown under natural conditions.

To analyze the allelic relationships among the E3, FT3, and ft3 alleles, two F₂ populations were produced. One population of 122 individuals was developed from the cross between Harosoy and 6-22-FT3, and the other population of 206 individuals was derived from a cross between Harosoy and 6-22-ft3. These populations were sown on May 30, 2007, at the National Institute of Agrobiological Sciences, Tsukuba, Japan (36°02' N, 140°11' E) and grown under natural-day-length conditions.

The photosensitivity of NILs (NILs 1-146, 6-22 and Harosoy and Harosoy-e3; L62-667) was investigated under fluorescence long-day (FLD) conditions. Normal cool white lamps (150 µmol sec^–1 m^–2) with a 16-hr-light and 8-hr-dark photoperiod at a constant temperature of 30° were used in a growth cabinet. Approximately 7–12 plants (3 plants/pot) were evaluated for their flowering phenotype.

A mutant line, carrying a null allele of the GmPhyA3 gene, and the wild type (Bay) were grown in the glasshouse at Hokkaido University, Sapporo, Japan (43°07' N, 141°39' E). The seeds of both lines were sown on January 22, 2008, and grown (three plants per pot) under normal-day-length conditions extended with extra lighting provided by mercury-vapor lamps (HF400X, Hitachi, Tokyo) from 7 AM to 9 PM daily. The genotypes of the NILs used in this study are listed in Table 1.

View this table: In this window In a new window   TABLE 1 Genotypes of plant materials including the NILs

 
For the phenotypic investigations, the dates from sowing to flowering were scored at the first flowering, which corresponded to the R1 developmental stage (FEHR et al. 1971). Investigations were performed on alternate days.

DNA isolation:

Genomic DNA from fresh trifoliate leaves of 1-week-old seedlings was extracted using the standard cetyltrimethyl ammonium bromide (CTAB) method (MURRAY and THOMPSON 1980).

AFLP analysis:

Templates for the AFLP reaction were prepared according to the method of VOS et al. (1995) and using 150 ng DNA for restriction enzyme digestion with EcoRI and MseI. Selective amplification was performed using combinations of EcoRI (E) primers and MseI (M) primers, each with three selective nucleotides. Nomenclature for the AFLP markers expressed as En₁Mn₂ ("n1" and "n2" indicate the primer codes) includes the letter E or M for the EcoRI or MseI primers, respectively, followed by a code representing the combinations of the three selective nucleotides. The amplification procedures, conditions for polyacrylamide gel electrophoresis, and the methods for detection and scoring of the polymorphic bands followed those described by HAYASHI et al. (2001). Bulked segregant analysis (BSA) was used to identify the AFLP markers tightly linked to the FT3 locus. Two DNA pools, an early flowering bulk and a late-flowering bulk from an RIL1-146 subpopulation, were used as DNA templates, and the AFLP marker E6M22 was identified. Additional polymorphic AFLP markers were identified from the rest of primers out of all of possible 4096 AFLP primer combinations using NILs 1-146 and 6-22.

Development of SCAR markers for fine mapping of the FT3 locus:

The detected polymorphic bands were cloned using the pGEM T-easy vector system (Promega K. K. Japan, Tokyo) and sequenced with an ABI PRISM 3100 avant Genetic Analyzer using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems Japan, Tokyo) following the manufacturer's instructions. Primer 3 (http://frodo.wi.mit.edu/) was used to design new primers for the SCAR markers, which were then used to construct a high-resolution map around the FT3 locus.

Construction of a physical contig of the FT3 region:

Two libraries of BAC and transformation-competent bacterial artificial chromosome (TAC) clones were constructed from the genomic DNA of Misuzudaizu. Library screening and end-sequencing were performed as described previously (XIA et al. 2005; WADAHAMA et al. 2008). Several BAC/TAC clones were screened using the SCAR markers.

The nucleotide sequences of two BAC/TAC clones (GM_TMiH_H17D12 and GMJMiB242F01) were determined according to the bridging shotgun method previously described (SATO et al. 2001). The generated sequences were assembled using Phred-Phrap programs (Philip Green, University of Washington, Seattle). A lower threshold of acceptability for the generation of consensus sequences was set at a Phred score of 20 for each base. These BAC/TAC sequences were used to develop new PCR-based markers and to predict the candidate gene for the FT3 locus.

DNA marker analysis:

For fine mapping, six DNA markers developed from the AFLP and BAC/TAC sequences listed in Table 2 were used to decide the genotypes of the progeny of an RHL. Genomic DNA (20–30 ng) was used as template, and the PCR reaction was performed using Ex-Taq (Takara Bio, Shiga, Japan) with 30 cycles at 96° for 30 sec, 58° for 30 sec, and 72° for X min (extension time for each primer pair is indicated in Table 2). PCR products were separated by 1.5% (w/v) agarose gel or 10% (w/v) polyacrylamide gel electrophoresis and visualized with ethidium bromide (EtBr).

View this table: In this window In a new window   TABLE 2 Marker information for fine mapping of the FT3 locus

 
For allelism tests between Harosoy and 6–22 NILs, one SSR marker located in the downstream spacer region of GmPhyA3 was identified. This marker showed a triallelic pattern among Harosoy, Misuzudaizu, and Moshidou Gong 503. PCR conditions were the same as those described above with an extension time of 1 min and with the following primers: forward, 5'-ATTAATTCGTTGACTCGGTACTCC-3', and reverse, 5'-GGACTTAGAATGGAGGGCATAAA-3'. PCR products were separated on a 10% (w/v) polyacrylamide gel and visualized with EtBr.

Analysis of GmPhyA3 expression and transcript structure:

Total RNA was extracted with the Trizol method (Invitrogen Japan K.K., Tokyo) from leaves of Moshidou Gong 503, Misuzudaizu, Harosoy, and Harosoy-e3. The 5' and 3' RACE method was used to determine the transcriptional initiation position and poly(A) site of the GmPhyA3 transcript. Details of the RACE–PCR method were described previously (SASSA et al. 2007). For the RT–PCR experiment, 1 µg of total RNA was used for first-strand cDNA synthesis with ReverTra Ace (TOYOBO, Osaka, Japan) and a standard oligo(dT₂₀) primer under the manufacturer's instructions, and then the cDNAs were diluted two times with PCR-grade water, and a 2-µl aliquot used as RT–PCR template. Specific primers for RT–PCR of GmPhyA3 were the following: P1, 5'-AACAAGGTGTGGCGATTAGG-3'; P2, 5'-GATGGGACCAGAATCAATCTTC-3'; P3, 5'-GTGTCAACGCCAGATTAGCA-3'; P4, 5'-TGCTTCCTTTCACTTTCTGATG-3'; and P5, 5'-CCTGATGCTATCAATGTCCTG-3'. Primer sequences for the soyActin control gene were the following: forward, 5'-CGACCTCGACATACTGGTGTTAT-3', and reverse, 5'-TGCCATATAGATCCTTTCTGATA-3'. The PCR reaction using Ex-Taq consisted of 30 cycles at 96° for 30 sec, 58° for 30 sec, and 72° for 3 min. PCR products were separated by 1.5% (w/v) agarose gel electrophoresis and visualized with EtBr.

Analysis of GmPhyA3 gene structure:

Genomic DNA extracted from Misuzudaizu, Moshidou Gong 503, Harosoy, Harosoy-e3, and Bay was used as template to determine the GmPhyA3 gene structure from the transcriptional initiation point to the poly(A) site. As the size of this gene extended from 9 to 11 kbp, several 3- to 4-kbp fragments were independently amplified, and their sequences were determined using the primer walking method.

Construction of soybean mutant libraries:

Seeds of the Bay soybean cultivar were separately treated with two different mutagens [X-ray or ethyl methanesulfonate (EMS)]. For X-ray treatment, dry seeds were irradiated with 200 Gy X-rays at an exposure rate of 3 Gy/min. For EMS treatment, seeds were soaked in a 0.35% (w/v) EMS solution for 12 hr and then rinsed in tap water for 8 hr. M2 seeds were obtained from self-fertilized M1 plants. Green leaves were harvested from individual M2 plants for DNA preparation. Genomic DNAs were purified using diatomaceous earth columns, followed by CTAB extraction. Pooled DNAs from eight individuals were used for mutant screening.

Mutant screening:

Targeting-induced local lesions in genomes (TILLING) screening was essentially carried out as previously described (MCCALLUM et al. 2000), except that 2% (w/v) agarose gel electrophoresis was used to separate CEL I-digested DNA fragments with GelRed stain (Biotium, Hayward, CA). Primers for TILLING screening were the same as the P1 and P2 primers used in RT–PCR analysis. The PCR reaction, using Pfu DNA polymerase, consisted of 40 cycles at 96° for 30 sec, 65° for 30 sec, and 72° for 1 min. CEL I was purified from celery stalks as described previously (YANG et al. 2000).

Data analysis:

Data were analyzed using R software (http://cran.r-project.org/) for one-way classification analysis of variance (ANOVA) without the assumption of equal variance. Multiple regression analysis was applied to estimate the additive and dominance effects of the FT3 locus, details of which were described previously (WATANABE et al. 2004). Phylogenic analysis of the phytochrome protein was performed using the neighboring-joining method with the program MEGA 3.1 (http://www.megasoftware.net).

Development of two RHLs for the FT3 locus:

Most RILs (F_8:10) showed a similar variance in their phenotypic distributions, whereas RIL1-146 showed a larger phenotypic variance (SD = 2.28) than the parents (estimated SD values of Misuzudaizu and Moshidou Gong 503 were 1.19 and 1.61, respectively). Using BSA analysis, a polymorphic AFLP marker, E6M22, was detected between the early flowering bulk and the late-flowering bulk derived from the progeny of RIL1-146. Mapping results and QTL analysis for flowering time using the RILs suggested that this marker located to the LOD peak position of the QTL assigned to FT3 (Figure 1). It indicated that RIL1-146 harbored some heterozygous region, including the FT3 locus, in the previous generation (F₇) and showed phenotypic segregation in the next generation (F_8:10). As a result of marker analysis, a region covering 5 cM (GM043-E6M22) including the FT3 locus was found to segregate in the progeny of RIL1-146. The plants heterogeneous for this region, designated as RHL1-146, generated NILs1-146-FT3 and -ft3 from their progeny. The difference of NILs was highly significant (P < 0.001) with flowering times of 58.2 ± 1.55 (n = 47) for 1-146-ft3 and 69.1 ± 1.21 (n = 70) for 1-146-FT3. These results indicated that the progeny derived from RHL1-146 would be suitable for fine mapping of the FT3 locus.

View larger version (30K): In this window In a new window Download PPT slide   FIGURE 1.— Detection of the location of the FT3 locus using RILs and identification of the segregating regions of two RHLs, 6-22 and 1-146, determined by DNA markers mapped on LG L. Solid and dotted lines indicate the LOD scores calculated by composite interval mapping and interval mapping for QTL, respectively. Information regarding the LOD peak position, corresponding to the FT3 locus, is summarized in the box. Shaded bars indicate the heterozygous region of two RHLs. PVE is the ratio of phenotypic variance explained by this QTL.

 
In contrast, RIL6-22 also showed an apparent segregation for growth habit controlled by the Dt1 locus. DNA marker analysis showed that the heterozygous region in RHL6-22 extended for 40 cM (from Dt1 to Satt373) including the FT3 locus (Figure 1). Although phenotypic segregation was expected in the progeny of this RHL as well, the variation in flowering days was rather small (SD = 1.25); thus NILs carrying a homozygous allele for the Dt1 and a different allele for FT3 (NILs 6-22-FT3 and -ft3) were developed on the basis of the genotypes of DNA markers mapped to this region.

Screening of AFLP markers tightly linked to the FT3 locus and construction of a physical contig including the FT3 locus:

Two groups of NILs (NILs6-22 and NILs1-146) were used to develop the AFLP markers tightly linked to the FT3 locus. Of all the possible 4096 primer pairs, only six fragments showed constant polymorphism between the contrasting genotypes of FT3/FT3 and ft3/ft3 in NILs1-146 and NILs6-22. The positions of these markers were confirmed using the RILs (data not shown). These polymorphic bands were excised from the gel, then sequenced, and converted to codominant SCAR markers. Using the SCAR marker developed from the AFLP marker E6M22, one BAC clone and one TAC clone were screened from two independent genomic DNA libraries. These two clones, GMJMiB242F01 (40 kb) and GM_TMiH_H17D12 (93 kb), were then subjected to shotgun sequence analysis. The two clones were found to overlap for >27 kb of their length. To narrow the location of the FT3 locus, a total of six DNA markers, including three AFLP-derived markers (markers 1, 3, and 6) and three PCR-based markers developed from the BAC/TAC sequences (markers 2, 4, and 5), were used in the following experiments (Table 2).

Fine mapping of the FT3 locus:

A population of 897 plants derived from seven RHL1-146 plants was used for precise mapping of the FT3 locus. The probability of double recombination between the two markers (marker 1 and marker 6 in Table 2) was not taken into consideration because these markers were mapped within a few centimorgans of the RILs. No recombination between these markers was found in 883 plants. Differences among these genotypes were significant (P < 0.001), and the numbers of the FT3 homozygous late-flowering alleles (n = 208, 68.3 ± 1.1) and heterozygous (n = 441, 66.2 ± 1.4) and ft3 homozygous early flowering alleles (n = 234, 62.2 ± 1.7) fitted a 1:2:1 segregation ratio (² =1.53, P < 0.5). These results suggested the presence of a single QTL for flowering time within a small heterozygous region in RHL1-146. The additive effect and the dominance effect of this QTL were estimated to be 3.0 and 0.98 days, respectively. Furthermore, the ratio of genetic variance explained by the FT3 locus accounted for 70.7% of the total variance. On the other hand, 14 plants showed recombination between these markers (Figure 2 and supporting information, Figure S1), and the recombination points were determined by the genotype of markers 2–5. The region covering six DNA markers spanning the FT3 locus was estimated to be 1.2 cM.

View larger version (15K): In this window In a new window Download PPT slide   FIGURE 2.— Phenotypic segregations observed in the progeny of recombinant individuals. The phenotypes and genotypes of each recombinant are shown in the left panel. The genotypes Misuzudaizu homozygous and Moshidou Gong 503 homozygous and heterozygous alleles are indicated by A, B, and H, respectively. Asterisks indicate the recombination points detected in each recombinant. The phenotypic segregation of the offspring is shown in the middle panel in a box-plot format. The interquartile region, median, and range are indicated by a box, bold vertical line, and horizontal dotted line, respectively, for each recombinant. This segregation pattern completely corresponded to the genotype of marker 3. The progenies of some of the lines showing an obvious segregating pattern in the middle panel were classified on the basis of the genotype of marker 3 and are shown in the right panel. Details related to this figure are summarized in Table S1.

 
The precise FT3 genotype for each recombinant could be determined with the segregating patterns classified into three types: early flowering, late-flowering, and segregating phenotypes (Figure 2 and Table S1). The FT3 genotypes in each recombinant completely coincided with the genotypes of marker 3 that originated from the closest AFLP marker E6M22 to the LOD peak position (Figure 1). Moreover, recombination points occurred on both sides of marker 3 (plant no. 04478_rec and no. 04814_rec in Figure 2) and corresponded to both sides of the TAC clone, GM_TMiH_H17D12. These results suggested that the gene responsible for the FT3 locus was restricted to the physical region covered by GM_TMiH_H17D12.

Candidate gene for the FT3 locus:

To predict the genes located on GM_TMiH_H17D12, the Rice Genome Automated Annotation System (http://ricegaas.dna.affrc.go.jp/) was used for annotation of the sequence. This system is composed of various programs for gene prediction and gene structural analysis. A total of 11 genes were predicted and are listed in Table S2. Previous studies had suggested that the FT3 locus may be identical to the maturity locus E3 (YAMANAKA et al. 2001) and that the E3 gene that showed a large effect on flowering time under FLD conditions had some association with a photoreceptor (COBER et al. 1996b). Considering these findings, one gene highly similar to that encoding phytochrome A was considered to be the gene responsible for the FT3 locus. To confirm this assumption, differences in this gene between the two parental lines were investigated. At first, the 5' and 3' RACE method was used to obtain the full-length sequence of this phytochrome A gene, which we hereafter refer to as GmPhyA3, since two other phytochrome A genes had been previously designated as GmPhyA1 (AB370252) and GmPhyA2 (AB370253) by LIU et al. (2008).

GmPhyA3 obtained from Misuzudaizu (GmPhyA3-Mi) was found to encode a protein composed of 1130 amino acids. A BLAST search found that GmPhyA3-Mi displayed normal features of phytochrome A, including a chromophore-attached domain, two PAS domains, and a histidine kinase domain as conserved domains (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). These features together with its gene structure are shown in Figure 3. Although some association was expected between the flowering phenotype and the GmPhyA3 expression pattern in leaves, it was not significant in the real-time PCR experiments using NILs (data not shown). Compared to GmPhyA3-Mi, the Moshidou Gong 503's GmPhyA3 (GmPhyA3-Mo) gene showed a large insertion in the fourth intron and one single nucleotide polymorphism (SNP) for a nonsynonymous amino acid substitution in the third exon. This SNP corresponded to the polymorphism detected by the AFLP marker E6M22 (Figure 3A). The inserted sequence was 2.5 kbp in length and was expected to encode a protein of 513 amino acids using the Genscan program (http://genes.mit.edu/GENSCAN.html). A BLAST search of this inserted sequence indicated that a part of this sequence was highly similar to that of the non-long-terminal-repeat (LTR) retrotransposon reverse transcriptase element, but did not resemble the Ty1/copia or Ty1/gypsy sequences detected in the e4 allele (LIU et al. 2008). Moreover, this inserted sequence showed a similar short sequence on both sides of the inserted position (Figure 3B). A BLAST search of the soybean genome sequence database (Phytozome; http://www.phytozome.net/soybean) revealed that this type of retrotransposon showed repeats with 97–99% similarity over 280 loci. Although this non-LTR type of retrotransposon might not affect the GmPhyA3 expression level, the functional significance of this insertion on GmPhyA3 remains unclear.

View larger version (24K): In this window In a new window Download PPT slide   FIGURE 3.— Genomic structure of the GmPhyA3 gene cloned from parental lines and expression analysis. (A) Open boxes, shaded boxes, and horizontal lines indicate exons, UTRs, and introns, respectively. The positions of the TATA-like sequence, initiation codon, and stop codon are indicated. The deleted region detected in Harosoy-e3 is denoted by a dotted line. The line with arrowheads at both ends indicates the amplified region analyzed in the RT–PCR experiment and the primer names. The numbers above the Misuzudaizu allele indicate the nucleotide position from the transcriptional start point as determined by the RACE experiment. The solid box on the Misuzudaizu allele indicates the AFLP fragment (E6M22) detected in NILs (Figure 2). The position of the large inserted sequence observed in GmPhyA3-Mo and -E3 is denoted by a right-pointing arrow (B). The integrated points of this insertion were compared between GmPhyA3-Mi (Mi_FT3) and GmPhyA3-Mo, and the integrated start and end points are indicated as Mo_Ins_st and Mo_Ins_end, respectively. Boxed sequences with asterisks indicate identical nucleotides in the three sequences, and the numbers shown above and at the bottom indicate the position of the nucleotide in the Misuzudaizu and Moshidou Gong 503 alleles. (C) Expression analysis of GmPhyA3 using leaves of Harosoy and Harosoy-e3. Genomic DNA and synthesized cDNA were used as templates. Each amplified region corresponds to the region with the arrowheads in A. Estimated fragment sizes are indicated on the left side of the gel.

 
To collect allelic information about GmPhyA3, the genes from Harosoy and Harosoy-e3 were subsequently isolated (referred to hereafter as GmPhyA3-E3 and GmPhyA3-e3, respectively). Surprisingly, the four GmPhyA3 genes displayed different organization. While a large retrotransposon-like insertion sequence was observed in GmPhyA3-E3, similar to that in GmPhyA3-Mo, the amino acid sequences encoded by GmPhyA3-Mi and -E3 were identical. On the other hand, a large deletion of 13.33 kbp at a position after the third exon was detected in GmPhyA3-e3; this may result in a nonfunctional phytochrome protein as the histidine kinase domain, which might play an important role in signal transduction, was partially deleted. However, the 3' RACE and RT–PCR experiments revealed GmPhyA3-e3 expression in the leaves (Figure 3C). Additional information about the SNPs in the four lines and physical position of all markers used in this study are summarized in Table S3 and Table S4, respectively.

The nucleotide sequence of GmPhyA3-Mo had only a single base-pair substitution compared with GmPhyA3-Mi, as described above, and resulted in the substitution of a glycine to an arginine residue in the kinase domain at position 1050. To investigate the significance of this substitution, GmPhyA3 was compared with several phytochrome genes identified in other plant species. Phylogenic analysis showed that these phytochrome A genes displayed a high level of similarity among plant species (Figure 4A). The glycine residue was highly conserved in >10 plant species, including monocots and dicots (Figure 4B). This fact suggests that the substitution of the arginine residue observed in GmPhyA3-Mo was unique and that the glycine residue might be essential for the correct functioning of phytochrome A.

View larger version (38K): In this window In a new window Download PPT slide   FIGURE 4.— Phylogenic analysis of phytochrome A proteins. (A) Phylogenic tree of the phytochrome A protein was constructed by the neighbor-joining method with a 1000-replication bootstrap confidence value. (B) Multiple alignment of phytochrome A amino acid sequences of Arabidopsis, pea, sorghum, tomato, potato, rice, and wheat, and GmphyA3-Mi and SOYPHYA for soybean focusing on the amino acid substitution detected between GmPhyA3-Mi and GmPhyA3-Mo. ClustalW was used to align these sequences. The position from the N terminus of GmPhyA3-Mi is shown on the alignment. Highly conserved amino acids are indicated by solid and shaded backgrounds, depending on the level of identity. The open arrowhead at the bottom of the alignment indicates the glycine residue at position 1050 substituted to an arginine residue in GmPhyA3-Mo.

 
The natural variations detected in GmPhyA3 make it difficult to understand their allelic relationships. To clarify the effects of the substitution observed in GmPhyA3-Mo and the insertion of the non-LTR retrotransposon, we performed testcrosses.

Allelism tests among the E3, FT3, and ft3 alleles:

To elucidate the relationship between the alleles and the variation in GmPhyA3, two populations from crosses between Harosoy and each of NILs 6–22 were developed. Because 6-22-FT3 and 6-22-ft3 were considered to have common alleles at other maturity loci and a common growth habit character with Harosoy (Table 1), a simple phenotypic segregation pattern controlled by Mendelian inheritance was expected in the F₂ populations. The genetic effects of the E3, FT3, and ft3 alleles on the flowering time were evaluated by one-way ANOVA based on the genotype of the SSR marker close to GmPhyA3. As a result, only the crossing population of Harosoy and 6-22-ft3 showed a significant difference (P < 0.001; Table 3). This indicated that the E3 and FT3 alleles had the same effect. The large insertion-like retrotransposon observed in GmPhyA3-E3 and -Mo therefore might have no effect on the phenotype, whereas the one-amino-acid substitution observed in the GmPhyA-Mo might have weakened the effect of the FT3 allele.

View this table: In this window In a new window   TABLE 3 Allelism tests among Harosoy, 6-22-FT3, and -ft3

 
Although the testcross results provided information about the allelic relationships, it was still necessary to confirm that GmPhyA3 is the gene responsible for the flowering-time QTL. Since COBER's study (1996b) indicated that the E3 allele exerted a large effect under FLD, the sensitivity to FLD conditions between the three NILs and the mutant line for the GmPhyA3 gene was evaluated.

Photosensitivity under fluorescence long-day conditions:

Three groups of NILs (Harosoy and -e3, 6-22-FT3 and -ft3, 1-146-FT3 and -ft3) were grown under FLD conditions (16-hr light and 8-hr dark photoperiod). All the combinations showed more radical differences (Table 4). While the flowering days of each line varied because of their different genetic backgrounds, the effect of the E3/FT3 allele was enhanced under FLD conditions in all the NILs. Additionally, one mutant line, with a 40-bp deletion in the middle of the first exon of the GmPhyA3 gene and screened from the mutant libraries, was compared with the wild type (Figure 5A). Although the sequence of GmPhyA3 originating from the wild-type plant (Bay) was identical to that of GmPhyA3-E3, this nonfunctional GmPhyA3 mutant line flowered 15 days earlier than the wild-type plant under extended mercury-vapor lamp with high red/far-red (R/FR) conditions like FLD (Figure 5B).

View this table: In this window In a new window   TABLE 4 Differences in photoperiod sensitivity among NILs under FLD conditions

 

View larger version (62K): In this window In a new window Download PPT slide   FIGURE 5.— Gene structure and phenotypic comparisons between wild type (Bay) and the GmPhyA3 mutant. (A) The structure of the GmPhyA3 gene is as described in Figure 4. The deleted region is in the middle part of the first exon of the mutant allele. The sequence of the 40-bp deletion and the corresponding translated amino acid sequence in the wild-type plant are displayed. As a result of the deletion, a stop codon following the 36 amino acids at the deletion site appears in the mutant allele and is shown in the box. (B) Mutant line and wild-type plants at 50 days after sowing. The mutant line and wild-type plants flowered at 33 and 48 days, respectively. In this photo, the stage of the wild-type plant corresponded to the time just after flowering (right top), while the mutant line plant displayed maturing pods on the node (left top).

 
Overall, the findings in this study demonstrate that (1) the position of a candidate gene for the FT3 locus could be narrowed to a region encompassed by a single TAC clone; (2) polymorphisms were detected in GmPhyA3 among all the parental lines; (3) E3 and FT3 alleles are identical and can perceive specific light conditions; and (4) mutations in GmPhyA3 result in the early flowering phenotype because of the loss of function to recognize the high R/FR long-day condition. These results strongly suggest that GmPhyA3 is the gene responsible for the soybean flowering and maturity locus E3/FT3.

In this study, two RHLs, 6-22 and 1-146, that were selected from the RILs consisting of 156 lines, were used to identify and characterize the gene responsible for the maturity locus E3. The ratio of residual heterozygosity within the RILs (F_8:10) was theoretically <1.6%. Although other heterozygous regions in their genome might remain in both RHLs, the genotypes of other flowering QTL were homozygous, and the influences from other segregated loci on the phenotypic segregation of flowering time might have decreased. Both RHLs harboring the common heterozygous FT3/ft3 genotype could be expected to show phenotypic segregation for flowering time in their progenies. However, only the progeny of RHL1-146 displayed the expected segregation pattern. This phenomenon could be attributed to epistasis between the FT1 and FT3 loci. Statistical analysis showed that the recessive allele, ft1, reduced the effect of the FT3 allele (YAMANAKA et al. 2000; WATANABE et al. 2004). Moreover, an allelism test between Harosoy and 6-22-ft3, both carrying the recessive ft1 (e1) allele, revealed a very small effect in the variation of flowering time (Table 3). It is therefore important to consider the segregation of the genetic markers flanking the target QTL as well as the existence of epistasis between multiple loci in further fine mapping of a QTL.

Although the recessive ft1 allele could suppress the effect of the FT3 allele under natural day-length conditions, specific light conditions with a high R/FR light enhanced the effect of the E3/FT3 allele. Considering the previous assumptions about the soybean maturity locus and our results, it is reasonable to assume that phytochrome A might be the gene associated with the E3 gene (COBER et al. 1996b). Phytochrome A plays an important role not only as a far-red light photoreceptor, but also as a red-light photoreceptor in many aspects of plant development. Recently, it was reported that a high irradiance of red light induced photoprotection of PhyA protein against light-induced degradation in Arabidopsis (FRANKLIN et al. 2007). Quadruple mutants for the phytochrome family, except for PhyA, were able to respond to R-mediated de-etiolation of seedlings and survive to flowering under continuous red light with high photon irradiance. These facts indicated that PhyA acts as a red-light sensor (FRANKLIN et al. 2007). The phyA mutant in rice displayed insensitivity to FR light in etiolated seedlings, but showed no significant differences from the wild type when grown under natural day-length conditions (TAKANO et al. 2001). Moreover, overexpression of oat PhyA in transgenic rice showed no differences from the wild-type rice plants (CLOUGH et al. 1995). Nevertheless, combinations of other phytochrome mutants could dramatically alter the flowering time. The phyA phyB and phyA phyC double mutants grown under natural day-length conditions showed an earlier flowering phenotype, and the PhyA effect was masked under the presence of other functional photoreceptors (TAKANO et al. 2005). This epistatic interaction observed in rice might be comparable with the epistasis between the FT1 (E1) and FT3 (E3) loci. Moreover, it is likely that GmPhyA3 may have acquired the ability to perceive red light as a result of subfunctionalization after genome duplication in soybean. However, there is no critical evidence for this, and more experiments will be necessary to verify this possibility.

In soybean, the first PhyA reported (accession nos. L34844 and L34842; COPE and PRATT 1992) was mapped on linkage group O using a SNP marker at the same position as GmPhyA1 (CHOI et al. 2007). GmPhyA2, the gene responsible for the E4 locus, was mapped on LG I (LIU et al. 2008). Therefore, together with GmPhyA3, at least three PhyA genes have been identified in the soybean genome. This redundancy of PhyA genes is the result of the complex genome constitution of soybean. Paleopolyploidy and gene duplication in soybean is well known (reviewed by SHOEMAKER et al. 2006). The homeologous duplicated regions were investigated by gel-blot analysis with RFLP markers (SHOEMAKER et al. 1996; TSUBOKURA et al. 2008) and EST-derived SSR markers (HISANO et al. 2007). GmPhyA1 and -2 were located on LG I and O, and these linkage groups shared the homeologous duplicated region (SHOEMAKER et al. 1996; HISANO et al. 2007; LIU et al. 2008; TSUBOKURA et al. 2008). Information obtained from the database LIS, together with previous studies, has shown that a part of LG L and N might be homeologous. Moreover, a BLAST search in the soybean genome database with GmPhyA3 revealed that another PhyA gene was detected on LG N (data not shown). Although this PhyA has a deletion in the third exon, the sequence similarity of these PhyA genes supports the homeologous relationship between LG L and N.

The E4 locus was identified with natural day length extended to 20 hr with incandescent lighting (ILD) that had a low R/FR ratio (BUZZELL and VOLDENG 1980; COBER et al. 1996b; ABE et al. 2003). The insensitivity caused by the e4 allele under ILD conditions required the e3 allele (COBER et al. 1996b). Moreover, Harosoy-e4 and Harosoy-e3e4 became etiolated under continuous exposure to far-red light. Nevertheless, this insensitivity is predominant and not equivalent to that of dark-grown seedlings, suggesting that the GmPhyA1 protein may have some role in seedling de-etiolation. In contrast, Harosoy-e3 did not affect seedling growth under far-red light conditions (LIU et al. 2008). It is likely that the GmPhyA1, -2, and -3 proteins display some overlapping but distinct functions in many aspects of soybean growth as a result of genome duplication. In particular, the opposite effect observed in GmPhyA2 and -3 is very important and intriguing since GmPhyA2 (E4) exerts a strong effect on responses to low R/FR conditions, whereas GmPhyA3 (E3) responds to high R/FR conditions for soybean flowering time and maturity. At present, limited information is available on the physiological activity of soybean phytochrome A proteins and their relationship to other clock gene products and interactions with other proteins in soybean. To some extent, a high level of homology between duplicated genes may hamper the functional analysis of the individual GmPhyA genes. Nevertheless, the soybean duplicated and subfunctionalized phyA genes provide an intriguing case study for understanding complex signaling cascades via phytochrome A and for adaptation in large cultivated regions. To elucidate the control of flowering in soybean, it is necessary to isolate various genes for the associated loci and to analyze their functions and interactions.

We thank M. Takano at the National Institute of Agrobiological Sciences (Tsukuba, Japan) for valuable suggestions for our study and R. L. Nelson at the U. S. Department of Agriculture Argricultural Research Station at the University of Illinois for supplying seeds of the Harosoy and Harosoy-e3 isolines. This study was partly supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences, by research fellowships from the Japan Society for the Promotion of Science for Young Scientists (17-2554), and by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation, DD-2040).

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.108.098772/DC1.

Sequence data described in this article have been deposited at the DDBJ Data Libraries under accession nos. AB468152–AB468155 for the genomic sequences and cDNA of GmPhyA3 from Misuzudaizu (FT3), Moshidou Gong 503 (ft3), Harosoy (E3), and Harosoy-e3, respectively, and under accession nos. AP010916 and AP010917 for the shotgun sequences of GM_TMiH_H17D12 and GMJMiB242F01R, respectively. Additionally, DNA markers and BAC end sequences used in this study were registered under accession nos. AB462634–AB462641 and AB465249–AB465258, respectively.

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Communicating editor: B. BEAVIS

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