| Abstract: | 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.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. |
