**1. Introduction**

50 Soybean – Genetics and Novel Techniques for Yield Enhancement

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The change from vegetative to reproductive growth is a critical developmental transition in the life of plants. Various external cues, such as photoperiod and temperature, are known to initiate plant flowering under the appropriate seasonal conditions. Endogenous cues include a system of juvenile to adult transition that affects competence to flower. To understand the molecular mechanism of flowering, extensive studies have been performed using model plants, *Arabidopsis thaliana* and rice (*Oryza sativa*), and these have revealed the numerous regulatory network components associated with flowering (Jung & Muller, 2009; Amasino, 2010). The general concept of the photoperiodic induction of flowering (photoperiodism) and the range of response types among plant species was established by Garner and Allard (1920). Among the external cues, light is the most important, being received by several photoreceptors including phytochromes, cryptochromes and phototropins. The role of phytochromes, that is the R-light- and FR-light- absorbing photoreceptors, in flowering has been investigated in several plant species. In *Arabidopsis*, a quantitative long-day (LD) plant, a phyA mutant flowered later in either long-day or short-day (SD) conditions with a night break (Johnson et al., 1994; Reed et al., 1994). In rice, a SD plant, the phyA monogenic mutant exhibited the same flowering time as the wild type under LD conditions, while, in the phyB and phyC mutant backgrounds, the flowering was greatly accelerated relative to phyB and phyC monogenic mutants (Takano et al., 2005). In pea, a LD plant, loss- or gainof-function phyA mutants displayed late or early flowering phenotypes, respectively (Weller et al., 1997, 2001). Day length is found to be perceived by leaves by Knott (1934). Because flowering occurs in the shoot apical meristem (SAM), the leaves must transmit a signal to the SAM and this signal is referred to as florigen (Chailakhyan, 1936). In *Arabidopsis*, three genes, *CONSTANS* (*CO*), *GIGANTEA* (*GI*) and *FLOWERING LOCUS T* (*FT*) were found to be involved in the production of a flowering promoter in LD conditions (Koornneef et al., 1991; Kardailsky et al., 1999). FT protein is now known to be florigen, and CO and GI are key players in the activation of FT expression. CO is a zinc-finger protein that

Positional Cloning of the Responsible Genes for Maturity Loci *E1*, *E2* and *E3* in Soybean 53

Flowering time is a very important trait which is related to productivity, adaptability and domestication. Soybean breeders have attempted to modify flowering and maturity to expand growing areas for soybean. Molecular identification of *E* loci and flowering network of soybean is useful for efficient breeding to control adaptability and increase yield of soybean. We have identified flowering-time quantitative loci (QTL), *FT1*, *FT2* and *FT3*, and found to correspond to *E1*, *E2* and *E3*, respectively (Yamanaka et al., 2001). We successfully identified the responsible genes for the *E1* (Xia et al., unpublished), *E2* (Watanabe et al., 2009) and *E3* (Watanabe et al., in press) by positional cloning strategy. In this chapter, we will describe the process of identification of responsible genes for the *E1*, *E2* and *E3* loci with variation of alleles and propose a tentative major flowering time pathway in soybean.

As flowering time is a quantitative trait, we employed QTL analysis (Tanksley, 1993) to dissect the genetic factors for flowering time into individual components by using recombinant inbred lines (RIL) derived from Misuzudaizu, a Japanese variety, and Moshidou Gong 503, a weedy line from China. To identify the underlying molecular basis for each QTL, map-based cloning method was performed because molecular or biochemical information for soybean flowering was very few or totally not available. Although NILs are usually used for fine mapping of each QTL, developing NILs is time-consuming and laborious process especially in soybean. Alternatively, we have proposed fine mapping using residual heterozygous lines (RHLs) (Yamanaka et al., 2005). An RHL selected from an RIL population harbors a heterozygous region where the target QTL is located but contains a homozygous background for most other regions of the genome. The progenies of the RHL are expected to show a simple phenotypic segregation based on the effects of the target QTL at the heterozygous region (Fig. 1). A similar term, heterogeneous inbred family (HIF), was used by Tuinstra et al. (1997) to identify the QTL associated with seed weight in sorghum. The RHL strategy has already been used to identify loci underlying pathogen resistance in soybean (Njiti et al., 1998; Meksem et al., 1999; Triwitayakorn et al., 2005). Genotypes of a trait in recombinants identified in the progenies of RHL, could be determined in the next

The probability of discovering RHLs for a target QTL depends on the heterozygosity ratio in a population and the size of the population. If p is the ratio of hetrozygosity of any population with size n, then the probability of detecting k individuals with a heterozygous genotype is supposed as nCk pk (1-p)n-k based on a binomial distribution. In the case of an F7 generation of RILs, the ratio of heterozygosity (p) is 0.0156 and with a population size of 200 (n), the probability of detecting at least one RHL is more than 0.95. We propose that QTL analysis using the F6-F8 RIL population in combination with the RHL strategy is useful for dissecting genetic factors for an agronomic trait into each QTL where the homozygous ratio is sufficiently high to evaluate traits with replication and the heterozygosity ratio is not so

In progenies of an RHL, we can identify NILs for the target QTL. New DNA markers in the heterozygous region were developed using NILs, bulked segregant analysis (BSA) in progenies of the RHL, and sequences of bacterial artificial chromosome (BAC) clones covering the target QTL. We usually developed amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) and sequence characterized amplified region (SCAR) markers. Genetic analyses of flowering phenotypes and DNA markers were performed in the

low and will allow the identification of a sufficient number of RHLs.

**2. Strategy for fine mapping and positional cloning** 

generation.

functions as a transcription factor (Putterill et al., 1995), and GI is a large protein involved in circadian clock function (Fowler et al., 1999; Park et al., 1999). FT is a small protein with some resemblance to RAF kinase inhibitors (Kardailsky et al., 1999; Kobayashi et al., 1999) that is produced in leaves and moves to the SAM (Corbesier et al., 2007; Jaeger & Wigge, 2007; Mathieu et al., 2007; Tamaki et al., 2007; Notaguchi et al., 2008). The rice orthologs of *Arabidopsis CO* and *FT* genes, *Heading date 1* (*HD1*) and *Heading date 3a* (*Hd3a*), respectively, have been identified (Yano et al., 2000; Kojima et al., 2002; Hayama et al., 2003). The promotion of flowering in *Arabidopsis* in LD conditions results from activation of *FT* by *CO*, while the delay in flowering in rice in LD conditions results from repression of *Hd3a* by *Hd1* (Izawa et al., 2000; Kojima et al., 2002; Roden et al., 2002; Hayama et al., 2003). A *CO*/*FT* module is likely to be conserved throughout the plant kingdom. CYCLING DOF FACTORS (CDFs) exhibit circadian cycling and bind to *CO* promoter and repress *CO* expression. The abundance of CDFs is controlled by FLAVIN-BINDING, KELCH REPEAT, F-BOX PROTEIN1 (FKF1) that appears to be involved in the ubiquitin-mediated degradation of CDFs. GI protein physically interacts with FKF1 and stabilizes it promoting CDF degradation and subsequent *CO* expression (Imaizumi et al., 2005.; Sawa et al., 2007; Fornara and Coupland, 2009; Imaizumi, 2009). Despite the conserved functions of *FT* orthologs, their expression may be controlled by different systems in different species. Non-*CO*/*FT* pathways have been proposed for several plants, such as morning glory (*Pharbitis nil*) (Hayama et al., 2007) and tomato (Ben-Naim et al., 2006; Lifschitz et al., 2006). In rice, *Early heading date 1* (*Ehd1*) has been found to promote flowering by inducing *FT*-like gene expression only under SD conditions independently of *Hd1* (Doi et al., 2004). There is no *Ehd1* ortholog in *Arabidopsis*.

Soybean is a typical SD plant whose photoperiodic sensitivity was discovered by Garner and Allard in 1920. Compared to the model plants, photoperiodic control of flowering in soybean is far less understood. The eight loci, *E1* to *E8*, conditioning flowering has been genetically identified (Bernard, 1971; Buzzel, 1971; Buzzel and Voldeng, 1980; McBlain and Bernard, 1987; Bonato and Vello, 1999; Cober and Voldeng, 2001; Cober et al., 2010). At each of these loci, two alleles have been identified, and except for *E6*, the recessive alleles at the *E* loci condition early flowering under both LD and SD conditions. The partially dominant alleles at the *E* loci delay flowering under LD conditions. Near-isogenic lines (NILs) for *E* loci have been developed and used for studies to elucidate the flowering in soybean (Saidon et al., 1989a,b; Upadhyay et al., 1994a,b; Cober et al., 1996a). Among these *E* loci, *E1*, *E3*, *E4* and *E7* are known to be involved in the response to the phtoperiod (Buzzell, 1971; Buzzell and Voldeng, 1980; McBlain et al., 1987; Cober et al., 1996b; Cober and Voldeng, 2001; Abe et al., 2003). The *E3* locus was first identified with the use of fluorescent lamps to extend day length. The *e3e3* recessive homozygote can initiate flowering under LD conditions where the day length was extended to 20 hr using fluorescent lamps (FLD) with a high red to far-red (R: FR) ratio (Buzzell, 1971). The *E4* locus was identified by extending the natural day length to 20 hr with incandescent lamps with a low R: FR ratio (Buzzell and Voldeng, 1980). The insensitivity of *e4e4* genotype to LD conditions with a low R: FR ratio is necessary of *e3e3* background (Buzzell and Voldeng, 1980; Saindon et al., 1989b; Cober et al. 1996b). The *E1* and *E7* loci are involved in the control of insensitivity to artificially induced LD conditions in the *e3* and *e4* backgrounds (Cober et al., 1996b; Cober and Voldeng 2001). Of the known *E* loci, the *E1* locus is considered to have the largest effect on time to flowering under field conditions (Stewart et al., 2003).

Flowering time is a very important trait which is related to productivity, adaptability and domestication. Soybean breeders have attempted to modify flowering and maturity to expand growing areas for soybean. Molecular identification of *E* loci and flowering network of soybean is useful for efficient breeding to control adaptability and increase yield of soybean. We have identified flowering-time quantitative loci (QTL), *FT1*, *FT2* and *FT3*, and found to correspond to *E1*, *E2* and *E3*, respectively (Yamanaka et al., 2001). We successfully identified the responsible genes for the *E1* (Xia et al., unpublished), *E2* (Watanabe et al., 2009) and *E3* (Watanabe et al., in press) by positional cloning strategy. In this chapter, we will describe the process of identification of responsible genes for the *E1*, *E2* and *E3* loci with variation of alleles and propose a tentative major flowering time pathway in soybean.
