**5. Conclusion**

68 Soybean – Genetics and Novel Techniques for Yield Enhancement

Fig. 15. Fine mapping of the *FT1/E1* locus. *E1* homozygous, *e1* homozygous and

genotype of each recombinant was identified by progeny test.

**4. Putative pathway of flowering time in soybean** 

heterozygous genotypes are shown by solid, open and meshed boxes, respectively. The *FT1*

The *FT1* locus was genetically mapped into the semi-central domain of linkage group C2 (Fig. 3) and was included in the pericentromeric region of chromosome 06 (http://www.phytosome.net/). In the heterochromatic regions, the ratio of physical to genetic distance is 3.5Mb/cM in comparison of 197 Kb/cM in euchromatic regions (Schmutz et al., 2010). The responsible gene for *FT1*/*E1* locus is characterized by relatively lower mRNA abundance. In fact, no EST data of the *FT1*/*E1* gene could be retrieved from public databases. The gene encodes a novel small protein and is unique in the sense of no apparent orthologs in model plants *Arabidopsis* or rice. We are analyzing the ligands of this protein

The responsible gene for the *E4* locus was identified as *GmPhyA2* through the candidate gene approach (Liu et al., 2008). At the *e4* allele, a Ty1/copia-like retrotransposon was inserted in exon 1 of the gene, which resulted in dysfunction of the gene and photoperiod insensitivity. Similarly, natural and artificial mutations of *GmPhyA3* resulted in weak or complete loss of photoperiod sensitivity (Watanabe et al., 2009). The *FT* homologs in soybean have been identified (Kong et al., 2010) and two of them, *GmFT2a* and *GmFT5a*, were highly upregulated under SD conditions and showed diurnal expression patterns with the highest expression 4h after dawn. Under LD conditions, expression of *GmFT2a* and *GmFT5a* was downregulated and did not follow a diurnal pattern. Ectopic expression analysis in *Arabidopsis* confirmed that both *GmFT2a* and *GmFT5a* had the same function as *Arabidopsis FT*. A double-mutant (*e3e3 e4e4*) for *GmPhyA2* and *GmPhyA3* expressed high levels of *GmFT2a* and *GmFT5a* under LD conditions (18-h light) with an R: FR ratio of 1.2, and it flowered slightly earlier under LD than the wild type (*E3E3 E4E4*) grown under SD. The expression levels of *GmFT2a* and *GmFT5a* were regulated by PHYA-mediated

Satt365 SSRA SSR8 Satt557 End5

4/1442 1/1442 0/1442 1/1442

WBb238P13 WBb139N16 WBb10K2 WBb55K17

> WBb20D6 WBb106D7 WBb104J15

WBb120K2

*FT1/E1* locus

060-052 060-250 060-285 060-828 060-946 060-784 060-1057

Genetic mapping

WBb168L14 WBb220D05

WBb33C20 WBb115J24

Physical contigs

DNA marker

Hetero *E1 e1 E1* Hetero Hetero *e1 E1* homozygous allele *e1* homozygous allele Heterozygous allele

Line *FT1* genotype

and the interaction with DNA sequences.

We successfully identified the responsible genes for the *E1*, *E2* and *E3* by positional cloning strategy and proposed a tentative flowering time gene network in soybean based on interaction of these genes. We used RHLs derived from RIL for fine mapping a single QTL effectively. An RHL harbors a heterozygous region where the target QTL is located and a homozygous background in most other regions of the genome. Novel DNA markers tightly linked to the locus were developed based on AFLP between the NILs of the locus derived from an RHL. A large-scale population derived from RHLs was used to locate the target

Positional Cloning of the Responsible Genes for Maturity Loci *E1*, *E2* and *E3* in Soybean 71

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locus precisely. We developed manual large-scale genotyping of seeds, in which powdered cotyledon was obtained by drilling a hole on the surface of seed without any damage to the embryonic axis. Recombinants carrying crossovers in the target region were selected based on genotypes of DNA markers around the region. Genotypes of the flowering time locus of recombinants were determined by progeny test and identified the cosegregated region based on these genotypes. Physical contigs were constructed with BAC/TAC clones screened by SCAR markers converted from these AFLP fragments. By sequencing the BAC contig covering the cosegregated region, we identified the candidate genes. Confirmation of the responsible gene was performed by investigation of association between natural and induced variation of the candidate gene structures and flowering time. Mutant screening was carried out with TILLING using X-ray irradiated or EMS treated mutant libraries. The interactions between the identified genes were analyzed using several NILs and segregating population for the *E* loci. A tentative flowering time network in soybean was proposed taking into consideration the possible functions of responsible genes for *E1*, *E2*, *E3* and *E4* loci and *GmFT*s. Further characterization of other *E* loci is necessary to reveal the molecular mechanism of flowering in soybean.

Recently, soybean genome sequence has been reported (Shumutz et al., 2010) and a large number of SSR (Song et al., 2010) and SNP (Hyten et al., 2010a; Lam et al., 2010) markers has been developed. New high-throughput sequencing technologies, and multiplex assays for genotyping a huge number of SNPs have become available. These technologies and information will accelerate the identification of responsible genes for agriculturally important loci. But methods and materials to precisely locate the target loci in the genome are still important. Moreover, variation of regional genome structure and gene content (Kim et al., 2010 ; William et al., 2010; Xia et al., unpublished) will need the sequencing of genome clones covering the target region.
