A map-based cloning strategy employing a residual heterozygous line reveals that the GIGANTEA gene is involved in soybean maturity and flowering

Abstract

Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In soybean (Glycine max), a flowering quantitative trait locus, FT2, corresponding to the maturity locus E2, was detected in recombinant inbred lines (RILs) derived from the varieties "Misuzudaizu" (ft2/ft2; JP28856) and "Moshidou Gong 503" (FT2/FT2; JP27603). A map-based cloning strategy using the progeny of a residual heterozygous line (RHL) from the RIL was employed to isolate the gene responsible for this quantitative trait locus. A GIGANTEA ortholog, GmGIa (Glyma10g36600), was identified as a candidate gene. A common premature stop codon at the 10th exon was present in the Misuzudaizu allele and in other near isogenic lines (NILs) originating from Harosoy (e2/e2; PI548573). Furthermore, a mutant line harboring another premature stop codon showed an earlier flowering phenotype than the original variety, Bay (E2/E2; PI553043). The e2/e2 genotype exhibited elevated expression of GmFT2a, one of the florigen genes that leads to early flowering. The effects of the E2 allele on flowering time were similar among NILs and constant under high (43°N) and middle (36°N) latitudinal regions in Japan. These results indicate that GmGIa is the gene responsible for the E2 locus and that a null mutation in GmGIa may contribute to the geographic adaptation of soybean.

Figure 1.—

Detection of the QTL designated FT2 using RILs and the identification of the heterozygous region of RHL 6-8 determined by DNA markers mapped on LG-O [chromosome (Chr.) 10]. Genetic linkage map, marker names, and genetic distances (cM) are displayed on the left side of the graph. Solid (2001) and dotted (1999) lines indicate the LOD scores for QTL analysis of flowering time, using 2 years of data on RILs and calculated by composite interval mapping (Watanabe et al. 2004). Representative information relating to the LOD peak position corresponding to the FT2 locus, the additive effect of the Misuzudaizu allele, and the phenotypic variance explained by the QTL (PVE) are summarized in the upper-right section of the graph. The heterozygous region of RHL 6-8 is indicated by the solid bar.

Figure 2.—

The genotype of each recombinant plant and estimation of QTL among the marker loci. (A) The genotypes of each plant were analyzed using the marker located on this physical region. The genotypes of A, B, and H indicate the Misuzudaizu homozygous and the Moshidou Gong 503 homozygous and heterozygous genotypes, respectively. (B) One-way classification ANOVA was performed to estimate the position of the QTL. Phenotypic values of each plant investigated in the experimental field in 2006 are shown at the left side of the line name. The representation of each marker genotype is summarized at the bottom. The statistical values and probability calculated using ANOVA are shown below the graphical genotypes of each marker. The highest statistical value is obtained for marker 4 surrounded by the dotted line.

Figure 3.—

Phylogenic analysis of GIGANTEA proteins. A phylogenic tree of GIGANTEA proteins, constructed by the NJ method with a p-distance and a 1000-replication bootstrap confidence value, is shown. Amino acid sequences of A. thaliana (AAT80910; Fowler et al. 1999), P. sativum (ABP81863; Hecht et al. 2007), Medicago truncatula (ABE81212), O. sativa (NP_001042220; Ohyanagi et al. 2006), H. vulgare (AAW66946; Dunford et al. 2005), T. aestivum (AAQ11738; Zhao et al. 2005), Lolium perenne (ABF83898), and Lemna paucicostata (BAD97864) were used.

Figure 4.—

Gene structure of GmGIa in NIL 6-8-ft2 (ft2/ft2), NIL 6-8-FT2 (FT2/FT2), and the GmGIa mutant line (E2-mut). (A) Exons, a part of the 3′ UTR, and introns of the GmGIa gene in the 24- to 45-kbp region of MiB300H01 are indicated by solid boxes, an open box and lines, respectively. Units on the nucleotide sequence scale shown by the vertical lines represent 1 kbp. The location in the 5th intron of marker 4, originating from AFLP E60M38, is represented by the shaded bar. (B) The substitution of nucleotides and amino acid residues (in parentheses) in the coding sequence are displayed above the boxes that represent each exon. Nucleotide and amino acid changes in the case of nonsynonymous and nonsense mutations from the ft2 to the FT2 allele, on the basis of the start codon position, are shown. In the early flowering ft2 allele, a single nucleotide substitution causing a premature stop codon was discovered in the 10th exon. The mutant line, harboring a nucleotide deletion in the same exon as the ft2 allele, had a truncated GI protein that similarly caused the early flowering phenotype under natural day-length conditions. The truncated protein lengths of the three alleles are compared at the bottom.

Figure 5.—

Expression analysis of GmFT2a between NILs of E2 under natural day-length conditions. Two pairs of NILs were used. Quantitative RT-PCR experiments for GmFT2a were performed using RNA isolated from trifoliolate leaves sampled at 9:00 AM 4 weeks after sowing. A representative polyacrylamide gel demonstrating the expression levels is shown. Each bar indicates the average expression level of GmFT2a compared with that of the GmTubulin gene with three independent replications and time to flowering; standard deviations of each line are shown below the bar graph.