The soybean stem growth habit gene Dt1 is an ortholog of Arabidopsis TERMINAL FLOWER1

Abstract

Classical genetic analysis has revealed that the determinate habit of soybean (Glycine max) is controlled by a recessive allele at the determinate stem (Dt1) locus. To dissect the molecular basis of the determinate habit, we isolated two orthologs of pea (Pisum sativum) TERMINAL FLOWER1a, GmTFL1a and GmTFL1b, from the soybean genome. Mapping analysis indicated that GmTFL1b is a candidate for Dt1. Despite their high amino acid identity, the two genes had different transcriptional profiles. GmTFL1b was expressed in the root and shoot apical meristems (SAMs), whereas GmTFL1a was mainly expressed in immature seed. The GmTFL1b transcript accumulated in the SAMs during early vegetative growth in both the determinate and indeterminate lines but thereafter was abruptly lost in the determinate line. Introduction of the genomic region of GmTFL1b from the indeterminate line complemented the stem growth habit in the determinate line: more nodes were produced, and flowering in the terminal raceme was delayed. The identity between Dt1 and GmTFL1b was also confirmed with a virus-induced gene silencing experiment. Taken together, our data suggest that Dt1 encodes the GmTFL1b protein and that the stem growth habit is determined by the variation of this gene. The dt1 allele may condition the determinate habit via the earlier loss in GmTFL1b expression concomitant with floral induction, although it functions normally under the noninductive phase of flowering. An association test of DNA polymorphisms with the stem growth habit among 16 cultivars suggested that a single amino acid substitution in exon 4 determines the fate of the SAM after floral induction.

Figure 1.

Morphology of the top of stems and plant architecture in determinate and indeterminate soybean NILs. A, Determinate NIL #6-22dt1 (dt1/dt1). A terminal raceme with a few flower buds (arrow) was produced at the tip of the main stem. B, Indeterminate NIL #6-22Dt1 (Dt1/Dt1). A cluster of axillary racemes (arrow) was surrounded by unexpanded leaflets. C, Plant morphology at maturity in the progeny of RHL #6-22. The #6-22dt1 (dt1/dt1) plants were short with a few nodes because of stem termination after stage R1, whereas #6-22Dt1 (Dt1/Dt1) plants were tall and produced more nodes. Plants with intermediate phenotypes were estimated to be heterozygous for the Dt1 locus, based on the FLP in intron 1 of GmTFL1b.

Figure 2.

Structures, predicted amino acid sequences, and phylogenetic tree of soybean TFL1 orthologs GmTFL1a and GmTFL1b. A, Exon/intron structures of GmTFL1a and GmTFL1b. Exon (boxes) and intron sizes are in bp. B, Multiple alignment of the predicted amino acid sequences of GmTFL1a, GmTFL1b, L. japonicus LjCEN/TFL1 (Guo et al., 2006), pea PsTFL1a (Foucher et al., 2003), Arabidopsis TFL1 (Bradley et al.,1997), Arabidopsis ATC (Mimida et al., 2001), and A. majus CEN (Bradley et al., 1996). Highly conserved amino acids are in black, dark gray, or light gray depending on the level of identity (darker = higher level). Numbers on the right refer to amino acid positions in relation to the N-terminal Met of each sequence. Arrowheads above the sequences refer to differences between GmTFL1a and GmTFL1b. The arrow indicates an amino acid substitution from Arg (R) in the Dt1 allele to Trp (W) in the dt1 allele. C, Phylogenetic relationships of TFL1/CEN proteins constructed using the neighbor-joining method with the program ClustalW. Bootstrap values of 1,000 replicates are indicated at the branches of the tree.

Figure 3.

Genetic map positions of soybean TFL1 orthologs GmTFL1a and GmTFL1b. GmTFL1a (in linkage group N) and GmTFL1b (in linkage group L) were mapped using RILs derived from a cross between determinate G. max (TK) and indeterminate G. soja (H4) lines.

Figure 4.

Frequency of plants with various numbers of nodes produced after the first flower appearance to show cosegregation between stem growth habit and GmTFL1b. A, Progeny of RHL #6-22. B, RILs derived from a cross between determinate G. max (TK) and indeterminate G. soja (H4) lines. Plants/lines homozygous for the determinate parent-derived GmTFL1b allele (white bars) were almost clearly separated from those having the indeterminate parent-derived allele (gray bars for heterozygote and black bars for homozygote) in the number of nodes produced after the first flower appearance.

Figure 5.

Expression analysis of GmTFL1a and GmTFL1b by RT-PCR. A, Gene expression in root (R), cotyledon (C), leaflet (L), stem tip (S), flower (F), pod (P), and immature cotyledon (IC) of the #6-22Dt1 plants. Transcripts of the β-tubulin gene were amplified as a control. For both paralogs, primers were designed to anneal the 3′ untranslated region and exon 3, which amplify DNA fragments of different sizes from cDNA and genomic DNA, to confirm that no amplification occurred from genomic DNA contaminants in the RNA sample. Note that the expression of GmTFL1a was high in the immature cotyledon but quite low in the stem tip and absent in the root, whereas GmTFL1b was expressed only in the root and the stem tip. B, Expression of GmTFL1a and GmTFL1b in stem tip from 15 to 30 DAE. GmTFL1a in the determinate (D) and indeterminate (I) lines was expressed at a high level only at a later stage of growth after flowering. On the other hand, GmTFL1b was expressed at earlier stages of growth through flowering (25 DAE) in the indeterminate line but was abruptly lost after 15 DAE in the determinate line.

Figure 6.

Complementation of the dt1 allele by transformation. A, Morphology at the stem tip of determinate cv KA, B, The T2 plants carrying the GmTFL1b genomic region. KA plants formed a terminal raceme at the tip of the main stem, whereas T2 plants formed terminal and axillary racemes with unexpanded leaflets. C, Number of nodes produced after the first flower appearance (stage R1). KA plants (dt1) and GFP-transformed T3 plants (GFP) produced no additional nodes after stage R1, whereas the T2 plants (A5-2 and B4-1), on average, produced 3.2 nodes (A5-2) and 3.5 nodes (B4-1). D, Number of days from stage R1 until a flower in the terminal raceme opened. In KA and GFP plants, flowering started at node 5 or 6, and a flower in the terminal raceme opened 2 d later, whereas T2 plants took longer for the flower in the terminal raceme to open. E, Plant morphology of transgenic plants. T2 plants (A5-2-4 and A5-2-8) were taller and produced more nodes than did KA and GFP plants.

Figure 7.

VIGS-induced suppression of vegetative activity of terminal buds. A, Reduction by VIGS of numbers of nodes produced after stage R1. The #6-22Dt1 plants mock inoculated (Dt1) and infected by the virus carrying the GFP sequence (GFP) produced an average of 3.6 and 4.6 nodes, respectively. On the other hand, of the 21 #6-22Dt1 plants infected by the virus carrying the GmTFL1b constructs (VIGS), four produced only one node after floral initiation (1-node) and six formed two nodes (2-node). The number of plants tested is given in parentheses above the bars. B, Pod size at lower nodes (white bars) and terminal node (black bars). All plants produced pods of approximately 4 cm at nodes 5 and 6, independent of the Dt1 genotypes and treatments. C, Pods of #6-22dt1 plants (dt1) were almost the same size as those at lower nodes. D, Stem tip morphology for the #6-22Dt1 plants mock inoculated (Dt1) and those infected by the virus carrying the GFP sequence (GFP). Those plants maintained vegetative activity and did not form any pods at the stem tip. VIGS-induced 1-node and 2-node plants produced pods of 2.0 cm or more at the terminal node. E, Suppression of the GmTFL1b expression in root by VIGS. Expression as evaluated with real-time PCR was low in #6-22dt1 plants (dt1) compared with the #6-22Dt1 plants mock inoculated (Dt1) and infected by the virus carrying the GFP sequence (GFP). GmTFL1b expression in VIGS plants (VIGS) was similar to levels in the determinate plants. Two plants were analyzed for each treatment. Data represent means for three replications with se.

Figure 8.

Association of stem growth habit with DNA polymorphisms of GmTFL1b. A, Genotypes for 16 early-maturing cultivars at three linked DNA polymorphisms detected consistently between determinate and indeterminate lines. B, The number of nodes produced after stage R1. Based on the deduced genotypes for three DNA markers, the cultivars were classified into three haplotypes: dt1 haplotype (white bars), Dt1 haplotype (black bars), and a new haplotype (gray bars). Cultivars are as follows: 1, Rokujunichimame; 2, Ishikarishiro 1 go; 3, Aochi (Natsu); 4, Wasekeburi; 5, Iwateyagi 1 go; 6, Wasekin; 7, Baihualudadou; 8, Keshuang; 9, Chashouryu; 10, Chamoshidou; 11, Kairyokimusume; 12, Baihua; 13, Shauilihong; 14, Lindiansuoyiling; 15, Chuandou; 16, Zihua 1. Harosoy (Dt1) and its NIL for dt1 (dt1) are included for comparison.