WO2013187554A1 - In GENE FOR CONTROLLING NUMBER OF SEEDS PER POD IN SOYBEAN AND USES THEREOF - Google Patents

Abstract

The present invention relates to ln gene produced by single nucleotide substitution in EAR motif of leaf shape-controlling Ln (broad leaflet) gene from soybean (Glycine max), a recombinant vector comprising the ln gene gene, a host cell transformed with recombinant vector, a method of controlling the number of seeds per pod by using the ln gene, a method of producing a plant with controlled number of seeds per pod by using the ln gene, a transformed plant with controlled number of seeds per pod that is produced by the method, seeds thereof, and a composition for controlling the number of seeds per pod in a plant comprising the recombinant vector comprising the ln gene.

Description

[Corrected under Rule 26 19.09.2012] In GENE FOR CONTROLLING NUMBER OF SEEDS PER POD IN SOYBEAN AND USES THEREOF

The present invention relates to ln gene for controlling the number of seeds per pod in soybean and uses thereof. More specifically, it relates to ln gene produced by single nucleotide substitution in EAR motif of leaf shape-controlling Ln (broad leaflet) gene from soybean (Glycine max), a recombinant vector comprising the ln gene gene, a host cell transformed with recombinant vector, a method of controlling the number of seeds per pod by using the ln gene, a method of producing a plant with controlled number of seeds per pod by using the ln gene, a transformed plant with controlled number of seeds per pod that is produced by the method, seeds thereof, and a composition for controlling the number of seeds per pod in a plant comprising the recombinant vector comprising the ln gene.

Leaves and flowers develop continuously at the flanks of the shoot apical meristem in flowering plants. A single mutation often causes pleiotropic phenotypes during leaf and flower development, suggesting that a common regulatory circuit is involved in the production of leaves and flowers. For example, Leafy/UNIFLOLIATA regulates both leaf and flower morphogenesis in the pea. Conversely, combinations of floral homeotic mutations result in the conversion of floral organs to leaf-like structures. The pea mutant crispa (cri), which is defective in the PHANTASTICA (PHAN) gene, has a reduced leaflet width-to-length ratio and exerts pleiotropic effects, including longer internodes, reduced peduncle length, and lower seed production per pod. However, the molecular genetic basis of the relationship between leaf/flower development and pods or fruits, which are a mature form of the flower, is poorly understood.

Seed yield is determined by the number of seeds per unit area and seed weight. During soybean production, the number of seeds per unit area is a product of the number of plants per unit area, the number of pods per plant, and the number of seeds per pod (NSPP). The NSPP is primarily determined by the number of ovules per placenta as well as the frequency of embryonic abortions. Soybean ovaries contain from one to four ovules, indicating that the NSPP is determined at the early stage of flower development. A quantitative trait loci (QTL) analysis using three recombinant inbred populations derived from reciprocal crosses of three cultivars demonstrated that the average NSPP in soybean is determined genetically by multiple significant QTL that account for approximately 50% of the heritable variation. Those QTL were linked to the Ms1, Ms6, or St5 genes for male and female sterility and Lf1 and ln for leaflet number and leaflet shape, respectively, although further molecular genetic analyses have not been conducted. Among the genes associated with NSPP, the ln gene, which was originally named for the narrow leaflet-determining gene in soybean, has been more frequently studied, because the leaflet-shape trait locus has long been suggested to be tightly linked to the trait locus controlling NSPP or to exert a major pleiotropic effect on increasing the level of NSPP. Nevertheless, the NSPP trait is governed not only by a single ln or its tightly linked gene, but also by additional modifying gene(s) in the ln genetic background (Jeong et al., 2011, Theor. Appl. Genet. 122, 865-874).

Previous agronomic studies for the ln locus using isogenic lines, diverse cultivars, and broad (Ln/Ln), heterozygous (Ln/ln), and narrow (ln/ln) leaflet types have repeatedly found that broad and narrow leaflet soybean genotypes have similar seed yields, but narrow leaflet plants consistently produce smaller seeds than those of broad leaflet plants. Although soybean yield has generally been described in terms of the total soybean seed weight per unit area, the fact that the narrow leaflet plants tend to produce a greater number of seeds has garnered a great deal of attention, particularly in the development of soybean cultivars for sprouts. The number of seeds is a critical yield component for soybean production sprouts, and this has resulted in a narrow leaflet shape in most of the sprout soybean cultivars recently developed in Korea. Collectively, the causes of this association might include either the tight linkage of genes that control independent traits or the pleiotropic effects of the target gene for both traits.

Cloning and functional understanding of loci regulating the yield components may provide molecular genetic tools for improving yield, one of the most complex plant traits. In this study, we performed map-based cloning of the ln locus, a regulator of leaflet shape and NSPP. Our results indicated that the leaflet-shape and NSPP traits are pleiotropic effects of the ln gene. Consequently, our results should facilitate development of new soybean cultivars with high yield potential.

Although US Patent No. 7,973,212 discloses 'Soybean plants having superior agronomic performance and methods for their production', there has been no report regarding a plant having controlled number of seeds per pod as described in the present invention, in which the plant is transformed with a recombinant vector comprising ln gene for controlling the number of seeds per pod.

The present invention is devised in view of the needs described above. Specifically, during the screening process for obtaining a high-yield variety, inventors of the present invention found that ln gene, which is a recessive gene of Ln (broad leaflet) gene of soybean (Glycine max), is generated by nonsynonymous nucleotide substitution due to single nucleotide substitution in EAR motif of Ln gene. It was further confirmed by the inventors that said gene causes not only the narrow leaflet shape but also pleiotropic effects to increase the number of seeds per pod. It was still further confirmed that when the Glycine max Ln gene or ln gene were transformed into jag-3, a mutant of Arabidopsis thaliana, they complemented the functions of their A. thaliana homologue, the JAG gene and thus the present invention was completed accordingly.

To solve the aforementioned problems, the present invention provides ln gene consisting of a nucleotide sequence of SEQ ID NO: 1, which is produced by single nucleotide substitution in EAR motif of Ln (broad leaflet) gene from soybean (Glycine max).

Further, the present invention provides a recombinant vector comprising the ln gene.

Further, the present invention provides a host cell transformed with the recombinant vector.

Further, the present invention provides a method of controlling the number of seeds per pod by transforming a plant cell with the recombinant vector comprising the ln gene.

Further, the present invention provides a method of producing a plant with controlled number of seeds per pod by transforming a plant cell with the recombinant vector comprising the gene.

Further, the present invention provides a transformed plant with controlled number of seeds per pod that is produced by the method, and seeds thereof.

Still further, the present invention provides a composition for controlling the number of seeds per pod in a plant comprising the recombinant vector comprising the ln gene produced by single nucleotide substitution in EAR motif of Ln gene from soybean (Glycine max).

By using the ln gene of the present invention that is produced by single nucleotide substitution in EAR motif of Ln gene from soybean (Glycine max), a transformed plant with increased number of seeds per pod can be obtained, and therefore it would be useful for development of a plant with increased production yield.

Figure 1 shows molecular cloning of ln. (a) Chromosomal location of ln determined by genetic linkage mapping on chromosome (Chr) 20. (b) Fine mapping of ln. Vertical lines indicate microsatellite and SNP markers. Markers are indicated above the lines. The numbers of recombinants left in the chromosomal interval still containing ln after the evaluation of the marker are indicated below the lines. (c) Sequence map between markers Ln_44k and Ln_57k. Mutations found between the mapping parents are labeled below the map: insertion/deletion polymorphisms are indicated by arrowheads (▲) and single nucleotide substitutions by asterisks (*). (d) JAG gene structure showing five exons (gray boxes), four introns (white boxes), and 1.5 kb of putative promoter region. Start and stop codons (vertical lines) and a 6-bp alternative splicing site (arrow) of the gene are labeled. (e) Amino acid sequence of the JAG protein. Position and nature of the ln (asterisk) mutation is indicated. Two amino acids changed by an alternative splicing of 6 bp between second intron and third exon are boxed. Putative conserved motifs are in bold font. The EAR repression motif is underlined by a solid line. A putative nuclear localization sequence is underlined by a dotted line. A proline-rich motif is underlined by a wave line.

Figure 2 shows alignment of amino acid sequences of Glycine max JAG1 and its homeolog JAG2, AtJAG (GenBank Accession no. AAR30036) and NUBBIN (NP_172797) from Arabidopsis thaliana, LYRATE (ACC99356) from Solanum lycopersicum, and ZmJAG (NP_001147366) from Zea mays. Boxed amino acids denote conserved residues. The EAR motif, putative nuclear localization signal (NLS) sequence, single C2H2-type zinc finger motif, and proline-rich motif are indicated.

Figure 3 shows the ln phenotype and its expression pattern in plant tissues. (a) Trifoliolate leaf of V94-5152 (Ln). (b) Trifoliolate leaf of Sowon (ln). (c) Pod types occurring in Ln allele plants. Predominant 2-seeded pod is indicated by asterisk (*). (d) Pod types occurring in ln allele plants. Predominant 3-seeded pod is indicated by asterisk (*). (e) Ln flower. (f) ln flower. (g) Ln epidermal cells. (h) ln epidermal cells. Scale bar: 50 mm (A and B); 10 mm (c and d); 2 mm (e and f); 20 ㎛ (g and h). (i) RT-PCR analysis of GmJAG1 in different tissues of ln and Ln allele plants. Numbers refer to PCR cycles. RNA was extracted from tissues of field-grown plants. Amplification of actin was used as a control.

Figure 4 shows functional analysis of GmJAG1 and Gmjag1 alleles in the Arabidopsis jag-3 mutant. (a) Confirmation of expression of soybean GmJAG1 and Gmjag1 alleles in the transgenic Arabidopsis jag-3 lines by RT-PCR analysis. RNA was extracted from inflorescence tissues of PAtJAG::gGmJAG1 transgenic plants, PAtJAG::gGmjag1 transgenic plants, wild-type Landsberg erecta (Ler), jag-3 mutant. (b) Ler flower. (c) jag-3 flower. (d) PAtJAG::gGmJAG1 flower. (e) PAtJAG::gGmjag1 flower. (f) Ler rosette leaves. (g) jag-3 rosette leaves. Leaves were excised from 4-week-old plants (e-g). The first leaves are seen left. (h) PAtJAG::gGmJAG1 rosette leaves. (i) PAtJAG::gGmjag1 rosette leaves. (j) Fully elongated fruit of Ler. (k) Fully elongated fruit of jag-3. (l) Fully elongated fruit of PAtJAG::gGmJAG1. (m) Fully elongated fruit of PAtJAG::gGmjag1. (n) Dehiscent fruit of Ler. (o) Dehiscent fruit of jag-3. (p) Dehiscent fruit of PAtJAG::gGmJAG1. (q) Dehiscent fruit of PAtJAG::gGmjag1. A valve was removed from each dehiscent silique to display the seeding pattern (n-q). Scale bar: 1 mm (b-e, j-q); 10 mm (f-i).

Figure 5 shows functional analysis of 35S::GmJAG1, 35S::Gmjag1, PGmJAG1::gGmJAG1, PGmjag1::gGmjag1, PAtJAG::gGmJAG1ΔEAR, and PAtJAG::gGmJAG2 gene constructs in the Arabidopsis jag-3 mutant. (a) Confirmation of expression of the introduced gene constructs in the transgenic Arabidopsis jag-3 lines by RT-PCR analysis. RNA was extracted from inflorescence tissues of 35S::GmJAG1, 35S::Gmjag1, PGmJAG1::gGmJAG1, PGmjag1::gGmjag1, and PAtJAG::gGmJAG1ΔEAR transgenic plants and wild-type Landsberg erecta (Ler) and jag-3 mutant. (b) Confirmation of expression of the introduced gene constructs in the transgenic Arabidopsis jag-3 lines by RT-PCR analysis. RNA was extracted from inflorescence tissues of PAtJAG::gGmJAG2 transgenic plants, wild-type Landsberg erecta (Ler), and jag-3 mutant. (c) 35S::GmJAG1 flower. (d) 35S::Gmjag1 flower. (e) PGmJAG1::gGmJAG1 flower. (f) PGmjag1::gGmjag1 flower. (g) Ler flower. (h) jag-3 flower. (i) PAtJAG::gGmJAG1ΔEAR flower. (j) PAtJAG::gGmJAG2 flower. Scale bar: 1 mm (c-j).

Figure 6 shows RT-PCR analysis of GmJAG2 expression in different tissues of ln and Ln allele plants. Numbers refer to PCR cycles. RNA was extracted from tissues of field-grown plants. Amplification of actin was used as a control.

In order to achieve the purpose described above, the present invention provides the ln gene produced by single nucleotide substitution in EAR motif of Ln (broad leaflet) gene from soybean (Glycine max).

According to the present invention, the single nucleotide substitution in EAR motif of the Ln gene may be preferably nonsynonymous nucleotide substitution. More preferably, according to nonsynonymous nucleotide substitution by which the nucleotide GAT corresponding to the amino acid D in the EAR motif (LDLNNLP) is substituted with CAT, the aspartic acid (Asp, D) may be substituted with histidine (His, H).

The ln gene may consist of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. Further, homologues of the nucleotide sequence are also included within the scope of the present invention. The homologues mean a nucleotide sequence which may have a different nucleotide sequence but have a similar functional characteristic compared to the nucleotide sequence of SEQ ID NO: 1. Specifically, the gene may comprise a nucleotide sequence which has sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% compared to the nucleotide sequence of SEQ ID NO: 1. Said "sequence homology %" for a certain polynucleotide is identified by comparing a comparative region with two sequences that are optimally aligned. In this regard, a part of the polynucleotide in comparative region may comprise an addition or a deletion (i.e., a gap) compared to a reference sequence (without any addition or deletion) relative to the optimized alignment of the two sequences.

Also provided by the present invention is a recombinant vector comprising the aforementioned ln gene.

The term "recombinant" indicates a cell which replicates a heterogeneous nucleotide or expresses said nucleotide, a peptide, a heterogeneous peptide, or a protein encoded by a heterogeneous nucleotide. Recombinant cell can express a gene or a gene fragment in a form of a sense or anti-sense, that are not found in natural state of cell. In addition, a recombinant cell can express a gene that is found in natural state, provided that said gene is modified and re-introduced into the cell by an artificial means.

The term "vector" is used herein to refer DNA fragment (s) and nucleotide molecules that are delivered to a cell. Vector can replicate DNA and be independently reproduced in a host cell.

According to the present invention, the aforementioned ln gene sequence may be incorporated into the recombinant expression vector. The term "recombinant vector" means bacteria plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vectors. In general, any plasmid and vector can be used if it can be replicated and stabilized in a host. Important characteristic of the recombinant vector is that it include a replication origin, a promoter, a marker gene, and a translation control element.

The expression vector comprising the ln gene sequence and appropriate signals for controlling transcription and translation can be constructed by a method that is well known to a person skilled in the art. Examples of the method include in vitro DNA recombination, DNA synthetic techniques, and in vivo recombination, or the like. To achieve mRNA synthesis, the aforementioned DNA sequence can be effectively linked to an appropriate promoter in an expression vector. The expression vector may also comprise a ribosome binding site as a translation initiation site and a transcription terminator.

The preferred example of recombinant vector according to the present invention is Ti-plasmid vector which can transfer a part of itself, i.e., so-called T-region, to a plant cell when the vector is present in an appropriate host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vector (see, EP 0 116 718 B1) are currently used for transferring a hybrid gene to protoplasts that can produce a new plant by appropriately inserting a plant cell or hybrid DNA to a plant genome. Especially preferred form of Ti-plasmid vector is a so-called binary vector which has been disclosed in EP 0 120 516 B1 and USP No. 4,940,838. The binary vector which may be used for the present invention may be any kind of a binary vector comprising RB (right border) and LB (left border) of T-DNA which can transform a plant when it is present with the Ti plasmid of Agrobacterium tumefaciens. Preferably, pBI101 (Cat#: 6018-1, Clontech, USA), pBIN19 (Genbank Deposit number: U09365), pBI121, pCAMBIA vector or the like that are commonly used in the field can be used. Other appropriate vectors that can be used for introducing the DNA of the present invention to a host plant can be selected from a double-stranded plant virus (e.g., CaMV), a single-stranded plant virus, and a viral vector which can be originated from Gemini virus, etc., for example a non-complete plant viral vector. Use of said vector can be especially advantageous when a plant host cannot be appropriately transformed.

The recombinant vector preferably comprises at least one selection marker. Said selection marker is a nucleotide sequence having a property which allows a selection based on a common chemical method. Any kind of gene that can be used for the differentiation of transformed cells from non-transformed cells can be a selection marker. Example includes, a gene resistant to herbicide such as glyphosate and phosphinotricin, and a gene resistant to antibiotics such as kanamycin, hygromycin, chloramphenicol, G418, or bleomycin, and aadA gene, but not limited thereto.

According to the recombinant vector of the present invention, the promoter can be CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter, or histone promoter, but not limited thereto. The term "promoter" indicates a region of DNA located upstream of a structure gene, and it corresponds to a DNA molecule to which an RNA polymerase binds to initiate transcription. The term "plant promoter" indicates the promoter that can initiate transcription in a plant cell. The term "constitutive promoter" indicates the promoter that is active under most environmental conditions and cell growth or differentiation state. Since selection of a transformant can be made for various tissues at various stages, the constitutive promoter may be preferred for the present invention. Thus, selection property is not limited by a constitutive promoter.

In the above-described recombinant vector of the present invention, any kind of a typical terminator can be used. Examples thereof include nopalin synthase (NOS), rice -amylase RAmy1 A terminator, and a terminator for Octopine gene of Agrobacterium tumefaciens, phaseoline terminator, rrnB1/B2 terminator of E. coli, etc., but are not limited thereto. Regarding the necessity of terminator, it is generally known that such region can increase a reliability and an efficiency of transcription in plant cells. Therefore, the use of terminator is highly preferable in view of the context of the present invention.

Also provided by the present invention is a host cell transformed with the aforementioned recombinant vector.

Any kind of a host cell known in the pertinent art can be used if stable and continuous cloning and expression of the vector of the present invention can be achieved within prokaryotic cells. Examples include strains belonging to the genus Bascillus such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bascillus subtilus, Bascillus thuringiensis, and the like, Salmonella typhimurium, intestinal flora and strains such as Serratia marcescens and various Pseudomonas Spp. and the like.

In addition, when the vector of the present invention is transformed in an eukaryotic cell, a host cell such as Saccharomyce cerevisiae, an insect cell, a human cell (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell line), a plant cell line and the like can be used. Preferably, the host cell is a plant cell.

When the host cell is a prokaryotic cell, transfer of the vector of the present invention into a host cell can be carried out according to CaCl₂ method, Hanahans method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)), and an electroporation method, etc. In addition, when the host cell is an eukaryotic cell, the vector of the present invention can be transferred into a host cell according to a gene gun-mediated transformation (bombardment), Agrobacterium-mediated transformation, a microscopic injection method, calcium phosphate precipitation method, an electroporation method, a liposome-mediated transformation, and DEAE-dextran treatment method, a gene bombardment method, etc.

Also provided by the present invention is a method of controlling the number of seeds per pod comprising a step of expressing the ln gene by transforming a plant cell with the recombinant vector comprising the ln gene, which is produced by single nucleotide substitution in EAR motif of the Ln gene from soybean.

The method of the present invention comprises a step of transforming a plant cell with the recombinant vector of the present invention and the transformation of a plant means any method which can transfer DNA to a plant. Such transformation is not necessarily required to have a period for regeneration and/or tissue culture. Transformation of a plant is now generally carried out not only for a dicot plant but also for a monocot plant. In principle, any method for transformation can be used for introducing a heterologous DNA of the present invention to a progenitor cell. Transformation can be carried out according to any method selected from a calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), an electroporation method for protoplasts (Shillito R. D. et al., 1985 Bio/Technol. 3, 1099-1102), a microscopic injection method for plant components (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), a particle bombardment method for various plant components (DNA or RNA-coated) (Klein T.M. et al., 1987, Nature 327, 70), or a (non-complete) viral infection method in Agrobacterium tumefaciens mediated gene transfer by plant invasion or transformation of fully ripened pollen or microspore (EP 0 301 316), etc. According to the present method, Agrobacterium mediated DNA transfer is preferred. In particular, so-called binary vector technique as disclosed in EP A 120 516 and USP No. 4,940,838 can be preferably adopted for the present invention.

According to the method of one embodiment of the present invention, the ln gene may consist of the nucleotide sequence of SEQ ID NO: 1, but not limited thereto.

Also provided by the present invention is a method of producing a plant with controlled number of seeds per pod, comprising steps of: transforming a plant cell with the recombinant vector comprising the ln gene which is produced by single nucleotide substitution in EAR motif of the Ln gene from soybean (Glycine max); and regenerating a plant from the transformed plant cell.

The method for transforming a plant cell is as described above.

The aforementioned method of the present invention also comprises a step of regenerating the transformed plant from the transformed plant cell. As for the method for regenerating a transformed plant from a transformed plant cell, any method well known in the field may be used.

Also provided by the present invention is a transformed plant with controlled number of seeds per pod that is produced by the aforementioned method, and seeds thereof. Preferably, the plant may have a narrow leaflet shape other than a leaflet shape of a wild type, i.e., a broad leaflet, and it may also be a plant with increased number of seeds per pod.

The plant according to one embodiment of the present invention can be a dicot plant. Preferably, it may be a plant of Solanaceae, Cruciferae, Rosaceae, Leguminosae, or Cucurbitaceae. More preferably, it may be soybean (Glycine max), but not limited thereto.

Also provided by the present invention is a composition for controlling the number of seeds per pod in a plant comprising the recombinant vector comprising the ln gene produced by single nucleotide substitution in EAR motif of Ln gene from soybean (Glycine max).

According to one embodiment of the present invention, the ln gene may consist of the nucleotide sequence of SEQ ID NO: 1. The composition of the present invention comprises, as an effective component, the ln gene from soybean (Glycine max) and the plant transformed with this gene may exhibit leaves with a narrow leaflet shape and increased number of seeds per pod.

The present invention will now be described in greater detail with reference to the following examples. However, it is only to specifically exemplify the present invention and in no case the scope of the present invention is limited to the examples.

Materials and Methods

1. Plant Materials

A BC₃F₂ population was developed from four self-fertilized BC₃F₁ hybrids made between an elite narrow leaflet soybean cultivar for sprouts (the female parent, Sowon) and a broad leaflet soybean cultivar (the male parent, V94-5152) (hereafter, referred to as the SV population). A total of 309 F₂ individuals were previously used to establish linkage relationships among molecular markers, the number of seed per pod and the narrow leaflet-shape trait presumed to be determined by the ln gene (Jeong et al., 2011, Theor. Appl. Genet. 122, 865-874).

To further dissect the 66 kb genomic region detected in the previous study (Jeong et al., 2011, Theor. Appl. Genet. 122, 865-874), 4219 F₃ seedlings derived from 162 F₂ plants heterozygous for both Ln_at004 and Ln_atre04 were grown and screened for recombinants between Ln_at004 and Ln_atre04. Plants with the homozygous genotype at one marker and heterozygous genotype at the other locus were selected, and a total of 17 individuals were grown for phenotypic evaluation.

The genetic correlation between markers and leaf shape was assessed in a collection of 71 presumably diverse soybean cultivars or wild accessions listed in Table 1. Among them, 14 variants were further analyzed by sequencing; Sowon, Pungsannamul, Myeongjunamul, IT182932, IT178535, IT191201, IT184014, and PI549049 collected or developed in Korea, V94-5152 and Williams 82 developed in the United States, and four wild soybean accessions PI378691 (Japan), PI407290 (China), PI423991 (Russia), and PI518282 (Taiwan) collected in the Japan, China, Russia, and Taiwan.

Table 1 Leaflet shapes of cultivated and wild soybean accessions and their genotype at marker Ln-AH locus

Accession or cultivar	Leaflet shape	Nucleotide base at Ln-AH locus	Species	Collection site of Glycine soja and G. max x G. soja
Pungsannamul	Narrow	C	Glycine max
Sowon	Narrow	C	G. max
Myeongjunamul	Narrow	C	G. max
Bokwang	Narrow	C	G. max
Saeal	Narrow	C	G. max
Jangyeob	Narrow	C	G. max
Eunha	Narrow	C	G. max
Someyng	Narrow	C	G. max
T136	Narrow	C	G. max
IT183014	Narrow	C	G. max x G. soja	Kyunggi, Korea
IT178535	Narrow	C	G. max x G. soja	Chungnam, Korea
IT191201	Narrow	C	G. max x G. soja	Kyunggi, Korea
V94-5152	Broad	G	G. max
Williams 82	Broad	G	G. max
Daewon	Broad	G	G. max
Jangmi	Broad	G	G. max
Jinpum2	Broad	G	G. max
Ilpumgeomjeong	Broad	G	G. max
Nokchae	Broad	G	G. max
Sodam	Broad	G	G. max
Jinpum	Broad	G	G. max
Daewon	Broad	G	G. max
Taekwang	Broad	G	G. max
Saeol	Broad	G	G. max
Dangkyung	Broad	G	G. max
Cheongja	Broad	G	G. max
T243	Broad	G	G. max
T210	Broad	G	G. max
Essex	Broad	G	G. max
Hwangkeum	Broad	G	G. max
SS2-2	Broad	G	G. max
T202	Broad	G	G. max
L29	Broad	G	G. max
Jack	Broad	G	G. max
Pureun	Broad	G	G. max
Wye	Broad	G	G. max
Namhae	Broad	G	G. max
Hutcheson	Broad	G	G. max
PI96983	Broad	G	G. max
Williams	Broad	G	G. max
Muhan	Broad	G	G. max
T181	Broad	G	G. max
T117	Broad	G	G. max
Ogden	Broad	G	G. max
T201	Broad	G	G. max
Kwangan	Broad	G	G. max
York	Broad	G	G. max
Evans	Broad	G	G. max
Geomjeong2	Broad	G	G. max
Peking	Broad	G	G. max
Marshall	Broad	G	G. max
Samnam	Broad	G	G. max
Lee68	Broad	G	G. max
T244	Broad	G	G. max
T176	Broad	G	G. max
T54	Broad	G	G. max
Jinyul	Broad	G	G. max
T245	Broad	G	G. max
T245	Broad	G	G. max
IT182932	Broad	G	G. soja	Kyunggi, Korea
PI378691	Broad	G	G. soja	Miyazaki, Japan
PI407290	Broad	G	G. soja	Jilin, China
PI423991	Broad	G	G. soja	Amur, Russia
PI518282	Broad	G	G. soja	Taiwan
PI 549046	Broad	G	G. soja	Shaanxi, China
IT183035	Broad	G	G. soja
IT184246	Broad	G	G. soja
IT184237	Broad	G	G. soja
IT182987	Broad	G	G. soja
IT184175	Broad	G	G. soja
IT138072	Broad	G	G. soja

2. DNA Isolation and Marker Analysis

Young trifoliolate leaf tissues from soybean accessions were collected. Soybean genomic DNA was isolated as described previously by Saghai Maroof et al. (1984, Proc. Natl. Acad. Sci. USA, 81, 8014-8081). For quick preparation from the F_2:3 line plants for the SV population, soybean genomic DNA was isolated using a FastDNA^® Kit in accordance with the manufacturer's protocols (MP Biomedicals, Solon, OH, USA) from a single young leaflet. The extracted DNA was dissolved in 200 ㎕ water and then 2 ㎕ of the solution was used in a 20 ㎕ PCR to amplify DNA fragments for marker genotyping. Microsatellite and single nucleotide polymorphism (SNP) markers were carried out as previously described (Jeong and Saghai Maroof, 2004, Plant Breed. 123, 305-310).

3. PCR of Genomic DNA and Sequencing

Genomic DNA isolation, PCR primer design, PCR amplification, PCR fragment purification, and sequencing of PCR fragments were conducted as described previously (Jeong and Saghai-Maroof, 2004, Plant Breed. 123, 305-310). In brief, the polymerase chain reaction (PCR) mixture contained 20 ng of total genomic DNA, 1x PCR buffer (10 mM Tris-HCl, 50 mM KCl, pH 8.3), 2.5 mM MgCl₂, 100 nM of each forward/reverse primer, 0.16 mM of each dNTP and 0.25 unit of Taq polymerase in a total volume of 20 ㎕. Standard PCR was conducted as follows: a denaturation step at 94℃ for 5 min, 34 cycles at 94℃ for 30 sec, 43-58℃ for 30 sec and 72℃ for 30 sec and an extension step at 72℃ for 5 min and followed by a 4℃ soak. Alignment of the nucleotide and amino acid sequences was performed using ClustalW implemented in BioEdit.

4. Vector Construct for Transformation

For the PAtJAG::gGmJAG1 vector, the JAG promoter was amplified first from Arabidopsis Ler ecotype using primers pAtJAG (SEQ ID NO: 2) and XhoI-pAtJAG (SEQ ID NO: 3). The promoter was then cloned into the pENTR/D-TOPO entry vector (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. The genomic fragments of GmJAG1 (Ln) were amplified from soybean cultivar V94-5152 using primers XhoI-soyJAG-1 (SEQ ID NO: 4) and SoyJAG (SEQ ID NO: 5). The DNA fragment was then cloned into the pENTR/D-TOPO entry vector. The genomic GmJAG1 in V94-5152 was subcloned into the PAtJAG entry vector using AscI and XhoI sites. The PAtJAG::gGmJAG1 cassette was transferred to the destination vector pHGWL7 using the attL ｘ attR reaction as described in the Gateway cloning technology instruction manual (Invitrogen). For the PAtJAG::gGmjag1 vector, the same procedure was used using genomic DNA from Sowon (ln) for the genomic fragment of ln.

For the 35S::gGmJAG1 vector, the genomic DNA fragment of Ln was amplified from V94-5152 (Ln) by PCR using primers p35S-So (SEQ ID NO: 6) and SoyJAG (SEQ ID NO: 5). The resulting genomic DNA was cloned into the pENTR/D-TOPO entry vector following the manufacturer's protocol. The attL ｘ attR reaction between the entry vector and the destination vector pH2GW7 was performed to place the gGmJAG1 downstream of a Cauliflower Mosaic Virus (CaMV) 35S promoter as described in the Gateway cloning technology instruction manual. For the 35S::gGmjag1 vector, the same procedure was used using genomic DNA from Sowon (ln) for the genomic fragment of ln.

For the PGmJAG1::gGmJAG1 vector, the JAG promoter region was amplified first from V94-5152 by PCR using primers SoyJAG-p (SEQ ID NO: 7)and SoyJAG-B (SEQ ID NO: 8). The promoter region was then cloned into the pENTR/D-TOPO entry vector. The coding region of GmJAG1 was amplified from V94-5152 using a SoyJAG forward primer (SEQ ID NO: 9) and SoyJAG reverse primer (SEQ ID NO: 5). The resulting genomic DNA was cloned into the pENTR/D-TOPO entry vector following the manufacturer's protocol. The gGmJAG1 was then subcloned into the PGmJAG1 entry vector using NotI and BglⅡ sites. The PGmJAG1::gGmJAG1 was transferred to the the destination vector pHGWL7 using the attL ｘ attR reaction. For the PGmjag1::gGmjag1 vector, the same procedure was used using genomic DNA from Sowon (ln) for the genomic fragment of ln except subcloning of the gGmjag1 into the PGmjag1 entry vector using NotI and XbaI sites.

For the PAtJAG::gGmJAG1ΔEAR vector, the genomic fragment of the EAR-motif-deleted GmJAG1 was amplified from the V94-5152 using primers XhoI-deltaEAR (SEQ ID NO: 10) and SoyJAG (SEQ ID NO: 5). This DNA fragment was then cloned into the pENTR/D-TOPO entry vector. The deletion EAR modified JAG gene was subcloned into the pAtJAG entry vector using AscI and XhoI sites. The PAtJAG::gGmJAG1ΔEAR cassette was transferred to the destination vector pHGWL7 using the attL ｘ attR reaction.

For the PAtJAG::gGmJAG2 vector, the genomic fragment of GmJAG2, the GmJAG1 homeolog, was amplified from V94-5152 using primers Xho1-hm-soyJAG (SEQ ID NO: 11) and hm-soyJAG (SEQ ID NO: 12). The genomic fragment was then cloned into the pENTR/D-TOPO entry vector. Then gGmJAG2 was subcloned into pAtJAG entry vector using AscI and XhoI sites. The gateway cassette of PAtJAG::gGmJAG2 was transferred into pHGWL7 by attL ｘ attR reaction.

5. Arabidopsis Transformation

The gene constructs were introduced into Agrobacterium tumefaciens strain GV3101. Transgenic plants were generated by vacuum infiltration of Arabidopsis Ler mutant jag-3 recipient plants using the A. tumefaciens strain GV3101, and plants were selected using resistance against the antibiotic hygromycin.

6. Southern Blot Analysis

Eight micrograms of the genomic DNA from both V94-5152 and Sowon were digested overnight with restriction enzymes, fractionated on 1% agarose gel, alkaline-transferred onto Hybond N+ nylon membranes (Amersham Pharmacia, Piscataway, USA), and probed using biotin-labeled GmJAG1 gene probes amplified using SoyJAG-2and SoyJAG-2 listed in Table 1. DNA probe preparation was conducted using the Nick Translation System (Invitrogen, Carlsbad, CA), in accordance with the manufacturer's instructions. The biotin-labelled DNA was then detected via chemiluminescence using streptavidin alkaline phosphatase and CDP-Star ^® (Applied Biosystems, Bedford, MA). Hybridization, membrane washing, and detection were all conducted in accordance with the manufacturer's instructions. The size of the DNA band was compared with a 1 Kb DNA Ladder (Bioneer, Daejeon, Korea).

7. RNA Extraction and One Step RT-PCR

Expanded leaf, young leaf, meristem, flower and young pod were frozen in liquid nitrogen and stored in the freezer until used. Tissues were ground using a mortar and pestle and total RNA was extracted using the RNeasy Plant Mini kit (Qiagen GmbH, Germany). Before cDNA synthesis, all RNA samples were treated using RNase free RQ DNase (Promega). cDNA was synthesized by one step RT-PCR (Qiagen GmbH, Germany) using gene specific primers as described by the manufacturer. Level of cDNA was adjusted by RT-PCR product of the soybean actin11. To verify GmJAG expression, the PCR product was generated using the following primer sets; cJAG-sp forward primer (SEQ ID NO: 13), cJAG-sp reverse primer (SEQ ID NO: 14) for GmJAG1 and cJAG-hsp forward primer (SEQ ID NO: 15), cJAG-hsp reverse primer (SEQ ID NO: 16) for GmJAG2. The RT-PCR products were separated on 1.5% agarose gel electrophoresis with ethidium bromide staining.

Example 1. Physical Map

Our fine genetic map was used to delimit the genomic region controlling both the leaflet shape and NSPP traits between microsatellite markers Ln_at004 and Ln_atre04 on soybean chromosome 20, which has a sequence length of 66 kb in the soybean genome, corresponding to 0.7 cM (Jeong et al., 2011, Theor. Appl. Genet. 122, 865-874) (Figure 1a). To further dissect the 66 kb genomic region, 4219 F₃ seedlings derived from 162 F₂ plants heterozygous for both Ln_at004 and Ln_atre04 were grown and screened for recombination between Ln_at004 and Ln_atre04. As a result, 17 recombinants with the genotype homozygous at one marker and heterozygous at the other were selected. The recombinants and their progeny were grown for a phenotypic evaluation of leaflet shape and NSPP traits. By further genotyping the recombinant plants using markers Ln_43k, Ln_44k, Ln_attre, Ln_AH, Ln_54k and Ln_57k, the ln locus was localized to the interval between Ln_44k and Ln_57k (Figure 1b). Three markers, Ln_attre, Ln_AH, and Ln_54k, cosegregated with ln. One recombination event between Ln_44k and ln and one recombination event between Ln_57k and ln were detected. Thus, the mutation responsible for the derivation of both narrow leaflet shape and high NSPP value in soybean was delimited to a 12.6-kb region between Ln_44k and Ln_57k (Figure 1c).

Only one gene (Glyma20g25000.1) has been annotated in this 12.6 kb region of the Glyma1.0 soybean gene annotation database (accessible at Phytozome v5.0, http://www.phytozome.net, accessed August 2011) (Figure 1c,d). The gene was predicted to be homologous to Arabidopsis thaliana JAGGED (JAG) belonging to the zinc finger (C2H2 type) family (Ohno et al., 2004, Development, 131, 1111-1122), and its coding region was physically located between 34,688,514 and 34,690,379 base pairs (bp) on the Gm20 pseudomolecule of soybean chromosome 20 (http://www.photozome.net). A BLAST search against the soybean genome sequence revealed that Glyma20g25000.1 has a close paralog gene, Glyma10g42020.1, on the Gm10 of soybean chromosome 10. Glyma20g25000.1 and Glyma10g42020.1 were named GmJAG1 and GmJAG2, respectively.

BLAST searches of the 12.6 kb against GenBank est database hit more than 10 ESTs, including GD767462 and GD866768, corresponding to the 5' untranslated and 3' untranslated regions of the annotated gene, respectively. However, alignment of those ESTs did not reveal a full-length open reading frame (ORF) and suggested different coding sequences from the annotated Glyma20g25000.1 and Glyma10g42020.1. Thus, the ORF of GmJAG1 was determined by cloning and then by sequencing reverse-transcription polymerase chain reaction (RT-PCR) products from RNA isolated from the shoot tips of 'sowon' and 'V94-5152'. A comparison of the RT-PCR product sequences revealed that GmJAG1 contains an alternative splicing site of 6-bp (two amino acids) on the 5' side of the third exon (Figure 1d). Thus, the GmJAG1 gene encodes 256 or 258 amino acid proteins. GmJAG1 and GmJAG2 showed 94% identity at the nucleotide sequence level of their predicted coding regions (Figure 1e).

Four conserved domains were readily identified in the predicted GmJAG1 and GmJAG2 sequences when compared with AtJAG and its Arabidopsis paralog NUBBIN, Solanum lycopersicum JAG, and Zea mays JAG: an EAR motif, a putative nuclear localization signal sequence, a single C2H2-type zinc finger motif, and a proline-rich motif (Figure 1e and Figure 2). The putative nuclear localization signal sequence and proline-rich motif are poorly conserved in NUBBIN and ZmJAG. A 1-bp substitution or change from aspartic acid (Ln) to histidine (ln), which is the only difference between the ORF sequences of the GmJAG1 genes in V94-5152 (Ln) and Sowon (ln) plants, occurred at the EAR motif. As the conserved EAR motif contained a nonsynonymous mutation, it could be hypothesized that the mutation might be responsible for the phenotypic differences between the Ln and ln plants. Outside of these described domains, amino acid sequence conservation between GmJAG1 and AtJAG/SlJAG was low, indicating possible functional dissimilarities (Figure 2). Nevertheless, the intron-exon structure of GmJAG1 (3134 bp) was completely conserved relative to AtJAG and SlJAG (Figure 1d and Figure 2).

A comparison of the 12.6-kb sequences between the mapping parents revealed eight mutations (Figure 1c,d). These included five mutations in the putative promoter region within 1.5 kb from the 5' untranslated exon, one mutation in the 5' untranslated exon, one mutation in the second exon, and one mutation in the 3' downstream of the stop codon.

Example 2. The ln Phenotype and Its Expression

Differences in leaflet shapes and pod types between Ln and ln soybean plants have been repeatedly observed in different soybean genetic backgrounds (Jeong et al., 2011, Theor. Appl. Genet. 122, 865-874); Ln plants tends to have broader leaflet shapes (Figure 3a,b) and higher NSPP than ln plants (Figure 3c,d). As the mutations of the Arabidopsis JAG gene, soybean homolog of which is presumed to encode Ln by physical mapping described above, were reported to display defects in floral morphology and cell number and shape (Ohno et al., 2004, Development, 131, 1111-1122), we examined morphology of flowers and number and shape of leaflet epidermal cells. However, we did not observe any differences in flowers (Figure 3e,f) or epidermal cells (Figure 3g,h) between Ln and ln plants.

To investigate whether promoter region mutations are responsible for the soybean leaflet shape and NSPP, JAG transcript levels in different soybean tissues of V94-5152 (Ln) and Sowon (ln) plants were examined using semi-quantitative RT-PCR. The patterns of RNA accumulation in both Ln and ln plants were quite similar to each other. The JAG1 transcript was detected during vegetative and reproductive development in the shoot apex and in open flowers, as observed for AtJAG in different tissues of Arabidopsis (Ohno et al., 2004, Development, 131, 1111-1122). In soybean, a low level of RNA transcript was detected in young ln pod tissues, but not in those of Ln (Figure 3i). These results indicate that the mutations in the promoter region of GmJAG1 did not affect mRNA expression of the gene. Thus, the one single nucleotide substitution mutation in the genic region of the GmJAG1 gene, which led to a single amino acid change, likely determined the phenotypic difference between the Ln ('V94-5152' cultivar) and ln ('sowon' cultivar) genotypes.

Example 3. Complementation of the Arabidopsis jag-3 Mutant with GmJAG1

To validate the function of GmJAG1 for broad leaflet and low NSPP (vs. Gmjag1 for narrow leaflet and high NSPP), we introduced the V94-5152 GmJAG1 allele driven by the Arabidopsis JAG promoter (PAtJAG::gGmJAG1) into an Arabidopsis jag loss-of-function mutant (jag-3). The jag-3 mutant expresses a defective truncated JAG protein by disruption of the 3'-splice acceptor sequence (Ohno et al., 2004, Development, 131, 1111-1122). The presence of the soybean GmJAG1 allele in the transgenic lines was detected by PCR analysis and sequencing of PCR fragments and then further verified by RT-PCR (Figure 4a). The transgene showed substantial rescue of the jag-3 phenotypes in 34 of 107 primary transformants (Figure 4d,h). Notably, the transformation of GmJAG1 converted narrow floral organs of the Arabidopsis jag-3 mutant into relatively broad floral organs, nearly the same characteristics as wild-type Arabidopsis Landsberg erecta (Ler) (Figure 4b,d). Serrated leaves (fifth leaf) in the PAtJAG::gGmJAG1 transgenics appeared slightly later than those in jag-3 (fourth leaf) but appeared slightly earlier than in the wild-type (seventh leaf) (Figure 4f,h). These results suggest that, despite substantial divergence in amino acid sequence, GmJAG and Arabidopsis JAG show a high degree of functional homology.

The question remained whether the one nonsynonymous nucleotide substitution (Gmjag1 allele) detected at the EAR motif region of GmJAG1 locus in the cultivated soybean had no or diminished functions relative to the GmJAG1 allele for leaflet shape and NSPP. To address this question, we introduced the Sowon Gmjag1 allele driven by the Arabidopsis JAG promoter (PAtJAG::gGmjag1) into the jag-3 mutants and obtained 63 transgenic (Gmjag1) lines. Expression of the Gmjag1 allele was confirmed by RT-PCR (Figure 4a). All PAtJAG::gGmjag1 transgenics showed phenotypes nearly identical to that of the Arabidopsis jag-3 mutant (Figure 4e,i). These results substantiated that the mutation at the EAR motif is sufficient for the conversion of GmJAG1 to Gmjag1.

Although JAG promotes the formation of the valve margin that facilitates the detachment of the valves from the replum during development of Arabidopsis fruit, a mature form of the flower, which is also referred to as silique, overall morphological differences between siliques of Arabidopsis JAG and jag plants has been poorly examined. As the NSPP trait is one of the agronomically important traits in the soybean ln mutant, it was necessary to examine changes in silique morphology, seeding pattern in the siliques, length of siliques, and the number of seeds per silique in the Arabidopsis wild type, jag-3, GmJAG transgenics, and Gmjag transgenics. The siliques in wild-type plants were longer and thicker (Figure 4j) than those in the jag-3 plant (Figure 4k). However, seeds were more densely packed in the siliques in jag-3 (Figure 4o) than those in the wild type (Figure 4n). The GmJAG1 transgenic lines showed substantial rescue of the jag-3 silique phenotypes (Figure 4l,p). However, the siliques in the Gmjag1 transgenic lines were similar in morphology to those of jag-3 (Figure 4m,q). Silique length was measured for each genotype and seeds from one side of the septum after removing a valve in each silique were counted for each genotype. Silique length and number of seeds in siliques were significantly longer and higher in the wild-type than those in jag-3. The number of seeds per unit silique length was significantly lower in the wild type than that in jag-3. Silique length and number of seeds in the GmJAG1 transgenic lines were significantly shorter and lower than in the wild type but significantly longer and higher than those in jag-3. Collectively, our results suggest that introducing GmJAG1 into the jag-3 mutant partially rescued the wild-type silique phenotypes by restoring silique length and seed distribution.

Example 4. Functional Divergence between Soybean JAG1 and Arabidopsis JAG

Our genetic and physical maps and complementation tests indicated that the single nucleotide substitution at the EAR motif of GmJAG1 was likely the causal mutation responsible for the phenotypic difference between Ln and ln soybean plants. However, more mutations were observed at the GmJAG1 promoter region and the sequence conservation between the Arabidopsis JAG and G. max JAG1 proteins was low outside of the conserved motifs, making it crucial to determine the extent to which the two proteins and their promoters are functionally similar. To address this question, we introduced several different gene constructs into the Arabidopsis jag-3 mutant. First, we compared the effect of GmJAG1 and Gmjag1 misexpression in the jag-3 genetic background with the published effects of Arabidopsis JAG overexpression (Ohno et al., 2004, Development, 131, 1111-1122) (Figure 5). The 35S::GmJAG1 transgenics displayed phenotypes highly reminiscent of PAtJAG::gGmJAG1 transgenic plants but did not display aberrant phenotypes, including fusion of rosette leaves and stipule outgrowth from the lateral margins at the base of the leaf observed in JAG overexpressing Arabidopsis. The 35S::Gmjag1 transgenics showed phenotypes nearly identical to that of the Arabidopsis jag-3 mutant. These results demonstrate that although Arabidopsis JAG and G. max JAG1 maintain common essential functions observed in the initial lateral organ development, the substantial divergence in amino acid sequence might have led to diminished function of G. max JAG1 in ectopic tissues. Second, to investigate whether the promoter region that had more mutations than coding regions between GmJAG1 and Gmjag1 maintained any functional conservation between soybean and Arabidopsis, V94-5152 GmJAG1 (PGmJAG1::gGmJAG1) and Sowon Gmjag1 (PGmjag1::gGmjag1) driven by their own promoter, respectively, were introduced into the Arabidopsis jag-3 mutant. We obtained 20 PGmJAG1::gGmJAG1 and 57 PGmjag1::gGmjag1 transgenics. RT-PCR did not detect expressed GmJAG1 mRNA (Figure 5a), and all transgenics showed phenotypes nearly identical to that of the Arabidopsis jag-3 mutant (Figure 5e,f). The results indicate that the promoter region of GmJAG1 was no longer functional in Arabidopsis due to substantial divergence. Third, an EAR-deleted version of GmJAG1 (PAtJAG::gGmJAG1ΔEAR) was transformed into the Arabidopsis jag-3 mutant. We obtained 49 PAtJAG::gGmJAG1ΔEAR transgenic lines. All PAtJAG::gGmJAG1ΔEAR transgenics showed phenotypes nearly identical to those of the Arabidopsis jag-3 mutant and PAtJAG::gGmjag1 transgenic lines (Figure 5). The results substantiated that the mutation at the EAR motif is sufficient for the conversion of GmJAG1 to Gmjag1 and also indicated that GmJAG1 without the EAR motif is nonfunctional.

Example 5. Evolutionary Diversification of JAG in Soybean

The BLAST search indicated that GmJAG1 and its homeolog GmJAG2 are located on large duplicated blocks between soybean chromosome 20 and 10, respectively, in the soybean genome, which were presumably generated by a soybean-lineage-specific paleoallotetraploidy event dated to approximately 13 million years ago. Therefore, these two genes are a homeologous pair. The presence of only two copies of the GmJAG homolog was substantiated by Southern blot analysis. The sequences of the GmJAG2 promoter and coding region, determined from Sowon and V94-5152, were identical, indicating that GmJAG2 is not responsible for the phenotypic difference between Sowon and V94-5152. These results indicate that GmJAG2 may be nonfunctional. To address this question, we introduced the GmJAG2 gene driven by the Arabidopsis JAG promoter (PAtJAG::gGmJAG2) into the Arabidopsis jag-3 mutant. Surprisingly, 13 of 35 PAtJAG::gGmJAG2 transgenics showed phenotypes identical to PAtJAG::gGmJAG1 transgenic plants relative to the wild-type Arabidopsis (Figure 5j).

The expression patterns of GmJAG1 and GmJAG2 were compared to better understand their functional diversity. Semi-quantitative RT-PCR was used to profile GmJAG2 expression in different tissues and at different developmental stages. Consistent with expression patterns of Arabidopsis JAG, GmJAG1 was mainly expressed in the meristem and flowers (Figure 3i). By way of contrast, the GmJAG2 transcript was found in broader types of tissues than that of GmJAG1 (Figure 6). In addition to the shoot apex and open flowers, the GmJAG2 transcript was detected in young pod tissues of both ln and Ln plants, and it was detected in young leaf tissues of Ln plants but not in those of ln plants (Figure 6). These results indicate that the GmJAG2 gene was sub- or neo-functionalized.

Allelic variation of the GmJAG1 gene in the wild and cultivated soybean populations was evaluated in the 59 accessions containing a broad leaflet type and in 12 accessions containing a narrow leaflet type. The genotypes scored by a marker Ln_AH generated from the 1-bp substitution mutation at the EAR correlated perfectly with the genotypes scored by leaflet types (Table 1). We observed no additional allelic variation presumed to affect the amino acid sequence encoded by the GmJAG1 gene from the comparison of sequences of the GmJAG1 gene determined from 14 accessions selected from the above 71 accessions.

Claims (13)

1. ln gene consisting of a nucleotide sequence of SEQ ID NO: 1, which is produced by single nucleotide substitution in EAR motif of Ln (broad leaflet) gene from soybean (Glycine max).
2. A recombinant vector comprising the gene of Claim 1.
3. A host cell transformed with the recombinant vector of Claim 2.
4. A method of controlling the number of seeds per pod comprising a step of expressing ln gene by transforming a plant cell with a recombinant vector comprising the ln gene produced by single nucleotide substitution in EAR motif of Ln gene from soybean (Glycine max).
5. The method according to Claim 4, characterized in that the ln gene consists of a nucleotide sequence of SEQ ID NO: 1.
6. A method of producing a plant with controlled number of seeds per pod, comprising steps of:

   transforming a plant cell with the recombinant vector comprising the ln gene which is produced by single nucleotide substitution in EAR motif of the Ln gene from soybean (Glycine max); and

   regenerating a plant from the transformed plant cell.
7. A transformed plant with controlled number of seeds per pod that is produced by the method of Claim 6.
8. The transformed plant according to Claim 7, characterized in that the plant is a dicot plant.
9. The transformed plant according to Claim 8, characterized in that the dicot plant is a plant of Solanaceae, Cruciferae, Rosaceae, Leguminosae, or Cucurbitaceae.
10. The transformed plant according to Claim 8, characterized in that the dicot plant is a soybean (Glycine max).
11. Seed of the plant of Claim 7.
12. A composition for controlling the number of seeds per pod in a plant comprising the recombinant vector comprising the ln gene produced by single nucleotide substitution in EAR motif of Ln gene from soybean (Glycine max).
13. The composition according to Claim 12, characterized in that the ln gene consists of a nucleotide sequence of SEQ ID NO: 1.

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