Patent Publication Number: US-2015082496-A1

Title: Rice gene, gs3, exerting primary control over grain length and grain weight

Description:
This application is a divisional of co-pending U.S. application Ser. No. 12/159,964, filed Dec. 1, 2008; which is a national stage application under 35 §371 of International Application No. PCT/CN2007/000002 filed Jan. 4, 2007, the entire disclosure of which is specifically incorporated herein by reference, which claims the priority of Chinese Patent Application Ser. No. 200610018107.4, filed Jan. 5, 2006. 
    
    
     TECHNICAL FIELD 
     The present invention relates to biotechnology of plants. Particularly, the present invention relates to the gene cloning of GS3, a major QTL regulating grain weight and grain length, which is located in the pericentrometric region of chromosome 3 in rice. 
     BACKGROUND OF INVENTION 
     Grain size of rice is an important economic trait because: (1) grain size is a major determinant of grain weight, which is one of the three components of grain yield, and therefore, grain size is an important trait for yield; (2) grain size is also an important trait of rice appearance because grain weight is positively correlated with several characters including grain length, grain width and grain thickness (Evans, 1972, Rice Breeding, Los Banos, International Rice Research Institute, Manila, pp. 499-511). In China, the USA and some Asian countries, Indica rice with long and slender grain is generally preferred by most consumers. In China, a length/width ratio of 2.8 is adopted as an enforced threshold for a national standard for high quality rice. Thus, understanding the genetic basis and molecular mechanisms of grain size is important in improving both rice yield and quality. 
     In addition, grain size also plays an important role in the study of evolution of cultivars. It is generally believed that wild type relatives are usually small and round in shape, and are thus favored under natural selection. After a long-term domestication and selection by humans, the shape of grain particles has changed significantly. Therefore, the study of the genetic basis of grain size provides clues for the study of the evolution of cultivars. 
     Grain size is a typical quantitative trait and is complex in its genetic basis. Utilization of molecular marker technology is able to separate and locate QTL (Quantitative Trait Loci) controlling quantitative traits, thus separating the complex quantitative traits to simple Mendelian factors for studies. By using aforesaid methods, many QTLs regulating rice grain size were identified in recent years. Among these QTLs, a major QTL (referred to as GS3 in the present invention) located in the pericentrometric region of rice chromosome 3 was detected by many researchers (Huang et al., 1997, Mol. Breed. 3:105-113; Redoñ a and Mackill, 1998, Theor. Appl. Genet. 96:957-963; Kubo et al., 2001, Rice Genet. Newsl. 18:26-28; Thomson et al., 2003, Theor. Appl. Genet. 107:479-493; Aluko et al., 2004, Theor. Appl. Genet. 109:630-639). Using F 2:3  and a recombinant inbred line population derived from a cross between Zhenshan 97 and Minghui 63, the present invention has detected a major QTL several times that is present in the GS3 locus regulating both the grain length and the grain weight. This QTL explains over 55% of the total variation of grain length, as well as approximately 20% of the total variation of grain weight (Yu et al., 1997, Proc. Natl. Acad. Sci. USA 94:9226-9231; Li et al., 2000, Theor. Appl. Genet. 101:248-254; Tan et al., 2000, Theor. Appl. Genet. 101:823-829; Xing et al., 2001, Acta. Bot. Sin. 43:721-726; Xing et al., 2002, Theor. Appl. Genet. 105:248-257; Hua et al., 2002, Genetics 162:1885-1895). These results indicate that GS3 gene can be stably expressed in various genetic backgrounds and different environments. Therefore, GS3 gene is greatly potent and a good prospect for the improvement of both yield and quality traits of rice. The high resolution mapping of GS3 gene and the cloning of the corresponding gene provide new genetic resources for the improvement of both yield and quality in rice breeding. 
     High resolution mapping of the quantitative traits in common population groups is very difficult because it is not easy to determine whether a major QTL or multiple minor QTL are detected (Yano et al., 1997, Plant Molecular Biology 35:145-153) in said population groups. Secondly, in said population groups, multiple QTLs affecting the same trait are separated, therefore the interference caused and the affects of the environmental factors greatly limit the resolution of the location of a QTL. Special population groups can be established from high resolution mapping of a QTL. A common approach is to establish a near isogenic line (NIL) of an interesting QTL and to eliminate most background differences besides the interesting QTL locus to make said QTL presented as typical Mendelian genetics. Said approach has played an important role in many high resolution mapping and gene cloning of QTLs. Li et al. mapped GS3 to a region of 93.8-kb in length (Li et al., 2004, Genetics 168:2187-2195). Said results have provided a basis for the separation of mapping and the cloning of the GS3 gene. 
     Upon aforesaid purposes, the present inventors have isolated and cloned a major gene GS3 regulating grain weight and grain length in rice by the approach of mapping and cloning, therefore providing new genetic resources for the improvement of both yield and quality in rice breeding, and also providing clues for the study on the evolution of cultivars. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to the isolation and cloning of the whole DNA fragment encoding a major gene regulating both grain weight and grain length in rice. The present invention also relates to the improvements in both yield and quality of rice using said gene. Said gene is named GS3. 
     The invention established a near isogenic line (NIL) of GS3. GS3 was finely mapped to a region of 7.9-kb in length using the approach of mapping and cloning. With the help of the sequence information of a whole cDNA in aforesaid region, the invention predicted and analyzed the gene structure of GS3 and the protein encoded by GS3. It was found that GS3 comprises 5 exons and encodes 232 amino acids. It was predicted based on bioinformatics that said protein contains conserved domains including a PEBP-like domain, a transmembrane domain, a cysteine-rich domain of TNFR (tumour necrosis factor receptor)/NGFR (nerve growth factor receptor) and a VWFC (von Willebrand factor type C) homologous domain. In addition, the inventors sequenced and compared three large grain species (Minghui 63, a indica rice in China; H94, from Shanghai Agrobiological Gene Center; 93-11, a rice variety that has been completely sequenced) and 3 small grain species (Zhenshan97, a indica rice in China; Chun 7, from Shanghai Agrobiological Gene Center; Niponbare, a rice variety that has been completely sequenced). It was found that there was only a single base variation in said region of 7.9-kb in length between large grain species and small grain species. Said variation was located in the end of the second exon of GS3, wherein a cysteine codon (TGC) in small grain species is mutated to the terminator codon TGA in large grain species. Said mutation causes GS3 to be prematurely terminated in large grain species and leads to the loss of 178 amino acids, including part of the PEBP-like domain and three other conserved domains. It was found through bioinformatics analysis that the VWFC domain regulates growth and development signals in combination with the growth regulator TGF-β family members. In the large grain species, GS3 lacks the VWFC domain, therefore it loses its capacity to regulate growth and development signaling, which finally leads to the change in grain length and grain weight. 
     The present invention has the following advantages: 
     (1) it is the first cloning of a gene that highly affects the grain length and grain weight in rice, therefore providing new gene resources for high yield and good quality in rice breeding, and providing a good technology example for the cloning of homologous genes in other species;
 
(2) the gene cloned in the present invention provides evidence for the study of domestication and molecular evolution of rice and other species.
 
    
    
     
       DESCRIPTION OF FIGURES 
         FIG. 1  shows the technical flowchart of the present invention; 
         FIG. 2  shows the six cultivars used in sequence alignment; 
         FIG. 3  shows the frequency distribution of 1,000-grain weight, grain length, grain width and grain thickness in the random BC 3  F 2  subpopulation; wherein 
       (a) frequency distribution of 1,000-grain weight;
 
(b) frequency distribution of grain length;
 
(c) frequency distribution of grain width;
 
(d) frequency distribution of grain thickness;
 
         FIG. 4  shows the map of the GS3 locus on the molecular linkage map of chromosome 3 (unit in cM); 
         FIG. 5  shows the maps of the gene region, wherein 
       (a) The high-resolution map of the GS3 gene. The numbers between molecular markers indicate the numbers of recombination events detected between the GS3 locus and respective markers. Genebank no. OSJNBa0030J19 is a BAC clone of Nipponbare encompassing the GS3 locus;
 
(b) Organization of the GS3 gene. The positions of exons (black boxes), 5′ and 3′ UTR (hatched boxes), translation start codon (ATG), translation stop codon (TGA), and one common single nucleotide mutation in the second exon between the two grain-length groups are indicated, in which a substitution of cysteine (TGC) in the small grain group mutated to the translation stop codon (TGA) in the large grain group;
 
(c) Predicted sequence of the GS3 gene expression product. The position of the amino acid change in large grain group (cysteine to stop codon) is indicated by an asterisk. The PEBP-like domain is indicated by dashed underline, the transmembrane region by single solid underline, the TNFR/NGFR family cysteine-rich domain by double underline, and the VWFC domain is boxed;
 
         FIG. 6  shows the organization of the predicted GS3 protein indicating the localization of the various conserved domains including the PEBP-like domain, the transmembrane domains, the cysteine-rich domain of TNFR/NGFR and the VWFC homologous domain, wherein the PEBP-like domain is located inside the membrane, while the cysteine-rich domain of TNFR and the VWFC homologous domain are located outside the membrane. 
     
    
    
     EXAMPLES 
     A near isogenic line of rice GS3 gene was established using Minghui 63 as the recurrent parent and Chuan 7 as the donor parent. Mapping and effect evaluation of GS3 gene were carried out on a random BC 3  F 2  subpopulation consisting of 201 BC 3 F 2  individuals. By further analysis on 1,384 BC 3 F 2  individuals with large grain phenotype using CAPS markers, GS3 was finally mapped between the two CAPS markers GS63 and SF19 (designed by the inventors; see Table 2), which was approximately 7.9 kb in distance. Based on a whole length cDNA sequence, the GS3 gene structure was predicted. In addition, the possible function of GS3 gene was predicted based on the bioinformational technology. It was found by sequence alignment of the 7.9 kb fragment that a mutation was presented in all the large grain species which led to deficiency of 178 amino acids. Such deficiency could lead to the loss of the gene function. It implied the essential reason of the change in grain size, and at the same time, proved the correctness of setting GS3 as the target gene ( FIG. 1 ). 
     The following Examples further define the present invention and describe the methods for the isolation and cloning of GS3 gene, as well as the methods for the detection of the base mutation between GS3 alleles by sequence alignment. Based on the following recitation and the Examples, the skilled in the art are able to confirm the essential features of the present invention, and are able to make various changes and adjustments on the present invention to apply it on different uses and conditions without deviating from the concepts and scope of the present invention. 
     Example 1 
     Establishment of a Near Isogenic Lines of Rice GS3 Gene 
     1. Backcrossing and Screening 
     As shown in  FIG. 2 , successive crossing was carried out using Minghui 63 (large grain) as the recurrent parent and Chuan 7 (small grain) as the donor parent. Positive selection of GS3 was carried out in F 1 , BC 1 F 1  and BC 2 F 1 , that is, selecting individual plants whose targeting region was Minghui63/Chuan7 heterozygous genotype for the following backcrossing. The targeting region was determined in a region between two known SSR (Simple Sequence Repeat) markers RM282 and RM16. In BC 3 F 1 , in addition to positive selection, surveillance was also carried out in the genetic backgrounds besides the targeting region. Individual plants with a genetic background closest to Minghui63 were selected for the following experiments. Referring to the published rice genetic linkage map (Temnykh et al., 2000, Theor. Appl. Genet. 100:697-712; Temnykh et al., 2001, Genome Res. 11:1441-1452), 125 SSR markers with known polymorphism in the parents and evenly distributed on the 12 rice chromosomes were selected for surveillance of genetic backgrounds. One plant (BC 3 F 1 -19) was finally selected, whose genotype in the RM282 and RM16 region was Minghui63/Chuan7 heterozygous genotype, while whose genotype in said 125 SSR markers, only about 20% (25 pairs) of the markers were Minghui63/Chuan7 heterozygous genotype, and the rest thereof were all Mingui63 homozygous genotype. The progeny (BC 3 F 2  and BC 3 F 3 ) of said individual plant were used in the following experiments. 
     2. SSR Methods 
     The standard PCR protocol followed the methods taught in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Translated by Jin Dong Yan et al., Science Press. In the PCR, a 20 μl reaction system was used, which contained: 20-50 ng DNA template, 10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100, 1.8 mM MgCl 2 , 0.1 mM dNTP, 0.2 μM primers (primers of RM282 and RM16 as mentioned above) and 1 U Taq DNA polymerase. Conditions for PCR included: 94° C. predenature for 4 min; 94° C. 1 min, 55° C. 1 min, 72° C. 1 min, 34 cycles; 72° C. elongation 10 min PCR products were separated on a 6% acrylamide gel and then silver-stained (Bassam et al., 1991, Anal. Biochem. 196: 80-83). 
     Example 2 
     Mapping and Effect Evaluation of GS3 in the Random Subpopulation 
     1. Measurements of Traits of Large and Small Grain 
     Grain particles were air-dried and stored at room temperature for at least 3 months before testing in order to make sure the dryness and water contents thereof were relatively identical. Ten randomly chosen full filled grains from each plant were lined up closely in a way that each lay head to head, tail to tail, with no overlap and no gap in between. Said grains were arranged length-wise to measure the grain length using a vernier caliper, and then were lined up closely side by side, that is, arranged by breadth to measure the grain width using a vernier caliper. Grain thickness was determined for each grain individually using a vernier caliper, and the values were averaged and used as the measurements for the plant. Grain weight was calculated based on 200 randomly chosen fully filled grains and converted to 1,000-grain weight. 
     201 individuals of BC 3 F 2  derived from the BC 3 F 1 -19 individual plant were randomly selected to form a random subpopulation. Distributions of grain weight, grain length, grain width and grain thickness were studied in Minhui 63, Chuan 7 and said random subpopulation of BC 3 F 2 . It was found that all the traits were significantly different between the two parents (Table 1). In the random subpopulation, both grain length and 1,000-grain weight expressed a discontinuous distribution. The plants were classified as long grain and short grain based on a boundary of 8.50-9.50 mm in grain length or 20.5-21.5 g in 1,000-grain weight (Table 1 and  FIG. 3 ). Grain length concurred completely with grain weight, such that long grains were heavier than short grains, and vice visa. However, grain width and thickness showed normal distributions ( FIG. 3 ). For simplicity, in the present invention the large and heavier grains are referred to as long grain, and the opposite type as short grain. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Descriptive statistics of the traits for the two parents and the long grains and short grains in the random subpopulation 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Parent (mean ± SD) 
                 MM 
                 CC 
                 MC 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Trait 
                 Minghui 63 
                 Chuan 7 
                 Mean ± SD 
                 Range 
                 Mean ± SD 
                 Range 
                 Mean ± SD 
                 Range 
               
               
                   
               
               
                 1,000-grain weight (g) 
                 28.6 ± 0.6  
                 12.5 ± 0.4  
                 25.6 ± 2.0 
                 21.5-29.8 
                 17.5 ± 1.3  
                 14.2-20.0 
                 19.0 ± 1.2  
                 14.0-20.5 
               
               
                 Grain length (mm) 
                  9.91 ± 0.099 
                  6.30 ± 0.089 
                 10.25 ± 0.29 
                  9.64-10.73 
                 7.32 ± 0.26 
                 6.86-7.84 
                 7.72 ± 0.25 
                 7.24-8.50 
               
               
                 Grain width (mm) 
                 2.80 ± 0.03 
                 2.48 ± 0.02 
                  2.72 ± 0.13 
                 2.43-2.96 
                 2.82 ± 0.12 
                 2.45-3.06 
                 2.85 ± 0.09 
                 2.56-3.04 
               
               
                 Grain thickness (mm) 
                 2.13 ± 0.06 
                 1.68 ± 0.03 
                  2.03 ± 0.09 
                 1.81-2.21 
                 1.95 ± 0.10 
                 1.45-2.10 
                 1.99 ± 0.06 
                 1.79-2.13 
               
               
                   
               
            
           
         
       
     
     2. Design of Molecular Markers 
     Some SSR markers used in the present invention are publicly known. In addition, 11 pairs of Indel (Insert/Deletion) and CAPS (cleaved amplified polymorphic sequence) markers which shows polymorphism between Minghui 63 and Chuan 7 were designed based on the genome DNA sequence of Japonica Rice, Nipponbare and India Rice, 93-11. Said Indel and CAPS markers were used in the high resolution mapping analysis of GS3. The DNA sequences of said markers are listed in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Indel and CAPS markers (primers) developed for the mapping of the GS3 gene locus 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Annealing 
                   
               
               
                   
                   
                   
                   
                 Temp. 
                 Restriction 
               
               
                 Marker 
                 Type 
                 Forward primer (5′-3′) 
                 Reverse primer (5′-3′) 
                 (° C.) 
                 enzyme 
               
               
                   
               
               
                 GS06 
                 Indel 
                 AGCAAAGCTGGAACGAAGAG 
                 TAAATTACGCCGTGTCAACG 
                 55 
                   
               
               
                   
                   
                 (SEQ ID NO. 4) 
                 (SEQ ID NO. 5) 
                   
                   
               
               
                   
               
               
                 GS09 
                 Indel 
                 GCAACCAAGTCCACGCTAAT 
                 TAGCCGAAGATCAGCCTCCT 
                 57 
                   
               
               
                   
                   
                 (SEQ ID NO. 6) 
                 (SEQ ID NO. 7) 
                   
                   
               
               
                   
               
               
                 GS47 
                 CAPS 
                 GATTATTGGAGACGGGACGA 
                 GACGGCATGACCACTCTTTT 
                 55 
                 HapII 
               
               
                   
                   
                 (SEQ ID NO. 8) 
                 (SEQ ID NO. 9) 
                   
                   
               
               
                   
               
               
                 GS52 
                 CAPS 
                 AGCTTTGGTGTCGTTCTGCT 
                 CCGACTTGGAGAGAATGGAA 
                 55 
                 BglI 
               
               
                   
                   
                 (SEQ ID NO. 10) 
                 (SEQ ID NO. 11) 
                   
                   
               
               
                   
               
               
                 GS56 
                 CAPS 
                 GCTGTGTTGTCCTTTGCTGA 
                 CCAATAAACCCCACTGCAAC 
                 55 
                 BglI 
               
               
                   
                   
                 (SEQ ID NO. 12) 
                 (SEQ ID NO. 13) 
                   
                   
               
               
                   
               
               
                 GS61 
                 CAPS 
                 CTTTACAAAACCGGCGGTAA 
                 TGAAGCGGACCTAGCATTTT 
                 53 
                 BclI 
               
               
                   
                   
                 (SEQ ID NO. 14) 
                 (SEQ ID NO. 15) 
                   
                   
               
               
                   
               
               
                 GS63 
                 CAPS 
                 AAGAACGACTACGCGCATCT 
                 CCATCGCTCTCTTTCCTCAG 
                 53 
                 HhaI 
               
               
                   
                   
                 (SEQ ID NO. 16) 
                 (SEQ ID NO. 17) 
                   
                   
               
               
                   
               
               
                 GS64 
                 CAPS 
                 CAACACCAGCAACGAACAAC 
                 ACGAGGGATTATCAGCCATT 
                 55 
                 EcoRI, 
               
               
                   
                   
                 (SEQ ID NO. 18) 
                 (SEQ ID NO. 19) 
                   
                 HapII 
               
               
                   
               
               
                 GS65 
                 CAPS 
                 CGGTATGCCAAGTTGAATGA 
                 TTGCCGCAGTAAACAAGAAG 
                 55 
                 HhaI, 
               
               
                   
                   
                 (SEQ ID NO. 20) 
                 (SEQ ID NO. 21) 
                   
                 HapII 
               
               
                   
               
               
                 SF18 
                 CAPS 
                 CCTTCAGTAAGAGAGATGTG 
                 AGTTGATGGTTTTGTGGGAT 
                 57 
                 BclI 
               
               
                   
                   
                 (SEQ ID NO. 22) 
                 (SEQ ID NO. 23) 
                   
                   
               
               
                   
               
               
                 SF19 
                 CAPS 
                 TCTGCTTGCGGTTATCTGTA 
                 TTAGGTCCCTTTTCTCGTCC 
                 57 
                 SacI 
               
               
                   
                   
                 (SEQ ID NO. 24) 
                 (SEQ ID NO. 25) 
                   
                   
               
               
                   
               
               
                 Remark: 
               
               
                 Markers (primers) GS63 and SF19 were designed by the inventors. 
               
            
           
         
       
     
     3. QTL Mapping and Effect Evaluation of GS3 
     Said random subpopulation was subjected to genotype analysis using 6 SSR markers (MRG5959, MRG0164, MRG5881, MRG2646, RM411, RM16) and 2 Indel markers (G506 and G509). Mapmaker/Exp 3.0 (Lincoln et al. 1992, Whitehead Institute Technical Report, Whitehead Institute, Cambridge, Mass., USA) was used to establish a partial genetic linkage map in GS3 region. QTL analysis on the traits including grain length, width, thickness and 1,000-grain weight of the random subpopulation was conducted using the program Mapmaker/QTL 1.1 at a threshold of LOD 3.0. QTL analysis indicated that a QTL found in the interval between GS09 and MRG5881 had effects simultaneously on grain weight, grain length, grain width and grain thickness, and contributed 83.4%, 95.6%, 19.8% and 12.1% of the phenotypic variation on these traits, respectively. The allele from Minghui 63 contributed to the increase of grain weight, grain length and grain thickness, but to the decrease of grain width. Moreover, the QTL also showed different modes of gene actions on the traits, such that partial dominance was observed for 1000-grain weight, grain length and grain thickness, while overdominance was detected for grain width. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Effects of the QTL (in the interval GS09- 
               
               
                 MRG5881) on grain shape and weight 
               
            
           
           
               
               
               
               
               
            
               
                 Traits 
                 LOD 
                 A″ 
                 D b   
                 Var. % c   
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1,000-grain weight (g) 
                 72.8 
                 −4.08 d   
                 −2.52 d   
                 83.4 
               
               
                 Grain length (mm) 
                 129.2 
                 −1.47 d   
                 −1.06 d   
                 95.6 
               
               
                 Grain width (mm) 
                 8.9 
                 0.05 d   
                 0.08 d   
                 19.8 
               
               
                 Grain thickness (mm) 
                 5.3 
                 −0.04 d   
                 0.004 e   
                 12.1 
               
               
                   
               
               
                   a  Additive effect of Chuan 7 allele 
               
               
                   b Dominance effect of Chuan 7 allele 
               
               
                   c Percentage of total phenotypic variance explained by the QTL 
               
               
                   d Significant at P &lt; 0.0001 in t test 
               
               
                   e Not significant at P &lt; 0.05 
               
            
           
         
       
     
     4. Progeny Test 
     Each plant in said random subpopulation was bred to 20 families (BC 3  F 3 ) which were subjected to progeny test. Progenies of 56 in 201 families were uniformly long grains, while 61 families were uniformly short grains and 84 families had both long and short grains. The ratio of the three groups fit well to the expected ratio (1:2:1) of single locus Mendelian segregation (χ2=5.67,P&gt;0.05) in the χ2 test. The results indicated that in this BC 3  F 2  subpopulation, the grain size was controlled by a major gene, and the small size allele is dominant over the large size allele. The three distinct phenotypic classes corresponded to the three genotypes of the BC 3  F 2  individuals at the GS3 locus: homozygote for the Minghui 63 allele (long grain), homozygote for the Chuan 7 alleles (short grain), and heterozygote. Using the three phenotypic classes as a marker, GS3 was directly mapped into a 1-cM region delimitated by an Indel marker GS09 and SSR marker MRG5881 ( FIG. 4 ). 
     Example 3 
     High Resolution Mapping of GS3 
     1. CAPS Analysis 
     9 CAPS markers used for the high resolution locating are listed in Table 2. The amplification product amplified by said markers had a size of around 1-kb. The PCR reaction system was identical with the SSR reaction system mentioned above. The amplification condition of the PCR was as below: 94° C. predenature 4 min; 94° C. 1 min, 53° C.˜57° C. 1 min, 72° C. 1.5 min, 34 cycles; 72° C. elongation 10 min. The digestion of the amplicons was carried out in a 20 μl reaction system containing: 10 μl PCT product, 1 U restriction digestive enzyme (from Takara Ltd., Japan). Additional components were as described in the manual provided by Takara Ltd. After digestion in 37° C. for 3-5 hours, 10 μl of the digestion product was subjected to separation by electrophoresis in a 1.5% agrose gel, which was then observed using UV after EB staining. 
     2. Analysis of the Recombinant Plant and High Resolution Mapping of GS3 
     To further narrow down the GS3 containing genomic region, 1,384 plants with long grain phenotype (long grain, 9.7 mm or longer) from the BC 3  F 2  population of 5,740 individuals derived from BC 3 F 1 -19 individual plant were selected for recombinant screening. 
     All of the 1,384 selected plants were screened using an SSR marker MRG5881 and Indel marker GS09, which identified a total of 55 recombinants which were further confirmed to be very large size singles by progeny test. Using 9 designed CAPS markers to screen the 55 recombinants, it was found that five recombination events were resolved between GS47 and GS3, four identified between GS52 and GS3, four between GS56 and GS3, three between GS61 and GS3, and two between GS65 and GS3 ( FIG. 5   a ). In particular, the assay revealed one recombination event between GS63 and GS3 and two recombination events between SF19 and GS3. In addition, GS64 and SF18 were found to co-segregate with the GS3 locus. Therefore, the genomic region containing the GS3 locus was narrowed down to the DNA fragment bounded by GS63 and SF19, which corresponded to approximately 7.9-kb in length in the genome sequence of Nipponbare and 93-11 ( FIG. 5   a ). 
     Example 4 
     Gene Structure and Predicted Function Analysis of GS3 
     (1) Gene Structure Analysis of GS3 
     A full-length cDNA, which is named osigcea013f09t3, from the plumule of an indica cultivar Guangluai 4 (provided by Shanghai Agrobiological Gene Center) was identified in the 7.9-kb fragment ( FIG. 4   b ) between GS63 and SF19 (which were designed by the applicants; refer to Table 2). The nucleotide sequence is shown in SEQ ID NO:1. The cDNA sequence of the cloned GS3 gene of the present invention is 953 bp in length, which matches well with the region between positions 1.6 and 7.3 kb of the 7.9-kb fragment. Allowing for the regulatory regions on both ends, this is considered as the only candidate gene for GS3. 
     The structure of GS3 gene was obtained by comparing the sequences of said total cDNA with the genomic DNA sequence of Nipponbare. GS3 gene is 5,363 bp in length from the translation start codon to the termination codon. It comprises 5 exons and 4 introns. The starting exon is 117 bp in length, while the second exon is 53 bp, third exon 45 bp, fourth exon 54 bp, terminal exon 430 bp in length, respectively. The first intron is 1,472 bp in length, while the second intron is 1,439 bp, third intron 83 bp, fourth intron 1,671 bp in length, respectively (Table 5b). Therefore, the open reading frame of GS3 gene is 699 bp in length and encodes 232 amino acids. The sequence of said gene is shown in SEQ ID NO:1. 
     (2) Function Prediction of GS3 
     Prediction for the structure of GS3 protein was carried out with InterProScan. It was revealed that the protein encoded by GS3 gene consists of 232 amino acids and comprises several conserved domains. There is a PEBP (phosphatidylethanolamine-binding protein)-like domain in amino acid 12-65 at the 5′ terminus. A transmembrane region is located at amino acid 97-117. The region of amino acid 116-1557 is a TNFR (tumor necrosis factor receptor)/NGFR (nerve growth factor receptor) family cysteine-rich domain. The 3′ terminal cysteine-rich region shows the characters of the conserved amino acid sequence of the von Willebrand factor type C (VWFC) domain, which is typically 60-80 amino acid in length and comprises ten cysteines, especially is characterized in that it contains a C2XXC3XC4 sequence located in the middle and a C8C9XXC10 sequence at the 3′ terminal end (wherein C represents cysteine; the number represents the order of said conserved cysteines; X indicates any amino acid) ( FIG. 5   c  and  FIG. 6 ). 
     VWFC domain is represented in a number of extracellular matrix proteins. Some studies show that VWFC binds to members of the transforming growth factor TGF-β superfamily, thus disrupting the receptor binding sites of TGF-b superfamily proteins and preventing activation of the TGF-b receptor, such that it regulates the growth factor signaling pathway (Abreu et al., 2002, Gene 287:39-47; O&#39;Leary et al., 2004, J. Biol. Chem. 279:53857-53866). 
     Example 5 
     Detection of Base Pair Variation Between GS3 Alleles by Sequence Alignment 
     (1) Sequencing 
     Two large grain species (Minghui 63 and H94) and two small grain species (Chuan 7 and Zhenshan 97) were sequenced in the target genomic DNA region. DNA fragments from these cultivars were amplified using 10 pairs of primers whose amplicons were partially overlaping with each other. The amplification was carried out with high fidelity LA-Taq (TakaRa, Dalian, China). The PCR products were cloned into pGEM-T vector (Promega, USA) according to the manufacturer&#39;s specification. The cloned product was transformed into  E. coli  DH10B (Invitrogen, USA) and positive clones were screened with blue-white methods. The T7-R and SP6-F universal primers (Shanghai Sangon Biological Engineering Technology and Services Co. LTD.) and the Big Dye Terminator Cycle Sequencing v3.1 (Applied Biosystems, Foster City, Calif., USA) were used for sequencing from both ends of the subclones. Sequence contigs were assembled using the computer program SEQUENCHER 4.1 (Gene Codes Corporation, USA). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Primers used in the sequence alignment. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Size of 
                 Annealing 
               
               
                 Marker 
                 Forward primer (5′-3′) 
                 Reverse primer (5′-3′) 
                 Amplicon 
                 Temp. (° C.) 
               
               
                   
               
               
                 GS63 
                 AAGAACGACTACGCGCATCT 
                 CCATCGCTCTCTTTCCTCAG 
                  704 bp 
                 53 
               
               
                   
                 (SEQ ID NO. 16) 
                 (SEQ ID NO. 17) 
                   
                   
               
               
                   
               
               
                 SF26 
                 GTCTGAGGAAAGAGAGCGAT 
                 AAGCAAGCCAAGGGAAATGT 
                 1091 bp 
                 60 
               
               
                   
                 (SEQ ID NO. 26) 
                 (SEQ ID NO. 27) 
                   
                   
               
               
                   
               
               
                 SF15 
                 AGCAAAAAAGGTGAAGGACG 
                 CAAAGGGAATAACAAGGCAG 
                 1295 bp 
                 57 
               
               
                   
                 (SEQ ID NO. 28) 
                 (SEQ ID NO. 29) 
                   
                   
               
               
                   
               
               
                 SF16 
                 CGAATAGGAAGTCAATGGC 
                 GTCGTACCCGCCTTAGTTGA 
                 1159 bp 
                 55 
               
               
                   
                 (SEQ ID NO. 30) 
                 (SEQ ID NO. 31) 
                   
                   
               
               
                   
               
               
                 SF28 
                 TGCCCATCTCCCTCGTTTAC 
                 TGTTCGTTGCTGGTGTTG 
                 1065 bp 
                 55 
               
               
                   
                 (SEQ ID NO. 32) 
                 (SEQ ID NO.33) 
                   
                   
               
               
                   
               
               
                 GS64 
                 CAACACCAGCAACGAACAAC 
                 ACGAGGGATTATCAGCCATT 
                 1155 bp 
                 55 
               
               
                   
                 (SEQ ID NO. 18) 
                 (SEQ ID NO. 19) 
                   
                   
               
               
                   
               
               
                 SF18 
                 CCTTCAGTAAGAGAGATGTG 
                 AGTTGATGGTTTTGTGGGAT 
                 1245 bp 
                 57 
               
               
                   
                 (SEQ ID NO. 22) 
                 (SEQ ID NO. 23) 
                   
                   
               
               
                   
               
               
                 SF45 
                 AACCTTCTCTTCCTACCCTT 
                 TCAGCAATCACGTACTCATC 
                 1137 bp 
                 55 
               
               
                   
                 (SEQ ID NO. 34) 
                 (SEQ ID NO. 35) 
                   
                   
               
               
                   
               
               
                 SF19 
                 TCTGCTTGCGGTTATCTGTA 
                 TTAGGTCCCTTTTCTCGTCC 
                 1224 bp 
                 57 
               
               
                   
                 (SEQ ID NO. 24) 
                 (SEQ ID NO. 25) 
               
               
                   
               
            
           
         
       
     
     The size of the PCR products depended on the sequence of Nipponbare and varied among different species. 
     (2) Sequence Alignment 
     Sequence alignment was carried out on three large grain varieties (Minghui 63, H94, 93-11) and three small grain varieties (Zhenshan97, Chun 7, Niponbare) ( FIG. 2 ). The GS3 region sequences of Nipponbare and 93-11 were from the BAC clone OSJNBa0030J19 and contig Ctg009226, respectively. Sequence alignment was conducted using the computer program Vector NTI 9 (InforMax™ Corporation, USA). 
     Although many nucleotide changes were observed among the six cultivars in the 7.9-kb region, there was only one common single nucleotide mutation detected between these two different grain-length groups, which indicated said mutation was the essential reason causing the grain size change. Further studies showed that said nucleotide mutation was located at the second exon of the GS3 gene, in which a cysteine codon (TGC) in the small-grain group was mutated to a termination codon (TGA) in the large-grain group ( FIG. 4   b ). This premature termination resulted in a 178-amino acids truncation in the C-terminus of the predicted protein in the large-grain group, which eliminated part of the PEBP-like domain and all the other three conserved domains. Such nonsense mutation was clearly in agreement with the recessive nature of the long grain phenotype, indicating that long grains resulted from the loss of the function of the protein otherwise producing short grains.