GW5 - Revision history

Mafeifei at 14:47, 8 June 2014

2014-06-08T14:47:14Z

Mafeifei at 14:37, 8 June 2014

2014-06-08T14:37:48Z

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Mafeifei at 14:32, 8 June 2014

2014-06-08T14:32:43Z

Mafeifei at 14:25, 8 June 2014

2014-06-08T14:25:11Z

Mafeifei at 14:22, 8 June 2014

2014-06-08T14:22:01Z

Mafeifei at 14:17, 8 June 2014

2014-06-08T14:17:46Z

Mafeifei at 13:26, 8 June 2014

2014-06-08T13:26:18Z

Mafeifei at 13:23, 8 June 2014

2014-06-08T13:23:01Z

Mafeifei at 13:21, 8 June 2014

2014-06-08T13:21:03Z

Mafeifei at 13:00, 8 June 2014

2014-06-08T13:00:39Z

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 In an effort to identify the functional partners of GW5, we carried out a yeast two-hybrid screen using full-length GW5 as the bait. A prey library was constructed using mRNAs derived from the young panicle of IR24 before heading, at which time GW5 expression was detected. We repeatedly found that polyubiquitin protein interacts with GW5. This result was found 14 times in about 200 candidate positive clones on synthetic growth medium without leucine, tryptophan, histidine and adenine. X-gal filter lift assays also detected a clear interaction between GW5 and polyubiquitin (Figure 6B). This result suggests that the GW5 protein may play an important role in regulating the grain shape through involvement with the ubiquitin-proteasome pathway<ref name="ref1" />.
 [[File:ma5.jpg|center|thumb|1000px|'''Figure 5.''' Sub-cellular localization of GW5. (A) and (C) Bright field images of onion epidermal cells. (B) GFP by itself (pCAMBIA 1302 vector) localizes to both cytoplasm and nucleus. (D) GW5-GFP fusion protein is localized only to the nucleus. Scale bars:50 mm.]]
-[[File:ma6.jpg|thumb|1000px|'''Figure 6.''' GW5 interacts with polyubiquitin in yeast two-hybrid assay. (A) AH109 yeast cells expressing pGBKT7-GW5/pGADT7(top), pGBKT7/pGADT7-polyubiquitin (middle) and pGBKT7-GW5/pGADT7-polyubiquitin (bottom) were selected on synthetic growth medium without Leu and Trp. (B) X-gal filter lift assay showing that GW5 interacts with polyubiquitin.]]
+[[File:ma6.jpg|center|thumb|1000px|'''Figure 6.''' GW5 interacts with polyubiquitin in yeast two-hybrid assay. (A) AH109 yeast cells expressing pGBKT7-GW5/pGADT7(top), pGBKT7/pGADT7-polyubiquitin (middle) and pGBKT7-GW5/pGADT7-polyubiquitin (bottom) were selected on synthetic growth medium without Leu and Trp. (B) X-gal filter lift assay showing that GW5 interacts with polyubiquitin.]]
 ==evolution and fine mapping of GW5==
 ===Stability of QTL for rice grain width and gene action of identified QTL===
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 ===Dissecting QTL qGW-5 into a single gene, GW5===
-To dissect qGW-5 into a single gene, we analyzed four genetic models that could possibly lead to the observed distortion of segregation in the BC4F2 population. Wide rice grain could be controlled by one of the following models: (1) two recessive linked genes at the qGW-5 locus, (2) a recessive gene linked with gamete sterility gene(s), (3) a recessive gene linked with gene(s) for weak germination or seedling viability, or (4) a recessive gene linked with hybrid partial sterility gene(s). If case 1 is correct, genotypes of the markers near qGW-5 including Asominori(A)/A, A/CSSL28(L), and L/L should show the normal 1:2:1 ratio in the BC4F2 population. However, the ratios of 8 SSR markers in the interval 20. 6–39.2cM near qGW-5 did not exhibit the predicted 1:2:1 ratio (Figure 5, A1–D1). If case 2 is the correct model, the above ratios of 7.68:1, 7.05:1, 7.36:1, and 7.54:1 should correspond to 1:3.9:3.8, 1:3.7:3.4, 1:3.7:3.6, and 1:3.9:3.8 for A/A:A/L:L/L, respectively. But the observed actual ratios listed in Figure 5, A1–D1, clearly exclude case 2. Additionally, rice seeds with the genotype of A/A, A/L, or L/L had similar germination ability and seedling viability (supplemental Table 3), ruling out the model presented in case 3.
+To dissect qGW-5 into a single gene, we analyzed four genetic models that could possibly lead to the observed distortion of segregation in the BC4F2 population. Wide rice grain could be controlled by one of the following models: (1) two recessive linked genes at the qGW-5 locus, (2) a recessive gene linked with gamete sterility gene(s), (3) a recessive gene linked with gene(s) for weak germination or seedling viability, or (4) a recessive gene linked with hybrid partial sterility gene(s). If case 1 is correct, genotypes of the markers near qGW-5 including Asominori(A)/A, A/CSSL28(L), and L/L should show the normal 1:2:1 ratio in the BC4F2 population. However, the ratios of 8 SSR markers in the interval 20. 6–39.2cM near qGW-5 did not exhibit the predicted 1:2:1 ratio (Figure 11, A1–D1). If case 2 is the correct model, the above ratios of 7.68:1, 7.05:1, 7.36:1, and 7.54:1 should correspond to 1:3.9:3.8, 1:3.7:3.4, 1:3.7:3.6, and 1:3.9:3.8 for A/A:A/L:L/L, respectively. But the observed actual ratios listed in Figure 11, A1–D1, clearly exclude case 2. Additionally, rice seeds with the genotype of A/A, A/L, or L/L had similar germination ability and seedling viability, ruling out the model presented in case 3. Several observations support the fourth case. First, complete fertility was observed for Asominori, IR24, and CSSL28, whereas partial sterility occurred in Asominori/ CSSL28 F1 plants, which evidently resulted from hybrid partial sterility gene(s). Second, an indica–japonica hybrid partial sterility gene S31(t) mapped to 20.9–24.7 cM on chromosome 5 in our laboratory <ref name="ref9" /> was linked with qGW-5 located at 36.4–44.9 cM. Third, eight SSR markers in the 20.6–39.2cM region showed regularly progressive segregation distortions in the three genotypes, with ratios of LL 1 AL/AA from 7.33:1 at 39.2 cM to 14.79:1 at 20.6 cM (Figure 11, A1), indicating that the closer the distance from the SSR markers to the S31(t) gene, the stronger the degree of segregation distortion. Thus, the S31(t) gene played a crucial role in inducing the distortion of the ratios. In contrast, four SSR markers (RM163, RM430, RM506, and RM408) far from the qGW-5 locus showed a nearly normal ratio of 3:1 for LL 1 AL/AA (Figure 11, A1). Similar results were also observed in E10–E12 (Figure 5, B1–D1). Additionally, when 300 randomly selected BC4F2 plants were divided into three groups on the basis of the three genotypes of the RMw513 marker, 35, 37, 42, and 36 plants with the genotype A/A in E9–E12, respectively, showed wide rice grains (Figure 11, A2–D2), whereas all other narrowgrain plants presented the genotype of A/L or L/L, indicating that the distorted segregation of marker genotypes was consistent with that of grain-width phenotype. Finally, all individuals in 35 BC4F3 families with the genotype A/A at the RMw513 locus showed wide grains without segregation.Of 2408 BC4F3 plants derived from 152 BC4F2 plants with the genotype A/L, the ratio of narrow- (2124) to wide-grain plants (284) was 7.48:1, consistent with that in the BC4F2 population (Figures 9 and 10). These findings clearly indicate that qGW-5 behaves like a single gene in the CSSL28/Asominori F2 population and that both wide rice grains and small length–width ratio are mainly controlled by a recessive qGW-5 allele, named GW5.
-Several observations support the fourth case. First, complete fertility was observed for Asominori, IR24, and CSSL28, whereas partial sterility occurred in Asominori/ CSSL28 F1 plants, which evidently resulted from hybrid partial sterility gene(s). Second, an indica–japonica hybrid partial sterility gene S31(t) mapped to 20.9–24.7 cM on chromosome 5 in our laboratory <ref name="ref9" /> was linked with qGW-5 located at 36.4–44.9 cM (Table 1). Third, eight SSR markers in the 20.6–39.2cM region showed regularly progressive segregation distortions in the three genotypes, with ratios of LL 1 AL/AA from 7.33:1 at 39.2 cM to 14.79:1 at 20.6 cM (Figure 5, A1), indicating that the closer the distance from the SSR markers to the S31(t) gene, the stronger the degree of segregation distortion. Thus, the S31(t) gene played a crucial role in inducing the distortion of the ratios. In contrast, four SSR markers (RM163, RM430, RM506, and RM408) far from the qGW-5 locus showed a nearly normal ratio of 3:1 for LL 1 AL/AA (Figure 5, A1). Similar results were also observed in E10–E12 (Figure 5, B1–D1). Additionally, when 300 randomly selected BC4F2 plants were divided into three groups on the basis of the three genotypes of the RMw513 marker, 35, 37, 42, and 36 plants with the genotype A/A in E9–E12, respectively, showed wide rice grains (Figure 5, A2–D2), whereas all other narrowgrain plants presented the genotype of A/L or L/L, indicating that the distorted segregation of marker genotypes was consistent with that of grain-width phenotype. Finally, all individuals in 35 BC4F3 families with the genotype A/A at the RMw513 locus showed wide grains without segregation.Of 2408 BC4F3 plants derived from 152 BC4F2 plants with the genotype A/L, the ratio of narrow- (2124) to wide-grain plants (284) was 7.48:1, consistent with that in the BC4F2 population (Figures 3 and 4). These findings clearly indicate that qGW-5 behaves like a single gene in the CSSL28/Asominori F2 population and that both wide rice grains and small length–width ratio are mainly controlled by a recessive qGW-5 allele, named GW5.
+[[File:ma205.jpg|center|thumb|1000px|'''Figure 11.'''The ratios of three genotypes of SSR markers and frequency distributions of grain width in 300 BC4F2 individuals across four environments (E9–E12). A1–D1 represent the ratios of three genotypes of 12 SSR markers in E9–E12, respectively;]]
 === Environmental impact on the effect of the GW5 gene===
-Since wide rice grain was controlled by the GW5 gene in the BC4F2 population, the environmental impact on the effect of the GW5 gene could be evaluated by comparing the average grain width of BC4F2 individuals with the genotype of GW5/ GW5 among the E9–E12 environments. The largest effect of the GW5 gene occurred in E9 and E11, with average grain widths of 3.36 ±0.05 and 3.36±0.04 mm, and 2.88 ±0.04 and 2.88 ±0.05 mm for paddy rice and brown rice, respectively; GW5 had the smallest effect in E10, in which paddy rice and brown rice had average grain widths of 3.20±0.05 and 2.66±0.04 mm, respectively. Moreover, significant differences of grain width were observed between E9/E11 and E12, as well as E12 and E10 (Table 3), thus showing that the environment had a significant impact on the effect of the GW5 gene. Likewise, a similar environmental impact was also found on the effect of the GW5 gene (supplemental Table 4).
+Since wide rice grain was controlled by the GW5 gene in the BC4F2 population, the environmental impact on the effect of the GW5 gene could be evaluated by comparing the average grain width of BC4F2 individuals with the genotype of GW5/ GW5 among the E9–E12 environments. The largest effect of the GW5 gene occurred in E9 and E11, with average grain widths of 3.36 ±0.05 and 3.36±0.04 mm, and 2.88 ±0.04 and 2.88 ±0.05 mm for paddy rice and brown rice, respectively; GW5 had the smallest effect in E10, in which paddy rice and brown rice had average grain widths of 3.20±0.05 and 2.66±0.04 mm, respectively. Moreover, significant differences of grain width were observed between E9/E11 and E12, as well as E12 and E10, thus showing that the environment had a significant impact on the effect of the GW5 gene. Likewise, a similar environmental impact was also found on the effect of the GW5 gene .
 === Fine mapping of the GW5 gene in a recombination hotspot region on chromosome 5:===
-For fine mapping, 805 wide-grain homozygotes of the total 6781 BC4F2 individuals were used to calculate the recombination frequency between the GW5 gene and the surrounding molecular markers. Using previously published SSR and EST markers, we detected three SSR markers (RM3328, RM3322, and RM5874) and one EST marker (C53703) on one side of the gw-5 gene, and one SSR marker (RM5994) on the other side (Figure 6C). Ten polymorphic SSR markers were then designed using DNA sequences of seven BAC/PAC contigs in Nipponbare (supplemental Table 2S and Figure 6B). Among the markers, RMw530 and RMw513 had 23 and 6 recombinants with the GW5 gene in the 805 homozygotes, respectively (Figure 6C). Thus, the GW5 gene was located in the 1.8-cM genetic region on chromosome 5. Searching for the contigs OJ1097_A12 (harboring RMw530) and B1007D10 (carrying RMw513) in the Nipponbare genome (http://www.gramene.org), we found that RMw530 and RMw513 reside at 5,309,078 and 5,358,806 bp on chromosome 5, respectively. Thus, the gw-5 gene was narrowed down to a 49.7-kb genomic region (Figure6C).
+For fine mapping, 805 wide-grain homozygotes of the total 6781 BC4F2 individuals were used to calculate the recombination frequency between the GW5 gene and the surrounding molecular markers. Using previously published SSR and EST markers, we detected three SSR markers (RM3328, RM3322, and RM5874) and one EST marker (C53703) on one side of the GW5 gene, and one SSR marker (RM5994) on the other side (Figure 12C). Ten polymorphic SSR markers were then designed using DNA sequences of seven BAC/PAC contigs in Nipponbare ( Figure 12B). Among the markers, RMw530 and RMw513 had 23 and 6 recombinants with the GW5 gene in the 805 homozygotes, respectively (Figure 12C). Thus, the GW5 gene was located in the 1.8-cM genetic region on chromosome 5. Searching for the contigs OJ1097_A12 (harboring RMw530) and B1007D10 (carrying RMw513) in the Nipponbare genome (http://www.gramene.org), we found that RMw530 and RMw513 reside at 5,309,078 and 5,358,806 bp on chromosome 5, respectively. Thus, the gw-5 gene was narrowed down to a 49.7-kb genomic region (Figure12C).The 17.1-cM genetic region (RM5874–RM5994) can be divided into 10 intervals, with ratios of physical-togenetic distance of 226.3, 261.7, 183.9, 68.9, 27.6, 40.0, 14.2, 257.3, 267.4, and 255.4 kb/cM (Figure 13). The GW5 gene was located in the RMw530–RMw513 interval with the kb/cM of 27.6. In rice, the genomewide average is estimated at 244 kb/cM <ref name="ref10" />, indicating that the crossover frequency between RMw530 and RMw513 was approximately nine times that of the wholegenome in rice.On the basis of the available sequence annotation, we found five predicted candidate genes in the 49.7-kb region (http://www.ncbi.nlm.nih.gov/BLAST; http://www.softberry.com). Of these genes, one had unknown function, and the other four encoded an auxin-responsive protein IAA16, a 20S proteasome b-subunit, a hexokinase 6, and the Ulp1 protease-like protein. This result will be useful in the molecular cloning and functional characterization of the GW5 gene.
-The 17.1-cM genetic region (RM5874–RM5994) can be divided into 10 intervals, with ratios of physical-togenetic distance of 226.3, 261.7, 183.9, 68.9, 27.6, 40.0, 14.2, 257.3, 267.4, and 255.4 kb/cM (Figure 7). The GW5 gene was located in the RMw530–RMw513 interval with the kb/cM of 27.6. In rice, the genomewide average is estimated at 244 kb/cM <ref name="ref10" />, indicating that the crossover frequency between RMw530 and RMw513 was approximately nine times that of the wholegenome in rice.On the basis of the available sequence annotation, we found five predicted candidate genes in the 49.7-kb region (http://www.ncbi.nlm.nih.gov/BLAST; http://www.softberry.com). Of these genes, one had unknown function, and the other four encoded an auxin-responsive protein IAA16, a 20S proteasome b-subunit, a hexokinase 6, and the Ulp1 protease-like protein. This result will be useful in the molecular cloning and functional characterization of the GW5 gene.
+[[File:ma206.jpg|center|thumb|1000px|'''Figure 12.'''Genetic and physical maps of the gw-5 gene on rice chromosome 5. (A) QTL mapping of the qGW-5 locus. (B) Six BAC/PAC contigs around the gw-5 locus. (C) Fine mapping of gw-5 using newly developed SSR markers. The red superscript a indicates that there are 167 recombinants of the total 805 homozygous plants between the gw-5 gene and the marker RM5874. In fact, the 167 recombinants included 113 wide-grain plants with the heterozygous band patterns of wide- and narrow-grain parents and 27 wide-grain plants with the band pattern of the narrow-grain parent. The recombination frequency ¼ (N1 1 N2/2)/N, where N is the total number of wide-grain plants surveyed, N1 is the number of wide-grain plants with the band pattern of the narrow-grain parent, and N2 is the number of wide-grain plants with the heterozygous band patterns. We marked the number of recombinants between RM5874 and the gw-5 gene as 167 to directly calculate the recombination frequency, [(27 3 2 1 113 ¼ 167)/(805 3 2)] 3 100 (cM). The other numbers marked in the row (no. of recombinants) were also calculated on the basis of this approach.]]
 ==Labs working on this gene==

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 ==evolution and fine mapping of GW5==
 ===Stability of QTL for rice grain width and gene action of identified QTL===
-The phenotypic distributions of rice grain width in the RIL populations grown in the E1–E4 environments (supplemental Table 1S) are shown in Figure 1. Variance among genotypes (G) was highly significant for grain width, but was not significant among environments (E). The significant G 3 E interaction explained 3.5% of the total phenotypic variation. Six additive effect QTL for rice grain width were identified and mapped to six chromosomes, with LOD values ranging from 7.68 to 23.77 (Table 1). Among the QTL, qGW-5 was consistently located in the Y1060L–R569 interval on chromosome 5 in populations grown across all four environments, and the average PVE by qGW-5 was 24.3%. The IR24 allele at the qGW-5 locus was found to reduce grain width by an average of 0.115 mm. Moreover, stability of qGW-5 was relatively high, as its QEI was not significant. Additionally, 11 pairs of epistatic QTL for rice grain width were detected and mapped on 10 chromosomes. Of these, three digenic interactions occurred between an additive effect QTL (qGW-1, qGW-4, or qGW-12) and a modifying factor (Table 2).Eighteen lines were selected from the total 66 CSSLs and used to analyze gene action of the six additive effect QTL. The donor IR24 segments harboring qGW-5 and qGW-9 were transferred into 2 (CSSL28 and 29) and 4 CSSLs (CSSL53–55 and 57), respectively<ref name="ref8" />. Significant difference in grain width of milled rice was observed between Asominori and each of CSSL28 and CSSL29 across all eight environments, indicating that the gene action of qGW-5 was significant and stable in the Asominori genetic background (Figure2). With qGW-9, significant difference in grain width between Asominori and each of the four target CSSLs was found only in a few environments. The direction of the effect of qGW-9 in CSSL53 and CSSL55 was consistent with that in the RIL population (Table 1), but opposite to that in CSSL54 (Figure 2). Thus, the gene action of qGW-9 was sensitive to both the genetic background and environmental conditions. Similar results were also observed for qGW-1, qGW-4, qGW-10, and qGW-12 (supplemental Figure S2). Therefore, the observed stability and gene action of these QTL indicates that qGW-5 is the most important genetic factor that controls rice grainwidth difference between the Asominori and IR24 parental lines.
+The phenotypic distributions of rice grain width in the RIL populations grown in the E1–E4 environments are shown in Figure 7. Variance among genotypes (G) was highly significant for grain width, but was not significant among environments (E). The significant G 3 E interaction explained 3.5% of the total phenotypic variation. Six additive effect QTL for rice grain width were identified and mapped to six chromosomes, with LOD values ranging from 7.68 to 23.77. Among the QTL, qGW-5 was consistently located in the Y1060L–R569 interval on chromosome 5 in populations grown across all four environments, and the average PVE by qGW-5 was 24.3%. The IR24 allele at the qGW-5 locus was found to reduce grain width by an average of 0.115 mm. Moreover, stability of qGW-5 was relatively high, as its QEI was not significant. Additionally, 11 pairs of epistatic QTL for rice grain width were detected and mapped on 10 chromosomes. Of these, three digenic interactions occurred between an additive effect QTL (qGW-1, qGW-4, or qGW-12) and a modifying factor.Eighteen lines were selected from the total 66 CSSLs and used to analyze gene action of the six additive effect QTL. The donor IR24 segments harboring qGW-5 and qGW-9 were transferred into 2 (CSSL28 and 29) and 4 CSSLs (CSSL53–55 and 57), respectively<ref name="ref8" />. Significant difference in grain width of milled rice was observed between Asominori and each of CSSL28 and CSSL29 across all eight environments, indicating that the gene action of qGW-5 was significant and stable in the Asominori genetic background (Figure2). With qGW-9, significant difference in grain width between Asominori and each of the four target CSSLs was found only in a few environments. The direction of the effect of qGW-9 in CSSL53 and CSSL55 was consistent with that in the RIL population, but opposite to that in CSSL54 (Figure 8). Thus, the gene action of qGW-9 was sensitive to both the genetic background and environmental conditions. Similar results were also observed for qGW-1, qGW-4, qGW-10, and qGW-12. Therefore, the observed stability and gene action of these QTL indicates that qGW-5 is the most important genetic factor that controls rice grainwidth difference between the Asominori and IR24 parental lines.
 ===QTL qGW-5 controlled both rice grain width and length–width ratio in the BC4F2 population===
 In E9, the phenotypic distributions of grain width of paddy and brown rice in the CSSL28/Asominori F2 population appeared to be bimodal with boundaries of 3.28 and 2.80 mm, respectively (Figure 3, A and B). The ratio of narrow- (1921) to wide-grain individuals (250) was 7.68:1, which does not fit with the expected segregation ratio of 3:1 for the inheritance of a single gene. Similarly, the ratio of small to large LWR individuals was 1:7.68 (Figure 3, E and F). Interestingly, all 250 small LWR individuals showed wide grains, and all 1921 large LWR plants presented narrow grains. Moreover, significantly negative correlations (r =-0.94** and-0.94**) were observed between grain width and LWR of paddy rice as well as brown rice in the total 2171 BC4F2 individuals. Furthermore, although LWR is a complex trait reflecting grain length and width, QTL for grain length had relatively small effects on the phenotypic variation of LWR in the BC4F2 population, as shown by Figure 3, C and D, which clearly indicated that minor factor(s) conferred the phenotypic variations of grain length. These results suggest that qGW-5 mainly affected both rice grain width and LWR in the BC4F2 population. This is further supported by the fact that the ratios of narrow to wide-grain individuals were 7.05:1, 7.36:1, and 7.54:1 in the CSSL28/Asominori F2 populations that included 1248 plants in E10, 2465 in E11, and 897 in E12 (Figure 4, A–C); their corresponding ratios of small to large LWR individuals were 1:7.05, 1:7.36, and 1:7.54, respectively.

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 Sequence analysis indicated that GW5 encodes a novel protein without significant homology to any proteins of known biochemical function. The protein is predicted to contain an NLS and an arginine-rich domain. To test the functionality of the predicted NLS, we fused the coding region of GW5 to the N-terminus of GFP. Transient expression in onion epidermal cells showed that the GW5-GFP fusion protein is exclusively localized to the nucleus (Figure 5).
 In an effort to identify the functional partners of GW5, we carried out a yeast two-hybrid screen using full-length GW5 as the bait. A prey library was constructed using mRNAs derived from the young panicle of IR24 before heading, at which time GW5 expression was detected. We repeatedly found that polyubiquitin protein interacts with GW5. This result was found 14 times in about 200 candidate positive clones on synthetic growth medium without leucine, tryptophan, histidine and adenine. X-gal filter lift assays also detected a clear interaction between GW5 and polyubiquitin (Figure 6B). This result suggests that the GW5 protein may play an important role in regulating the grain shape through involvement with the ubiquitin-proteasome pathway<ref name="ref1" />.
 ==evolution and fine mapping of GW5==
 ===Stability of QTL for rice grain width and gene action of identified QTL===

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 ===GW5 is associated with a 1212-bp deletion in the Asominori cultivar===
 For genetic analysis and isolation of the dominant narrow-grain QTL, named GW5, the F2 population was constructed from a cross between Asominori and CSSL28, and the QTL was dissected into a single gene <ref name="ref2" />. By means of linkage analysis using the genotype data of both the GW5 gene and simple sequence repeat (SSR) markers, the GW5 gene was mapped to an interval between markers RM3328 and RMw513 in 805 homozygotes (Figure 2A). Thus, the GW5 gene was mapped to a genomic region 2.7 cM in length, located 2.3 cM from RM3328 and 0.37 cM from RMw513. In this region, three bacterial artificial chromosome (BAC) contigs were found – OJ1725_E07, OJ1097_A12 and B1007D10 (Figure 2B) – according to the Nipponbare genome (http://www.gramene.org). For high-resolution mapping of GW5, five new cleaved-amplified polymorphic sequence (CAPS) markers were developed on one of the BACs (OJ1097_A12). The GW5 locus was pinpointed into an interval between the CAPS markers Cw5 and Cw6 in 2180 wide-grain homozygous individuals from the BC4F2 population (Figure 2C). Within this region, the 21-kb fragment of Asominori and the corresponding 22.2-kb fragments of CSSL28 and IR24 were sequenced. Further comparison of these sequences showed that the Asominori genome harbored a 1212-bp deletion(Figure 2D). Meanwhile, two Indel (insertion or deletion) markers – Indel1, including 775 bp of the 1.2 kb fragment, and Indel2, containing the aforementioned1.2 kb fragment and totaling 1897 bp in length – were designed and used to confirm the presence of GW5. Genetic analysis revealed that these two Indel markers co-segregated with GW5 in 2180 wide-grain progenies (Figure 2C). On the basis of the available sequence annotation (http://www.softberry.com; http://www.ncbi.nlm.nih.gov/BLAST), two open reading frames (ORFs) were predicted in the 21.0-kb target region of Asominori, and three ORFs were predicted in the corresponding region of CSSL28. However, no sequence differences were found between these lines for ORF1 and ORF3. ORF1 encodes a protein showing high similarity to ubiquitin-proteaselike protein, whereas ORF3 encodes an unknown protein containing a calmodulin-binding motif. Interestingly, we found that the 22.2-kb region of CSSL28 contains a third ORF between ORF1 and ORF3, designated ORF2, which is located in the deleted region of Asominori (Figure 2E). Motif scan analysis showed that the predicted product of ORF2 harbors a nuclear localization signal (NLS) and an arginine-rich domain (http://hits.isb-sib.ch/cgi-bin/motif-scan). These results suggest that ORF2, which is present in CSSL28 but absent in Asominori, likely corresponds to GW5.
-[[File:ma2.jpg|thumb|1000px|'''Figure 2.'''Figure 2 Fine mapping of GW5. This map was constructed on the basis of publicly available rice sequences. The CAPS and Indel markers developed in this work are indicated. (A) GW5 was mapped to a region between markers RM3328 and RMW513 in 805 recessive individuals. (B) Three BAC contigs, OJ1725_E07, OJ1097_A12 and B1007D10, cover the GW5 locus in the Nipponbare genomic sequence. (C) GW5 was narrowed down to a 21-kb genomic DNA region between CAPS markers Cw5 and Cw6, and it co-segregated with the Indel1 marker in 2 180 recessive individuals. (D) Compared with IR24, 1.2-kb genomic DNA is deleted in Asominori. (E) Three ORFs are predicted in the candidate region harboring GW5 (http://softberry.com). The predicted ORF2 is located in the deleted region in Asominori.]]
+[[File:ma2.jpg|thumb|1000px|'''Figure 2.''' Fine mapping of GW5. This map was constructed on the basis of publicly available rice sequences. The CAPS and Indel markers developed in this work are indicated. (A) GW5 was mapped to a region between markers RM3328 and RMW513 in 805 recessive individuals. (B) Three BAC contigs, OJ1725_E07, OJ1097_A12 and B1007D10, cover the GW5 locus in the Nipponbare genomic sequence. (C) GW5 was narrowed down to a 21-kb genomic DNA region between CAPS markers Cw5 and Cw6, and it co-segregated with the Indel1 marker in 2 180 recessive individuals. (D) Compared with IR24, 1.2-kb genomic DNA is deleted in Asominori. (E) Three ORFs are predicted in the candidate region harboring GW5 (http://softberry.com). The predicted ORF2 is located in the deleted region in Asominori.]]
 ===The deletion prevails in wide-grain rice cultivars and defines a domestication-related rice gene===
-To further confirm the identity of GW5, we randomly selected 46 rice lines, including Asominori, IR24 and 44 other cultivars, to determine the co-segregation relationship between the absence of the candidate GW5 gene and the wide-grain phenotype (Table 4). The 46 rice lines were divided into two groups: group I contained 23 narrow-grain varieties (grain width ranging from 2.40 to 2.85 mm) and group II consisted of 23 wide-grain ones (3.30-3.92 mm) (Table 4). Segregation analysis of the Indel1 marker showed that the 775-bp target fragment was observed only in group I cultivars, but not in group II cultivars (Figure 3A and 3B). Similarly, PCR analysis with the Indel2 marker detected a 1 897-bp fragment in group I cultivars, but only a 697-bp product in group II cultivars (Figure 3C and 3D). These results suggested that the 1.2-kb fragment containing the GW5 gene was absent in all 23 wide-grain cultivars examined here but was present in the narrow-grain ones. The strict correlation between the grain-width phenotype and deletion of the 1.2-kb genomic DNA strongly supports the notion that the candidate ORF2 located on the deleted 1.2-kb fragment represents GW5. To address how this deletion in GW5 prevails in rice domestication, we further analyzed the 46 cultivars. Group I contained 21 indica species (grain width ranged from 2.40 to 2.84 mm) and two slender japonica varieties (one 2.74 mm wide, the other 2.85 mm wide), whereas most japonica (3.30-3.92 mm) fell within group II, with the exception of two wide indica lines (3.46 and 3.48 mm). PCR analysis showed that GW5 was deleted in all group II lines, including both japonica and indica subspecies. We thus conclude that the deletion is highly correlated with the grain-width phenotype among the japonica rice in group II. Our results suggest that GW5 was kept in most japonica cultivars during rice domestication and that this gene may underlie the relatively wide- and short-grain phenotype <ref name="ref6" />.
+To further confirm the identity of GW5, we randomly selected 46 rice lines, including Asominori, IR24 and 44 other cultivars, to determine the co-segregation relationship between the absence of the candidate GW5 gene and the wide-grain phenotype. The 46 rice lines were divided into two groups: group I contained 23 narrow-grain varieties (grain width ranging from 2.40 to 2.85 mm) and group II consisted of 23 wide-grain ones (3.30-3.92 mm). Segregation analysis of the Indel1 marker showed that the 775-bp target fragment was observed only in group I cultivars, but not in group II cultivars (Figure 3A and 3B). Similarly, PCR analysis with the Indel2 marker detected a 1 897-bp fragment in group I cultivars, but only a 697-bp product in group II cultivars (Figure 3C and 3D). These results suggested that the 1.2-kb fragment containing the GW5 gene was absent in all 23 wide-grain cultivars examined here but was present in the narrow-grain ones. The strict correlation between the grain-width phenotype and deletion of the 1.2-kb genomic DNA strongly supports the notion that the candidate ORF2 located on the deleted 1.2-kb fragment represents GW5. To address how this deletion in GW5 prevails in rice domestication, we further analyzed the 46 cultivars. Group I contained 21 indica species (grain width ranged from 2.40 to 2.84 mm) and two slender japonica varieties (one 2.74 mm wide, the other 2.85 mm wide), whereas most japonica (3.30-3.92 mm) fell within group II, with the exception of two wide indica lines (3.46 and 3.48 mm). PCR analysis showed that GW5 was deleted in all group II lines, including both japonica and indica subspecies. We thus conclude that the deletion is highly correlated with the grain-width phenotype among the japonica rice in group II. Our results suggest that GW5 was kept in most japonica cultivars during rice domestication and that this gene may underlie the relatively wide- and short-grain phenotype <ref name="ref6" />.
 ===GW5 is expressed in slender-grain rice===
-The sequence of 9311 (a narrow-grain indica rice cultivar) in the cognate region is similar to that in the GW5 region in CSSL28. Analysis of the indica rice genome sequence showed that GW5 represents a single copy gene without any expressed sequence tags (ESTs) or cDNA support. However, we noticed that expression of the candidate ORF was detected in the tiling microarray database of 9311 (GenBank Acc: CL971152) <ref name="ref7" />. To further confirm the expression of GW5, we performed RT-PCR analysis using mRNAs derived from the young panicle of CSSL28 (Figure 4A). We obtained RT-PCR products with primer combinations of F6 and R2, as well as with F6 and R3 (Figure 4B and Table 3). These PCR products were confirmed by sequencing analyses. Therefore, GW5 is expressed in CSSL28.
+The sequence of 9311 (a narrow-grain indica rice cultivar) in the cognate region is similar to that in the GW5 region in CSSL28. Analysis of the indica rice genome sequence showed that GW5 represents a single copy gene without any expressed sequence tags (ESTs) or cDNA support. However, we noticed that expression of the candidate ORF was detected in the tiling microarray database of 9311 (GenBank Acc: CL971152) <ref name="ref7" />. To further confirm the expression of GW5, we performed RT-PCR analysis using mRNAs derived from the young panicle of CSSL28 (Figure 4A). We obtained RT-PCR products with primer combinations of F6 and R2, as well as with F6 and R3 (Figure 4B). These PCR products were confirmed by sequencing analyses. Therefore, GW5 is expressed in CSSL28.
 ===GW5 physically interacts with polyubiquitin===
 Sequence analysis indicated that GW5 encodes a novel protein without significant homology to any proteins of known biochemical function. The protein is predicted to contain an NLS and an arginine-rich domain. To test the functionality of the predicted NLS, we fused the coding region of GW5 to the N-terminus of GFP. Transient expression in onion epidermal cells showed that the GW5-GFP fusion protein is exclusively localized to the nucleus (Figure 5).

@@ Line 26: / Line 26: @@
 ==Initial characterization of GW5==
 ===Characterization of the narrow rice line, CSSL28===
-Grain width is quantitatively inherited; therefore, it is difficult to analyze the mechanism of grainformation using conventional methods. To dissect the loci that control grain width into several single genes, 71 recombinant inbred lines (RILs) were derived from a cross between Asominori and IR24 by single-seed descent <ref name="ref4" />. Sixty-six chromosome segment substitution lines (CSSLs) with largely Asominori background, named CSSL1-66, were produced by nonselectively crossing and backcrossing 19 selected RILs with Asominori to produce the BC3F1 generation<ref name="ref5" />. One of these CSSLs, CSSL28 (narrow-grain rice), shows a slender-grain phenotype, because it harbors a chromosomal segment between RFLP (restriction fragment length polymorphism) markers C263 and R2289 (Figure 1). This segment derives from IR24 (narrow-grain rice) and is substituted into the Asominori (wide-grain rice) genomic background. CSSL28 shows a 16.4% reduction in grain width of paddy rice, and its 1000-grain weight is reduced by 18.7% compared with that of Asominori, mainly because of the reduced grain width<ref name="ref3" /> (Table 1).
+Grain width is quantitatively inherited; therefore, it is difficult to analyze the mechanism of grainformation using conventional methods. To dissect the loci that control grain width into several single genes, 71 recombinant inbred lines (RILs) were derived from a cross between Asominori and IR24 by single-seed descent <ref name="ref4" />. Sixty-six chromosome segment substitution lines (CSSLs) with largely Asominori background, named CSSL1-66, were produced by nonselectively crossing and backcrossing 19 selected RILs with Asominori to produce the BC3F1 generation<ref name="ref5" />. One of these CSSLs, CSSL28 (narrow-grain rice), shows a slender-grain phenotype, because it harbors a chromosomal segment between RFLP (restriction fragment length polymorphism) markers C263 and R2289 (Figure 1). This segment derives from IR24 (narrow-grain rice) and is substituted into the Asominori (wide-grain rice) genomic background. CSSL28 shows a 16.4% reduction in grain width of paddy rice, and its 1000-grain weight is reduced by 18.7% compared with that of Asominori, mainly because of the reduced grain width<ref name="ref3" /> .
 [[File:ma1.jpg|thumb|1000px|'''Figure 1.'''qGW5 controls grain width and weight in CSSL28. (A)Genotype of the narrow grain line, CSSL28 (a segment substitution between the RFLP markers C263 and R2289 on chromosome 5). The black bar indicates the fragment from IR24 in the Asominori genomics background. (B) Phenotype of the paddy rice grain, IR24 (top), Asominori (middle) and CSSL28 (bottom). (C) Phenotype of the brown rice grain. Scale bar: 3 mm. ]]
 ===GW5 is associated with a 1212-bp deletion in the Asominori cultivar===
-For genetic analysis and isolation of the dominant narrow-grain QTL, named GW5, the F2 population was constructed from a cross between Asominori and CSSL28, and the QTL was dissected into a single gene <ref name="ref2" />. By means of linkage analysis using the genotype data of both the GW5 gene and simple sequence repeat (SSR) markers, the GW5 gene was mapped to an interval between markers RM3328 and RMw513 in 805 homozygotes (Figure 2A). Thus, the GW5 gene was mapped to a genomic region 2.7 cM in length, located 2.3 cM from RM3328 and 0.37 cM from RMw513. In this region, three bacterial artificial chromosome (BAC) contigs were found – OJ1725_E07, OJ1097_A12 and B1007D10 (Figure 2B) – according to the Nipponbare genome (http://www.gramene.org). For high-resolution mapping of GW5, five new cleaved-amplified polymorphic sequence (CAPS) markers were developed on one of the BACs (OJ1097_A12). The GW5 locus was pinpointed into an interval between the CAPS markers Cw5 and Cw6 in 2180 wide-grain homozygous individuals from the BC4F2 population (Figure 2C and Table 2). Within this region, the 21-kb fragment of Asominori and the corresponding 22.2-kb fragments of CSSL28 and IR24 were sequenced. Further comparison of these sequences showed that the Asominori genome harbored a 1212-bp deletion(Figure 2D). Meanwhile, two Indel (insertion or deletion) markers – Indel1, including 775 bp of the 1.2 kb fragment, and Indel2, containing the aforementioned1.2 kb fragment and totaling 1897 bp in length – were designed (Table 3) and used to confirm the presence of GW5. Genetic analysis revealed that these two Indel markers co-segregated with GW5 in 2180 wide-grain progenies (Figure 2C). On the basis of the available sequence annotation (http://www.softberry.com; http://www.ncbi.nlm.nih.gov/BLAST), two open reading frames (ORFs) were predicted in the 21.0-kb target region of Asominori, and three ORFs were predicted in the corresponding region of CSSL28. However, no sequence differences were found between these lines for ORF1 and ORF3. ORF1 encodes a protein showing high similarity to ubiquitin-proteaselike protein, whereas ORF3 encodes an unknown protein containing a calmodulin-binding motif. Interestingly, we found that the 22.2-kb region of CSSL28 contains a third ORF between ORF1 and ORF3, designated ORF2, which is located in the deleted region of Asominori (Figure 2E). Motif scan analysis showed that the predicted product of ORF2 harbors a nuclear localization signal (NLS) and an arginine-rich domain (http://hits.isb-sib.ch/cgi-bin/motif-scan). These results suggest that ORF2, which is present in CSSL28 but absent in Asominori, likely corresponds to GW5.
+For genetic analysis and isolation of the dominant narrow-grain QTL, named GW5, the F2 population was constructed from a cross between Asominori and CSSL28, and the QTL was dissected into a single gene <ref name="ref2" />. By means of linkage analysis using the genotype data of both the GW5 gene and simple sequence repeat (SSR) markers, the GW5 gene was mapped to an interval between markers RM3328 and RMw513 in 805 homozygotes (Figure 2A). Thus, the GW5 gene was mapped to a genomic region 2.7 cM in length, located 2.3 cM from RM3328 and 0.37 cM from RMw513. In this region, three bacterial artificial chromosome (BAC) contigs were found – OJ1725_E07, OJ1097_A12 and B1007D10 (Figure 2B) – according to the Nipponbare genome (http://www.gramene.org). For high-resolution mapping of GW5, five new cleaved-amplified polymorphic sequence (CAPS) markers were developed on one of the BACs (OJ1097_A12). The GW5 locus was pinpointed into an interval between the CAPS markers Cw5 and Cw6 in 2180 wide-grain homozygous individuals from the BC4F2 population (Figure 2C). Within this region, the 21-kb fragment of Asominori and the corresponding 22.2-kb fragments of CSSL28 and IR24 were sequenced. Further comparison of these sequences showed that the Asominori genome harbored a 1212-bp deletion(Figure 2D). Meanwhile, two Indel (insertion or deletion) markers – Indel1, including 775 bp of the 1.2 kb fragment, and Indel2, containing the aforementioned1.2 kb fragment and totaling 1897 bp in length – were designed and used to confirm the presence of GW5. Genetic analysis revealed that these two Indel markers co-segregated with GW5 in 2180 wide-grain progenies (Figure 2C). On the basis of the available sequence annotation (http://www.softberry.com; http://www.ncbi.nlm.nih.gov/BLAST), two open reading frames (ORFs) were predicted in the 21.0-kb target region of Asominori, and three ORFs were predicted in the corresponding region of CSSL28. However, no sequence differences were found between these lines for ORF1 and ORF3. ORF1 encodes a protein showing high similarity to ubiquitin-proteaselike protein, whereas ORF3 encodes an unknown protein containing a calmodulin-binding motif. Interestingly, we found that the 22.2-kb region of CSSL28 contains a third ORF between ORF1 and ORF3, designated ORF2, which is located in the deleted region of Asominori (Figure 2E). Motif scan analysis showed that the predicted product of ORF2 harbors a nuclear localization signal (NLS) and an arginine-rich domain (http://hits.isb-sib.ch/cgi-bin/motif-scan). These results suggest that ORF2, which is present in CSSL28 but absent in Asominori, likely corresponds to GW5.
 ===The deletion prevails in wide-grain rice cultivars and defines a domestication-related rice gene===
 To further confirm the identity of GW5, we randomly selected 46 rice lines, including Asominori, IR24 and 44 other cultivars, to determine the co-segregation relationship between the absence of the candidate GW5 gene and the wide-grain phenotype (Table 4). The 46 rice lines were divided into two groups: group I contained 23 narrow-grain varieties (grain width ranging from 2.40 to 2.85 mm) and group II consisted of 23 wide-grain ones (3.30-3.92 mm) (Table 4). Segregation analysis of the Indel1 marker showed that the 775-bp target fragment was observed only in group I cultivars, but not in group II cultivars (Figure 3A and 3B). Similarly, PCR analysis with the Indel2 marker detected a 1 897-bp fragment in group I cultivars, but only a 697-bp product in group II cultivars (Figure 3C and 3D). These results suggested that the 1.2-kb fragment containing the GW5 gene was absent in all 23 wide-grain cultivars examined here but was present in the narrow-grain ones. The strict correlation between the grain-width phenotype and deletion of the 1.2-kb genomic DNA strongly supports the notion that the candidate ORF2 located on the deleted 1.2-kb fragment represents GW5. To address how this deletion in GW5 prevails in rice domestication, we further analyzed the 46 cultivars. Group I contained 21 indica species (grain width ranged from 2.40 to 2.84 mm) and two slender japonica varieties (one 2.74 mm wide, the other 2.85 mm wide), whereas most japonica (3.30-3.92 mm) fell within group II, with the exception of two wide indica lines (3.46 and 3.48 mm). PCR analysis showed that GW5 was deleted in all group II lines, including both japonica and indica subspecies. We thus conclude that the deletion is highly correlated with the grain-width phenotype among the japonica rice in group II. Our results suggest that GW5 was kept in most japonica cultivars during rice domestication and that this gene may underlie the relatively wide- and short-grain phenotype <ref name="ref6" />.