Verification and dissection of one quantitative trait locus for grain size and weight on chromosome 1 in rice

Grain size and weight are the key traits determining rice quality and yield and are mainly controlled by quantitative trait loci (QTL). In this study, one minor QTL that was previously mapped in the marker interval of JD1009-JD1019 using the Huanghuazhan/Jizi1560 (HHZ/JZ1560) recombinant inbred line (RIL) population, qTGW1-2, was validated to regulate grain size and weight across four rice-growing seasons using twenty-one near isogenic line (NIL)-F2 populations. The twenty-one populations were in two types of genetic background that were derived from the same parents HHZ and JZ1560. Twelve F9, F10 or F11 NIL-F2 populations with the sequential residual heterozygous regions covering JD1009-RM6840 were developed from one residual heterozygote (RH) in the HHZ/JZ1560 RIL population, and the remaining nine BC3F3, BC3F4 or BC3F5 NIL-F2 populations with the sequential residual heterozygous regions covering JD1009-RM6840 were constructed through consecutive backcrosses to the recurrent parent HHZ followed with marker assistant selection in each generation. Based on the QTL analysis of these genetic populations, qTGW1-2 was successfully confirmed to control grain length, width and weight and further dissected into two QTLs, qTGW1-2a and qTGW1-2b, which were respectively narrowed down to the marker intervals of JD1139-JD1127 (~ 978.2-kb) and JD1121-JD1102 (~ 54.8-kb). Furthermore, the two types of NIL-F2 populations were proved to be able to decrease the genetic background noise and increase the detection power of minor QTL. These results provided an important basis for further map-based cloning and molecular design breeding with the two QTLs in rice.


Results
Validation and dissection of qTGW1-2 using ten RH-derived NIL-F 2 populations. Descriptive statistics of TGW, GL and GW in the ten RH-derived populations in F 9 and F 10 are shown in Table 1. Three traits in all the ten populations showed continuous distributions with low skewness and kurtosis, which suggested the typical phenotypic distributions of quantitative traits.
Based on the genotype and phenotype data, qTGW1-2 for TGW, GL and GW was identified in all the three populations, and the enhancing alleles for the three traits in all the three populations were all derived from JZ1560 (Table 1). In YC1-1, qTGW1-2 showed the additive effects of 1.66 g, 0.11 mm and 0.06 mm for TGW, GL and GW, and explained 19.3%, 24.4% and 33.9% of phenotypic variations (R 2 ). In YC1-2, the additive effects for TGW, GL and GW were 1.59 g, 0.11 mm and 0.04 mm, explaining 23.8%, 26.3% and 18.9% of phenotypic variations. Similar additive effects in the same direction and similar R 2 values confirmed the existence of qTGW1-2 in the common segregating region of YC1-1 and YC1-2. Compared with the additive effects for TGW in YC1-1 and YC1-2, a relatively smaller additive effect of 0.92 g was detected in the remaining population YC1-3. We further compared the segregating regions of JD1009-RM6840, JD1009-JD1090 and JD1018-RM6840 in YC1-1, YC1-2 and YC1-3 populations. The segregating regions of YC1-1 and YC1-2 covered all or part of the segregating region of YC1-3. These results suggested that there might be two or more QTLs with the same direction of additive effects in the primary interval of qTGW1-2.
To confirm our conjecture, the segregating region responsible for qTGW1-2 should be subdivided into smaller heterozygous segments. Seven recombinant plants with sequential segregating regions covering the interval JD1009-RM6840 were selected from YC1-1, YC1-2 and YC1-3 populations and selfed to develop F 10 RH-derived F 2 populations, namely YC2-1, YC2-2, YC2-3, YC2-4, YC2-5, YC2-6 and YC2-7 (Table 2; Fig. 1). The seven F 10 populations carried smaller segregating regions. TGW, GL and GW of each plant in the seven populations were measured and showed continuous segregations in each population (Table 1). Combined the genotype and phenotype information of each plant in the seven populations, seven segmental linkage maps were constructed (Fig. 2b). QTL analysis results for TGW, GL and GW using these populations are shown in Table 3. Except for YC2-4, QTLs responsible for TGW, GL and GW were detected in the remaining six populations. Since YC2-1 and YC2-2 showed the common segregating region (JD1139-JD1022) and no overlapping segregating region with YC2-5 and YC2-6, these results indicated that there should be two QTLs in the target interval of qTGW1-2. Furthermore, the segregating region of YC2-3 overlapped with the common segregating region (JD1139-JD1022), and similar additive effects for TGW, GL and GW were observed in YC1-1, YC1-2 and YC1-3. The result indicated that one QTL controlling TGW, GL and GW was located in the common segregating region of YC2-1, YC2-2 and YC2-3 flanked by JD1139 and JD1062, corresponding to a 1.2-Mb region in the Nipponbare genome. We designated this QTL as qTGW1-2a. Due to the fact that no QTL for all the three traits detected in YC2-4, the other QTL for TGW, GL and GW should be located in the common segregating region of YC2-5, YC2-6 and YC2-7 but outside the segregating region of YC2-4, with the interval flanked by JD1052 and JD1102, corresponding to a 235.1-kb region in the Nipponbare genome. We named this QTL as qTGW1-2b.
Validation and dissection of qTGW1-2 using seven NIL-F 2 populations in BC 3 F 3 and BC 3 F 4 . For further validation and dissection of qTGW1-2, we conducted another experiment using advanced backcross populations with the genetic background of HHZ. Three BC 3 F 3 and four BC 3 F 4 populations with the sequential segregating regions covering the interval JD1009-RM6840 were established and planted in three rice-growing seasons ( Table 2; Fig. 1). For each plant of the seven populations, TGW, GL and GW were measured, and the distribution tendencies of the three traits are shown in Fig. 3 www.nature.com/scientificreports/ tions in all the populations, but differentiation between the HHZ and JZ1560 homozygous genotypes was also observed. Except for in YD2-2 population, concentrations of the HHZ and JZ1560 homozygous plants were obviously distributed in the low-and high-value areas for TGW, GL and GW in YD1-1, YD1-2, YD1-3, YD2-1, YD2-3 and YD2-4 populations, which suggested qTGW1-2 was segregated in these populations (Fig. 3). Similar frequency distribution of the three traits of the HHZ and JZ1560 homozygous plants was observed in YD2-2 population.
Combined the genotype and phenotype information of each plant in the seven populations, seven segmental linkage maps were constructed (Fig. 4). Results of QTL analysis for TGW, GL and GW using YD1-1, YD1-2 and YD1-3 populations were presented in Table 4. Significant QTL effects for the three traits were observed in all the three BC 3 F 3 populations. For TGW, GL and GW, the enhancing alleles were derived from JZ1560 in all the three populations. In YD1-1, the additive effects were 0.54 g for TGW, 0.15 mm for GL and 0.02 mm for GW, explaining 22.8%, 38.6% and 16.9% of the phenotypic variances, respectively. In YD1-2, the additive effects were 0.87 g for TGW, 0.18 mm for GL and 0.02 mm for GW, explaining 45.6%, 39.6% and 13.6% of the phenotypic variations, respectively. In YD1-3, the additive effects were 0.72 g for TGW, 0.08 mm for GL and 0.03 mm for GW, explaining 31.4%, 13.4% and 22.7% of the phenotypic variances, respectively. The segregating region of YD1-2 overlapped the segregating regions of YD1-1 and YD1-3, and larger additive effects for TGW and GL www.nature.com/scientificreports/ were detected in YD1-2 than that in YD1-1 and YD1-3 populations (Fig. 4a). The results suggested there might be more than one QTL in the whole segregating region, which is consistent with the results of QTL analysis in the three F 9 RH-derived populations. For further delimitation of qTGW1-2, we conducted QTL analysis of four BC 3 F 4 populations with sequential segregating regions covering the interval JD1009-RM6840, which were developed from four recombinant plants in the YD1-1, YD1-2 and YD1-3 populations (Table 4). Significant QTL effects were detected in YD2-1, YD2-3 and YD2-4 but not in YD2-2. In YD2-1, the additive effects were 0.40 g for TGW, 0.07 mm for GL and 0.02 mm for GW, explaining 24.2%, 15.0% and 12.3% of the phenotypic variances, respectively. In view of nonsignificant QTL effects detected in YD2-2, the YD2-1 population was segregated for qTGW1-2a only. Thus, for the region of qTGW1-2a, the segregating region of YD2-2 was excluded and the cross-over region on the left of the InDel marker JD1127 should be included. In YD2-3 and YD2-4, significant QTL effects were identified for all the three traits. The enhancing alleles were all derived from JZ1560 with the additive effects of 0.52 g and 0.47 g for TGW, 0.06 mm and 0.04 mm for GL, 0.02 mm and 0.03 mm for GW in YD2-3 and YD2-4 populations, respectively. Considering that the similar additive effects of the three traits were detected in YD2-3 and YD2-4, we confirmed that the QTL qTGW1-2b should be located in the common segregating region and two cross-over regions flanked by JD1023 and RM6840 (Fig. 4b), which is in corresponding to the results of QTL analysis in the seven F 10 RH-derived populations.
Fine-mapping of qTGW1-2b using four newly developed NIL-F 2 populations. For further delimitation of qTGW1-2b, two RH-derived populations in F 11 and two backcross populations in BC 3 F 5 with sequential segregating regions covering the interval JD1117-RM6840 were developed from the last generation (Table 2; Fig. 1). Descriptive statistics of TGW, GL and GW in the two RH-derived populations in F 11 are shown in Table 5. Three traits showed the same continuous distribution with low skewness and kurtosis as the previous RH-derived populations. Similarly, the distribution tendencies of the three traits are descripted in Fig. 5. The plants with HHZ and JZ1560 homozygous genotypes concentrated to low-and high-value areas, which indicated that qTGW1-2b was segregated in the two BC 3 F 5 populations. Table 1. Phenotypic performance of TGW, GL and GW in ten RH-derived populations in F 9 or F 10. TGW, 1000-grain weight (g); GL, grain length (mm); GW, grain width (mm). www.nature.com/scientificreports/ Genotypic compositions of the two F 11 and two BC 3 F 5 populations in the segregating regions are shown in Fig. 6. The segregating region covered the whole original interval of qTGW1-2b. QTL analysis results for TGW, GL and GW using these populations are shown in Table 6. Significant QTL effects were detected in YD3-1 and YD3-2 but not in YC3-1 and YC3-2. In YD3-1, the additive effects were 0.46 g for TGW, 0.06 mm for GL and 0.02 mm for GW, explaining 36.7%, 29.1% and 20.3% of the phenotypic variances, respectively. In YD3-2, the additive effects for TGW, GL and GW were 0.46 g, 0.05 mm and 0.03 mm, explaining 44.1%, 16.3% and 30.6% of phenotypic variations, respectively. Similar additive effects with the enhancing alleles derived from JZ1560 in the same direction comfirmed the existence of qTGW1-2b. In view of non-significant QTL effects detected in YC3-1 and YC3-2, qTGW1-2b was delimited to the common segregating region of YD3-1 and YD3-2 flanked by JD1121 and JD1102 which corresponded to a 54.8-kb region in the Nipponbare genome.
Candidate gene analysis of qTGW1-2b. According to Rice Genome Annotation Project (http:// rice. plant biolo gy. msu. edu), there are nine candidate genes in the 54.8-kb region for qTGW1-2b. Gene ID and products are listed in Table 7. Based on 100× whole genome re-sequencing of HHZ and JZ1560 23 , we found five genes showing polymorphic sites between the two parents in the target 54.8-kb region. Among them, two genes showed 3-bp or 5-bp deletion in the UTR of the genes whereas one gene showed a 5-bp deletion in the intron. Only two genes showed polymorphic sites in the coding region between the two parents. LOC_Os01g72480 showed a 9-bp insertion in the first exon in JZ1560, which encodes a C3HC4-type RING finger E3 ubiquitin ligase of the RING/U-box superfamily, whereas LOC_Os01g72500 encoding a retrotransposon protein showed a 3-bp deletion in the first exon in JZ1560. According to the 9-bp sequence difference in LOC_Os01g72480, one InDel marker JD1160 was designed to genotype YC3-1, YC3-2, YD3-1 and YD3-2 segregating populations. As shown in Fig. 6, genotypes of YD3-1 and YD3-2 that showed significant additive effects were heterozygous at the polymorphic site of JD1160. In the segregating populations, the JZ1560 homozygous plants with 9-bp insertion in the first exon of LOC_Os01g72480 were obviously distributed in the high-value areas for all the three grain traits, whereas the HHZ homozygous plants were distributed in the low-value areas (Fig. 5), indicating that the 9-bp insertion might result in the phenotypic viriations of TGW, GL and GW.

Discussion
In the past decade, significant progress has been achieved in the isolation and functional characterization of QTLs for yield traits in rice, especially for grain size and weight 30,31,35 . To date, more than 500 QTLs for rice grain weight distributing throughout the 12 chromosomes (http:// www. grame ne. org) have been identified in the primary mapping. Only 20 of them with major effects for grain size and weight have been cloned, which distributed on the eight chromosomes except for chromosomes 1, 10, 11 and 12. Majority of QTLs controlling grain size and www.nature.com/scientificreports/ weight show minor effects and are difficult to be cloned. In the present study, one minor QTL for grain weight and GL, qTGW1-2, was validated and dissected. No QTL for grain size and weight has been validated or cloned in the target region of qTGW1-2. qTGW1-2 is a stably expressed QTL with minor effects for TGW, GL and GW. In this study, the minor effects of qTGW1-2 on TGW, GL and GW were stably observed across different populations, generations, rice-growing seasons and locations. Although the values of additive effects appeared to be changed in different experiments, the enhancing allele was derived from JZ1560 and the direction of additive effects remained unchanged for all the three grain traits. qTGW1-2 was further dissected into qTGW1-2a and qTGW1-2b. Due to the clustering distribution of QTLs controlling grain size and weight, genetic dissection of QTL regions into different QTLs has been frequently reported [36][37][38][39][40] . Based on the results of QTL analysis using seven RH-derived populations in www.nature.com/scientificreports/ F 10 and four BC 3 F 4 populations, qTGW1-2 was dissected into two QTLs (qTGW1-2a and qTGW1-2b), which were responsible for all the three grain traits. Compared the target intervals of these two QTLs in F 10 RH-derived populations and BC 3 F 4 populations, qTGW1-2a was delimited to the marker interval flanked by JD1139 and JD1127 (~ 978.2-kb), and qTGW1-2b was narrowed down into a ~ 186.0 kb region flanked by JD1023 and JD1102. Subsequently, two RH-derived populations in F 11 and two BC 3 F 5 populations were constructed, and qTGW1-2b was successfully fine-mapped to an accurate region flanked by JD1121 and JD1102, corresponding to a 54.8-kb region in the Nipponbare genome. According to 100 × whole genome re-sequencing of HHZ and JZ1560, only two genes showed insertion/deletion polymorphic sites in the exons. LOC_Os01g72480 encoding a C3HC4-type RING finger E3 ubiquitin ligase of the RING/U-box superfamily, showed a 9-bp insertion in the first exon of JZ1560 compared to that of HHZ. It was reported that ubiquitin pathway is one of the most important regulatory pathways for seed development 35 . LOC_Os01g72500 showed a 3-bp deletion in the first exon and encodes a retrotransposon protein. No related evidence was reported that LOC_Os01g72500 involves in seed development. Therefore, LOC_Os01g72480 might be the possible candidate gene to regulate grain size and weight.
NIL-F 2 populations developed from RHs of the RIL population and advanced backcross individuals are ideal materials for identifying minor QTLs. In this study, these two types of populations were both constructed to validate and dissect the minor QTL, qTGW1-2. In our previous primary mapping, qTGW1-2 was detected to be a pleiotropic QTL for TGW and GL because qTGW1-2 for TGW and qGL1-2 for GL were mapped in the same marker interval JD1009-JD1019 across two years in the HHZ/JZ1560 RIL population. No significant QTL effect for GW was observed in the target interval of qTGW1-2. Compared with the QTL effects of qTGW1-2 in the primary mapping RIL population, qTGW1-2 showed small but significant additive effects on GW besides TGW and GL in the NIL-F 2 populations derived from both RHs and advanced backcross individuals. Furthermore, qTGW1-2 explained much higher phenotypic variation in NIL-F 2 populations. For TGW, qTGW1-2 explained 9.20%-10.72% of phenotypic variations in the HHZ/JZ1560 RIL population. In the present study, qTGW1-2 Table 3. QTLs detected for TGW, GL, and GW in ten RH-derived populations in F 9 and F 10. TGW, 1000-grain weight (g); GL, grain length (mm); GW, grain width (mm); A, additive effect of replacing a HHZ allele with a JZ1560 allele; D, dominance effect; R 2 , proportion of phenotypic variance explained by the QTL effect; ns, no significance. www.nature.com/scientificreports/ accounted for 17.0%-23.8% and 22.8%-31.4% of the total phenotypic variations in RH-derived F 9 populations and BC 3 F 3 populations, respectively. These results indicated that elimination of genetic background noise in the NIL-F 2 population increased the detection power of minor QTLs. Compared to the cloned QTLs, both qTGW1-2a and qTGW1-2b showed relatively smaller effects, which might increase the difficulty in map-based cloning of them. The most likely candidate gene of qTGW1-2b, LOC_Os01g72480 encoding an E3 ubiquitin ligase might control grain size and weight through the www.nature.com/scientificreports/ ubiquitin-proteasome pathway. In rice, GW2 and WTG1 encoding an E3 ubiquitin ligase and a deubiquitinase respectively also regulate grain size by ubiquitin regulatory pathway 9,41 . In this study, we successfully delimited qTGW1-2a and qTGW1-2b to the marker intervals of JD1139-JD1127 (~ 978.2-kb) and JD1121-JD1102 (~ 54.8kb) at the end of chromosome 1, respectively. The closely linked InDel markers, such as JD1160, JD1140 and JD1102, could be used in the marker assistant selection of qTGW1-2a and qTGW1-2b to improve rice varieties. Compared with the major QTLs, the breeders are hard to select the varieties which carry minor QTLs by the phenotypic identification. However, benefited from the development of gene markers or tightly linked markers, molecular marker assistant selection of the minor QTLs can be performed with high accuracy and efficiency in the early stage of breeding.

Data analysis.
Genetic maps of each population were constructed using Mapmaker/Exp 3.0, in which genetic distances between markers were presented in centiMorgans (cM) derived with Kosambi function. QTL analysis was performed with Windows QTL Cartographer 2.5, and the LOD value of 3.0 was taken as the threshold value.
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Conclusions
A total of 21 NIL-F 2 populations were constructed to validate and dissect a minor and stably expressed QTL, qTGW1-2. Based on the QTL analysis of these genetic populations, qTGW1-2 was successfully validated to control grain length, width and weight with the enhancing allele derived from JZ1560. Furthermore, qTGW1-2 was further dissected into two QTLs, qTGW1-2a and qTGW1-2b, which were respectively narrowed down to Table 6. QTLs detected for TGW, GL, and GW in two RH-derived populations in F 11 and two backcross populations in BC 3 F 5. TGW, 1000-grain weight (g); GL, grain length (mm); GW, grain width (mm); A, additive effect of replacing a HHZ allele with a JZ1560 allele; D, dominance effect; R 2 , proportion of phenotypic variance explained by the QTL effect; ns, no significance.  Table 7. Candidate genes in the target region of qTGW1-2b.

LOC_Os01g72440
Hypothetical protein None

DNA binding protein None
LOC_Os01g72460 NADPH quinone oxidoreductase None

LOC_Os01g72500
Retrotransposon protein 3-bp deletion in the first exon

LOC_Os01g72510
Eukaryotic aspartyl protease domain containing protein None

LOC_Os01g72520
Phosphoesterase family protein 5-bp deletion in the first intron