Tillering architecture directly impacts crop yield, yet the precise regulatory networks governing high-order tiller development remain largely undefined. Here, we report that the HD-ZIP III transcription factor LF1 orchestrates high-order tillering in rice. Evasion of miRNA165/166-mediated repression leads to the ectopic expression of LF1, which triggers excessive high-order tiller outgrowth. Mechanistically, LF1 forms a negative feedback loop with the core tillering regulator MOC1, wherein LF1 directly activates MOC1 transcription and enhances its protein stability to promote axillary meristem initiation, while MOC1 reciprocally triggers LF1 degradation. Concurrently, LF1 directly activates the strigolactone (SL) biosynthesis genes D10 and D27, functioning as a hormonal brake to prevent excessive tiller bud elongation. Furthermore, natural variations in the LF1 promoter strongly correlate with indica-japonica tillering differentiation. Together, our findings elucidate a homeostatic network integrating transcriptional regulation, protein stability, and hormone signaling to shape plant architecture.

Leaf angle is a key determinant of plant architecture and grain yield in rice, yet the complex genetic regulatory network remains unclear. Here, we reveal that the HD-ZIP III transcription factor LF1 mediates brassinosteroid (BR) signaling to modulate leaf angle. A miRNA165/166-resistant, gain-of-function lf1 mutant displays an enlarged leaf angle driven by elongated adaxial parenchymal cells and diminished abaxial sclerenchyma. At the transcriptional level, the BR-activated transcription factor OsBZR1 directly induces LF1, which in turn upregulates OsOFP8, forming an OsBZR1-LF1-OsOFP8 hierarchical transcriptional cascade that mediates BR signaling to regulate leaf angle development. At the post-translational level, LF1 protein stability is fine-tuned by reciprocal post-translational modifications, wherein OsMAPK6-mediated phosphorylation at threonine residue at position 148 promotes LF1 accumulation and transcriptional activity, directly counteracting its ubiquitination and degradation by the APC/CTAD1 complex. Genetic analyses demonstrate that LF1, OsMAPK6, and TAD1 function in a common pathway to regulate leaf angle development. Overall, our study establishes LF1 as a central hub coupling transcriptional and post-translational mechanisms to modulate BR-mediated leaf angle, providing targets for plant architecture improvement.

Single-segment substitution line (SSSL) platform serves not only as an ideal tool for quantitative trait locus (QTL) mapping but also as an effective strategy for achieving targeted substitution of chromosome segments across the genome in design breeding. Grain size, a critical determinant of rice yield, is generally governed by multiple QTL that primarily act by hull cell proliferation or cell expansion. However, the molecular connection between cell cycle and grain size remains poorly understood. Here, we map-cloned a novel QTL, qGL11, from the single-segment substitution line Z556 in Xihui 18 background. The long-grain allele qGL11Xinhui18 encodes OsCycT1;3, which forms dimer with OsCDKC;1 to phosphorylate RNA Pol II. This activation promotes cell cycle-related genes transcription during G1 phase, thereby maintaining normal grain cell division (proliferation index (PI) of 20.92%), and producing long-grain phenotype in Xihui 18. In contrast, a 125-bp indel of promoter variation in short-grain allele qGL11Huhan3 prolongs G1 phase, slowing the cell cycle (PI of 17.34%), and resulting in short-grains of Z556. Haplotype analysis suggests that qGL11 is a key QTL underlying the significant grain length divergence between indica (Hap II) and japonica (Hap I) subspecies. Notably, the hybrid Xidaxiang 6A/Z556 exhibited strong yield heterosis (53.6%), outperforming the control Yuxiang203. Our study elucidates that qGL11/OsCycT1;3 regulates grain length via OsCycT1;3–OsCDKC;1–RNA Pol II module to influence cell proliferation, providing a promising target for yield improvement.

As the outermost floral organ of the rice spikelet, the normal morphological development of the glume (lemma and palea) is fundamental to grain morphology, quality, and yield formation. However, the regulatory mechanisms governing rice lemma development remain incompletely understood. Here, we identified a rice mutant, drb4, which is defective in lemma polarity development and displays abnormal phenotypes including narrowed or filamentous lemmas. DRB4 is highly expressed in floral organs. It encodes a double-stranded RNA-binding protein containing three DSRM domains and localizes to both the cytoplasm and nucleus. In drb4, the synthesis of ta-siRNA, particularly tasiR-ARF, is significantly reduced, leading to the ectopic expression of their downstream target genes OsARFs on the adaxial of the lemma. Furthermore, biochemical and genetic evidence indicates that DRB4 interacts with OsDCL4 and jointly participates in the biosynthesis of tasiR-ARF. Eventually, it regulates the polarity development of the glume, especially the development of the lemma, by influencing the polarity expression of OsARFs in the glume. Our findings established a crucial DRB4-OsDCL4-tasiR-ARF-OsARF module that precisely controls rice lemma polarity, providing new insights into floral organ development.

As one of the world's most important cereals, rice (Oryza sativa L.) demands sustained yield improvement. However, this goal is challenging because yield components are complex quantitative traits governed by numerous minor-effect genes. To reveal this genetic complexity, Single Segment Substitution Lines (SSSLs) provide an ideal platform for gene identification and designed breeding. Here, we report on a Chromosome Segment Substitution Line (CSSL), Z799, containing 10 substitution segments from the restorer line R225 in the genetic background of Nipponbare. These substitution located on eight different chromosomes, with an average substitution length of 3.0 Mb. Z799 exhibited a complex yield-related phenotype relative to Nipponbare, with several traits being significantly altered. Genetic mapping in a secondary F2 population of Nipponbare/Z799 uncovered 27 QTL, but a more efficient SSSL-based approach, which yielded five lines (S1-S5), detected a total of 35 QTL. All five SSSLs significantly enhanced grain length through distinct QTL (qGL1, qGL3, qGL12-1, qGL12-2, qGL12-3) without compromising grain width. Mechanistically, microscopic observation of lemma cells revealed two divergent pathways: four QTL (qGL1, qGL3, qGL12-1, qGL12-2) increased grain length by promoting cell division, whereas qGL12-3 achieved the same effect by stimulating cell expansion. Our study thus not only identifies critical QTL for yield components but also elucidates their underlying cellular mechanisms, offering a platform for future gene cloning and designed breeding strategies.

Grain size is a pivotal factor that significantly influences grain yield. However, the genetic basis is mostly unknown. Here, we found that our previously identified wl1 mutant, which regulates leaf width development through the APC/CTAD1-WL1-NAL1 pathway, also exhibits a wide grain phenotype with increased cell expansion and proliferation in glume. Genetic analysis showed that the APC/CTAD1-WL1 module also regulates grain width in a common pathway. Further, WL1 can bind to the regulatory regions of Narrow Leaf 2 (NAL2) (a grain width positive regulatory gene) directly to repress its expression by downregulating histone acetylation levels of the chromatin to regulate grain width development. Meanwhile, we also found that WL1 participates in the cytokinin signaling pathway and regulates grain width by interacting with cytokinin B-type response regulator RRB2 and inhibiting its transcriptional activation activity on A-type response regulator OsRR6. In summary, this study established a pivotal WL1-mediated grain size regulation pathway, which is crucial for understanding grain development and improving crop yield.

Rice chromosome segment substitution lines (CSSLs) are ideal for creating natural variation and dissecting complex quantitative traits. In addition, it builds a bridge for molecular breeding and accurate identification of quantitative trait loci (QTLs). In this study, to construct an indica rice library of single-segment substitution lines (SSSLs) spanning the whole genome, a rice CSSL-Z691 carrying four substitution segments (4.07 Mb of average length) was identified by marker-assisted selection (MAS) from indica restorer line “Jinhui35” in the “Xihui18” genetic background. Compared with large panicle type Xihui18, seed setting ratio, grain width, and 1000-grain weight increased in Z691. In contrast, the number of primary branches, spikelet number per panicle, grain number per panicle, grain length, rate of length to width, and yield per plant decreased in Z691. Then, 11 QTLs were identified in the secondary F2 population from Xihui18/Z691. Again, four QTLs (qGW6, qGL4, qRLW4, and qGWT4) were validated by three SSSLs (S1–S3) developed in F3. In addition, 11 new QTLs were detected by the three SSSLs that were not identified in the F2 population. Moreover, the different QTLs in D1–D3 showed various genetic models. Some QTLs, e.g., qGWT6 (a = 0.96 g) and qGWT7 (a = −0.29 g), displayed independent inheritance, while others exhibited various epistatic interactions. Thus, it is vital to identify different QTLs and their genetic models. Resolving the epistasis effects among different QTLs is crucial for screening QTLs for breeding by design. Finally, qGL4 and qGW6 were fine-mapped to 160- and 240-kb intervals on chromosomes 4 and 6, and two candidate genes were determined by DNA sequencing. These results provide valuable genetic and breeding materials for cloning qGL4 and qGW6 and for future molecular breeding by design.

Sucrose (Suc) is transported from source leaves to sink tissues to sustain plant growth, development, and crop yield. However, the molecular mechanisms underlying carbohydrate partitioning still remain largely unclear. Here, we report a rice (Oryza sativa) mutant aberrant carbohydrate partitioning 1 (acp1), which hyperaccumulates carbohydrates in leaves and exhibits leaf chlorosis and premature senescence. ACP1 encodes a novel protein that contains two conserved domains of unknown function, DUF4220 and DUF594. Subcellular localization in rice and tobacco showed that ACP1 was localized in the endoplasmic reticulum. In situ expression analysis showed that ACP1 was mainly expressed in vascular bundles. Dye and sugar export experiments suggested that sugar trafficking through vascular tissues was impaired in the acp1 mutant. The acp1 mutant exhibits a significant cellulose deficiency in its leaves. Transmission electron microscopy experiments found that the abnormal cell wall ultrastructure in acp1. Furthermore, turgor pressure in source leaves of acp1 decreased compared with WT. Together, these results suggest that ACP1 plays a critical role in the partitioning of carbohydrates by regulating cell wall formation, which in turn affects the overall carbohydrate distribution and plant physiology.

Chromosomal segment substitution lines (CSSLs) serve as ideal materials for creating natural variation, mapping quantitative trait loci (QTL), and enabling breeding by design. In this study, Z668, a CSSL developed in the Nipponbare background with eight substituted segments from the indica restorer line R225 (average length 3.63 Mb), exhibited significant dwarfism compared to Nipponbare. Additionally, it showed a 39.9

Grain size is difficult to research due to its complex inheritance, which is usually controlled by multiple genes in traditional populations. Single segment substitution lines (SSSLs) are ideal materials for the genetic studying of these complex traits and breeding by design owing to their favorable quantitative trait loci (QTL) in the uniform genetic background. Here, we constructed a new rice (Oryza sativa L.) short-wide grain chromosomal segment substitution line Z525 with four substitution segments from R232 in the genetic background of Xihui18. Then, an F2 population from Xihui18/Z525 was used as a segregation population to map QTL for grain size by mixed linear model method. In total, four QTL were detected, and four SSSLs (S1-S4) and five dual-segment substitution lines (DSSLs, D1-D5) were developed, and within them 15 QTL (qGL5, qGW5, qRLW5, qGWT5, qGW3, qRLW3, qGWT3, qGL4, qGW4, qRLW4, qGWT4, qGL7, qGW7, qRLW7, and qGWT7) were associated with grain size. Again, the genetic models of various QTL for grain size were revealed. Interaction of qGW3 (a = 0.09 mm) and qGW4 (a = 0.04 mm) in D1 produced -0.04 mm of epistatic effect; qGW3 (a = 0.09 mm) and qGW5 (a = 0.16 mm) belonged to independent inheritance in D2. Finally, by overlapping substitution mapping and DNA sequencing, we identified a novel qGW5, different from the reported GW5 (5.73 Mb), within an estimated interval of 1.10 Mb of Chr.5. LOC_Os05g12260, LOC_Os05g12570, and LOC_Os05g13950 are possible candidates for qGW5.

Identifying quantitative trait loci (QTL) for yield traits using single-segment substitution lines (SSSL) is essential for both targeted breeding and functional analysis of key genes. Here, a wide-grain rice chromosome segment substitution line (CSSL), Z708, carrying four substitution segments from Jinhui35 in the genetic background of Xihui18, was used to identify the QTL associated with grain size. Seven QTL for yield-related traits (qGW4, qRLW4, qGWT4, qGW5, qRLW5, qGWT5, and qGPP5) were identified on the substitution segments of the fourth and fifth chromosomes of Z708. Subsequently, four SSSLs (S1-S4), which harbored 16 QTL for yield traits, were constructed using molecular marker-assisted selection. These lines (S1-S4) exhibited a significant increase in yield per plant compared to that of Xihui18. Among them, qGW4, which controls wide grains, belongs to a single dominant gene action in S1 based on the frequency distribution of grain width and chi-square test analysis. Finally, qGW4 was fine-mapped to the interval of 80-kb (minimum) and 310-kb (maximum) using both traditional fine mapping and overlapping substitution mapping of the newly constructed secondary SSSLs (S5-S8). Within this interval, four previously unreported candidate genes were predicted.

Leaves and glumes act as lateral organs and have essential effects on photosynthesis and seed morphology, thus affecting yield. However, the molecular mechanisms controlling their polarity development in rice still need further study. Here, we isolated a polarity defect of lateral organs 1(pdl1) mutant in rice, which exhibits twisted/filamentous-shaped leaves and cracked/filamentous-shaped lemmas caused by defects in polarity development. PDL1 encodes a SUPPRESSOR OF GENE SILENCING 3 protein localized in the cytoplasmic granules. PDL1 is expressed in the shoot apical meristem, inflorescence meristem, floral meristem, and lateral organs including leaves and floral organs. PDL1 is involved in the synthesis of tasiR-ARF, which may subsequently modulate the expression of OsARFs. Meanwhile, the expression levels of abaxial miR165/166 and the adaxial identity genes OSHBs were respectively increased and reduced significantly. The results of this study clarify the molecular mechanism by which PDL1-mediated tasiR-ARF synthesis regulates the lateral organ polarity development in rice.

LHC assembly is a fundamental process in forming a peripheral antenna system, which has a significant impact on photosynthesis. However, the molecular mechanism of the LHC assembly still needs to be further investigated in monocotyledonous plants. Here, we identified a bifunctional protein YGL9 in rice, a homolog of cpSRP43 in Arabidopsis, mediates LHC assembly by simultaneously regulating LHCPs transport and chlorophyll synthesis. Mutation of YGL9 exhibits a yellow-green leaf phenotype, with reduced LHCPs contents, impaired photosystem activity and reduced chlorophyll content. YGL9 interacts with cpSRP54 forming the cpSRP complex that transport LHCPs, and YGL9 also interacts with and stabilizes OsGUN4, which is an activator of MgCh and participates in the regulation of chlorophyll synthesis, to synergistically participate in chlorophyll synthesis. Further, genetic evidence demonstrates that YGL9 functions in the same pathway as cpSRP54 and OsGUN4 to regulate LHCPs transport and chlorophyll synthesis. Thus, our study reveals a cross-relationship between LHCPs transport and chlorophyll synthesis, and provides new insights into the LHC assembly process in monocotyledonous plants.

Seed size, a key determinant of rice yield, is regulated by brassinosteroid (BR); however, the BR pathway in rice has not been fully elucidated. Here, we report the cloning and characterization of the quantitative trait locus Rice Big Grain 1 (qRBG1) from single-segment substitution line Z499. Our data show that qRBG1(Z) is an unselected rare promoter variation that reduces qRBG1 expression to increase cell number and size, resulting in larger grains, whereas qRBG1 overexpression causes smaller grains in recipient Nipponbare. We demonstrate that qRBG1 encodes a non-canonical BES1 (Bri1-EMS-Suppressor1)/BZR1(Brassinazole-Resistant1) family member, OsBZR5, that regulates grain size upon phosphorylation by OsGSK2 (GSK3-like Kinase2) and binding to D2 (DWARF2) and OFP1 (Ovate-Family-Protein1) promoters. qRBG1 interacts with OsBZR1 to synergistically repress D2, and to antagonistically mediate OFP1 for grain size. Our results reveal a regulatory network controlling grain size via OsGSK2-qRBG1-OsBZR1-D2-OFP1 module, providing a target for improving rice yield.

The dynamic balance between the self-renewal and differentiation of stem cells in plants is precisely regulated by a series of developmental regulated genes that exhibit spatiotemporal-specific expression patterns. Several studies have demonstrated that the WOX family transcription factors play critical roles in maintaining the identity of stem cells in Arabidopsis thaliana. In this study, we obtained amiR-WOX9 transgenic plants, which displayed terminating prematurely of shoot apical meristem (SAM) development, along with alterations in inflorescence meristem and flower development. The phenotype of amiR-WOX9 plants exhibited similarities to that of wus-101 mutant, characterized by a stop-and-go growth pattern. It was also found that the expression of WUS in amiR-WOX9 lines was decreased significantly, while in UBQ10::WOX9-GFP transgenic plants, the WUS expression was increased significantly despite no substantial alteration in meristem size compared to Col. Therefore, these data substantiated the indispensable role of WOX9 in maintaining the proper expression of WUS. Further investigations unveiled the direct binding of WOX9 to the WUS promoter via the TAAT motif, thereby activating its expression. It was also found that WUS recognized identical the same TAAT motif cis-elements in its own promoter, thereby repress self-expression. Next, we successfully identified a physical interaction between WOX9 and WUS, and verified that it was harmful to the expression of WUS. Finally, our experimental findings demonstrate that WOX9 was responsible for the direct activating of WUS, which however was interfered by the ways of WUS binding its own promoter and the interaction of WUS and WOX9, thereby ensuring the appropriate expression pattern of WUS and then the stem cell stability. This study contributes to an enhanced comprehension of the regulatory network of the WOX9-WUS module in maintaining the equilibrium of the SAM.

Abstract Improving photosynthetic potential and light use efficiency is a crucial way to increase rice yield. The virescent-albino leaf 1 (val1) mutant, wild-type ‘Jinhui 10’ (WT), and VAL1 gene overexpression plants (VAL1-OE) were used to determine the physiological mechanisms of survival strategy in val1 mutant and improvement of photosynthetic potential in VAL1-OE through investigating leaf photosynthetic characteristics, photoprotection processes and yield. The results showed that, compared with WT, the expressions of key genes in photosynthetic pathway and chlorophyll contents in val1 mutant were both significantly lower at the early growth stage and higher at the late growth stage. Besides, the photosynthetic electron transport rate, the quantum yield of photosystem II and I, and carboxylation efficiency in val1 mutant were significantly lower than those in WT at the early growth stage, but significantly higher at the late growth stage. Furthermore, the non-photochemical quenching (NPQ) of val1 mutant was significantly higher than WT, thereby optimizing the heat dissipation pathway on the basis of reducing the chlorophyll contents and light absorption at the early growth stage. The NPQ and cyclic electron flow (CEF) were significantly higher in val1 mutant than WT at the late growth stage, resulting in the improvements of light use efficiency and photosynthetic acclimation under both low and high light conditions. Compared with WT, the expressions of key genes in photosynthetic pathway and chlorophyll contents were significantly higher in VAL1-OE. The photosynthetic electron transport rate, quantum yield of photosystem II and I, and the carboxylation efficiency in VAL1-OE were significantly higher than those in WT, improving light use efficiency and carboxylation efficiency of VAL1-OE. Overall, the val1 mutant survived by the optimal plant phenotype and leaf photoprotection pathway at the early growth stage, and improved photosynthetic potential and light use efficiency at the late growth stage. VAL1-OE improved light absorption by optimizing the dissipation pathway of excess light energy so as to increase light use efficiency and carboxylation efficiency. Cultivating phenotypic materials with high leaf area on the basis of high photosynthesis rate in VAL1-OE could be a breakthrough in high photosynthetic efficiency rice breeding.

Rice chromosomal segment substitution lines (CSSLs) are ideal materials for studying quantitative traits such as grain size. Here, a rice large-grain CSSL-Z403 was identified among progeny of the recipient Xihui18 and the donor Jinhui35 based on molecular marker-assisted selection. Z403 carried 10 substitution segments with average length of 3.01 Mb. Then, a secondary F-2 population derived from a cross between Xihui18 and Z403 was used to map quantitative trait loci (QTL) for grain size. Six QTLs distributed on chromosomes 5, 6, 7, 9 and 12 were detected. Finally four single-segment substitution lines (SSSLs) and two dual-segment substitution lines (DSSLs) carrying these target QTLs were constructed, and 10 novel QTLs were identified by four SSSLs. The large grain of Z403 was controlled at least by qGWT5, qGWT7, qGWT9 and qGWT12, and its grain weight was influenced through grain length QTL such as qGL5, qGL6, qGL9 and qGL12, as well as grain width QTL such as qGW5, qGW7, qGW9 and qGW12. Among 16 QTLs, four QTLs including qGL6, etc., might be novel compared with the reported documents. Again, positive or less negative epistatic effects between two non-allelic QTLs (additive effect > 0) may assist screening the genotype with larger grain size in further selection.

【Background】Food safety is key for ensuring national security.Rice is the staple food crop upon which people life depend.It is an important breeding target to improve its yield.Rice yield is composed of panicle number per plant,grain number per panicle and grain weight,among which grain weight relates closely to grain size and filling degree.However,these traits are controlled by multiple genes,and their genetic basis are complex.Chromosome segment substitution lines （CSSLs） can accurately dissect QTL for complex trait into a single Mendel’s factor,which is closely linked with the breeding work,so they are ideal materials for genetic research and breeding.【Objective】In the early stage,we fine-mapped a seed shattering gene SH6 using a rice chromosome segment substitution line Z481 carrying four substitution segments,However,there are still some significant differences in the panicle traits between Z481 and its recipient parent Nipponbare.It is important to understand how to distribute for these QTLs controlling panicle traits on 4 substitution segments of Z481 and then to dissect them into single segment substitution lines （SSSLs）for map-cloning of target QTL in theory meaning and for rice breeding by design in application value.【Method】Here,the secondary F 2 population constructed by crossing Nipponbare with Z481 was used to map QTL for these traits by mixed linear model （MLM）method in SAS9.3 statictic shoftware （P＜0.05）,and then by MAS method to develop SSSLs and dual-segment substitution lines（DSSLs） in F 3 derived from 42 F 2 indiviuals according to their genotypes and phynotypes.Finally,the additive effect and epistasis effect of QTL were analyzed using these SSSLs and DSSLs by ONE-WAY ANOVA,TWO-WAY ANOVA,LSD and Duncan’s multiple comparasion （P＜0.05） in IBM SPSS Statistics 25.0.【Result】12 QTLs controlling rice panicle traits are mapped from the secondary F 2 population constructed by Nipponbare/Z481,and 11 single segment substitution lines （S1-S11） and 3 dual-segment substitution lines （D1-D3） with each corresponding single substitution segment are developed.Among them,8 QTLs （q GL1,q GL3,q GL6,q G-W1,q GW3,qRLW1,q RLW3,qRLW6） can be verified by 11 SSSLs,indicating that these QTLs are genetically stable.In addition,33 QTLs such as q GL1-2,q GL1-3,q GL3-2 etc.are only detected by 11 single segment substitution lines.Among them,15QTLs such as q NSB1-1 etc.might be novel QTLs identified in the study.Furthermore,the epistasis effect between non-allelic QTLs was analyzed by three DSSLs and corresponding SSSLs,the results showed that pyramid of different QTL produce various epistasis effect.For example,the pyramid of q GL3 （a=1.26） and q GL6-2（a=0.86） yield epistasis effect of-0.77,according to the genetic model of DSSL,D2 with the genetic effect of 1.35 produce longer grain length than any of two SSSLs with q GL3 or qGL6-2;the pyramid of q GWT3-2 （a=3.18） and q GWT6-2 （a=3.39） produce epistasis effect of-5.46,making the 1000-grain weight of D2significantly smaller than that of the corresponding SSSLs due to its genetic effect of 1.11.【Conclusion】In total 45 QTLs for rice panicle traits are deteted on the 4 substitution segments of Z481 and then further dissected into 11 secondary SSSLs.SSSL have higher efficiency for QTL detection than the F 2 population.The additive effect and epistasis effect of these QTLs detected by SSSL and DSSL are necessary for breeders to predict the phenotype of the designed genotype according to these genetic informations and then to screen favorable SSSLs to breed by design.

Abstract Background Grain size is an important factor affecting yield, appearance and processing quality of rice. Therefore, it is of great significance to explore genes controlling grain size in rice. However, its genetic mechanisms are complex and belong to quantitative inheritance controlled by polygenes with minor effects. Chromosome segment substitution lines are ideal materials for natural variation creation, QTL dissection, functional analysis and pyramid breeding of favorable alleles. Results Z668 was identified containing 8 substitution segments from indica restorer line R225 in the genetic background of Nipponbare. Its average substitution length was 3.63 Mb. Compared with Nipponbare, Z668 plants exhibited significant dwarf, and the grain length, ratio of length to width and 1000-grain weight of Z668 was increased by 39.9%, 31% and 25.9%, respectively, and grain width of Z668 was decreased by 3.3%. Then, a total of 7 QTLs for grain size were identified from F2 populations derived from Nipponbare/Z668, including 3 for grain length (qGL3.4, qGL12-1, qGL12-2), 3 for ratio of length to width (qRLW3.4, qRLW12-1, qRLW12-2), and 1 for 1000-grain weight (qGWT7), which were found distributing on chromosomes 3, 7 and 12. Finally, 5 single segment substitution lines were developed and 13 QTLs (qGL1, qGL6, qGL7, qGL12-2, qGW7-1, qGW7-2, qRLW1, qRLW6, qRLW7-1, qRLW7-2, qRLW12-2, qGWT6 and qGWT7) were detected by them. Among them qGL1, qGL3.4, qGL12-1 and qGL12-2 might be new QTLs identified in the study, In addition, qGL3.4 controlling long grain displayed dominant inheritance by analysis of frequency distribution in F3 population developed by recombinant plants of single qGL3.4 locus and Chi-square test. And qGL3.4 was then fine mapped into the 300 kb region between SSR3 and SSR4 on chromosome 3 by 142 recessive plants with short-grains and 5 polymorphic SSR markers designed in the substitution interval RM5864 and RM5626 where qGL3.4 was located. By gene prediction, 7 genes were found to be associated with grain size. And through DNA amplification and sequencing as well as qRT-PCR analysis, the candidate1 and 3 were considered as preferred candidate genes for qGL3.4.

Single segment substitution line (SSSL) libraries are an ideal platform for breeding by design. To develop SSSLs-Xihui18 covering the whole genome, a novel rice chromosome segment substitution line (CSSL), Z783, carrying two substitution segments (average length of 6.55 Mb) on Chr.4 and Chr.9 was identified, which was a gap in the library previously. Z783 was developed from the progeny of recipient “Xihui18” (an indica restorer line) and donor “Huhan3” (a japonica cultivar) by advanced backcross combined molecular marker-assisted selection (MAS). It displayed multiple panicles and less spikelets and wide grains. Then, a F2 population derived from Xihui18/Z783 was used to map quantitative trait loci (QTLs) for yield-related traits by the mixed linear model method. Nine QTLs were detected (p < 0.05). Furthermore, three SSSLs were constructed by MAS, and all 9 QTLs could be validated, and 15 novel QTLs could be detected by these SSSLs by a one-way ANOVA analysis. The genetic analysis showed that qSSP4 for less spikelets and qGW9 for wide grain all displayed dominant gene action in their SSSLs. Finally, qSSP4 and qGW9 were fine-mapped to intervals of 2.75 Mb and 1.84 Mb, on Chromosomes 4 and 9, respectively. The results lay a solid foundation for their map cloning and molecular breeding by design.