The APC/CTAD1-WIDE LEAF 1-NARROW LEAF 1 pathway controls leaf width in rice.

Leaf morphology is one of the most important features of the ideal plant architecture. However, the genetic and molecular mechanisms controlling this feature in crops remain largely unknown. Here, we characterized the rice (Oryza sativa) wide leaf 1 (wl1) mutant, which has wider leaves than the wild-type due to more vascular bundles and greater distance between small vascular bundles. WL1 encodes a Cys-2/His-2-type zinc finger protein that interacts with Tillering and Dwarf 1 (TAD1), a co-activator of the anaphase-promoting complex/cyclosome (APC/C) (a multi-subunit E3 ligase). The APC/CTAD1 complex degrades WL1 via the ubiquitin-26S proteasome degradation pathway. Loss-of-function of TAD1 resulted in plants with narrow leaves due to reduced vascular bundle numbers and distance between the small vascular bundles. Interestingly, we found that WL1 negatively regulated the expression of a narrow leaf gene, NARROW LEAF 1 (NAL1), by recruiting the co-repressor TOPLESS-RELATED PROTEIN and directly binding to the NAL1 regulatory region to inhibit its expression by reducing the chromatin histone acetylation. Furthermore, biochemical and genetic analyses revealed that TAD1, WL1, and NAL1 operated in a common pathway to control the leaf width. Our study establishes an important framework for understanding the APC/CTAD1-WL1-NAL1 pathway-mediated control of leaf width in rice, and provides insights for improving crop plant architecture.

NONSTOP GLUMES1 Encodes a C2H2 Zinc Finger Protein That Regulates Spikelet Development in Rice.

The spikelet is an inflorescence structure unique to grasses. The molecular mechanisms underlying spikelet development and evolution are unclear. In this study, we characterized three allelic recessive mutants in rice (Oryza sativa): nonstop glumes 1-1 (nsg1-1), nsg1-2, and nsg1-3 In these mutants, organs such as the rudimentary glume, sterile lemma, palea, lodicule, and filament were elongated and/or widened, or transformed into lemma- and/or marginal region of the palea-like organs. NSG1 encoded a member of the C2H2 zinc finger protein family and was expressed mainly in the organ primordia of the spikelet. In the nsg1-1 mutant spikelet, LHS1 DL, and MFO1 were ectopically expressed in two or more organs, including the rudimentary glume, sterile lemma, palea, lodicule, and stamen, whereas G1 was downregulated in the rudimentary glume and sterile lemma. Furthermore, the NSG1 protein was able to bind to regulatory regions of LHS1 and then recruit the corepressor TOPLESS-RELATED PROTEIN to repress expression by downregulating histone acetylation levels of the chromatin. The results suggest that NSG1 plays a pivotal role in maintaining organ identities in the spikelet by repressing the expression of LHS1, DL, and MFO1.

Genetic Effects Analysis of QTLs for Rice Grain Size Based on CSSL-Z403 and Its Dissected Single and Dual-Segment Substitution Lines.

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 F2 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.

Development of Single-Segment Substitution Lines and Fine-Mapping of qSPP4 for Spikelets Per Panicle and qGW9 for Grain Width Based on Rice Dual-Segment Substitution Line Z783.

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.

SHORT-ROOT 1 is critical to cell division and tracheary element development in rice roots.

The exocyst is a key factor in vesicle transport and is involved in cell secretion, cell growth, cell division and other cytological processes in eukaryotes. EXO70 is the key exocyst subunit. We obtained a gene, SHORT-ROOT 1 (SR1), through map-based cloning and genetic complementation. SR1 is a conserved protein with an EXO70 domain in plants. SR1 mutation affected the whole root-development process: producing shorter radicles, adventitious roots and lateral roots, and demonstrating abnormal xylem development, resulting in dwarfing and reduced water potential and moisture content. SR1 was largely expressed in the roots, but only in developing root meristems and tracheary elements. The shortness of the sr1 mutant roots was caused by the presence of fewer meristem cells. The in situ histone H4 expression patterns confirmed that cell proliferation during root development was impaired. Tracheary element dysplasia was caused by marked decreases in the inner diameters of and distances between the perforations of adjacent tracheary elements. The membrane transport of sr1 mutants was blocked, affecting cell division in the root apical region and the development of root tracheary elements. The study of SR1 will deepen our understanding of the function of EXO70 genes in Oryza sativa (rice) and guide future studies on the molecular mechanisms involved in plant root development.

Identification, pyramid, and candidate gene of QTL for yield-related traits based on rice CSSLs in indica Xihui18 background.

Chromosome segment substitution line (CSSL) is important for functional analysis and design breeding of target genes. Here, a novel rice CSSL-Z431 was identified from indica restorer line Xihui18 as recipient and japonica Huhan3 as donor. Z431 contained six segments from Huhan3, with average substitution length of 2.12 Mb. Compared with Xihui18, Z431 increased panicle number per plant (PN) and displayed short-wide grains. The short-wide grain of Z431 was caused by decreased length and increased width of glume cell. Then, thirteen QTLs were identified in secondary F2 population from Xihui18/Z431. Again, eleven QTLs (qPL3, qPN3, qGPP12, qSPP12, qGL3, qGW5, qRLW2, qRLW3, qRLW5, qGWT3, qGWT5-2) were validated by six single-segment substitution lines (SSSLs, S1-S6) developed in F3. In addition, fifteen QTLs (qPN5, qGL1, qGL2, qGL5, qGW1, qGW5-1, qRLW1, qRLW5-2, qGWT1, qGWT2, qYD1, qYD2, qYD3, qYD5, qYD12) were detected by these SSSLs, while not be identified in the F2 population. Multiple panicles of Z431 were controlled by qPN3 and qPN5. OsIAGLU should be the candidate gene for qPN3. The short-wide grain of Z431 was controlled by qGL3, qGW5, etc. By DNA sequencing and qRT-PCR analysis, two best candidate genes for qGL3 and qGW5 were identified, respectively. In addition, pyramid of different QTLs in D1-D3 and T1-T2 showed independent inheritance or various epistatic effects. So, it is necessary to understand all genetic effects of target QTLs for designing breeding. Furthermore, these secondary substitution lines improved the deficiencies of Xihui18 to some extent, especially increasing yield per plant in S1, S3, S5, D1-D3, T1, and T2. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01284-x.

The CC-NB-LRR OsRLR1 mediates rice disease resistance through interaction with OsWRKY19.

Nucleotide-binding site-leucine-rich repeat (NB-LRR) resistance proteins are critical for plant resistance to pathogens; however, their mechanism of activation and signal transduction is still not well understood. We identified a mutation in an as yet uncharacterized rice coiled-coil (CC)-NB-LRR, Oryza sativa RPM1-like resistance gene 1 (OsRLR1), which leads to hypersensitive response (HR)-like lesions on the leaf blade and broad-range resistance to the fungal pathogen Pyricularia oryzae (syn. Magnaporthe oryzae) and the bacterial pathogen Xanthomonas oryzae pv. oryzae, together with strong growth reduction. Consistently, OsRLR1-overexpression lines showed enhanced resistance to both pathogens. Moreover, we found that OsRLR1 mediates the defence response through direct interaction in the nucleus with the transcription factor OsWRKY19. Down-regulation of OsWRKY19 in the rlr1 mutant compromised the HR-like phenotype and resistance response, and largely restored plant growth. OsWRKY19 binds to the promoter of OsPR10 to activate the defence response. Taken together, our data highlight the role of a new residue involved in the NB-LRR activation mechanism, allowing identification of a new NB-LRR downstream signalling pathway.

LF1 regulates the lateral organs polarity development in rice.

The patterning of adaxial-abaxial tissues plays a vital role in the morphology of lateral organs, which is maintained by antagonism between the genes that specify adaxial and abaxial tissue identity. The homeo-domain leucine zipper class III (HD-ZIP III) family genes regulate adaxial identity; however, little information is known about the physical interactions or transcriptionally regulated downstream genes of HD-ZIP III. In this study, we identified a dominant rice mutant, lateral floret 1 (lf1), which has defects in lateral organ polarity. LF1 encodes the HD-ZIP III transcription factor, which expressed in the adaxial area of lateral organs. LF1 can activate directly the expression of LITTLE ZIPPER family gene OsZPR4 and HD-ZIP II family gene OsHOX1, and OsZPR4 and OsHOX1 respectively interact with LF1 to form a heterodimer to repress the transcriptional activity of LF1. LF1 influences indole-3-acetic acid (IAA) content by directly regulating the expression of OsYUCCA6. Therefore, LF1 forms negative feedback loops between OsZPR4 and OsHOX1 to affect IAA content, leading to the regulation of lateral organs polarity development. These results reveal the cross-talk among HD-ZIP III, LITTLE ZIPPER, and HD-ZIP II proteins and provide new insights into the molecular mechanisms underlying the polarity development of lateral organs.

LATERAL FLORET 1 induced the three-florets spikelet in rice.

The spikelet is a unique inflorescence structure in grass. The molecular mechanisms behind the development and evolution of the spikelet are far from clear. In this study, a dominant rice mutant, lateral florets 1 (lf1), was characterized. In the lf1 spikelet, lateral floral meristems were promoted unexpectedly and could generally blossom into relatively normal florets. LF1 encoded a class III homeodomain-leucine zipper (HD-ZIP III) protein, and the site of mutation in lf1 was located in a putative miRNA165/166 target sequence. Ectopic expression of both LF1 and the meristem maintenance gene OSH1 was detected in the axil of the sterile lemma primordia of the lf1 spikelet. Furthermore, the promoter of OSH1 could be bound directly by LF1 protein. Collectively, these results indicate that the mutation of LF1 induces ectopic expression of OSH1, which results in the initiation of lateral meristems to generate lateral florets in the axil of the sterile lemma. This study thus offers strong evidence in support of the "three-florets spikelet" hypothesis in rice.

Association between sheath blight resistance and chitinase activity in transgenic rice plants expressing McCHIT1 from bitter melon.

No usable resources with high-level resistance to sheath blight (SB) have yet been found in rice germplasm resources worldwide. Therefore, creating and breeding new disease-resistant rice resources with sheath blight resistance (SBR) are imperative. In this study, we inoculated rice plants with hyphae of the highly pathogenic strain RH-9 of rice SB fungus Rhizoctonia solani to obtain eight stable transgenic rice lines harbouring the chitinase gene (McCHIT1) of bitter melon with good SBR in the T5 generation. The mean disease index for SB of wild-type plants was 92% and 37-44% in transgenic lines. From 24 h before until 120 h after inoculation with R. solani, chitinase activity in stable transgenic plants with increased SBR was 2.0-5.5 and 1.8-2.7 times that of wild-type plants and plants of a disease-susceptible stable transgenic line, respectively. The correlation between SBR and chitinase activity in McCHIT1-transgenic rice line plants was significant. This work stresses how McCHIT1 from bitter melon can be used to protect rice plants from SB infection.

Mutation of the OsSAC1 Gene, which Encodes an Endoplasmic Reticulum Protein with an Unknown Function, Causes Sugar Accumulation in Rice Leaves.

Sugars are the most abundant organic compounds produced by plants, and can be used to build carbon skeletons and generate energy. The sugar accumulation 1 (OsSAC1) gene encodes a protein with an unknown function that exhibits four N-terminal transmembrane regions and two conserved domains of unknown function, DUF4220 and DUF594. OsSAC1 was found to be poorly and specifically expressed at the bottoms of young leaves and in the developing leaf sheaths. Subcellular location results showed that OsSAC1 was co-localized with ER:mCherry and targeted the endoplasmic reticulum (ER). OsSAC1 has been found to affect sugar partitioning in rice (Oryza sativa). I2/KI starch staining, ultrastructure observations and starch content measurements indicated that more and larger starch granules accumulated in ossac1 source leaves than in wild-type (WT) source leaves. Additionally, higher sucrose and glucose concentrations accumulated in the ossac1 source leaves than in WT source leaves, whereas lower sucrose and glucose concentrations were observed in the ossac1 young leaves and developing leaf sheaths than in those of the WT. Much greater expression of OsAGPL1 and OsAGPS1 (responsible for starch synthesis) and significantly less expression of OscFBP1, OscFBP2, OsSPS1 and OsSPS11 (responsible for sucrose synthesis) and OsSWEET11, OsSWEET14 and OsSUT1 (responsible for sucrose loading) occurred in ossac1 source leaves than in WT source leaves. A greater amount of the rice plasmodesmatal negative regulator OsGSD1 was detected in ossac1 young leaves and developing leaf sheaths than in those of the WT. These results suggest that ER-targeted OsSAC1 may indirectly regulate sugar partitioning in carbon-demanding young leaves and developing leaf sheaths.

VIRESCENT-ALBINO LEAF 1 regulates leaf colour development and cell division in rice.

The de novo synthesis of purine nucleotides is crucial to all living organisms, but limited information is available for plants. In this study, we isolated a virescent-albino leaf 1 (val1) mutant of rice (Oryza sativa) that produces dynamic green-revertible albino and narrow-leaf phenotypes. In albino leaves, chloroplast development was defective, pigment contents were reduced, and cell division was impaired compared with the wild-type. Map-based cloning revealed that VAL1 encodes a phosphoribosylamine-glycine ligase (PurD), the second enzyme in the de novo purine biosynthesis pathway. Subcellular localization analysis demonstrated that VAL1 was localized in the chloroplast. Our results demonstrate that VAL1 is a crucial enzyme in the de novo purine biosynthesis pathway and is involved in regulating chloroplast development, chlorophyll metabolism, and cell division during leaf development in rice.

ABNORMAL VASCULAR BUNDLES regulates cell proliferation and procambium cell establishment during aerial organ development in rice.

To understand the molecular mechanisms of rice aerial organ development, we identified a mutant gene that caused a significant decrease in the width of aerial organs, termed ABNORMAL VASCULAR BUNDLES (AVB). Histological analysis showed that the slender aerial organs were caused by cell number reduction. In avb, the number of vascular bundles in aerial organs was reduced, whereas the area of the vascular bundles was increased. Ploidy analysis and the in situ expression patterns of histone H4 confirmed that cell proliferation was impaired during lateral primordia development, whereas procambium cells showed a greater ability to undergo cell division in avb. RNA sequencing (RNA-seq) showed that the development process was affected in avb. Map-based cloning and genetic complementation demonstrated that AVB encodes a land plant conserved protein with unknown functions. Our research shows that AVB is involved in the maintenance of the normal cell division pattern in lateral primordia development and that the AVB gene is required for procambium establishment following auxin signaling.

Map-based cloning and functional analysis of YGL8, which controls leaf colour in rice (Oryza sativa).

BACKGROUND: As the indispensable part of plant, leaf blade mainly functions as the production workshops where organic substance is produced by photosynthesis. Leaf colour mutation is a genetic phenomenon that has a high frequency and is easily identified. The mutations always exhibit negative impact on the development of plants in any of the different stages of growth. Up to now, numerous genes involved in leaf colour mutations have been cloned. RESULTS: In this study, a yellow-green leaf mutant, yellow-green leaf 8 (ygl8), with stable genetic phenotype, has been screened out in the progeny of an excellent indica restorer line Jinhui 10 with seeds treated by EMS. The levels of Chl a, Chl b and total chlorophyll were significantly lower in ygl8 than those in the WT throughout the whole growth period, while no clear change was noted in the Chl a/b ratio. Transmission electron microscopy demonstrated that the lamellae were clearly intumescent and intricately stacked in ygl8. Furthermore, compared with those of the WT, the stomatal conductance, intercellular CO2 concentration, photosynthetic rate and transpiration rate of ylg8 were all significantly lower. Map-based cloning results showed that Loc_Os01g73450, encoding a chloroplast-targeted UMP kinase, corresponded to Ygl8 and played an important role in regulating leaf colour in rice (Oryza sativa). Complementation of ygl8 with the WT DNA sequence of Loc_Os01g73450 led to restoration of the normal phenotype, and transgenic RNA interference plants showed a yellow-green colour. Analysis of the spatial and temporal expression of Ygl8 indicated that it was highly expressed in leaf blades and weakly expressed in other tissues. qRT-PCR also showed that the expression levels of the major Photosystem I core subunits plastome-encoded PsaA, PsaB and PsbC were significantly reduced in ygl8. The expression levels of nuclear-encoded gene involved in Chl biosynthesis HEMC, HEME, and PORA were also decreased when compared with the wild-type. CONCLUSIONS: Independent of Chl biosynthesis and photosystem, YGL8 may affect the structure and function of chloroplasts grana lamellae by regulating plastid genome encoded thylakoid membrane constitutive gene expression and indirectly influences Chl biosynthesis.

MULTI-FLORET SPIKELET1, which encodes an AP2/ERF protein, determines spikelet meristem fate and sterile lemma identity in rice.

The spikelet is a unique inflorescence structure of grass. The molecular mechanism that controls the development of the spikelet remains unclear. In this study, we identified a rice (Oryza sativa) spikelet mutant, multi-floret spikelet1 (mfs1), that showed delayed transformation of spikelet meristems to floral meristems, which resulted in an extra hull-like organ and an elongated rachilla. In addition, the sterile lemma was homeotically converted to the rudimentary glume and the body of the palea was degenerated in mfs1. These results suggest that the MULTI-FLORET SPIKELET1 (MFS1) gene plays an important role in the regulation of spikelet meristem determinacy and floral organ identity. MFS1 belongs to an unknown function clade in the APETALA2/ethylene-responsive factor (AP2/ERF) family. The MFS1-green fluorescent protein fusion protein is localized in the nucleus. MFS1 messenger RNA is expressed in various tissues, especially in the spikelet and floral meristems. Furthermore, our findings suggest that MFS1 positively regulates the expression of LONG STERILE LEMMA and the INDETERMINATE SPIKELET1 (IDS1)-like genes SUPERNUMERARY BRACT and OsIDS1.

CHIMERIC FLORAL ORGANS1, encoding a monocot-specific MADS box protein, regulates floral organ identity in rice.

The control of floral organ identity by homeotic MADS box genes is well established in eudicots. However, grasses have highly specialized outer floral organs, and the identities of the genes that regulate the highly specialized outer floral organs of grasses remain unclear. In this study, we characterized a MIKC-type MADS box gene, CHIMERIC FLORAL ORGANS (CFO1), which plays a key role in the regulation of floral organ identity in rice (Oryza sativa). The cfo1 mutant displayed defective marginal regions of the palea, chimeric floral organs, and ectopic floral organs. Map-based cloning demonstrated that CFO1 encoded the OsMADS32 protein. Phylogenetic analysis revealed that CFO1/OsMADS32 belonged to a monocot-specific clade in the MIKC-type MADS box gene family. The expression domains of CFO1 were mainly restricted to the marginal region of the palea and inner floral organs. The floral organ identity gene DROOPING LEAF (DL) was expressed ectopically in all defective organs of cfo1 flowers. Double mutant analysis revealed that loss of DL function mitigated some of the defects of floral organs in cfo1 flowers. We propose that the CFO1 gene plays a pivotal role in maintaining floral organ identity through negative regulation of DL expression.

Rolling-leaf14 is a 2OG-Fe (II) oxygenase family protein that modulates rice leaf rolling by affecting secondary cell wall formation in leaves.

As an important agronomic trait, leaf rolling in rice (Oryza sativa L.) has attracted much attention from plant biologists and breeders. Moderate leaf rolling increases the amount of photosynthesis in cultivars and hence raises grain yield. Here, we describe the map-based cloning of the gene RL14, which was found to encode a 2OG-Fe (II) oxygenase of unknown function. rl14 mutant plants had incurved leaves because of the shrinkage of bulliform cells on the adaxial side. In addition, rl14 mutant plants displayed smaller stomatal complexes and decreased transpiration rates, as compared with the wild type. Defective development could be rescued functionally by the expression of wild-type RL14. RL14 was transcribed in sclerenchymatous cells in leaves that remained wrapped inside the sheath. In mature leaves, RL14 accumulated mainly in the mesophyll cells that surround the vasculature. Expression of genes related to secondary cell wall formation was affected in rl14-1 mutants, and cellulose and lignin content were altered in rl14-1 leaves. These results reveal that the RL14 gene affects water transport in leaves by affecting the composition of the secondary cell wall. This change in water transport results in water deficiency, which is the major reason for the abnormal shape of the bulliform cells.

Detecting and pyramiding target QTL for plant- and grain-related traits via chromosomal segment substitution line of rice.

Introduction: Plant height and grain length are important agronomic traits in rice, exhibiting a strong effect on plant architecture and grain quality of rice varieties. Methods: Methods: A novel rice chromosomal segment substitution line (CSSL), i.e., CSSL-Z1357, with significantly increased plant height (PH) and grain length (GL) was identified from CSSLs constructed by using Nipponbare as a receptor and a restorer line Xihui 18 as a donor. Seven agronomic traits of PH, PL, GL, GW, GPP, SPP, and TGW were phenotyped, and REML implemented in HPMIXED of SAS were used to detect the QTL for these traits. Secondary CSSLs were screened out via marker-assisted selection (MAS) to estimate the additive and epistatic effects of detected QTLs, evaluating the potential utilization of pyramiding the target QTLs for yield and quality improvement of rice varieties. Results and Discussion: Results and Discussion: CSSL-Z1357 carried nine segments from Xihui 18 with an average segment length of 4.13 Mb. The results show that the long grain of CSSL-Z1357 was caused by the increased number of surface cells and the length of the inner glume. Thirteen quantitative trait loci were identified via the F2 population of Nipponbare/CSSL-Z1357, including three each for GL (qGL-3, qGL-6, and qGL-7) and PH (qPH-1, qPH-7, and qPH-12I), among which qGL-3 increased GL by 0.23 mm with synergistic allele from CSSL-Z1357. Additionally, three single (S1 to S3), two double (D1, D2), and one triple segment (T1) substitution lines were developed in F3 via MAS. Results show that pyramiding the segments from Chr.3 (qGL-3 and qPH-3), Chr.6 (qGL-6 and qPH-6), and Chr.7 (Null and qPH-7) tended to result in better phenotype of increased GL and PH and decreased grain width, providing a potential basis for enhancing grain yield and quality in rice breeding.

QTL Analysis of Z414, a Chromosome Segment Substitution Line with Short, Wide Grains, and Substitution Mapping of qGL11 in Rice.

Most agronomic traits of rice (Oryza sativa), such as grain length, are complex traits controlled by multiple genes. Chromosome segment substitution lines (CSSLs) are ideal materials for dissecting these complex traits. We developed the novel rice CSSL 'Z414', which has short, wide grains, from progeny of the recipient parent 'Xihui 18' (an indica restorer line) and the donor parent 'Huhan 3' (a japonica cultivar). Z414 contains four substitution segments with an average length of 3.04 Mb. Z414 displays seven traits that significantly differ from those of Xihui 18, including differences in grain length, width, and weight; degree of chalkiness; and brown rice rate. We identified seven quantitative trait loci (QTL) that are responsible for these differences in an F2 population from a cross between Xihui 18 and Z414. Among these, six QTL (qPL3, qGW5, qGL11, qRLW5, qRLW11, and qGWT5) were detected in newly developed single-segment substitution lines (SSSLs) S1-S6. In addition, four QTL (qGL3, qGL5, qCD3, and qCD5) were detected in S1 and S5. Analysis of these SSSLs attributed the short, wide grain trait of Z414 to qGL11, qGL3, qGL5, and qGW5. Substitution mapping delimited qGL11 within an 810-kb interval on chromosome 11. Sequencing, real time quantitative PCR, and cell morphology analysis revealed that qGL11 might be a novel QTL encoding the cyclin CycT1;3. Finally, pyramiding qGL3 (a = 0.43) and qGL11 (a = - 0.37) led to shorter grains in the dual-segment substitution line D2 and revealed that qGL11 is epistatic to qGL3. In addition, S1 and D2 exhibited different grain sizes and less chalkiness than Z414. In conclusion, the short grain phenotype of the CSSL Z414 is controlled by qGL11, qGL3, and qGL5. qGL11 might be a novel QTL encoding CycT1;3, whose specific role in regulating grain length was previously unknown, and qGL11 is epistatic to qGL3. S1 and D2 could potentially be used in hybrid rice breeding.

Identification, pyramid and candidate genes of QTLs for associated traits based on a dense erect panicle rice CSSL-Z749 and five SSSLs, three DSSLs and one TSSL.

BACKGROUND: Seed-set density is an important agronomic trait in rice. However, its genetic mechanism is complex. Chromosome segment substitution lines (CSSLs) are ideal materials for studying complex traits. RESULTS: A rice CSSL, Z749, with a dense and erect panicle phenotype, was identified among progeny of the recipient parent Nipponbare and the donor parent Xihui 18. Z749 carried seven substitution segments (average length 2.12 Mb). Compared with Nipponbare, Z749 showed significant increases in the numbers of primary (NPB) and secondary branches (NSB), number of spikelets (SPP) and grains per panicle (GPP), seed-set density (SSD), and decrease in panicle length (PL). A secondary F2 population derived from a cross between Nipponbare and Z749 was used to map quantitative trait loci (QTLs) for associated traits. Fifteen QTLs distributed on chromosomes 5, 7, 8, and 10 were detected. The QTL qPL7 might be an allele of OsFAD8 and the remaining 14 QTLs (e.g., qSSD5 and qSSD10 etc.) might be novel. Fourteen QTLs were verified using five single-segment substitution lines (SSSLs). The seed-set density of Z749 was controlled predominantly by one major QTL (qSSD10) and two minor QTLs (qSSD5 and qSSD8). The QTLs qSSD10, qSSD5, and qSSD8 were fine-mapped to intervals of 1.05, 1.46, and 1.53 Mb on chromosomes 10, 5, and 8, respectively. Analysis of QTL additive effects indicated that qSSD5, qSSD8, and qSSD10 from Xihui18 increased seed-set density of Z749 by 14.10, 11.38, and 5.11 spikelets per 10 cm panicle, respectively. Analysis of QTL epistatic effects revealed that pyramiding of qSSD5 and qSSD8, qSSD5 and qSSD10, qSSD8 and qSSD10, and qSSD5, qSSD8 and qSSD10 produced novel genotypes with increased seed-set density. CONCLUSIONS: Inheritance of seed-set density in Z749 was controlled predominantly by one major QTL (qSSD10) and two minor QTLs (qSSD5 and qSSD8). Then, they were fine-mapped to intervals of 1.05, 1.46, and 1.53 Mb on chromosomes 10, 5, 8, respectively. Two MAPK genes (OsMPK9 and OsMPK17) and one gene (candidate gene 6) involved in auxin metabolism might be candidate genes for qSSD5, and OsSAUR32 might be the candidate gene for qSSD8. Pyramiding of qSSD5, qSSD8, and qSSD10 enhanced seed-set density.