【Objective】 Starch degradation is involved in lodicule absorbing abundant water and swelling during rice floret opening, but the amylase genes associated with this process have not been identified yet. 【Method】 To identify the swelling of tissues during floret opening, the in vitro rice panicles absorbed diluted Fuchsin basic and the dye remains were observed after florets were closing again. The starch grain distribution in rice florets before and during anthesis from stage 11 to stage 14 (according to 14 stages of rice anther development) was detected using iodide staining. The spatial-temporal expression patterns of 10 α-amylase genes were detected by RT-PCR, RT-qPCR and GUS staining. 【Result】 Before floret opening, the stamens, pistils and lodicules are enclosed by the lemma and palea through marginal tissues of palea (mtp). Rapid swelling of the lodicules causes floret opening by separating the lemma from the palea. After the in vitro panicles absorbed diluted Fuchsin basic during floret opening, the dye remains were observed located in the joint between mtp and lodicules and filaments. Iodide staining showed that the starch grains were mainly located in the stamens and mtp and a small amount of starch grains in the lodicules at stage 12 (before floret opening), whereas the starch grains in the mtp and lodicules were almost completely degraded at stage 13-14 (during floret opening). RT-PCR showed that OsRAmy2A and OsRAmy3D began to express from stage 12 and were expressed with high levels at stage 13-14. The expression levels of the two genes decreased at DAP1 (1 day after pollination). OsRAmy3E and OsRAmy3F kept expressed during this process. The expression level of OsRAmy3E was higher than that of OsRAmy3F. The RT-qPCR analysis showed that the expression level of OsRAmy2A increased most dramatically at stage 13-14, followed by OsRAMy3A and OsRAMy3E. Further, the transgenic plants expressing the GUS reporter gene driven by the OsRAmy2A promoter were generated. The GUS signaling was located only in the lemma, palea and mtp at stage 12 and the expression of the GUS gene driven by the RAmy2A promoter was induced in the mtp, lodicules and filaments at stage 13-14. 【Conclusion】 These data indicated that starch grain degradation in the mtp and lodicules at stage 13-14 might be related with high expression levels of some α-amylase genes such as OsRAmy2A and OsRAmy3D, probably involved in controlling lodicules swelling and floret opening in rice.
【Objective】 Rice is an important food crop, providing staple food for more than half of the world’s population. Panicle traits are the main factors affecting rice yield. Discover the elite haplotype of the panicle regulation gene, and provide important germplasm and gene resources for pyramiding breeding. 【Method】 In this study, recombinant inbred lines (RILs) derived from a cross between SN265 and R99 were re-sequenced through high-throughput sequencing. QTL analysis and candidate gene identification were conducted on the grain number on the primary branch, the grain number on the secondary branch, and the grain shape. The sequences of candidate genes were compared using the long-read sequence assemblies of SN265 and R99. The combination of candidate genes that can maximize grain yield was selected among RILs. Finally, the super rice variety SN265 was improved using CRISPR/Cas9 gene editing technology. 【Result】 The R99 had significantly more grain number per panicle and grain number on the secondary branch, whereas SN265 had significantly more grain number on the primary branch. The grain of R99 is slender, and the grain of SN265 is short and round. The RILs were sequenced with approximately 6.25-fold depth. For parent lines, 30.0-fold depth and 32.0-fold depth data were generated for R99 and SN265, respectively. Subsequently, a bin map was constructed by 1456445 high-quality SNPs. The genetic map containing 3 569 recombinant blocks, with an average length of 58.17 kb. The QTL analysis detected a QTL on Chr.9 for grain number per panicle and grain number on both primary and secondary branch, a QTL on Chr.1 for grain number per panicle and grain number on the secondary branch, a QTL on Chr.5 for grain shape. The candidate gene prediction and sequence comparison showed that DEP1 regulated the grain number on both primary and secondary branches of rice, Gn1a mainly regulated the grain number on secondary branches of rice, and qSW5 mainly regulated the grain shape. The yield of the combination of Gn1aR99/DEP1SN265/qSW5SN265 alleles showed an advantage in yield performance among the RILs. We further conducted a molecular design breeding to SN265 by knocking out the Gn1a locus using CRISPR/Ca9 gene editing technology, and the grain number per panicle of the transgenic plants increased significantly compared to that of SN265. 【Conclusion】 This study used RILs derived from a XI/GJ cross and high-throughput sequencing technology to conduct QTL analysis of rice panicle traits, revealed the effects of DEP1, Gn1a, and qSW5 on grain number per panicle and grain shape, and clarified that Gn1aSN265/ DEP1R99/qSW5R99 was the best gene combination in RILs. The yield per plant was further improved by knocking out the Gn1a locus of SN265. This study provided important germplasm and gene resources for pyramiding breeding with elite alleles.
【Objective】 Rice yield is composed of effective panicle number per unit area, grains per panicle and grain weight, in which grain weight is mainly determined by grain morphology. Screening and identification of new grain type mutation materials and genes can lay a foundation for molecular design breeding of yield traits. 【Method】 A short and wide grain mutant short and widen grain1 (swg1) was identified in the mutant population of indica rice maintainer line Xida1B(XD1B) induced by ethyl methane sulfonate (EMS). The grain morphology and other agronomic characters were analyzed, and the glume was observed and analyzed by histocytology. Gene mapping was carried out by BSA method, and candidate genes were identified by genetic complementarity experiment. qRT-PCR was used to analyze the expression pattern of the gene and the expression level of other genes related to grain shape and cell development.【Result】 The analysis of agronomic characters showed that the grain length of swg1 mutant was significantly lower and the grain width was significantly higher than that of wild type, showing the phenotype of short and wide grains, and further histological and cytological analysis showed that the shortening of longitudinal cells of glume was the main reason for the shortening of grain length, while the increase of grain width was due to the increase of the number and size of transverse cells of glume at the same time. The results of genetic analysis showed that the mutation was controlled by a recessive single gene, and the candidate gene for SWG1 was determined to be LOC_Os07g42410 by map-based cloning and genetic complementary verification, which encoded a plant-specific transcription factor. qRT-PCR analysis showed that the expression of this gene had no obvious tissue specificity, and its expression is strong in stem, leaf and young panicle. According to the analysis of the expression of known genes related to grain shape, cell cycle and cell expansion, it was found that GS5 and GW8, which positively regulate the number and/or size of glume transverse cells to determine grain width, were significantly up-regulated in the mutants, while GW7/GL7 genes, which positively regulated the number and size of longitudinal cells and negatively regulated the number and size of transverse cells, were significantly down-regulated in the mutants. Some genes related to cell cycle and cell expansion also showed significant differences between mutants and wild types. 【Conclusion】 SWG1 encodes a plant-specific transcription factor, which affects glume cell proliferation and cell expansion by regulating grain shape genes GS5, GW8 and GW7/GL7, thus determining rice grain length and width.
【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 F2 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 F3 derived from 42 F2 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 F2 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 (qGL1, qGL3, qGL6, qG-W1, qGW3, qRLW1, qRLW3, qRLW6) can be verified by 11 SSSLs, indicating that these QTLs are genetically stable. In addition, 33 QTLs such as qGL1-2, qGL1-3, qGL3-2 etc. are only detected by 11 single segment substitution lines. Among them, 15 QTLs such as qNSB1-1 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 qGL3 (a=1.26) and qGL6-2(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 qGL3 or qGL6-2; the pyramid of qGWT3-2 (a=3.18) and qGWT6-2 (a=3.39) produce epistasis effect of -5.46, making the 1000-grain weight of D2 significantly 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 F2 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.
【Objective】 Spikelet is a unique floral organ of the grass family. In rice, the spikelet, as the basic unit and unique structure of inflorescence, has an important impact on the yield and quality. Therefore, studying the development of rice spikelets and floral organs can provide the foundation for the formation of rice yield and quality. 【Method】 Two rice allelic mutants, polarity defect of lateral organs 2-1 and polarity defect of lateral organs 2-2 (pdl2-1 and pdl2-2) with similar mutant phenotypes were obtained using Ethyl Methane Sulfonate (EMS) mutagenesis in the indica rice maintenance line Xinong 1B. Because of their phenotypic similarity, pdl2-1 (named pdl2) was selected as the material for further analysis. Microscopic observation and paraffin sectioning techniques were used to analyze their spikelet mutant phenotypes; agronomic trait examination was used to analyze the effect of lemma on rice yield; map-based cloning were used to verify the function of PDL2; in situ hybridization and real-time fluorescence quantitative PCR (RT-qPCR) were used to analyze the expression pattern of PDL2. 【Result】 The results of the phenotypic analysis showed that the lemma of pdl2 mutant was significantly narrower and could not be closely hooked to the palea compared with that of wild type, resulting in spikelet dehiscence and partially exposed inner whorl floral organs. However, the morphology and number of stamens, pistils, and lodicules were normal. Further paraffin section results showed that the reduced volume and number of silicified and vesicular cells in the mutant lemma, as well as the reduced spacing of vascular bundles, were responsible for the significant narrowing of the width of the lemma in pdl2. Agronomic traits examined showed that the mutation in the pdl2 lemma eventually caused the seeds to be teardrop-shaped and resulted in a significant decrease in yield traits such as seed setting rate and 1000-grain weight in the pdl2 mutant. Genetic analysis and map-based cloning showed that PDL2 is a single recessive nuclear gene, which is OsDCL4 gene localized on chromosome 4. PDL2 encodes a Dicer-like protein that plays an essential role in the rice ta-siRNA synthesis pathway, and the pdl2 mutant is a novel weak allelic mutation of OsDCL4 gene. The expression pattern analysis showed that the PDL2 gene was constitutively expressed in all whorls of floral organs. Mutation of PDL2 affected the ta-siRNA synthesis and the expression of genes related to the establishment of adaxial-abaxial polarity, thus leading to the disorder of adaxial-abaxial polarity establishment in the lemma of pdl2. And the characteristics of floral organs were normal. 【Conclusion】 Mutation of lemma degeneration gene PDL2 disrupts the establishment of lemma adaxial-abaxial polarity and affects the development of lemma and the formation of yield traits.
