Control of Grain Shape and Size in Rice by Two Functional Alleles of OsPUB3 in Varied Genetic Background.

Grain shape and size are key determinants of grain appearance quality and yield in rice. In our previous study, a grain shape QTL, qGS1-35.2, was fine-mapped using near-isogenic lines (NILs) derived from a cross between Zhenshan 97 (ZS97) and Milyang 46 (MY46). One annotated gene, OsPUB3, was found to be the most likely candidate gene. Here, knockout and overexpression experiments were performed to investigate the effects of OsPUB3 on grain shape and size. Four traits were tested, including grain length, grain width, grain weight, and the ratio of grain length to width. Knockout of OsPUB3 in NILZS97, NILMY46, and another rice cultivar carrying the OsPUB3MY46 allele all caused decreases in grain width and weight and increases in the ratio of grain length to width. Results also showed that the magnitude of the mutational effects varied depending on the target allele and the genetic background. Moreover, it was found that NILZS97 and NILMY46 carried different functional alleles of OsPUB3, causing differences in grain shape rather than grain weight. In the overexpression experiment, significant differences between transgenic-positive and transgenic-negative plants were detected in all four traits. These results indicate that OsPUB3 regulates grain shape and size through a complex mechanism and is a good target for deciphering the regulatory network of grain shape. This gene could be used to improve grain appearance quality through molecular breeding as well.

Abscisic Acid Improves Rice Thermo-Tolerance by Affecting Trehalose Metabolism.

Heat stress that occurs during the flowering stage severely decreases the rice (Oryza sativa L.) seed-setting rate. This damage can be reversed by abscisic acid (ABA), through effects on reactive oxygen species, carbohydrate metabolism, and heat shock proteins, but the exact role of trehalose and ATP in this process remains unclear. Two rice genotypes, namely, Zhefu802 (heat-resistant plant, a recurrent parent) and its near-isogenic line (faded green leaf, Fgl, heat-sensitive plant), were subjected to 38 °C heat stress after being sprayed with ABA or its biosynthetic inhibitor, fluridone (Flu), at the flowering stage. The results showed that exogenous ABA significantly increased the seed-setting rate of rice under heat stress, by 14.31 and 22.40% in Zhefu802 and Fgl, respectively, when compared with the H2O treatment. Similarly, exogenous ABA increased trehalose content, key enzyme activities of trehalose metabolism, ATP content, and F1Fo-ATPase activity. Importantly, the opposite results were observed in plants treated with Flu. Therefore, ABA may improve rice thermo-tolerance by affecting trehalose metabolism and ATP consumption.

Impaired Function of the Calcium-Dependent Protein Kinase, OsCPK12, Leads to Early Senescence in Rice (Oryza sativa L.).

Premature leaf senescence affects plant yield and quality, and numerous researches about it have been conducted until now. In this study, we identified an early senescent mutant es4 in rice (Oryza sativa L.); early senescence appeared approximately at 60 dps and became increasingly senescent with the growth of es4 mutant. We detected that content of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as activity of superoxide dismutase (SOD) were elevated, while chlorophyll content, soluble protein content, activity of catalase (CAT), activity of peroxidase (POD) and photosynthetic rate were reduced in the es4 mutant leaves. We mapped es4 in a 33.5 Kb physical distance on chromosome 4 by map-based cloning. Sequencing analysis in target interval indicated there was an eight bases deletion mutation in OsCPK12 which encoded a calcium-dependent protein kinase. Functional complementation of OsCPK12 in es4 completely restored the normal phenotype. We used CRISPR/Cas9 for targeted disruption of OsCPK12 in ZH8015 and all the mutants exhibited the premature senescence. All the results indicated that the phenotype of es4 was caused by the mutation of OsCPK12. Overexpression of OsCPK12 in ZH8015 enhanced the net photosynthetic rate (P n) and chlorophyll content. OsCPK12 was mainly expressed in green organs. The results of qRT-PCR analysis showed that the expression levels of some key genes involved in senescence, chlorophyll biosynthesis, and photosynthesis were significantly altered in the es4 mutant. Our results demonstrate that the mutant of OsCPK12 triggers the premature leaf senescence; however, the overexpression of OsCPK12 may delay its growth period and provide the potentially positive effect on productivity in rice.

qSE7 is a major quantitative trait locus (QTL) influencing stigma exsertion rate in rice (Oryza sativa L.).

Stigma exsertion is a key determinant to increase the efficiency of commercial hybrid rice seed production. The major quantitative trait locus (QTL) qSE7 for stigma exsertion rate was previously detected on the chromosome 7 using 75 Chromosome Segment Substitution Lines (CSSLs) derived from a cross between the high stigma exsertion indica maintainer XieqingzaoB (XQZB) and low stigma exsertion indica restorer Zhonghui9308 (ZH9308). The C51 line, a CSSL population with an introgression from XQZB, was backcrossed with ZH9308 to produce the secondary F2 (BC5F2) and F2:3 (BC5F2:3) populations. As a result, the Near Isogenic Line (NIL qSE7XB) was developed. Analysis indicated qSE7 acted as a single Mendelian factor and decreased the stigma exsertion. We hypothesized qSE7 regulates single, dual, and total stigma exsertion rate, provided experimental support. qSE7 was mapped and localized between RM5436 and RM5499 markers, within a physical distance of 1000-kb. With use of new insertion-deletion (InDel) markers and analysis of the heterozygous and phenotypic data, it was ultimately dissected to a 322.9-kb region between InDel SER4-1 and RM5436. The results are useful for additional identification and isolation of this candidate gene controlling stigma exsertion rate, and provide a basis for further fine mapping, gene cloning, and Marker Assisted Selection (MAS) breeding later.

Genetic Dissection of qPCG1 for a Quantitative Trait Locus for Percentage of Chalky Grain in Rice (Oryza sativa L.).

Rice is a pivotal cereal crop that provides the staple food for more than half of the world's population. Along with improvements in the standard of living, people not only pay attention to the grain yield but also to the grain quality. Chalkiness is one of the most important index of grain quality. In this study, qPCG1, a QTL for percentage of chalky grain, was mapped in an interval with a physical distance about 139 kb on chromosome 1 by residual heterozygous line (RHL) method. qPCG1 was incomplete dominant and the additive effect plays a major role and explained 6.8-21.9% of phenotypic variance within the heterogeneous region on chromosome 1. The effect of allele from Zhonghui9308 was decreasing the percentage of chalky grains (PCG). Microscope observation results indicated that there are great differences in the shape, structure and arrangement of starch granule between the chalky part and transparent part. Analysis of starch physicochemical properties showed that the total starch content, amylose content and chain length distribution of amylopectin changed while the protein contents were not apparently affected with the changed chalkiness. qPCG1 had little influence on main agronomic traits and it might be useful in rice breeding for it did not bring negative effect on grain yield while reducing the chalkiness.

Genetic dissection of yield traits in super hybrid rice Xieyou9308 using both unconditional and conditional genome-wide association mapping.

With the development and application of super rice breeding, elite rice hybrids with super high-yielding potential have been widely developed in last decades in China. Xieyou9308 is one of the most famous super hybrid rice varieties. To uncover the genetic mechanism of Xieyou9308's high yield potential, a recombinant inbred line (RIL) population derived from cross of XieqingzaoB and Zhonghui9308 was re-sequenced and investigated on the grain yield (GYD) and its three component traits, number of panicles per plant (NP), number of filled grains per panicle (NFGP), and grain weight (GW). Unconditional and conditional genome-wide association analysis, based on a linear mixed model with epistasis and gene-environment interaction effects, were conducted, using ~0.7 million identified SNPs. There were six, four, seven, and seven QTSs identified for GYD, NP, NFGP, and GW, respectively, with accumulated explanatory heritability varying from 43.06% to 48.36%; additive by environment interactions were detected for GYD, some minor epistases were detected for NP and NFGP. Further, conditional genetic mapping analysis for GYD given its three components revealed several novel QTSs associated with yield than that were suppressed in our unconditional mapping analysis.

Dissection of genetic architecture of rice plant height and heading date by multiple-strategy-based association studies.

Xieyou9308 is a certified super hybrid rice cultivar with a high grain yield. To investigate its underlying genetic basis of high yield potential, a recombinant inbred line (RIL) population derived from the cross between the maintainer line XieqingzaoB (XQZB) and the restorer line Zhonghui9308 (ZH9308) was constructed for identification of quantitative trait SNPs (QTSs) associated with two important agronomic traits, plant height (PH) and heading date (HD). By re-sequencing of 138 recombinant inbred lines (RILs), a total of ~0.7 million SNPs were identified for the association studies on the PH and HD. Three association mapping strategies (including hypothesis-free genome-wide association and its two complementary hypothesis-engaged ones, QTL-based association and gene-based association) were adopted for data analysis. Using a saturated mixed linear model including epistasis and environmental interaction, we identified a total of 31 QTSs associated with either the PH or the HD. The total estimated heritability across three analyses ranged from 37.22% to 45.63% and from 37.53% to 55.96% for the PH and HD, respectively. In this study we examined the feasibility of association studies in an experimental population (RIL) and identified several common loci through multiple strategies which could be preferred candidates for further research.

Genome sequence of Pseudomonas parafulva CRS01-1, an antagonistic bacterium isolated from rice field.

Pseudomonas parafulva (formerly known as Pseudomonas fulva) is an antagonistic bacterium against several rice bacterial and fungal diseases. The total genome size of P. parafulva CRS01-1 is 5,087,619 bp with 4389 coding sequences (CDSs), 77 tRNAs, and 7 rRNAs. The annotated full genome sequence of the P. parafulva CRS01-1 strain might shed light on its role as an antagonistic bacterium.