Changjie Yan

Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities

More than half of the worlds population uses rice as a source of carbon intake every day. Improving grain quality is thus essential to rice consumers. The three main properties that determine rice eating and cooking quality—amylose content, gel consistency, and gelatinization temperature—correlate with one another, but the underlying mechanism of these properties remains unclear. Through an association analysis approach, we found that genes related to starch synthesis cooperate with each other to form a fine regulating network that controls the eating and cooking quality and defines the correlation among these three properties. Genetic transformation results verified the association findings and also suggested the possibility of developing elite cultivars through modification with selected major and/or minor starch synthesis-related genes.

Mutations in the F‐box gene LARGER PANICLE improve the panicle architecture and enhance the grain yield in rice

Panicle architecture is one of the most important agronomical traits that directly contribute to grain yield in rice (Oryza sativa L.). We report herein an in-depth characterization of two allelic larger panicle (lp) mutants that show significantly increased panicle size as well as improved plant architecture. Morphological analyses reveal that panicles of two mutants produced more inflorescence branches, especially the primary branches, and contained more grains. Moreover, mutant plants also display more lodging resistance than the wild type. The grain yield per plant in mutants is also increased, suggesting that mutant plants have useful potential for high grain yield in rice breeding. Map-based cloning reveals that LARGER PANICLE (LP) encodes a Kelch repeat-containing F-box protein. RNA in situ hybridization studies display that LP expression was enriched in the branch primordial region. Subcellular localization analyses demonstrate that LP is an endoplasmic reticulum (ER) localized protein, suggesting that LP might be involved in ER-associated protein degradation (ERAD). Using yeast two-hybrid assay and bimolecular fluorescence complementation analysis, we confirm that LP is an F-box protein and could interact with rice SKP1-like protein in an F-box domain-dependent manner. Quantitative real-time PCR results show that OsCKX2, which encodes cytokinin oxidase/dehydrogenase, is down-regulated evidently in mutants, implying that LP might be involved in modulating cytokinin level in plant tissues. These results suggest that LP plays an important role in regulating plant architecture, particularly in regulating panicle architecture, thereby representing promising targets for genetic improvement of grain production plants.

ROLLED LEAF 9, encoding a GARP protein, regulates the leaf abaxial cell fate in rice

Leaves, the collective organ produced by the shoot apical meristem (SAM), are polarized along their adaxial–abaxial axis. In this study, we characterized two rice (Oryza sativa) allelic rolled-leaf mutants, rolled leaf 9-1 (rl9-1) and rl9-2, which display very similar phenotypes with completely adaxialized leaves and malformed spikelets. We cloned the RL9 gene by way of a map-based cloning strategy. Molecular studies have revealed that RL9 encodes a GARP protein, an orthologue of ArabidopsisKANADIs. RL9 is mainly expressed in roots, leaves, and flowers. The transient expression of a RL9–GFP (green fluorescent protein) fusion protein has indicated that RL9 protein is localized in the nucleus, suggesting that RL9 acts as a putative transcription factor.

ERECT PANICLE2 encodes a novel protein that regulates panicle erectness in indica rice.

Rice (Oryza sativa L.) inflorescence (panicle) architecture is an important agronomic trait for rice breeding. A number of high-yielding japonica rice strains, characterized by an erect panicle (EP) of their architecture, have been released as commercial varieties in China. But no EP-type indica varieties are released so far. Here, we identified two allelic erect-panicle mutants in indica rice, erect panicle2-1 (ep2-1) and erect panicle2-2 (ep2-2), exhibiting the characteristic erect panicle phenotype. Both mutants were derived from spontaneous mutation. We cloned the EP2 gene by way of a map-based cloning strategy, and a transgenic complementation test rescued the phenotype of ep2-1. Anatomical investigations revealed that the ep2 mutants have more vascular bundles and a thicker stem than that of wild-type plants, explaining the panicle erectness phenotype in ep2 mutants. It was shown that EP2 was specifically expressed in the vascular bundles of internodes by GUS staining and RT–PCR. EP2 encodes a novel plant-specific protein, which localizes to the endoplasmic reticulum with unknown biochemical function. In addition, EP2 also regulates other panicle characteristics, such as panicle length and grain size, but grain number per panicle shows little change, indicating that the mutation of the ep2 gene could be applied in EP-type indica rice breeding.

Fine Mapping and Isolation of Bc7(t), Allelic to OsCesA4

Several brittle culm mutants of rice were identified and characterized. In this study, we characterized a brittle mutant (bc7(t)) identified from japonica variety Zhonghua 11 by means of 60Co-gamma radiation. This mutant displays normal phenotype similar to its wild type plants except for the fragility of all plant body, with approximately 10% decrease in the cellulose content. The genetic analysis and gene fine mapping showed that the bc7(t) mutant was controlled by a recessive gene, residing on an 8.4 kb region of the long arm of chromosome 1. The gene annotation indicated that there was only one putative gene encoding cellulose synthase catalytic subunit (CesA) in this region, which was allelic to OsCesA4. Furthermore, the sequence analysis was carried out and 7 bases deletion in the junction of exon 10 and intron 10 was done in bc7(t) mutant, resulting in the change of reading frame and the consequent failure to generate functional protein. In addition, the result of RNA interference experiment showed that when the Bc7(t) was knocked down, the transplants exhibited fragility, similar to bc7(t) mutant. The finding of novel allele of OsCesA4 locus will facilitate the understanding of the mechanism of cell wall biosynthesis. The potential utilization of the bc7(t) mutant in animal food was discussed as well.

Development of gene-tagged markers for quantitative trait loci underlying rice yield components

Higher yields of rice have always been a predominant goal in rice breeding techniques. However, the inheritances of rice yield and its components are still unknown, and no information regarding suitable alleles can be directly provided for improving the rice yield level until three major quantitative trait loci (QTLs) have been cloned and functionally characterized. These QTLs contain Gn1a for grain number per panicle and GS3 and GW2 for grain weight. It has been proven that these three genes show a potential in improving the rice yield level. However, the distribution of suitable alleles on these three loci in rice cultivars and germ plasm are yet to be elucidated, this retards the progress of the utilization of suitable alleles in rice breeding techniques to produce higher yields. In the present study, we developed a set of gene-tagged markers based on the gene mutation sites Gn1a-M1 and Gn1a-M2 for Gn1a, GW2-HapI for GW2, and GS3-PstI for GS3. The results demonstrated that these STS markers could clearly differentiate between the different alleles at each gene locus. A survey of the allele distributions of the abovementioned three genes was performed with 156 cultivars. It was observed that the 5150-Gn1a allele was absent on the Gn1a locus and only two type alleles (Ha-Gn1a and Ko-Gn1a) were present, of which 54.3% indica and 21.5% japonica cultivars contained the Ha-Gn1a allele. Two alleles (MH-GS3 and ZS-GS3) were detected on the GS3 locus, and 48.6% indica and 9.9% japonica cultivars harbored the suitable allele MH-GS3. Further, all the cultivars contained the FA-GW2 allele on GW2, whereas the WY-GW2 allele was not found. These results further suggested that some of the alleles residing in the indica subspecies have introgressed into the japonica group with a very low frequency. The gene-tagged markers developed in the present study can be directly used as a tool for marker-aided selection (MAS) in rice breeding techniques to produce higher yields.

Fine mapping of a semidwarf genesd-g in indica rice (Oryza sativa L.)

The semidwarf genesd-g which has been used in indica rice breeding in southern China is a new one, nonallelic tosd-1. To mapsd-g, an F2 population derived from the cross between Xinguiaishuangai and 02428 was constructed. Thesd-g was roughly mapped between two microsatellite markers RM440 and RM163, with genetic distances of 0.5 and 2.5 cM, respectively. Then nine new polymorphic microsatellite markers were developed in this region. Thesd-g was further mapped between two microsatellite markers SSR5-1 and SSR5-51, with genetic distances of 0.1 and 0.3 cM, respectively, while cosegregated with SSR418. A BAC contig was found to span thesd-g locus, the region being delimited to 85 kb. This result was very useful for cloning of thesd-g gene.

Genetic Effect of PUL Allelic Variation on Rice Cooking and Eating Qualities

Pullulanase (PUL) is a type of starch debranching enzymes involved in starch biosynthesis. To test the effect of PUL allelic variation on cooking and eating qualities in rice (Oryza sativa L.), 2 sets of near-isogenic lines (NILs) for PUL gene were developed by crossing and backcrossing Guichao 2 (indica) and Suyunuo (japonica glutinous) and selected with the help of the gene-specific STS marker on PUL gene. The NILs had no significant differences in amylose content, gel consistency, and starch crystalline structure compared with their corresponding recurrent parents. In contrast, the starch paste viscosity and starch thermal property changed obviously between the NILs and their recurrent parents. It was concluded that PUL gene plays an important role in determining rice grain cooking and eating qualities, and functional differentiation occurred at the PUL locus between indica variety Guichao 2 and japonica variety Suyunuo. The gene-specific molecular marker for PUL gene developed on the basis of genomic sequence diversity could be directly applied in rice good quality breeding program.