Strategy and utilization of a herbicide resistance gene in two-line hybrid rice

Hybrid rice plays an important role in China's aim to improve rice production as it accounts for some 50% of rice planting area but produces about 60% of the total rice grain. However, the existing three-line system used in hybrid rice production has its limitations. The two-line system, which makes use of photoperiod-sensitive genic male-sterile (PGMS) and thermo-sensitive genic male-sterile (TGMS) lines to generate the male-sterile parental line, was developed to overcome some of these limitations. The sterility of the male-sterile line of two-line hybrid rice, however, fluctuates when the temperature-sensitive phase of fertility encounters abnormal low temperatures during hybrid seed production, which induces selfing and decreases the purity of hybrid. We describe here the strategy of utilizing a herbicide resistance gene in two-line hybrid rice to eliminate this fluctuation in the sterility of the P/TGMS lines during hybrid seed production and reports the development of the herbicide resistance restorer line Bar68-1 and its herbicide-resistant early season hybrid rice Xiang125s/Bar68-1. When the restorer line and its derived hybrid are herbicide resistant, the selfed seeds can be removed easily from the hybrid by herbicide spraying. A herbicide resistance gene bar was transferred into a restorer line by particle bombardment. The resulting transgenic restorer line Bar68-1 and its hybrid Xiang125 s/Bar68-1 inherited stable herbicide resistance. The purity of Xiang125s/Bar68-1 was increased by spraying the seed bed with herbicide, which resulted in a significant increase in yield, grain quality, and disease resistance in comparison to the controls in a regional trial.     

Characterization and fine mapping of a novel rice albino mutant low temperature albino 1

Albino mutants are useful genetic resource for studying chlorophyll biosynthesis and chloroplast development and cloning genes involved in these processes in plants.Here we report a novel rice mutant low temperature albino 1(lta1) that showed albino leaves before 4-leaf stage when grown under temperature lower than 20℃,but developed normal green leaves under temperature higher than 24℃or similar morphological phenotypes in dark as did the wild-type(WT).Our analysis showed that the contents of chlorophylls and chlorophyll precursors were remarkably decreased in the ltal mutant under low temperature compared to WT.Transmission electron microscope observation revealed that chloroplasts were defectively developed in the albino lta1 leaves,which lacked of well-stacked granum and contained less stroma lamellae.These results suggested that the lta1 mutation may delay the light-induced thylakoid assembly under low temperature.Genetic analysis indicated that the albino phenotype was controlled by a single recessive locus.Through map-based approach,we finally located the Lta1 gene to a region of 40.3 kb on the short arm of chromosome 11.There are 8 predicted open reading frames(ORFs) in this region and two of them were deleted in lta1 genome compared with the WT genome.The further characterization of the Ltal gene would provide a good approach to uncover the novel molecular mechanisms involved in chloroplast development under low temperature stress.     

Characterization of a zebra mutant of rice with increased susceptibility to light stress

The rice zebra mutant TCM248 is a single recessive mutant. This mutant develops transverse-striped leaves with green and white sectors under alternate light/dark growth conditions. Mutants that were grown under a higher light intensity during the light period showed a more intense striped phenotype. The white tissues contained abnormal chloroplasts with few internal membrane structures, while the green tissues in the mutants contained normal chloroplasts. The white tissue contained only trace amounts of Chls and carotenoids, and mRNA accumulation of nuclear genes encoding chloroplast proteins (rbcS, cab) was strongly suppressed compared to that in the wild type plants. A series of growth condition shift experiments demonstrated that the mutant displayed the striped phenotype only if it was exposed to the alternate light/dark growth conditions during a limited stage of early leaf development. These data suggest that the zebra gene is involved in the acquisition of photoprotective capacity of the plants and that this gene functions at an early stage of chloroplast differentiation.     

The Endosperm-Specific Expression of <i>YUCCA</i> Genes Enhances Rice Grain Filling

Grain filling is a crucial process that affects yield in rice (Oryza sativa L.). Auxin biosynthesis and signaling are closely related to rice yield; therefore, it is important to understand the effects of auxin biosynthesis on rice grain filling to improve crop yield. In this study, we used physiological and molecular strategies to identify the roles of auxin in rice grain filling. Exogenous application of auxin (IAA) or auxin analogues (2, 4-D) to young spikelets and flag leaves improved the seed-setting rate and yield per spike. Furthermore, real-time quantitative PCR assays confirmed that nine members of the OsYUCCA family of auxin biosynthetic genes were upregulated during grain filling, implication that auxin biosynthesis plays a major role in grain development. The specific expression of either Arabidopsis AtYUCCA1 or OsYUCCA2 in the endosperm or leaves resulted in increased expression of OsIAA genes and auxin content of seeds, as well as increased grain filling and seed-setting rate. This result establishes that the auxin content in grains and leaves is important for grain development. Our findings further highlight the potential applications for improving rice yield by elevating targeted gene expression in specific tissues.     

Development of insect-resistant transgenic indica rice with a synthetic cry1C* gene

Stemborers and leaffolders are two groups of lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, a cry1C* gene encoding Bacillus thuringiensis (Bt) δ-endotoxin was synthesized by codon optimization as the first step towards gene stacking in our resistance management strategy of transgenic rice. Agrobacterium-mediated transformation of this gene into Minghui 63 (Oryza sativa L.), an elite indica CMS restorer line, produced 120 independently transformed plants, 19 of which had a single-copy transgene. Preliminary screening of T₁ families of these 19 transformants in the field identified five lines showing a high level of resistance to leaffolders (Cnaphalocrocis medinalis) and stemborers. Hybrids were produced by crossing these five lines with Zhenshan 97A, the male-sterile line for Shanyou 63, the most widely cultivated hybrid in China. These five lines and their hybrids were highly resistant to yellow stemborer (Tryporyza incertulas) as revealed by an insect bioassay. The content of Cry1C* protein varied considerably among the five lines as well as among the corresponding hybrids. T1c-19, a line showing the highest content of Cry1C* protein, and its hybrid were tested in the field for insect resistance and agronomic performance and found to be highly resistant to stemborers and leaffolders throughout the growth period, resulting in a significantly increased grain yield compared with the respective controls. These results indicate that T1c-19 can be used for production of insect-resistant hybrid rice and as a germplasm for gene stacking to produce rice plants with two toxins.     

一种紫色水稻的遗传及其在光敏不育系育种中应用的研究

研究表明,本院获得的一种紫色水稻的植株色遗传受控于C、 A、Pl3个独立基因座位上显性基因的互补作用,另有一独立的显性基因对Pl基因的表达起抑制作用。由于该抑制基因在籼稻中的高频率存在,因而,紫稻与一般绿稻品种杂交F1多表现为绿株。紫稻光敏不育(株)系的不育性表达和配合力表现,均可达到与普通光敏不育系相似的水平。本文还讨论了选育籼型紫稻光敏不育系设想的可行性。 Abstract:The inheritance of a purple rice in crosses to green rices was investigated.A group of dominant and interactive genes,C,A and Pl,was found to control the expression of the trait and the other independent inhibitor I-Pl-1 to inhibit the effect of the gene Pl.Because of the wide existence of the gene I-Pl-1 in indica rice,most of F1 plants of the crosses between purple rice and green cultivars were green.The primary study indicated that,for the purple photoperiod-sensitive genic male-sterile lines,the degrees of the sterility and its stability in the sterile stage,and of the fertility in the fertile stage,and the combining ability levels of the purple rice were as high as the degrees and levels for the green rice.A tentative idea on breeding purple photoperiod-sensitive male-sterile lines of indica rice was suggested and its feasibility and advantage was discussed.     

Gene mapping related to yellow green leaf in a mutant line in rice (Oryza sativa L.)

A mutant, which derived from the restorer line Jinhui10 treated with EMS, showed completely yellow green leaves, and it had low chlorophyll content and poor agronomic characteristics during the growing stage. The F1 plants from the cross between normal × the mutant showed normal green leaves, and the segregation ratio of normal to yellow green leaves was 3 : 1 in F₂ population. It indicated that the trait was controlled by a single recessive nuclear gene, temporarily designated asygl3. The geneygl3 was mapped between RM468 and RM3684 with genetic distances 8.4 cM and 1.8 cM on chromosome 3. This result would be used as genetic information for fine mapping and map-based cloning ofygl3 gene.     

Characterization and fine mapping of a shortened uppermost internode mutant in rice

Internode elongation is an important agronomic trait in rice that is associated with lodging, yield, flooding adaptation, and hybrid seed production. We identified a novel rice mutant line showing a shortened uppermost internode among the rice Ac/Ds insertional mutant population and named it shortened uppermost internode 4 (sui4). Interestingly, T1 plants of this line segregated into three phenotypes: extremely shortened uppermost internode-type, normal wild-type, and intermediate-type. The phenotypes of F1 plants and F2 plants from the cross of sui4 with its original variety, Dongjin, indicated that the SUI4 gene shows incomplete dominance or semidominance. Because the Ds genotypes did not co-segregate with the sui4 phenotypes, we performed mapping of this gene with 273 F2 plants from a cross between sui4 and Milyang23. Primary mapping revealed that the SUI4 locus was located between the S07012 and S07015 markers on rice chromosome 7. Further fine mapping with 36 F3 lines derived from F2 plants that have recombination in this region narrowed down the location of SUI4 to the 1.1-Mbp interval of RM1253–S07015.     

Effect of culture methods on the regeneration of albino rice (Oryza sativa L.) plantlets

In our study, we investigated the effects of regeneration conditions on both green and albino rice plants (Oryza sativa L.). The regeneration frequency of an albino cell line was compared to a normal cell line obtained from mature seed under two kinds of culture conditions; namely, the static culture on semi-solid regeneration medium and the suspension culture in liquid regeneration medium. The albino cell line, from which only albino plantlets were regenerated, was induced from the albino leaf segments. There were no significant differences in the regeneration frequencies between normal and albino calli on the semisolid regeneration medium. On the other hand, the frequency of regeneration of albino calli was significantly lower than that of the control specifically in the liquid regeneration medium.     

Characterization and fine mapping of the rice premature senescence mutant ospse1

Premature senescence can limit crop productivity by limiting the growth phase. In the present study, a spontaneous premature senescence mutant was identified in rice (Oryza sativa L.). Genetic analysis revealed that the premature senescence phenotype was controlled by a recessive mutation, which we named Oryza sativa premature senescence1 (ospse1). The ospse1 mutants showed premature leaf senescence from the booting stage and exhibited more severe symptoms during reproductive and ripening stages. Key yield-related agronomic traits such as 1,000-grain weight and seed-setting rate, but not panicle grain number, were significantly reduced in ospse1 plants. Chlorophyll content, net photosynthetic rate, and transpiration rate of ospse1 flag leaves were similar to the wild-type plants in vegetative stages, but these parameters decreased steeply in the mutant after the heading stage. Consistent with this, the senescence-associated genes OsNYC1 and OsSgr were up-regulated in ospse1 mutant during premature leaf senescence. The ospse1 locus was mapped to a 38-kb region on chromosome 1 and sequence analysis of this region identified a single-nucleotide deletion in the 3′ region of an open reading frame (ORF) encoding a putative pectate lyase, leading to a frame shift and a longer ORF. Our results suggested that the premature senescence of the ospse1 may be regulated by a novel mechanism mediated by pectate lyase.     

Map-Based Cloning of zb7 Encoding an IPP and DMAPP Synthase in the MEP Pathway of Maize

IspH is a key enzyme in the last step of the methyl-D-erythritol-4-phosphate (MEP) pathway. Loss of function of IspH can often result in complete yellow or albino phenotype in many plants. Here, we report the characterization of a recessive mutant of maize, zebra7 (zb7), showing transverse green/yellow striped leaves in young plants. The yellow bands of the mutant have decreased levels of chlorophylls and carotenoids with delayed chloroplast development. Low temperature suppressed mutant phenotype, while alternate light/dark cycle or high temperature enlarged the yellow section. Map-based cloning demonstrated that zb7 encodes the IspH protein with a mis-sense mutation in a conserved region. Transgenic silencing of Zb7 in maize resulted in complete albino plantlets that are aborted in a few weeks, confirming that Zb7 is important in the early stages of maize chloroplast development. Zb7 is constitutively expressed and its expression subject to a 16-h light/8-h dark cycle regulation. Our results suggest that the less effective or unstable IspH in zb7 mutant, together with its diurnal expression, are mechanistically accounted for the zebra phenotype. The increased IspH mRNA in the leaves of zb7 at the late development stage may explain the restoration of mutant phenotype in mature stages.     

Young Leaf Chlorosis 1, a chloroplast-localized gene required for chlorophyll and lutein accumulation during early leaf development in rice

Chlorophyll (Chl) and lutein are the two most abundant and essential components in photosynthetic apparatus, and play critical roles in plant development. In this study, we characterized a rice mutant named young leaf chlorosis 1 (ylc1) from a ⁶⁰Co-irradiated population. Young leaves of the ylc1 mutant showed decreased levels of Chl and lutein compared to those of wild type, and transmission electron microscopy analysis revealed that the thylakoid lamellar structures were obviously loosely arranged. Whereas, the mutant turns green gradually and approaches normal green at the maximum tillering stage. The Young Leaf Chlorosis 1 (YLC1) gene was isolated via map-based cloning and identified to encode a protein of unknown function belonging to the DUF3353 superfamily. Complementation and RNA-interference tests confirmed the role of the YLC1 gene, which expressed in all tested rice tissues, especially in the leaves. Real-time PCR analyses showed that the expression levels of the genes associated with Chl biosynthesis and photosynthesis were affected in ylc1 mutant at different temperatures. In rice protoplasts, the YLC1 protein displayed a typical chloroplast location pattern. The N-terminal 50 amino acid residues were confirmed to be necessary and sufficient for chloroplast targeting. These data suggested that the YLC1 protein may be involved in Chl and lutein accumulation and chloroplast development at early leaf development in rice.     

Genome sequencing reveals agronomically important loci in rice using MutMap

The majority of agronomic traits are controlled by multiple genes that cause minor phenotypic effects, making the identification of these genes difficult. Here we introduce MutMap, a method based on whole-genome resequencing of pooled DNA from a segregating population of plants that show a useful phenotype. In MutMap, a mutant is crossed directly to the original wild-type line and then selfed, allowing unequivocal segregation in second filial generation (F(2)) progeny of subtle phenotypic differences. This approach is particularly amenable to crop species because it minimizes the number of genetic crosses (n = 1 or 0) and mutant F(2) progeny that are required. We applied MutMap to seven mutants of a Japanese elite rice cultivar and identified the unique genomic positions most probable to harbor mutations causing pale green leaves and semidwarfism, an agronomically relevant trait. These results show that MutMap can accelerate the genetic improvement of rice and other crop plants.