Characterization and mapping of a white panicle mutant gene in rice

A spontaneous white panicle mutant was found from the F6 progenies of an indicajaponica cross.The mutant exhibits white stripes on its basal leaves while the panicles,rachis and pedicel are milky white colored at flowering stage.Genetic analysis in an F2 population from the cross of Zhi7/white panicle mutant indicates that the white panicle phenotype is controlled by a single recessive nuclear gene,tentatively termed as wp(t).Using microsatellite markers,the wp(t) gene was anchored between the markers of SSR101 and SSR63.9 with a map distance of 2.3 and 0.8cM,respectively,and co-segregated with the marker of SSR17 on rice chromosome 1.     

Genetic analysis and fine mapping of the pubescence gene GL6 in rice (Oryza sativa L.)

The pubescence of the leaf blade surface is an important agronomic characteristic for rice morphology and significantly influences rice growth as well as physiological characteristics. This characteristic was analyzed in F1 and F2 plants derived by crossing cultivar 75-1-127 with the indica cultivar Minghui 63, as well as the glabrous cultivar Lemont and indica cultivar 9311. Results indicated that the pubescence of the leaf blade surface was a dominant trait and controlled by a single gene. The GL6 gene was primarily mapped on rice chromosome 6 with recessive F2 population derived from 75-1-127/Minghui 63 by combining bulked segregation analysis and recessive class analysis using the Mapmaker3.0/MapDraw software. The genetic distances between the simple sequence repeat markers RM20491 and RM20547 were 7.2 and 2.2 cM, respectively. The GL6 gene was fine mapped in the interval between InDel-106 and InDel-115 at genetic distances of 0.3 and 0.1 cM, respectively. The large, recessive F2 population was derived from 75-1-127/Minghui 63. A high-resolution genetic and physical map of GL6 was constructed. Derived from the map-based sequences published by the International Rice Genome Sequencing Project, the GL6 gene was localized at an interval of 79 (japonica) and 116.82 kb (9311) bracketed by InDel-106 and InDel-115 within the BAC accession numbers AP008403 and AP005760. Seven annotated genes (japonica) and eight annotated genes (9311) were present. The basis was further set for GL6 cloning and function analysis.     

Genetic analysis and gene cloning of a triangular hull 1 (tri1) mutant in rice (Oryza sativa L.)

Grain shape and size are two key factors that determine rice yield and quality. In the present study, a rice triangular hull mutant (tri1) was obtained from the progeny of japonica rice variety Taipei 309 treated with 60Co γ-rays. Compared to the wild type, the tri1 mutant presents a triangular hull, and exhibits an increase in grain thickness and protein content, but with a slight decrease in plant height and grain weight. Genetic analysis indicated that the mutant phenotype was controlled by a recessive nuclear gene which is stably inherited. Using a map-based cloning strategy, we fine-mapped tri1 to a 47-kb region between the molecular markers CHR0122 and CHR0127 on the long arm of chromosome 1, and showed that it co-segregates with the molecular marker CHR0119. According to the rice genome sequence annotation there are six predicated genes within the mapped region. Sequencing analysis of the mutant and the wild type indicated that there was a deletion of an A nucleotide in exon 3 of the OsMADS32 gene, which could result in a downstream frameshift mutation and premature termination of the predicted polypeptide. Both semi-quantitative and real-time RT-PCR analyses showed that this gene expressed highly in young inflorescences, while expressed at very low levels in other tissues. These results implied that the OsMADS32 gene could be a candidate of TRI1. Taken together, the results of this study lay the foundation for further investigation into the molecular mechanisms regulating rice caryopsis development.     

Mapping and characterization of a tiller-spreading mutant<Emphasis Type="Italic">lazy-2</Emphasis> in rice

Tiller angle of rice is an important agronomic trait that contributes to breed new varieties with ideal architecture. In this study, we report mapping and characterization of a rice mutant defective in tiller angle. At the seedling stage, the newly developed tillers of the mutant plants grow with a large angle that leads to a “lazy“ phenotype at the mature stage. Genetic analysis indicates that this tillerspreading phenotype is controlled by one recessive gene that is allelic to a reported mutant la. Therefore, the mutant was named la-2 and la renamed la-1. To map and clone LA, we constructed a large mapping population. Genetic linkage analysis showed that the LA gene is located between 2 SSR markers RM202 and RM229. By using the 6 newly-developed molecular markers, the LA gene was placed within a 0.4 cM interval on chromosome 11, allowing us to clone LA and study the mechanism that controls rice tiller angle at the molecular level.     

Genetic analysis and molecular mapping of a presenescing leaf gene psl1 in rice (Oryza sativa L.)

A rice psl1 (presenescing leaf) mutant was obtained from a japonica variety Zhonghua 11 via radiation of 60Co-γ in M2 generation. Every leaf of the mutant began to wither after it reached the big-gest length,while the leaves of the wild variety could keep green for 25―35 d. In this study,genetic analysis and gene mapping were carried out for the mutant identified. The SSR marker analysis showed that the mutant was controlled by a single recessive gene (psl1) located on chromosome 2. Fine mapping of the psl1 locus was conducted with 34 new STS markers developed around psl1 anchored region based on the sequence diversity between Nippon-bare and 93-11. The psl1 was further mapped be-tween two STS markers,STS2-19 and STS2-26,with genetic distances of 0.43 and 0.11 cM,respectively,while cosegregated with STS2-25. A BAC contig was found to span the psl1 locus,the region being delim-ited to 48 kb. This result was very useful for cloning of the psl1 gene.     

Genetic analysis and molecular mapping of a presenescing leaf gene psll in rice （Oryza sativa L.）

A rice psl1 （presenescing leaf） mutant was obtained from a japonica variety Zhonghua 11 via radiation of ^60Co-γ in M2 generation. Every leaf of the mutant began to wither after it reached the biggest length, while the leaves of the wild variety could keep green for 25--35 d. In this study, genetic analysis and gene mapping were carried out for the mutant identified. The SSR marker analysis showed that the mutant was controlled by a single recessive gene （psl1） located on chromosome 2. Fine mapping of the psl1 locus was conducted with 34 new STS markers developed around psl1 anchored region based on the sequence diversity between Nipponbare and 93-11. The psl1 was further mapped between two STS markers, STS2-19 and STS2-26, with genetic distances of 0.43 and 0.11 cM, respectively, while cosegregated with STS2-25. A BAC contig was found to span the psl1 locus, the region being delimited to 48 kb. This result was very useful for cloning of the psl1 gene.     

Morphological and physiological analysis of narrow and striped leaf 1 （nsl1） mutant of rice （Oryza sativa L.） and the gene mapping

The shape and color of rice leaves are impor- tant agronomic traits that directly influence the proportion of sunlight energy utilization and ultimately affect the yield and quality. A new mutant exhibiting stable inheritance was identified as derived from ethyl methane sulfonate （EMS）-treated restorer Jinhui 10, tentatively named as narrow and striped leaf 1 （nsll）. The nsll displayed pale white leaves at the seeding stage and then white striped leaves in parallel to the main vein at the jointing stage. Meanwhile, its leaf blades are significantly narrower than the control group of Jinhui 10. The chloroplast structures of cells in the white striped area of the nsll mutant break down, and the photosynthetic pigments are significantly lower than that of the wild type. Moreover, fluorescence parameters, such as Fo, Fv/Fm, ФpsⅡ, qP, and ETR, in the nsll mutant are significantly lower than those of the wild type, and the photosynthetic efficiency is also significantly decreased. These changes in leaf color and shape, together with physiological changes in the nsll, result in smaller plant height and a decrease in the most important agro- nomic traits, such as the number of grains per panicle, grain weight, etc. Genetic analysis shows that the narrow and striped traits of the nsll mutant are controlled by a single recessive nuclear gene, which is located between InDel 16 and InDel 12 in chromosome 3. The physical distance is 204 kb. So far, no similar genes of such leaf color and shape in this area have been reported, This study has laid asolid foundation for the gene cloning and function analysis of NSL 1.     

Identification and gene mapping of a narrow and upper-albino leaf mutant in rice (Oryza sativa L.)

The leaf blade consists of color and shape traits. Studies of leaf-blade development are important for improvement of rice yield and quality because it is an essential organ for photosynthesis. A narrow and upper-albino leaf mutant (nul1) was identified from among progeny of the indica restorer line Jinhui10 raised from seeds treated with ethyl methane sulfonate. Under field conditions, the mutant displayed narrow and upper-albino leaf blades with significantly decreased photosynthetic pigment contents throughout their development. The narrow-leaf trait is caused by a decreased number of small veins. In contrast to the wild type, the growth period was extended by approximately 8 d and agronomic traits, such as effective panicle number, percentage seed set and 1000-grain weight, declined significantly in the nul1 mutant. Genetic analysis suggested that the narrow and upper-albino leaf characteristics showed coseparation and were controlled by one recessive gene. The Nul1 gene was mapped onto chromosome 7 between the Indel marker Ind07-1 and the Simple Sequence Repeat marker RM21637. The physical distance between the markers was 75 kb and eight genes were annotated in this region based on the rice Nipponbare genome sequence. These results provide a foundation for cloning and function analysis of Nul1.     

Fine mapping of cisc(t), a gene for cold-induced seedling chlorosis, and identification of its candidate in rice

The seedlings of indica rice cultivar Dular are susceptible to chlorosis under low temperature conditions. Our previous studies indicated that low temperature-induced seedling chlorosis is controlled by a recessive gene, located between SSR markers RM257 and RM242, on the long arm of chromosome 9. We temporarily named the gene cisc(t). Using a large F₂ population derived from a cross between Dular and the japonica cultivar Lemont, which displays a normal green color at low temperatures, cisc(t) was fine mapped to within a 12-kb interval. There is only one annotated gene in this interval, which encodes a pentatricopeptide repeat (PPR) protein. Sequence analysis indicated that 8 bases were deleted at the 60th base in the Dular allele, resulting in a frame-shift mutation and loss of function of the gene. This is consistent with the chlorosis mutant phenotype of Dular. In addition, previous studies have shown that many chlorosis mutants of seedlings are related to PPR proteins. Hence, we presume that the PPR gene is the candidate for cisc(t).     

利用CRISPR /Cas9技术快速创制香型“秀水134”水稻

以目前上海市主栽的高产常规水稻"秀水134"为材料,利用CRISPR/Cas9技术成功敲除甜菜碱醛脱氢酶2基因,获得了两种类型纯合突变体植株.采用表达载体特异性结合的引物检测T_1代转基因植株,成功获得6株不携带载体骨架的转基因植株.定量PCR分析显示,突变体植株甜菜碱醛脱氢酶2基因表达量极显著低于野生型对照(p0.01),但突变体植株成熟种子香味物质2-乙酰-1-吡咯啉(2AP)含量极显著高于野生型对照(p0.01).比较野生型对照与突变体植株的主要农艺性状和产量性状,两者间都没有显著差异(p0.05).本研究可为加快高产香型水稻在上海及周边地区的推广应用,以及为今后利用CRISPR/Cas9技术快速培育其他高产香型水稻新品种研究奠定基础.     

Identification of a novel tillering dwarf mutant and fine mapping of the TDDL（T） gene in rice （Oryza sativa L.）

Rice plant architecture is an important agronomic trait that affects the grain yield. To understand the molecular mechanism that controls plant architecture, a tillering dwarf mutant with darker-green leaves derived from an indica cultivar IR64 treated with EMS is characterized. The mutant, designated as tddl（t）, is nonallelic to the known tiilering dwarf mutants. It is controlled by one recessive nuclear gene, TDDL（T）, and grouped into the dn-type dwarfism according to Takeda＇s definition. The dwarfism of the mutant is independent of gibberellic acid based on the analyses of two GA-mediated processes. The independence of brassinosteroid （BR） and naphthal-3-acetic acid （NAA） of the tddl（t） mutant, together with the decreased size of parenchyma cells in the vascular bundle, indicates that the TDDL（7） gene might participate in another hormone pathway. TDDL（T） is fine mapped within an 85.51 kb region on the long arm of rice chromosome 4, where 20 ORFs are predicted by RiceGAAS （http：//ricegaas.dna.affrc. go.jp/rgadb/）. Further cloning of TDDL（T） will benefit both marker assisted selection （MAS） of plant architecture and dissection of the molecular mechanism underlying tillering dwarf in rice.