Comparative analysis of the 100 kb region containing the Pi-k(h) locus between indica and japonica rice lines

We have recently cloned a pathogen inducible blast resistance gene Pi-kh from the indica rice line Tetep using a positional cloning approach. In this study, we carried out structural organization analysis of the Pi-kh locus in both indica and japonica rice lines. A 100 kb region containing 50 kb upstream and 50 kb down- stream sequences flanking to the Pi-kh locus was selected for the investigation. A total of 16 genes in indica and 15 genes in japonica were predicted and anno- tated in this region. The average GC content of indica and japonica genes in this region was 53.15% and 49.3%, respectively. Both indica and japonica sequences were polymorphic for simple sequence repeats having mono-, di-, tri-, tetra-, and pentanucleotides. Sequence analysis of the specific blast resistant Pi-kh allele of Tetep and the susceptible Pi-kh allele of the japonica rice line Nipponbare showed differences in the number and distribution of motifs involved in phosphorylation, resulting in the resistance phenotype in Tetep.     

中国春小麦株高、育性近等基因系的建立及应用

以矮败小麦和中国春小麦为材料,经过杂交和连续回交,得到了中国春小麦遗传背景的分别表现矮秆不育、矮秆可育、高秆不育、高秆可育的近等基因系。根据近等基因系各成员系的株高表现,计算出矮秆基因Rht10的降秆强度是69.8%。借助于赤霉酸处理,在幼芽期就可分出矮败中国春小麦后代的不育株与可育株。 Abstract：　Use Dwarfing Male-sterile Wheat and cv. Chinese Spring as parents, after cross and continuously back cross, the isogenic lines with Chinese Spring background were developed. These lines include dwarfing male-sterile line, dwarfing fertile line, tall male-sterile line and tall fertile line. The dwarfing intensity of gene Rht10 was calculated to be 69.8% according the differences between the isogenic lines. Treated with GA3solution, the male-sterile and fertile plants in Chinese Spring Dwarfing Male-sterile Wheat can be identified clearly when they are seedlings.     

中国春小麦株高、育性近等基因系的建立及应用

以矮败小麦和中国春小麦为材料,经过杂交和连续回交,得到了中国春小麦遗传背景的分别表现矮秆不育、矮秆可育、高秆不育、高秆可育的近等基因系。根据近等基因系各成员系的株高表现,计算出矮秆基因Rht10的降秆强度是69.8%。借助于赤霉酸处理,在幼芽期就可分出矮败中国春小麦后代的不育株与可育株。 Abstract：　Use Dwarfing Male-sterile Wheat and cv. Chinese Spring as parents, after cross and continuously back cross, the isogenic lines with Chinese Spring background were developed. These lines include dwarfing male-sterile line, dwarfing fertile line, tall male-sterile line and tall fertile line. The dwarfing intensity of gene Rht10 was calculated to be 69.8% according the differences between the isogenic lines. Treated with GA3solution, the male-sterile and fertile plants in Chinese Spring Dwarfing Male-sterile Wheat can be identified clearly when they are seedlings.     

Elemental Content in Brown Rice by Inductively Coupled Plasma Atomic Emission Spectroscopy Reveals the Evolution of Asian Cultivated Rice

The phylogenetic relationship for classification traits and eight mineral elements in brown rice （Oryza sativa L.） from Yunnan Province in China was carried out using microwave assisted digestion followed by inductively coupled plasma atomic emission spectroscopy, and the analytical procedures were carefully controlled and validated. In general, the results show that the mean levels of K, Ca, Mg, Fe and Cu in brown rice for 789 accessions of rice landraces was distinctly lower than that of improved cultivars. They further demonstrate that Ca plays an important role in the differentiation of subspecies indica-japonica, especially to enhance adaptation of cold stress, and that five mineral elements in brown rice enhance the eurytopicity from landrace to improved cultivar. Hierarchical cluster analysis, using average linkage from SPSS software based on eight mineral elements in brown rice, showed that Yunnan rice could be grouped into rice landrace and improved cultivar, with the rice landrace being further clustered into five subgroups, and that, interestingly, purple rice does not cluster with either of the groups. Our present data confirm that indica is the closest relative of late rice and white rice, and that they constitute rice landraces together, whereas japonica is the closest relatives of non-nuda, early-mid and glutinous rice. It is further shown that japonica, non-nuda, early-mid, glutinous, white and red rice might be more primitive than indica, nuda, late, non-glutinous and purple rice, respectively.     

Characterization and Fine Mapping of a Novel Rice Narrow Leaf Mutant nal9

A narrow leaf mutant was isolated from transgenic rice （Oryza sativa L.） lines carrying a T-DNA insertion. The mutant is characterized by narrow leaves during its whole growth period, and was named nal9 （narrow leaf 9）. The mutant also has other phenotypes, such as light green leaves at the seedling stage, reduced plant height, a small panicle and increased tillering. Genetic analysis revealed that the mutation is controlled by a single recessive gene. A hygromycin resistance assay showed that the mutation was not caused by T-DNA insertion, so a map-based cloning strategy was employed to isolate the nal9 gene. The mutant individuals from the F2 generations of a cross between the nal9mutant and Longtepu were used for mapping. With 24 F2 mutants, the nal9 gene was preliminarily mapped near the marker RM156 on the chromosome 3. New INDEL markers were then designed based on the sequence differences between japonica and indica at the region near RM156. The nal9 gene was finally located in a 69.3 kb region between the markers V239B and V239G within BAC OJ1212_C05 by chromosome walking. Sequence and expression analysis showed that an ATP-dependent CIp protease proteolytic subunit gene （CIpP） was most likely to be the nal9 gene. Furthermore, the nal9 mutation was rescued by transformation of the CIpP cDNA driven by the 35S promoter. Accordingly, the CIpP gene was identified as the NAL9 gene. Our results provide a basis for functional studies of NAL9 in future work.     

The breeding of two polyploid rice lines with the characteristic of polyploid meiosis stability

Polyploidization is a basic feature of plant evolution. Nearly all of the main food, cotton and oil crops are polyploid. When ploidy levels increase, yields double; this phenomenon suggested a new strategy of rice breeding that utilizes wide crosses and polyploidization dual advantages to breed super rice. Because low seed set rates in polyploid rice usually makes it difficult to breed, the selection of Ph-liked gene lines was emphasized. After progenies of indica-japonica were identified and selected, two poly- ploid lines, PMeS-1 and PMeS-2 with Polyploid Meiosis Stability (PMeS) genes were bred. The proce- dure included seven steps: selecting parents, crossing or multiple crossing, back-crossing, doubling chromosomes, identifying the polyploid, and choosing plants with high seed set rates that can breed themselves into stable lines. The characteristics of PMeS were determined by observing meiotic be- haviors and by cross-identification of seed sets. PMeS-1 and PMeS-2, (japonica rice), have several characteristics different from other polyploid rice lines, including a higher rate of seed set (more than 65%, increasing to more than 70% in their F1 offspring); and stable meiotic behaviors (pairing with bi- valents and quarivalents nearly without over-quarivalent in prophase, nearly without lagging chromo- somes in metaphase and without micronuclei in anaphase and telophase). The latter was obviously different from control polyploid line Dure-4X, which displayed abnormal meiotic behaviors including a higher rate of multivalents, univalents and trivalents in prophase, lagging chromosomes in metaphase and micronuclei in anaphase and telophase. There were also three differences of the breeding method between PMeS lines and normal diploid lines: chromosomes doubling, polyploidism identifying and higher seed set testing. The selection of PMeS lines is the first step in polyploid rice breeding; their use will advance the progress of polyploid rice breeding, which will in turn offer a new way to breed super rice.     

LSCHL4 from Japonica Cultivar,Which Is Allelic to NAL 1, Increases Yield of Indica Super Rice 93-11

The basic premise of high yield in rice is to improve leaf photosynthetic efficiency and coordinate the sourcesink relationship in rice plants. Quantitative trait loci （QTLs） related to morphological traits and chlorophyll content of rice leaves were detected at the stages of heading to maturity, and a major QTL （qLSCHL4） related to flag leaf shape and chlorophyll content was detected at both stages in recombinant inbred lines constructed using the indica rice cultivar 93-11 and the japonica rice cultivar Nipponbare. Map-based cloning and expression analysis showed that LSCHL4 is allelic to NAL1, a gene previously reported in narrow leaf mutant of rice. Overexpression lines transformed with vector carrying LSCHL4 from Nipponbare and a near-isogenic line of 93-11 （NIL-9311） had significantly increased leaf chlorophyll content, enlarged flag leaf size, and improved panicle type. The average yield of NIL-9311 was 18.70% higher than that of 93-11. These results indicate that LSCHL4 had a pleiotropic function. Exploring and pyramiding more high-yield alleles resem- bling LSCHL4 for super rice breeding provides an effective way to achieve new breakthroughs in raising rice yield and generate new ideas for solving the problem of global food safety.     

Introduction of a rice blight resistance gene,Xa21, into five Chinese rice varieties through anAgrobacterium-mediated system

A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3 : 1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transfor-mants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.     

Agrobacterium-Mediated Multiple Gene Transformation in Rice Using a Single Vector

The homodimeric hemoglobin gene (VHb), the trans-zeatin synthetase gene (tzs), the modified 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS), a selectable marker gene (hpt), and a reporter gene (gus), as linked expression cassettes, were stacked into the T-DNA region of a binary vector and introduced simultaneously into immature embryos of the rice (Oryza sativa L.) varieties Xiushui-11, Qiufeng,Youfeng, and Hanfeng by Agrobacterium tumefaciens. A total of 1 153 transgenic lines was obtained through selection for hygromycin B resistance. Approximately 90.2% of the transgenic lines harbored all the transgenes. Integration of multiple transgenes occurred at one to three genetic loci. Expression analysis revealed that the transgenes were coexpressed and inherited in a simple Mendelian fashion in transgenic plants and the frequency of coexpression was approximately 85%. On the basis of the cointegration and coexpression of the transgenes, most transgenic families were considered to be useful in a breeding program.     

水稻感光性和光敏不育性的发育遗传关系

在人工气候室控制的光长与温度下,用光敏不育系7001S与早中熟粳稻(Oryza sativa ssp,japoni-ca)感光性弱的“秋光”、“有芒早粳”和“CPSLO-l7”3个品种分别杂交,分析了杂种F_1、F_2植株育性与感光性的表现,以及两者的关系。结果表明:这3个组合的恢复亲本均属于具两对光敏不育的恢复基因的品种。F_1植株均倾向晚熟亲本,说明控制感光性强的基因属于显性。F_2植株在长日照下表现为不育株的感光性均倾向感光性强的亲本;且有不少不育株为晚熟超亲;在少数弱感光性的植株中没有不育株。说明光敏不育基因与感光性基因关系密切,可能两者有连锁关系,光敏不育基因要在感光性基因表达的基础上才能表达。     

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.     

Genotyping the Heading Date of Male-Sterile Rice Line Ⅱ-32A

Ⅱ-32A, an elite male-sterile line of rice (Oryza sativa L.), has been widely used for the production of hybrid rice seed in China. Heading date in most combinations using Ⅱ-32A shows transgressive inheritance or similarity to the latter parent, but the genotype of Ⅱ-32A with respect to major genes for heading time is unknown. This limits the further exploitation of this sterile line in breeding and hybrid seed production. Using a number of major gene heading date isogenic lines and heading date QTL near-isogenic lines, we genetically analyzed Ⅱ-32B under both long- and short-day conditions. We show that Ⅱ-32B carries two photoperiod-sensitive genes, E1 and E3, a recessive late-heading gene, ef-1, and a photoperiod-sensitive allele, Se-1u. In addition we identified in Ⅱ-32B a recessive inhibitor for E1 or Se-1n and other modified photoperiod-sensitive genes. The heading-date constitution of Ⅱ-32A was determined to be E1e2E3Se-1Uef-1i-Se-1.     

Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production

The pentatricopeptide repeat (PPR) gene family represents one of the largest gene families in higher plants. Accumulating data suggest that PPR proteins play a central and broad role in modulating the expression of organellar genes in plants. Here we report a rice (Oryza sativa) mutant named young seedling albino (ysa) derived from the rice thermo/photoperiod-sensitive genic male-sterile line Pei'ai64S, which is a leading male-sterile line for commercial two-line hybrid rice production. The ysa mutant develops albino leaves before the three-leaf stage, but the mutant gradually turns green and recovers to normal green at the six-leaf stage. Further investigation showed that the change in leaf color in ysa mutant is associated with changes in chlorophyll content and chloroplast development. Map-based cloning revealed that YSA encodes a PPR protein with 16 tandem PPR motifs. YSA is highly expressed in young leaves and stems, and its expression level is regulated by light. We showed that the ysa mutation has no apparent negative effects on several important agronomic traits, such as fertility, stigma extrusion rate, selfed seed-setting rate, hybrid seed-setting rate, and yield heterosis under normal growth conditions. We further demonstrated that ysa can be used as an early marker for efficient identification and elimination of false hybrids in commercial hybrid rice production, resulting in yield increases by up to approximately 537 kg ha(-1).     

有利基因和有利的基因互作能够提高籼粳杂种育性

利用一套籼粳交ＤＨ系与两份具有广亲和性的水稻光（温）敏核不育系测交,构建两个测交Ｆ１群体,同时用９２个多型性的ＲＦＬＰ标记分析了杂交亲本和另外３０个不同类型的籼、粳品种（对照组）的基因型。利用对照组籼或粳表型与分子标记的关联性,检测出４１个与籼粳分化高度相关的ＲＦＬＰ标记。结果表明,８７．８％的这种标记参与了对杂种优势显著或极显著的影响,表明在水稻的演化过程中,参与适应性进化的基因与控制经济产量的     

光敏核不育水稻农垦58S与其衍生不育系的叶绿体DNA的比较

扩增了光敏核不育系农垦58S及从它衍生出来的5个两用核不育系叶绿体DNA的ORF（open reading frame）100、ORF29-TrnC^GCA、rps16（ribosomal proteins16）基因内含子和TrnT^UGU-TrnL^UAA（tRNA^Thr（UGU）-tRNA^-Leu（UAA）等4个片段,并测定了其序列。研究结果表明,粳型光敏核不系农垦58S的叶绿体为粳型。农垦58S衍生的核不育系中,粳型核不育系7001S以及3个籼型核不育系1103S、培矮64S和广占63S的叶绿体DNA为粳型,与选育者提供的细胞质系谱一致。籼型核不育系W6154S的叶绿体DNA为籼型,与选育者提供的细胞质系谱不一致,推断选育者在选育过程中更换过细胞质（曾用不育系作过父本）。5个粳型叶绿体DNA的rps16基因内含子和TrnT^UGU-TrnL^UAA间区的序列相互之间有1-2个单核苷酸的变异。     

中籼杂交水稻亲本多态性的AFLP分析

对15个籼型杂交水稻亲本进行AFLP分析,结果表明：亲本间遗传距离小,在0.0589-0.3305之间,平均为0.2033。15个亲本按类平均法可聚为两类,Ⅰ类为不育系,Ⅱ类为恢复系。其中Ⅱ类又分为两个亚类,Ⅱ－1不含明恢63血缘、Ⅱ－2全部含明恢63血缘。Ⅰ/Ⅱ－1与Ⅰ/Ⅱ－2间的遗传距离无明显差异,揭示恢复系的遗传基础较一致,这可能是当前的品种不能超过籼优63的重要原因之一。要提高水稻的杂种优势,需丰富亲本的遗传基础,扩大其遗传差异。     

Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice

Potassium antimonate was used to locate Ca²⁺ in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice (Oryza sativa L. japonica). During the development of fertile anthers, abundant calcium precipitates accumulated in the anther walls and on the surface of pollen grains and Ubish bodies at the late developmental stage of the microspore, but not in the cytoplasm of pollen grains. Following the accumulation of starch grains in pollen, calcium precipitates on pollen walls diminished and increased in parenchymatous cells of the connective tissue. In sterile anthers, calcium precipitates were abundant in the middle layer and endothecium, but not in the tapetum, as was found in fertile anthers. A special cell wall was observed between the tapetum and middle layer of sterile anthers that appeared to relate to distinctive calcium accumulation patterns and poor pollen wall formation in the loculi. The formation of different patterns of antimonate-induced calcium precipitates in the anthers of photoperiod-sensitive genic male-sterile rice indicates that anomalies in the distribution of calcium accumulation correlate with the failure of pollen development and pollen abortion. Received: 30 May 1997 / Accepted: 5 July 1997     

以水稻雄性不育系为母本不经幼胚培养获得Oryza sativa×O.officinalis杂种的研究

利用亚洲栽培稻(Oryza sativa L.)的核质互作型和光敏感型雄性不育系为母本,药用野生稻(O.officinalis Wall)为父本进行杂交,首次在未经幼胚培养的条件下得到了杂种种子。杂种F_1生长优势强,分蘖力和发根力等超过双亲,并具有父本的植株散生、高位分蘖、小穗具芒、穗分枝散生、落粒性和感光性强等特性。其酯酶同工酶谱中含有双亲的全部酶带。花粉母细胞中染色体数为2n=24,在减数分裂中期Ⅰ。基本上全部为单价体。杂种自交不孕。本研究结果表明,利用雄性不育系进行异源种间的远缘杂交具有简化杂交过程以便大量配组、增加异源遗传物质导入机会和杂种可靠、便于鉴定等优点。本研究不但对探讨水稻远缘杂交的新途径有一定意义,而且为雄性不育系的利用展开了新的视野。     

Effect of Photoperiod Treatments on Phytochrome a Levels and Its mRNA Abundance in Photoperiod-Sensitive Genic Male-Sterile Rice Mutant and the Wild Type

The effect of photoperiod treatments on phytochrome A (Phy A) level and its mRNA abundance in the leaves of a photoperiod-sensitive genic male-sterile mutant (Nongken 58S) and its wild type ("Nongken 58') of Oryza sativa L. was investigated. The top two leaves of each rice shoot were harvested at the end of the last dark phase of 10 cycles during photoperiod-sensitive stage for fertility alteration of the mutant. Phy A was measured by a sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal and mouse monoclonal antibodies. Compared with longday (LD) treatment,short day (SD) resulted in 38.5% increase of relative Phy A content in the mutant, only 18.5% increase in the wild type. In an extended darkness (25 h), the accumulation of Phy A also appeared to be more rapid in the mutant seedlings than in its wild type. RNA dot blot analyses with RPA3 (a cDNA clone of rice Phy A) as a probe showed:the abundances of Phy A mRNA in top leaves of Nongken 58S and "Nongken 58" were obviously higher in SD than those in ID at the end of dark phase of 5 d and 10 d photoperiod treatments. Moreover, under SD Phy A mRNA contents in Nongken 58S were more than those in "Nongken 58" during the whole photoperi- od-sensitive stage for fertility alteration. In addition, after 10 cycles of end-of-day far-red irradiations (EOD FR), the heading and flowering date of the mutant was delayed for 2 d. However, EOD FR had little or no effect on male fertility of the mutant.