Identification of natural variants affecting chlorophyll content dynamics during rice seedling development

Seedling vigour is an important characteristic in relation to crop growth and yield. Traits such as photosynthetic capacity and chlorophyll content contribute significantly to seedling establishment at the early growth stage in various crop species, including rice. A diverse panel of 227 rice varieties from several countries was evaluated to determine chlorophyll contents at multiple time points during the seedling stage using a soil–plant analysis development (SPAD) meter, a non‐destructive portable device. Using new statistical approaches, several chromosomal regions associated with variations in chlorophyll content in the third leaf at 13, 16 and 19 days after imbibition were detected. A single nucleotide polymorphism (SNP) cluster on the end of chromosome 11 was significantly associated with the onset of leaf senescence. This region was enriched with genes related to cell death and the stress response. We have identified rice germplasm showing delayed‐senescence phenotypes, these could be suitable donors and genetic resources for breeding, and the use of significant SNP markers associated with these traits could enhance the efficiency of their selection in breeding programmes.     

Genetic analysis of methionine content in indica-japonica hybrid rice (Oryza sativa L.) at different grain developmental stages

Genetics of methionine content in indica-japonica hybrid rice (Oryza sativa L.) was studied in 35 F₁ hybrids derived from crossing 7 male-sterile indica rice cultivars with 5 restorer japonica rice cultivars. Two genetic models and their corresponding statistical methods for quantitative traits of triploid endosperm in cereal crops were used for the analysis. One is the unconditional model, which refers to the analysis of cumulative measurements along the developmental stages, while another is the conditional model, which relates to analysis throughout the developmental stages. Results showed that methionine content of indica-japonica hybrid rice was controlled by expression of triploid endosperm nuclear genes, cytoplasm genes, diploid maternal plant nuclear genes, and their genotype-environment interaction effects. Dominant effects were important at the first three developmental stages, while additive effects were important at the next two stages of grain development under both unconditional and conditional analyses. With regard to the components of heritability, maternal and cytoplasm general heritabilities and their environmental interaction heritabilities were important. It was suggested that enhancing methionine content could be more efficient when selection is based on maternal plants in early breeding generations.     

二十一世纪水稻育种新战略Ⅱ．利用远缘杂交和多倍体双重优势进行超级稻育种

本文论述了远缘杂交和多倍体化结合利用基因组间和多倍本杂种优势，开创水稻育种新途径的策略。纵观水稻育种的历史，无论是常规杂交育种，还是杂交稻育种，其研究战略都是建立在有性生殖和二倍体基础上的，归根结底，都是利用栽培稻同一基因组（A基因组）内优良基因的重组以及从野生稻向栽培稻引入少数优良基因。从作物进化趋势看，水稻是二倍体，基因组小、DNA含量低、染色体小，增加基因组数，提高倍性水平，利用异源多倍体杂种优势将是水稻育种新途径。针对同源四倍体水稻结实率低的关键问题，可采取拉大亲缘关系距离、减少多价体形成，应用广亲和、无融合生殖基因等措施，从遗传机理上提高多倍体水稻结实率。按三步实施战略：一、选用极端类型籼粳稻、爪哇稻，诱导亚种间杂种多倍体；二、诱导亚洲栽培稻和非洲栽培稻以及非洲野生稻种间杂种多倍体；三、诱导AA基因组的栽培稻与其它不同基因组野生稻（BB、CC、BBCC、CCDD、DD、EE、FF、GG、HHJJ）的种间多倍体。在实施过程中发挥一特殊基因材料的特殊作用，如广亲和基因对于克服籼粳杂种一代不育性、特别是雌败育的作用，无融合生殖缺乏减数分裂发生和受精过程对于克服染色体配对分离而导致的杂种败育的作用，以及类似于小麦中存在的抑制部分同源染色体配对的Ph基因对于防止水稻异源多倍体的基因组间部分同源性配对形成多价体、染色体桥、落后染色体等现象的作用，为选育异源多倍体水稻、无融合生殖水稻提供可靠保证。近三年的实践已经初获良好的结果，利用无融合生殖水稻品系籼粳交，亚洲稻与美洲稻杂交已获得杂种优势明显、结实率高达85％的优良多倍体株系，为实现这一育种新战略打下了良好基础。     

5个水稻蛋白激酶的克隆、表达及活性研究

蛋白激酶在生长发育、表达调控、逆境反应、信号传导等几乎所有的生命过程中发挥着重要作用，蛋白激酶的研究具有重要的理论和应用价值。全基因组测序结果表明，水稻基因组中至少有1500个蛋白激酶。本研究利用已有的蛋白激酶序列，通过PCR扩增和重组克隆，将5个重要的蛋白激酶在酿酒酵母系统中进行了表达，它们的编号分别是：NM-189878，NM-194133，NM-190508，NM-197522和NM-197571，其中NM-194133具有较强的自我磷酸化活性。同源性分析表明，这些激酶在水稻中可能的作用包括：碳代谢的生物合成调控、种子形态发生、细胞分裂调控、生物和非生物刺激的反应调控等。这项工作为进一步的体外酶活分析及蛋白质－蛋白质相互作用研究等提供了基础，也为高通量克隆表达水稻蛋白激酶提供了一个有效的系统。     

Development and validation of class I SSR markers targeting (GATA) n repeat motifs in rice

SSR markers targeting (GATA) _n motifs are known to be highly polymorphic and useful in many organisms. (GATA) _n motif specific SSR markers covering the whole rice genome are not available. The present study was carried out with an objective to identify class I rice microsatellites in the rice genome with (GATA)_n motifs, in-silico, and validate their potential as molecular markers. A total of 243 such motifs were identified; 65 of these were present in the genic region, 59 were in the upstream region and the remaining motifs were found in the intergenic regions. Many of the (GATA) _n motifs were found within and/or upstream of genes associated with biotic or abiotic stress tolerance. A total of 230 PCR-based markers targeting all the class I (GATA) _n microsatellites were developed and 35 of these markers spread across the rice genome were validated in a set of 24 representative rice varieties belonging to five distinct cultivar groups. All the markers were polymorphic, with average polymorphism information content (PIC) value of 0.61, and the rice cultivars could be uniquely distinguished into different cultivar groups based on marker analysis. These informative markers targeting (GATA) _n motifs representing a new set of markers in rice will be highly useful for genetic studies and marker-assisted selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Passoupathy Rajendrakumar, Akshaya Kumar Biswal and Kannabiran Sakthivel contributed equally.     

黑龙江省稻瘟病生理小种及品种资源抗性鉴定

为了明确黑龙江省水稻品种资源稻瘟病抗性,挖掘优异种质资源,适时了解黑龙江省生理小种群体变化特征。采用中国生理小种命名方法,通过苗期喷雾接菌鉴定,将2013-2014年黑龙江省的稻瘟病菌株划分为7个群42个生理小种,优势小种为ZD5和ZD7,出现频率分别为19.77%和12.21%,总频率为31.98%;通过苗期抗病性表现,筛选出宽抗谱品种14份,这些品种携带2~7个抗稻瘟病基因,绥粳12+合江23(Pi9、Pi20、Pi33、Pi54、Pik)、牡丹江26+龙粳31(Pi9、Pi20、Pi33、Pi54、Pita、Pik)、牡丹江26+合江23(Pi9、Pi20、Pi33、Pi54、Pik)等29个在抗稻瘟病育种生产上将具有较好防病效果的组合,并且能够聚合多个抗性基因,提高抗性水平、拓宽抗谱;其中龙粳31与其他9个品种的配对组合均为最优组合,对稻瘟病具有较高抗性;这14份宽抗谱品种是抗稻瘟病育种较好的抗源材料;部分品种如垦稻15、龙粳23和牡丹江25,仅携带2个本研究鉴定的基因,这些品种可能是携带未知抗性基因的新抗源,可作为进一步鉴定和寻找抗性基因的试验材料。     

Genome‐Wide Identification of MicroRNAs Responsive to High Temperature in Rice (Oryza sativa) by High‐Throughput Deep Sequencing

MicroRNAs (miRNAs) are known to play important roles in plant growth and stress response. Heat stress is a severe abiotic stresses by adversely affecting plant growth and yield. To identify heat‐responsive miRNAs at the genome‐wide level in rice (Oryza sativa), we constructed two small RNA libraries from young panicles treated or not with heat conditions. Ion torrent sequencing of the two libraries identified 294 known miRNAs and 539 novel miRNAs. Differential expression analysis showed that 26 miRNAs were downregulated and 21 miRNAs were upregulated in response to heat stress. Among them, five heat‐responsive miRNAs, including miR162b, miR529a‐p5, PC‐5P‐62245‐9, miR171b and miR169n, were validated by quantitative real‐time polymerase chain reaction. A total of 44 target genes of the differentially expressed miRNAs were predicted. These target genes are most significantly overrepresented in the cell growth process. The results demonstrated that rice miRNAs play critical roles in the heat stress response. This study opens up a new avenue for understanding the regulatory mechanisms of miRNAs involvement in the heat stress response in rice.     

Identification of Quantitative Trait Loci Associated with Aluminum Tolerance in Rice (Oryza Sativa L.)

Summary Quantitative trait loci (QTL) analysis for Al tolerance was performed in rice using a mapping population of 98 BC₁F₁₀ lines (backcross inbred lines: BILs), derived from a cross of Al-tolerant cultivar of rice (Oryza sativa L. cv. Nipponbare) and Al-sensitive cultivar (cv. Kasalath). Three characters related to Al tolerance, including root elongation under non-stress conditions (CRE), root elongation under Al stress (SRE) and the relative root elongation (RRE) under Al stress versus non-stress conditions, were evaluated for the BILs and the parents at seedling stage. A total of seven QTLs for the three traits were identified. Among them, three putative QTLs for CRE (qCRE-6, qCRE-8 and qCRE-9) were mapped on chromosomes 6, 8 and 9, respectively. One QTL for SRE (qSRE-4) was identified on chromosome 4. Three QTLs (qRRE-5, qRRE-9 and qRRE-10) for RRE were detected on chromosomes 5, 9, 10 and accounted for 9.7–11.8% of total phenotypic variation. Interestingly, the QTL qRRE-5 appears to be syntenic with the genomic region carrying a major Al tolerance gene on chromosome 6 of maize. Another QTL, qRRE-9, appears to be similar among different rice populations, while qRRE-10 is unique in the BIL population. The common QTLs for CRE and RRE indicate that candidate genes conferring Al tolerance in the rice chromosome 9 may be associated with root growth rates. The existence of QTLs for Al tolerance was confirmed in substitution lines for corresponding chromosomal segments. These results also provide the possibilities of enhancing Al tolerance in rice through using marker-assisted selection (MAS) and pyramiding QTLs.     

Exploring diversity among rice germplasms based on their physiological traits responses to salinity

Salinity is one of the major abiotic stresses that severely effects rice production throughout the world. Previously, there have not been many studies which focused on studying diversity among rice germplasm based on specific physiological traits for salt tolerance. Our diversity study was based on physiological traits such as Na+ concentration, K⁺ concentration, K⁺/Na⁺ ratio, osmotic potential, and biomass which are major components determining salt tolerance. This study has systematically analyzed phenotypic data of 191 germplasms in two different salt concentrations apart from the control. The current study identified salt tolerant germplasms based on their response to a single physiological trait as well as a combination of different physiological traits. Some of the germplasm identified outperformed known salt tolerant cultivar Pokkali. This study identifies correlation among various physiological traits. The salt tolerant germplasms can be taken forward into developing better varieties by conventional breeding and exploring genes for salt tolerance.     

OsWD40过表达水稻根系响应盐胁迫的转录组分析

盐胁迫是影响水稻产量的主要因素之一,开展水稻耐盐机制的研究十分必要。为了揭示OsWD40基因参与耐盐的分子机制,以日本晴和OsWD40过表达水稻株系为材料,用浓度为200mmol/L的NaCl分别处理0、12、24和48h,对其根系进行转录组测序分析。结果显示,比较日本晴和OsWD40过表达株系在盐胁迫相同时间（ST0与NT0、ST12与NT12、ST24与NT24、ST48与NT48）的基因表达量,分别检测到1950、1646、3499和1522个差异表达基因。其中,盐胁迫处理24h的差异表达基因多于0、12和48h处理。对4个比较组的差异表达基因分别进行GO功能富集分析和KEGG代谢通路分析,发现差异表达基因主要富集在盐胁迫响应、脱落酸响应和转录调控等GO条目中,富集的重要代谢通路主要是植物激素信号转导,植物MAPK信号传导途径和苯丙烷生物合成、类黄酮生物合成相关的次生代谢途径等。同时,转录因子家族基因,如WRKY、MYB和bHLH等,在各比较组中呈现差异表达。由此推测,苯丙烷生物合成和类黄酮生物合成等植物次生代谢途径在OsWD40过表达水稻根系响应盐胁迫中发挥着重要作用,而且OsWD40可能介导响应脱落酸的基因转录调控,激活下游盐胁迫相关基因的表达。     

转CrylAc/CpTI栽培稻外源基因渗入普通野生稻中可稳定的遗传和表达

转基因水稻的研发以及环境释放,外源基因通过花粉逃逸到野生近缘种中,可能带来的生态效应愈来愈受到人们的关注.本研究采用人工杂交技术将CrylAc/CpTI双价抗虫基因从转基因栽培稻渗入普通野生稻(Oryza rufipogon Griff.)中,获得普通野生稻F1及分离后代F2、F3、F4、F5和回交后代BC1F1和BC...     

玉米HD-ZIP I亚家族基因鉴定及表达分析

转录因子是植物响应逆境胁迫的重要调节因子,在其整个生长发育过程中发挥着重要的作用。HD-ZIP家族蛋白是植物中特有的一大类转录因子,包含4个亚家族(HD-ZIP I～IV),其中HD-ZIP I亚家族成员主要参与干旱、渗透压等极端环境和ABA及乙烯等激素处理的响应过程。本文采用隐马可夫模型(HMM)在玉米参考基因组中鉴定到17个HD-ZIP I亚家族成员,这些基因不均匀分布于玉米6条染色体上,与水稻的亲缘关系要近于拟南芥。玉米HDZIP I亚家族基因在玉米7种组织中表现出多种表达模式,具有明显的组织表达特异性。另外, HD-ZIP I亚家族基因对高盐、淹水及冷害等不同的逆境胁迫处理呈现出不同的响应模式及响应程度差异。5种不同激素处理后,玉米HD-ZIP I亚家族基因也表现出复杂的响应模式。这些结果为进一步解析玉米HD-ZIP I亚家族基因的生物学功能和作用机理提供了一定的参考价值。     

转基因改良水稻抗稻瘟病的策略及其进展

利用转基因技术是改良水稻稻瘟病抗性的有效途径。已有研究证实通过某些抗病基因、抗真菌蛋白基因、杀菌肽基因的转基因,可以培育出获得对稻瘟病广谱抗性的水稻品种。本文就以上几个方面的进展评述了水稻稻瘟病抗性的分子改良策略。     

一个新的水稻逆境响应基因OsMsr1的表达与克隆

为深入了解植物的逆境反应机理和发现新的水稻耐逆基因, 采用Affymetrix水稻表达芯片(含51 279个转录本)分析了超级稻两优培九母本培矮64S不同生长发育时期、不同组织器官全基因组在低温、干旱、高温逆境胁迫下的表达水平, 筛选出众多表达水平显著升高与降低的基因。OsMsr1 (Oryza sativa L. multiple stresses responsive gene 1, GenBank登录号为EU284112) 是其中一个受高温、低温与干旱诱导、在各生长发育时期与组织器官均显著上调的基因, 用实时定量PCR方法对其表达水平进行了进一步的分析, 所得结果与基因芯片结果基本吻合, 因此, 此基因为一多逆境响应基因。用RT-PCR方法扩增获得了包含其完整ORF (open reading frame)的cDNA克隆。根据其ORF序列进行预测, 此基因编码一个包含89个氨基酸残基的小分子蛋白, 分子量约为10 kD, pI约为5。搜索有关数据库, 在水稻、玉米、小麦与拟南芥中找到有高相似性的基因, 但功能未知, 也未发现相同与类似的已知功能的基因保守结构域。对其可能的启动子序列分析, 发现5个与逆境反应有关的顺式作用元件。因此, 我们认为该基因为一新的水稻耐逆候选基因, 进一步的研究正在进行。     

Through the genetic bottleneck: O. rufipogon as a source of trait-enhancing alleles for O. sativa

This paper summarizes results from a decade of collaborative research using advanced backcross (AB) populations to a) identify quantitative trait loci (QTL) associated with improved performance in rice and to b) clone genes underlying key QTLs of interest. We demonstrate that AB-QTL analysis is capable of (1) successfully uncovering positive alleles in wild germplasm that were not obvious based on the phenotype of the parent (2) offering an estimation of the breeding value of exotic germplasm, (3) generating near isogenic lines that can be used as the basis for gene isolation and also as parents for further crossing in a variety development program and (4) providing gene-based markers for targeted introgression of alleles using marker-assisted-selection (MAS). Knowledge gained from studies examining the population structure and evolutionary history of rice is helping to illuminate a long-term strategy for exploiting and simultaneously preserving the well-partitioned gene pools in rice.     

一个新的水稻逆境响应基因OsMsr1的表达与克隆

为深入了解植物的逆境反应机理和发现新的水稻耐逆基因, 采用Affymetrix水稻表达芯片(含51 279个转录本)分析了超级稻两优培九母本培矮64S不同生长发育时期、不同组织器官全基因组在低温、干旱、高温逆境胁迫下的表达水平, 筛选出众多表达水平显著升高与降低的基因。OsMsr1 (Oryza sativa L. multiple stresses responsive gene 1, GenBank登录号为EU284112) 是其中一个受高温、低温与干旱诱导、在各生长发育时期与组织器官均显著上调的基因, 用实时定量PCR方法对其表达水平进行了进一步的分析, 所得结果与基因芯片结果基本吻合, 因此, 此基因为一多逆境响应基因。用RT-PCR方法扩增获得了包含其完整ORF (open reading frame)的cDNA克隆。根据其ORF序列进行预测, 此基因编码一个包含89个氨基酸残基的小分子蛋白, 分子量约为10 kD, pI约为5。搜索有关数据库, 在水稻、玉米、小麦与拟南芥中找到有高相似性的基因, 但功能未知, 也未发现相同与类似的已知功能的基因保守结构域。对其可能的启动子序列分析, 发现5个与逆境反应有关的顺式作用元件。因此, 我们认为该基因为一新的水稻耐逆候选基因, 进一步的研究正在进行。     

水稻抗坏血酸氧化酶基因家族生物信息学分析

抗坏血酸氧化酶(ascorbic acid oxidase,AAO)是多铜氧化酶家族中的一员,在植物生长发育过程中起重要作用。为深入研究水稻AAO基因家族的功能特征及表达模式,采用生物信息学从Phytozome的水稻数据库鉴定出14个AAO成员,并对其染色体定位、蛋白理化性质及二级结构、基因结构、保守基序、系统发生树和表达模式等方面进行预测分析。结果显示,14个OsAAO不均匀地分布在8条染色体上,多为碱性蛋白;蛋白二级结构以无规则卷曲为主要组成部分;基因结构和保守基序分析表明,OsAAO内含子数目变化差异较大,但氨基酸序列具有较强的保守性;系统发生树可分为3个亚家族,OsAAOs与玉米、高粱簇在一起,亲缘关系较近;另外表达模式分析发现,OsAAO在不同的组织部位表达水平具有差异,这表明其行使功能不同。这些结果为今后研究该基因家族的生物学功能和培育耐旱品种提供理论依据。     

水稻双组分系统基因干旱胁迫表达谱分析

利用全基因组表达芯片,系统分析干旱胁迫条件下水稻双组分信号系统(two component system,TCS)相关基因在其不同发育阶段、不同组织以及抗旱能力不同的株系中的表达谱变化特征.结果表明,双组分信号系统基因在干旱胁迫环境下的表达具明显的时空特异性,表现为不同组织的TCS基因干旱胁迫表达谱差异较大,而同一组织内表达谱相似,其中生殖生长期(幼穗分化期和孕穗期)两个叶片材料中TCS基因表达谱最为接近;与细胞分裂素信号传导负向调控相关的A型应答调控器在旱胁迫下表达下调,而与CK信号传导正向调控相关的B型应答调控器呈现明显上调趋势,推测与干旱胁迫下CK信号传导增强有关,同时乙烯受体基因在干旱胁迫环境下的表达下调,从激素间相互关系的角度更好地印证了上述推测;与拟南芥细胞分裂素受体基因(AHK2、AHK3和AHK5)序列相似的磷酸感应激酶基因HK5和HK3在干旱胁迫下表达上调,与拟南芥中不依赖细胞分裂素的AHK5序列接近的HK6则表达下调,进一步验证上述推测;干旱胁迫下抗旱能力不同的水稻株系其TCS基因表达谱没有明显差异,推测TCS基因差异表达可能源于对干旱胁迫反应,而其表达对抗旱能力的增强还有待进一步研究.     

Induced mutations – A new paradigm in plant breeding

The use of ionizing radiation, such as X-rays, gamma rays and neutrons and chemical mutagens for inducing variation, is well established. Induced mutations have been used to improve major crops such as wheat, rice, barley,cotton, peanuts, and beans, which are seed propagated. Since the establishment of the Joint FAO/IAEA Division of the Nuclear Techniques in Agriculture, more than 1800 cultivars obtained either as direct mutants or derived from their crosses have been released worldwide in 50 countries. In vegetatively propagated plants, many of mutants were derived from irradiating rooted stem cuttings, detached leaves, and dormant plants. According to the FAO/IAEA database, of the 465 mutants released among the vegetatively propagated plants, most are in the floricultural plants and a few in fruit trees. These include chrysanthemum, Alstroemeria, dahlia, bougainvillea, rose, Achimenes,begonia, carnation, Streptocarpus, and azalea. The irradiation of in vitro cultured date palm, apple, potato, sweet potato and pineapple now provides a means to treat large populations which would not have been possible before. Irradiation of micropropagated plants, axillary and adventitious buds, apical meristems, regenerative callus cultures, anthers and microspores, and somatic embryos provides a miniaturized version of trees and seeds in the Petridish instead of the field. During the last decade, the use of radio-actively labeled probes in recombinant DNA research for cloning and mapping plant genes and transgenesis, particularly for RFLP, micro satellite based DNA fingerprinting, has become a routine procedure. Many homeotic mutants that change floral development have been isolated in Arabidopsis, Petunia, Antirrhinum and Lycopersicon. Mutants of Arabidopsis are being used to analyze genes, which determine response to auxins, cytokinins, gibberellin, abscisic acid and ethylene in plant growth, floral development and senescence, fruit formation and ripening. These mutants are facilitating the isolation, identification and cloning of the genes, which would ultimately help in designing crops with improved yield, increased stress tolerance, longer shelf-life and reduced agronomic inputs. The identification and analysis of mutants by using molecular techniques of DNA fingerprinting and mapping with PCR based markers, such as RAPDs, AFLP and STMS, and mutant tagging shall bring a new dimension in gene technology. Already, mutations can be linked to changes in DNA sequences for some plant traits and to establish molecular maps in structural and functional genomics of crop plants. These in turn would lead to a rapid enhancement of crop yields and quality. This revised version was published online in July 2006 with corrections to the Cover Date.