An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves

Background

Although the genetic structure of rice germplasm has been characterized worldwide, few studies investigated germplasm from Thailand, the world’s largest exporter of rice. Thailand and the International Rice Research Institute (IRRI) have diverse collections of rice germplasm, which could be used to develop breeding lines with desirable traits. This study aimed to investigate the level of genetic diversity and structures of Thai and selected IRRI germplasm. Understanding the genetic structure and relationships among these germplasm will be useful for parent selection used in rice breeding programs.

Results

From the 98 InDel markers tested for single copy and polymorphism, 19 markers were used to evaluate 43 Thai and 57 IRRI germplasm, including improved cultivars, breeding lines, landraces, and 5 other Oryza species. The Thai accessions were selected from all rice ecologies such as irrigated, deep water, upland, and rainfed lowland ecosystems. The IRRI accessions were groups of germplasm having agronomic desirable traits, including temperature-sensitive genetic male sterility (TGMS), new plant type, early flowering, and biotic and abiotic stress resistances. Most of the InDel markers were genes with diverse functions. These markers produced the total of 127 alleles for all loci, with a mean of 6.68 alleles per locus, and a mean Polymorphic Information Content (PIC) of 0.440. Genetic diversity of Thai rice were 0.3665, 0.4479 and 0.3972 for improved cultivars, breeding lines, and landraces, respectively, while genetic diversity of IRRI improved and breeding lines were 0.3272 and 0.2970, respectively. Cluster, structure, and differentiation analyses showed six distinct groups: japonica, TGMS, deep-water, IRRI germplasm, Thai landraces and breeding lines, and other Oryza species.

Conclusions

Thai and IRRI germplasm were significantly different. Thus, they can be used to broaden the genetic base and trait improvements. Cluster, structure, and differentiation analyses showed concordant results having six distinct groups, in agreement with their development, and ecologies.     

Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

The discovery of thermo-sensitive genic male sterility(TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F_2 populations derived from crosses between IR68301 S, an indica TGMS rice line, and IR14632(tropical japonica), Supanburi 91062(indica) and IR67966-188-2-2-1(tropical japonica), respectively.Approximately 1:3 ratio between sterile and normal pollen of F_2 plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat(SSR) and insertion-deletion(InDel) markers were used to identify markers linked to the tms gene. The linkage analysis based on the three populations indicated that the tms locus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F_2 population, the results showed that the tms locus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from the tms gene to these two flanking markers were 1.10 and 0.82 cM, respectively.InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to the tms gene using F_2 populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S.     

Marker-Assisted Breeding of Thermo-Sensitive Genic Male Sterile Line 1892S for Disease Resistance and Submergence Tolerance

Rice line 1892S is an elite thermo-sensitive genic male sterile(TGMS)line for two-line hybrid rice production.However,1892S is susceptible to rice blast,bacterial blight and submergence.Here we reported the introduction of blast resistance(R)gene Pi9,bacterial blight R gene Xa21 and submergence tolerance gene Sub1A into 1892S genetic background through backcrossing and marker-assisted selection.The improved TGMS line 31892S and its hybrids conferred disease resistance to rice blast and bacterial blight,and showed submergence tolerance for over 14 d without significant loss of viability.The sterility-fertility conversion of 31892S was similar to that of 1892S.31892S and its derived hybrid rice had similar agronomic traits and grain quality with 1892S and the control hybrid rice,respectively.The newly developed 31892S provided an improved TGMS line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight,and submergence tolerance with no yield penalty or change in grain quality.     

Development ofNew InDel Marker to Detect Genotypes of Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility inRice简

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility (CMS) in japonica rice, a functional marker InDel-Rf-1a based on the difference of nucleotide sequence in Rf-1a locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-1arf-1a showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-1a locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F₂ segregation population, and three genotypes in Rf-1a locus could be distinguished distinctly. Therefore, the marker InDel-Rf-1a could be widely used for genetic identification and marker-assisted selection (MAS) in breeding japonica restorer lines.     

Fertility Expression of TGMS-Genes in the Backgrounds of indica CMS-lines, B-lines and R-lines of Hybrid Rice

The cytoplasmic male sterile (CMS-lines) and photoperiod/thermo-sensitive genic male sterile lines (P/TGMS-lines) are important genetic tools in hybrid rice breeding, which served as the female parents of the three-line and two-line hybrid rice breeding system, respectively. It has been reported that the CMS system possesses complete and stable sterility which is controlled by the interaction of the abortive cytoplasm and nucleus genes from their cognate B-lines [1] but TGMS-lines used t…     

Identification of CMS maintainers and restorers for hybrid rice development in Ghana

The cytoplasmic-genetic male sterility is the most widely used system for hybrid rice (Oryza sativa) seed production. It requires three lines: the two hybrid parents (A and R lines) and a third line (maintainer (B line)), which maintains the sterility of the female parent during seed increase. Studies were undertaken to identify maintainers and restorers for introduced cytoplasmic male sterile (CMS) lines using simple sequence repeat (SSR) markers and testcrosses. Molecular screening with SSR marker RM6100 grouped the assembled inbred lines into 30 potential restorers and 55 potential maintainers. One maintainer (WAS 127 -12-1-6-3-1) and 16 restorers were confirmed by testcrosses. WAS 127-12-1-2-3 had higher grain yield per plant in crosses with CMS1 (IR69625A) than the local checks (AgraRice and Jasmine 85) and could be used as a restorer to develop a locally adapted, high-yielding hybrid. Conversion of WAS 127 -12-1-6-3-1 into a local CMS line was stopped because of sterility instability in the BC₁F₁ generation. Continued testcrosses to the set of identified potential maintainers could help identify a stable one that can successfully be converted into a locally adapted CMS line.     

以淡绿叶为标记的籼型光-温敏核不育系M2S的选育

 以淡绿叶标记基因系IGM19与籼型光-温敏核不育系8902S杂交，再用8902S回交1次，通过多代选育获得了带淡绿色叶隐性标记性状，且育性转换特性与8902S明显不同的籼型光－温敏核不育系M2S。在杭州（30°05’N）自然条件下，M2S具有连续30 d以上的不育期（不育度＞99.5%），在秋季正常情况下，结实率可达51.8%。人工气候箱控制条件下，M2S在短日适温时结实正常，在长日（15.0 h）与所有处理温度（30.1℃、24.1℃和23.1℃）、高温（30.1℃）与所有处理日长（15.0 h、14.0 h和12.5 h）的光温组合下均完全不育或高度不育。结实率的方差分析结果表明，其育性转换的温度效应和光周期效应均不显著，仅光温工作效应显著，属典型的光温互作雄性不育系。M2S本身较稳定的不育性和带有隐性淡绿色叶的标记性状将有利于在两系法杂交水稻中应用。     

Marker-Assisted Selection of Xa21 Conferring Resistance to Bacterial Leaf Blight in indica Rice Cultivar LT2

Bacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive diseases in Asian rice fields. A high-quality rice variety, LT2, was used as the recipient parent. IRBB21, which carries the Xa21 gene, was used as the donor parent. The resistance gene Xa21 was introduced into LT2 by marker-assisted backcrossing. Three Xoo races were used to inoculate the improved lines following the clipping method. Eleven BC₃F₃ lines carrying Xa21 were obtained based on molecular markers and agronomic performance. The 11 lines were then inoculated with the three Xoo races. All the 11 improved lines showed better resistance to BLB than the recipient parent LT2. Based on the level of resistance to BLB and their agronomic performance, five lines (BC₃F₃ 5.1.5.1, BC₃F₃ 5.1.5.12, BC₃F₃ 8.5.6.44, BC₃F₃ 9.5.4.1 and BC₃F₃ 9.5.4.23) were selected as the most promising for commercial release. These improved lines could contribute to rice production in terms of food security.     

小麦隐性核不育保持系表达载体的构建及拟南芥遗传转化

利用小麦杂种优势能够创制稳定的雄性不育系。相比于细胞质雄性不育系和光温敏雄性不育系,细胞核雄性不育具有育性稳定、易恢复、不受环境影响等特点。小麦隐性核不育基因 ms1的克隆以及杂交制种技术(hybrid seed production technology, SPT)的开发,为雄性不育的保持和杂种优势利用奠定了基础。本研究将小麦花粉育性恢复基因 Ms1、花粉致死基因 ZmAA1、红色荧光蛋白基因 DsRed2及其特异性启动子和终止子连接到表达载体pGEII上,转化野生型拟南芥,得到6株转基因拟南芥。通过荧光显微镜观察拟南芥种子,发现野生型拟南芥种子表面平滑,不能发出红色荧光;而转基因种子表面呈现颗粒状,能够发出红色荧光。这说明表达载体构建成功,并能够在拟南芥中成功表达。这为创制人工小麦隐性核不育系奠定了理论和材料基础。     

Thoughts and Practice on Some Problems about Research and Application of Two-Line Hybrid Rice

The main problems about research and application of two-line hybrid rice were reviewed, including the confusing nomenclature and male sterile lines classification, the unclear characteristics of photoperiod and temperature responses and the unsuitable site selection for male sterile line and hybrid rice seed production. In order to efficiently and accurately use dual-purpose genic male sterile lines, four types, including PTGMS (photo-thermo-sensitive genic male sterile rice), TGMS (thermo-sensitive genic male sterile rice), reverse PTGMS and reverse TGMS, were proposed. A new idea for explaining the mechanism of sterility in dual-purpose hybrid rice was proposed. The transition from sterile to fertile was involved in the cooperative regulation of major-effect sterile genes and photoperiod and/or temperature sensitive ones. The minor-effect genes with accumulative effect on sterility were important factors that affected the critical temperature of sterility transfer. In order to make better use of dual-purpose lines, the characterization of responses to photoperiod and temperature of PTGMS should be made and the identification method for the characterization of photoperiod and temperature responses of PTGMS should also be put forward. The optimal ecological site for seed production could be determined according to the historical climate data and the requirements for the meteorological conditions during the different periods of seed production.     

模拟盛夏低气温条件下水温对水稻温敏不育系育性的影响

 在模拟盛夏低气温的条件下（日平均温度22.5或23.0℃），研究了8个温敏不育系在28.6、26.8和24.0℃处理时的不育性。结果表明高于温敏不育系临界温度的温水，可以保持温敏不育系的雄性不育性稳定。为利用水温作为一个有效手段防止盛夏低温引起的负效应提供了理论基础。     

Development of Novel Cytoplasmic Male Sterile Source from Dongxiang Wild Rice (Oryza rufipogon)

This study was conducted to develop and characterize a novel cytoplasmic male sterile(CMS)source which was identified from Dongxiang wild rice(Oryza rufipogon)by crossing Dongxiang wild rice as female with Zhongzao 35,an indica inbred variety,as male and continuous backcrossing with Zhongzao 35.Observation under optical microscope manifested that this novel CMS belonged to typical abortion type with less pollen compared with wild abortive type cytoplasm(CMS-WA).Sequential planting showed that this novel CMS has complete and stable male sterility.Testcross experiment showed that all the 24 tested materials including maintainer and restorer lines of CMS-WA and Honglian type cytoplasm(CMS-HL)and other indica inbred varieties are the maintainers with complete maintaining ability,suggesting that this novel CMS has fertility restoration totally different from CMS-WA and CMS-HL and belongs to a novel type of CMS.So far,we only discovered a unique fertility restoration source for this novel CMS.Inheritance analysis showed that the fertility restoration of this CMS was governed by three pairs of independent dominant genes.Prospect for application of this novel CMS system in hybrid rice breeding was also discussed.     

分子标记辅助选择改良宁恢8号的条纹叶枯病抗性

为改良宁恢8号的条纹叶枯病抗性,以含条纹叶枯病抗性基因Stv-bi的品种关东194为供体,通过杂交、回交,利用与条纹叶枯病抗性基因Stv-bi和恢复基因Rf1a共分离的分子标记进行辅助选择,将条纹叶枯病抗性基因Stv-bi导入宁恢8号,结合田间抗性和性状鉴定,至BC3F3育成10个条纹叶枯病抗性显著提高、农艺性状优良的改良系。选择其中4个综合性状较优的株系分别与不育系A25、A19和A70进行配组鉴定。结果表明,改良系-2所配组合的综合性状明显优于对照宁恢8号的相应组合。     

Genetic analyses of heading date of Japonica rice cultivars from Northeast China

Northeast regions of China (38–55°N latitude) play an important role in Japonica rice planting. Heading dates of 10 Japonica rice cultivars native to the Northeast of China were investigated and their sensitivities to photoperiod and temperature were analyzed. The results showed that these Japonica rice cultivars were insensitive to photoperiod but strongly sensitive to temperature, i.e. a high temperature can markedly shorten the heading date. Genetic analyses were conducted on these 10 cultivars using a set of heading date tester lines. All these Japonica cultivars carried a dominant early heading gene Ef-1, and most of these cultivars carried the photoperiod insensitivity allele e₁, and two types of alleles were presented at the Se-1 locus, including recessive photoperiod insensitivity gene Se-1^e and dominant strong photoperiod-sensitivity (PS) gene Se-1ⁿ. The PS of these cultivars carrying E₁ or Se-1ⁿ can be repressed or weakened by Ef-1 and the recessive allele hd2 they carried. These results provided a reasonable explanation to the adaptability of Japonica rice to the high latitude environment of the Northeast China, and could be useful for breeding new cultivars well adapted to the high latitude regions and expanding the rice cultivation range.     

Improvement of bacterial blight resistance of hybrid rice in China using the Xa23 gene derived from wild rice (Oryza rufipogon)

A novel bacterial blight (BB) resistance gene, Xa23, identified from Oryza rufipogon was introgressed into three popular restorer lines (Minghui63, YR293 and Y1671) for wild abortive cytoplasmic male sterility by marker-assisted backcross breeding approach in combination with artificial inoculation and stringent phenotypic selections. The three derived BB resistant restorer lines (Minghui63-Xa23, YR293-Xa23 and Y1671-Xa23) and their hybrid combinations with Zhenshan97A (Shanyou63-Xa23), NongfengA (Fengyou293-Xa23) and Zhong9A (Zhongyou1671-Xa23) demonstrated similar BB resistance spectrum as the donor parent, CBB23 (B). The newly developed BB resistant restorers and their derived hybrids were identical to their respective original versions for agronomic traits especially under disease free condition. However, under severe disease condition, the three BB resistant restorer lines exhibited significantly higher grain weight and spikelet fertility as compared to the respective original restorer lines thus further resulting in BB resistant hybrids with significantly higher grain yields than their respective popular original hybrids. The results indicated that the Xa23 gene could completely express its dominant broad spectrum resistance in different backgrounds of both restorer and male sterile lines across different growth stages, suggesting its immense breeding value in BB resistance improvement for hybrid rice. Moreover, a reasonable utilization and deployment of Xa23 gene for efficient control of BB disease in hybrid rice production was recommended.     

水稻新质源两用核不育系—CIS28—10

ＣＩＳ２８－１０是由水稻核质杂种（Ｃａｒｌｏｒｏ／ＩＲ２８）Ｂ９Ｆ４中的突变单株按系统选育程序选育而成的新质源两用核不育系．在湘潭，它的不育期达６０多天，育性稳定，对低温钝感。在遗传上其育性受一对隐性基因控制，遗传行为简单。     

温敏核不育基因在籼型三系遗传背景下的育性表达

用3个温敏核不育系培矮64S、6311S和360S与7个籼型质核互作型水稻雄性不育系及相应的保持系、3个恢复系配组,观察了51个组合的F1、19个F2及6个BC1的育性表现。结果表明：培矮64S温敏核不育性状由2对隐性基因控制,具有对质核互作型雄性不育系育性的强恢复基因;6311S温敏核不育性状由1对隐性基因控制,同时具有1对弱恢复基因;360S温敏核不育性状由1对隐性基因控制,但没有恢复基因。进一步利用4个细胞质雄性不育系/6311S组合F2群体中4个温敏核不育株与5个细胞质雄性不育系CMS配组,研究了杂交F1的育性,表明在可育细胞质背景下,三系恢复基因对温敏核不育基因的表达没有影响;在不育细胞质背景下,三系恢复基因是温敏核不育基因表达的关键。由此提出了选育不育细胞质背景的光温敏核不育系和温敏核不育背景的质核互作型不育系的策略。     

玉米不育系回交转育群体背景选择效果的研究

以玉米细胞质不育系cms-合344为供体亲本、辐63018为轮回亲本,构建BC_1和BC_2群体,研究利用SSR标记进行遗传背景选择加速回交转育不育系的效果。选用两亲本间多态性好的并覆盖玉米10条染色体的60个SSR标记进行轮回亲本的遗传背景分析。结果表明,对两群体遗传背景回复率分析,86株BC_1群体背景回复率平均值为74.41%,63株BC_2群体背景回复率平均值为87.36%,BC_1群体表型与分子标记辅助选择相似度均较高的株系有9株,BC_2有8株。对两群体进行不同标记数目分析,两群体中使用40个标记与总数60个标记所计算的背景回复率基本相同,相关系数分别达到0.96和0.95以上。     

两系法杂交水稻研究和应用中若干问题的思与行

指出了当前我国两系法杂交水稻研究和应用中面临的主要问题,即两系法杂交水稻体系中不育系的称谓和分类不规范,生产上利用的水稻两用核不育系育性温光反应特性研究不清楚,水稻两用核不育系繁殖和两系法杂交稻制种基地选择不当等。针对当前情形,结合多年育种实践,提出了解决这些问题的对策及措施。建议将水稻两用核不育系分为长光高温不育型、高温不育型、短光低温不育型、低温不育型等四种类型。提出了水稻光温不育机理设想,即水稻光温敏核不育系育性转换是主效不育基因与其位置相近的发育感光基因或(和)发育感温基因相互作用的结果,微效不育基因可影响两用核不育系的不育起点温度。只有深入研究光温敏型不育系育性的光温组合效应,且获得具体的光温指标,才能更好地指导两系法杂交水稻制种和不育系繁殖。提出了水稻两用核不育系育性光温特性的鉴定方法以及两系法杂交稻种子生产应根据不育系育性温光特性和历史气象资料来合理安排基地和时段。     

水稻苯达松敏感突变研究进展

采用γ-射线辐射诱变技术,日本和中国学者分别从粳稻和籼稻中获得了苯达松敏感致死突变体农林8号m和M8077S。水稻苯达松敏感突变也表现对磺酰脲类除草剂敏感,但对供试的其他除草剂没有差异反应。遗传分析表明,这两个突变体表现为等位的隐性单基因突变,对这两种除草剂的敏感是同一个基因突变的效应。对M8077S的突变位点进行了精细定位、基因克隆和互补试验,证明农林8号m和M8077S都是源于第3染色体上的一个细胞色素P450基因(CYP81A6)的单碱基缺失导致的移码突变:农林8号m中的CYP81A6自转录起始密码子ATG起第507位缺失了一个C,而M8077S中的CYP81A6第2058位缺失了一个G。水稻苯达松敏感突变用于两系法杂交水稻的除杂保纯,已经成功选育出3个具有苯达松敏感突变标记的光温敏雄性不育系(M8064S、苯88S、苯63S)以及相配套的3个杂交组合(丰两优2号、苯两优9号和苯两优639),分别通过了湖北和江西省品种审定;用于杂交水稻混植法制种,已经成功选育出具有苯达松敏感标记的恢复系(MC526)以及相配套的杂交组合(混制1号),制定了机械化混播制种的生产规程和技术要点。还展望了CYP81A6基因在抗除草剂育种、转基因水稻安全控制以及水稻化学杀雄方面的应用前景。