抗虫抗除草剂转基因水稻B1C893的获得与鉴定

利用农杆菌介导法将Cry1Ca#和Bar基因转化水稻恢复系R893,获得6株再生植株。经过分子鉴定,发现Bar基因和Cry1Ca#基因成功整合到水稻基因组并稳定表达。测定其中单拷贝且可育的转基因株系苗期不同器官中Cry1Ca蛋白的表达量,发现根系中最低而叶片中最高,其中最高的第6号株系叶片中含量达到35.07μg/g。获得的转基因株系对除草剂草铵膦和稻纵卷叶螟均具有良好的抗性。     

聚合白背飞虱和褐飞虱抗性基因创制杂交水稻恢复系

【目的】为了创制兼抗白背飞虱和褐飞虱的水稻恢复系，【方法】分别以抗褐飞虱材料B5（携带褐飞虱抗性基因Bph14和Bph15）及携带白背飞虱抗性位点qsI-4的籼型恢复系福恢7011为供体亲本，以骨干恢复系福恢676为轮回亲本，应用低世代分离群体田间表型结合单株鉴定与高世代稳定株系室内筛选和分子标记辅助选择相结合的方法，并对抗虫株系及其测交后代进行考查和农艺性状分析。【结果】选育出聚合Bph14、Bph15 和qsI-4的恢复系材料3份，携带2个抗虫基因的恢复系材料3份。其中6份恢复系的褐飞虱抗性鉴定结果均表现中抗以上。通过抗性鉴定和杂交后代农艺性状分析筛选出具有生产应用潜力的恢复系材料2份。【结论】为褐飞虱和白背飞虱抗性聚合新种质的创制和应用提供了基础材料。     

聚合白背飞虱和褐飞虱抗性基因创制杂交水稻恢复系

【目的】为了创制兼抗白背飞虱和褐飞虱的水稻恢复系,【方法】分别以抗褐飞虱材料B5(携带褐飞虱抗性基因Bph14和Bph15)及携带白背飞虱抗性位点qsI-4的籼型恢复系福恢7011为供体亲本,以骨干恢复系福恢676为轮回亲本,应用低世代分离群体田间表型结合单株鉴定与高世代稳定株系室内筛选和分子标记辅助选择相结合的方法,并对抗虫株系及其测交后代进行考查和农艺性状分析。【结果】选育出聚合Bph14、Bph15和qsI-4的恢复系材料3份,携带2个抗虫基因的恢复系材料3份。其中6份恢复系的褐飞虱抗性鉴定结果均表现中抗以上。通过抗性鉴定和杂交后代农艺性状分析筛选出具有生产应用潜力的恢复系材料2份。【结论】为褐飞虱和白背飞虱抗性聚合新种质的创制和应用提供了基础材料。     

聚合白背飞虱和褐飞虱抗性基因创制杂交水稻恢复系

【目的】为了创制兼抗白背飞虱和褐飞虱的水稻恢复系,【方法】分别以抗褐飞虱材料B5(携带褐飞虱抗性基因Bph14和Bph15)及携带白背飞虱抗性位点qsI-4的籼型恢复系福恢7011为供体亲本,以骨干恢复系福恢676为轮回亲本,应用低世代分离群体田间表型结合单株鉴定与高世代稳定株系室内筛选和分子标记辅助选择相结合的方法,并对抗虫株系及其测交后代进行考查和农艺性状分析。【结果】选育出聚合Bph14、Bph15和qsI-4的恢复系材料3份,携带2个抗虫基因的恢复系材料3份。其中6份恢复系的褐飞虱抗性鉴定结果均表现中抗以上。通过抗性鉴定和杂交后代农艺性状分析筛选出具有生产应用潜力的恢复系材料2份。【结论】为褐飞虱和白背飞虱抗性聚合新种质的创制和应用提供了基础材料。     

利用分子标记辅助选择改良水稻两系不育系C815S的褐飞虱抗性研究

以水稻品种B5(含褐飞虱抗性基因Bph14、Bph15)为供体亲本,目前生产上大面积应用的两系不育系C815S为受体亲本,利用分子标记辅助选择技术,将2个褐飞虱抗性基因Bph14和Bph15导入C815S,获得3个同时携带2个抗性基因的纯合改良株系。苗期群体鉴定和大田成熟期鉴定表明,3个改良不育株系的褐飞虱抗性均为3级,表现为抗;而受体亲本C815S为9级,表现为感。农艺性状考察表明,改良不育株系基本保持了受体亲本的优良性状,部分穗部性状还显著优于C815S。因此,利用分子标记辅助选择技术聚合Bph14和Bph15基因改良不育系褐飞虱抗性效果明显。     

抗虫抗除草剂转基因水稻恢复系B2A68的培育

利用人工优化合成的抗虫基因Cry2Aa#与抗除草剂的Bar基因构建的植物表达载体转化水稻恢复系D68,经过分子鉴定获得1个单拷贝的转基因水稻株系B2A68。B2A68和杂交种株1S/B2A68叶片中Cry2Aa蛋白的表达量在齐穗期分别为10.45～26.84和9.0～13.45μg/g。草铵膦抗性试验和抗虫性测定结果显示,转基因水稻B2A68既抗除草剂草铵膦,又抗稻纵卷叶螟。     

水稻抗褐飞虱遗传和育种研究

综述了水稻抗褐飞虱基因资源的鉴定及其分布特点、水稻抗褐飞虱主基因定位和QTL分析、水稻抗褐飞虱遗传的“基因对基因”关系以及水稻品种对褐飞虱的抗虫机制与褐飞虱对抗虫品种的适应等4个方面的研究进展，同时简述了抗褐飞虱基因的育种利用现状，并对今后抗褐飞虱遗传和育种利用研究作了展望。     

广谱抗稻瘟病种质75-1-127的褐飞虱抗性基因鉴定及分子标记辅助选择育种

【目的】水稻品系75-1-127携带广谱抗稻瘟病基因Pi9,已被广泛应用于抗稻瘟病水稻品种改良。笔者育种实践发现75-1-127表现出较强的褐飞虱抗性,因此鉴定该品系中的褐飞虱抗性基因并进行分子辅助选择育种。【方法】根据水稻品系B5中褐飞虱抗性基因Bph14和Bph15的序列,设计引物扩增75-1-127的基因组DNA,并对PCR产物进行测序分析。采用苗期集团法鉴定了75-1-127和B5的褐飞虱抗性表型。利用与Bph14与Bph15连锁的分子标记筛查了75-1-127为稻瘟病抗源回交转育的两系不育系后代,并鉴定了这些后代的稻瘟病抗性、褐飞虱抗性和主要农艺性状。【结果】75-1-127中含有与B5完全一致的Bph14和Bph15序列。75-1-127和B5苗期褐飞虱抗性均为1级。在以75-1-127为抗源改良的两系不育系中,携带Bph14、Bph15的单基因系或双基因系的褐飞虱抗性均得以改良,其中双基因聚合系的死苗率为8.5%,与供体亲本75-1-127以及阳性对照B5差异不显著,进一步证实75-1-127含有褐飞虱抗性基因。【结论】水稻品系75-1-127携带褐飞虱抗性基因Bph14和Bph15,可以作为抗源应用于水稻褐飞虱抗性育种。     

水稻抗褐飞虱基因研究进展与展望

褐飞虱严重影响水稻的产量和品质,抗虫品种的培育和种植是控制该虫害最安全有效的措施。水稻抗褐飞虱育种的关键是抗虫基因的发掘和合理利用。目前至少报道了34个褐飞虱抗性位点,其中28个主效抗性基因已被定位,显性基因Bph3、Bph14和Bph26已被成功克隆。介绍了水稻褐飞虱的生物型及抗性机理、褐飞虱抗性基因定位与克隆及抗虫基因在育种上的应用,并对水稻褐飞虱抗性育种面临的问题和应用前景进行了讨论。     

分子标记辅助选择改良水稻恢复系R1813稻瘟病、白叶枯病和褐飞虱抗性研究

R1813是湖南隆平种业有限公司选育的高产、抗倒、迟熟优良籼型两系恢复系,以R1813为父本,已经选配出一批组合通过审定。该研究利用分子标记辅助选择和回交转育技术,将MD12086-41携带的抗稻瘟病基因Pi9、抗白叶枯病基因Xa23、抗褐飞虱基因Bph14和Bph15渗入到R1813的背景中,选出3个携带Pi9、Xa23、Bph14和Bph15基因的近等基因系ST15005-1-106-9、ST15005-1-318-4和ST15005-1-318-14。抗性鉴定结果表明,这3个改良株系均表现抗稻瘟病、高抗白叶枯病和中抗褐飞虱,与R1813相比,3种病虫害的抗性都明显提高。田间试验和品质分析表明,3个改良株系的产量、主要农艺性状和稻米品质主要指标都与R1813相似。对少量测配组合的抗性鉴定、田间试验和品质分析表明,除了抗性有明显提高以外,产量、主要农艺性状、稻米品质主要指标都与R1813所配组合相似。     

转几丁质酶-葡聚糖酶基因水稻稻瘟病抗谱分析

用来源于云南各地属于24个稻瘟病菌（[i]Magnaporthe grisea[/i]）生理小种的33个菌株,对受体品种南29及其4个转几丁质酶 β-1,3-葡聚糖酶串联基因水稻T5代品系进行了温室接种鉴定。结果表明,不能侵染受体品种南29 的13个菌株也不能侵染转基因品系,可侵染受体品种南29 的20个菌株一般不能侵染转基因品系,转基因水稻对稻瘟病菌抗性范围较受体对照扩大,证明转几丁质酶 葡聚糖酶基因水稻对稻瘟病具有一定的广谱抗性;诱发条件下,转基因品系大田叶瘟病情指数为0～1级,穗瘟发病率为0.3%～10.23%,抗性较受体对照大幅度提高,表明这些品系在稻瘟病抗病育种中是有应用潜力的。     

分子标记辅助选择改良水稻恢复系香5的病虫抗性

香5是由湖北省农科院选育的优质两系杂交稻恢复系,所配组合广两优5号(广占63-4S/香5)于2013年通过了湖北省审定.利用回交和分子标记辅助选择技术,将供体亲本MD12086-1351中的抗稻瘟病基因Pi9、抗褐飞虱基因Bph14、Bph15和抗白叶枯病基因Xa23渗入到香5背景中,育成了3个同时携带Pi9、Bph14、Bph15和Xa23基因的新株系.鉴定结果表明,新株系的叶瘟抗性明显提高,穗颈瘟抗性部分提高,苗期抗褐飞虱,分蘖盛期高抗白叶枯病;产量、主要农艺性状、香味和稻米品质主要指标与香5相似.新株系所配的组合在产量、主要农艺性状上与香5所配的组合相似.表明新株系可以作为香5的替代系用于培育抗稻瘟病、抗褐飞虱和抗白叶枯病的两系杂交稻新组合.     

Analysis of Blast Resistance Genes and Neck Blast Resistance of japonica Rice Varieties/Lines Recently Developed in Jiangsu Province

【Objective】Evaluating the breeding potential of the known blast resistance (R) genes harbored in rice varieties is one of prerequisites for developing rice blast resistant varieties and R genes-based control of the disease epidemic.【Method】A total of 195 new japonica varieties/lines from Jiangsu Province were genotyped using functional markers corresponding to 14 rice blast R gene loci. In order to identify gene or gene combination with breeding potential against neck-blast, 158 japonica rice varieties were then artificially inoculated with blast strains as well as 17 advanced backcrossing lines containing Pigm gene in two different genetic backgrounds at booting stage. 【Result】Most varieties carried 2 to 5 R genes, and none of new varieties contained Pigm; the distribution frequencies of Pib, Pita and Pikh were relatively high and all exceeded 45%, the rest 10 genes were under 30%; Pid3, Pid2, Pia and Pb1 had apparently higher frequency in lines from regional tests than that in authorized and released varieties. Three genes with effects ordered as Pia > Pi3/5/i > Pita significantly contributed to neck-blast resistance, and significantly positive interaction effects were found between Pia and Pita, and between Pi3/5/i and Pita. Combination effect of Pia+Pita was significantly higher than that of Pi3/5/i+Pita, and the varieties harboring Pia+Pita reached resistant or highly resistant levels. The resistance of 17 advanced backcrossing lines carrying Pigm was all significantly improved as compared with corresponding controls, and all reached resistant level or even higher. 【Conclusion】 The results suggested that Pigm and the combination of Pia+Pita have critically important breeding potential in japonica rice breeding against neck blast in Jiangsu Province.     

水稻Pi9基因序列标记的开发及其抗瘟育种应用

利用广谱抗稻瘟病基因Pi9的3'UTR序列设计特异DNA标记pi9utr,通过分子标记辅助选择和连续回交育种实践,改良7份籼稻亲本(316B、金23B、R207、R228、R288、R389、明恢86)的稻瘟病抗性。结果表明:pi9utr是一个高效显性分子标记,除R207外,在其余6份受体亲本与Pi9供体亲本75-1-127间均存在明显而稳定的多态;在室内接种后的316B×75-1-127 BC4F1群体和R288×775-1-127 BC6F1群体中随机取样,进行稻瘟病抗性表型和基因型鉴定及Pi9基因表达分析,证明pi9utr对两个组合回交后代个体的抗病性辅助选择效率均为100%,且在所有抗病单株中均能检测到Pi9基因的高效表达,在所有感病单株中均没有检测到Pi9基因的表达。利用pi9utr在回交世代中的连续辅助选择,获得了3个组合的BC4F1及3个组合的BC6F2代群体,为选育抗稻瘟病新品种打下了基础。     

Development of rice introgression lines with brown planthopper resistance and KDML105 grain quality characteristics through marker-assisted selection

‘Khao Dawk Mali 105’ (KDML105), a Thai aromatic rice cultivar, has been accepted in markets as a prime jasmine rice with premium prices. It has been extensively used as a parental line to develop new cultivars for rainfed lowland areas in Thailand because of its favorable quality and fragrance. However, this cultivar is highly susceptible to brown planthopper (BPH), a phloem sap-feeding insect pest of rice. The main objective of this study was to combine KDML105 essential grain quality traits with BPH resistance from the donor cultivar, ‘Rathu Heenati’. The linkage drag between Bph3 and Wx^a alleles was successfully broken by phenotypic and marker-assisted selections. All introgression lines (ILs) developed in this study showed a broad spectrum resistance against BPH populations in Thailand and had KDML105 grain quality standards. Finally this study was revealed that the ILs can be directly developed into BPH resistant varieties or can be used as genetic resources of BPH resistance to improve rice varieties with the Wx^b allele in rice breeding programs.     

转bar基因水稻除草剂抗性遗传研究及其应用

试验用３个恢复系－测６４,明恢６３和特青作母本分别与３个抗除草剂转ｂａｒ基因水稻品种－Ｂｅｎｇａｌ－Ｈｕ１０,Ｃｙｐｒｅｓｓ ＰＢ－６和Ｇｕｌｆｍｏｎｔ杂交,对其后代（Ｆ１,Ｆ２和ＢＣ１）进行遗传分析表明,除草剂的抗性是受一对显性核基因控制,通过杂交可将这种基因转育到恢复系,配出抗除草剂杂交组合,应用这项技术可放宽对不育系完全雄性不育的严格要求,有利于优良不育系的培育及高产、优质,多抗新组合的选配     

Development of Marker-Free Transgenic Cry1Ab Rice with Lepidopteran Pest Resistance by Agrobacterium Mixture-Mediated Co-transformation

Cry1Ab gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains (EHA105 and EHA101) contained Hpt and Cry1Ab genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and Cry1Ab genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T₂ progenies were analyzed for Cry1Ab and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive (80.94%), 62 plants were also Cry1Ab co-transformants (15.05% in total frequency), and 42 plants among the 272 T₂ progenies were Cry1Ab positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene. Southern blot analysis of five independent marker-free T₂ transgenic lines co-transformed from Zhejing 22 showed that Cry1Ab gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.     

Strategy for Use of Rice Blast Resistance Genes in Rice Molecular Breeding

Rice blast is one of the most destructive diseases affecting rice production worldwide. The development and rational use of resistant varieties has been the most effective and economical measure to control blast. In this review, we summarized the cloning and utilization of rice blast resistance genes, such as Pi1, Pi2, Pi9, Pi54, Pigm and Piz-t. We concluded that three main problems in the current breeding of rice blast resistance are: availability of few R(resistance) genes that confer resistance to both seedling and panicle blast, the resistance effect of pyramided lines is not the result of a simple accumulation of resistance spectrum, and only a few R genes have been successfully used for molecular breeding. Therefore, novel utilization strategies for rice blast R genes in molecular breeding were proposed, such as accurately understanding the utilization of R genes in main modern rice varieties, creating a core resistant germplasm with excellent comprehensive traits, screening and utilizing broadspectrum and durable resistance gene combinations. Lastly, the trends and possible development direction of blast resistance improvement were also discussed, including new genes regulating resistance identified via GWAS(genome-wide association study) and improving rice blast resistance using genetic editing.     

利用CRISPR/Cas9技术创制抗稻瘟病香型早籼温敏核不育系

【目的】创制新型抗稻瘟病香型早籼温敏核不育系，为高产优质杂交水稻选育提供资源。【方法】利用CRISPP/Cas9技术在水稻稻瘟病基因Pi21、温敏不育基因TMS5和香味基因Badh2的第1外显子处设计靶位点，构建多基因表达载体pC1300-2×35S::g^TMS5-g^Badh2-g^Pi21，转化优质常规籼稻品种中早70，测序鉴定分析获得纯合阳性稳定株系。利用稻瘟病喷雾接种和打孔接种方法对稻瘟病基因Pi21的纯合突变株系进行稻瘟病抗性鉴定，利用GC-MS技术对Badh2纯合突变株系的香味物质2-乙酰-1-吡咯啉（2-AP）含量进行测定。【结果】在T₀转基因株系中，Pi21、TMS5和Badh2突变频率分别为87.5%、80.0%和87.5%，突变类型多为双等位突变。从T₁代中筛选不含载体骨架的纯合突变株系，获得两种三突变纯合株系。稻瘟病接种结果表明，与野生型相比，T₂代Pi21纯合变异株系的抗性显著提高。同时，接种后纯合突变体株系内相关防卫基因的表达量显著上调，ROS积累量也显著增加。tms5纯合变异株系表现出典型的温敏不育特性，TMS5基因的表达水平与野生型相比显著降低，高温下Ub_L404基因的表达水平明显高于野生型。与野生型相比，在Badh2纯合突变体植株中Badh2的表达水平显著下调，并且香味物质2-AP含量极显著增加。【结论】利用CRISPR/Cas9技术成功对Pi21、TMS5和Badh2基因同时进行定向编辑，获得了具有高抗稻瘟病的香型温敏不育系，为高抗、香型不育系材料的选育提供参考，加快高产优质杂交水稻的选育。     

光身杂交稻选育研究进展

利用美国光身稻种质资源,培育出野败型三系光身杂交稻配套亲本光香A、B和光身R及光身光温敏核不育系光S,并用其配组率先育成一批三系光身杂交稻组合,如光香优德87、光香优602、光香优613等;还选配出一批非光身的优质高产三系杂交稻新组合如光香优7号、光香优13和光香优6号以及两系杂交稻组合光两优113和光两优193等.光身杂交稻及其配套亲本的选育成功,将进一步促进水稻亚种间杂种优势利用,可望实现我国杂交稻育种在米质、产量、抗性和适应性方面新的突破.