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.     

稻瘟病抗性分子育种研究综述

对稻瘟病菌致病机理、抗性鉴定方法、抗源筛选、抗性基因定位和克隆及无毒基因研究、分子标记辅助育种和转基因育种等方面的研究进展进行了综述,同时对抗性基因的利用以及聚合育种进行了展望.     

水稻子预44和江南香糯基因组比较鉴定稻瘟病抗性相关基因

【目的】子预44是一具有广谱持久稻瘟病抗性的云南地方粳稻品种。为了鉴定子预44中候选稻瘟病抗性相关基因,【方法】本研究利用高通量测序技术（High-throughput sequencing）对子预44和感病水稻江南香糯进行了全基因组测序。而后使用软件GATK(3.4-46)对高质量测序结果进行SNP和InDel的检测和统计,进一步筛选出子预44和江南香糯DNA水平存在SNPs/InDels多态性抗病相关基因。【结果】通过Hiseq X10 PE150平台分别获得了4 118 170 045 bp和2 995 054 509 bp子预44和江南香糯的基因组数据,比对到参考基因组（Ensembl release 31）的比对率分别为98.56％和98.30％。在抗病水稻子预44和感病水稻江南香糯之间鉴定了922个纯合突变的差异抗病相关基因。结合基因定位结果,在子预44中鉴定了一个新的抗稻瘟病候选基因。【结论】 研究结果为子预44中抗稻瘟病新基因的克隆提供了参考,对子预44广谱持久抗瘟分子机制的研究奠定了基础。     

水稻子预44和江南香糯基因组比较鉴定稻瘟病抗性相关基因

【目的】子预44是一具有广谱持久稻瘟病抗性的云南地方粳稻品种。为了鉴定子预44中候选稻瘟病抗性相关基因，【方法】本研究利用高通量测序技术（High-throughput sequencing）对子预44和感病水稻江南香糯进行了全基因组测序。而后使用软件GATK(3.4-46)对高质量测序结果进行SNP和InDel的检测和统计，进一步筛选出子预44和江南香糯DNA水平存在SNPs/InDels多态性抗病相关基因。【结果】通过Hiseq X10 PE150平台分别获得了4 118 170 045 bp和2 995 054 509 bp子预44和江南香糯的基因组数据，比对到参考基因组（Ensembl release 31）的比对率分别为98.56％和98.30％。在抗病水稻子预44和感病水稻江南香糯之间鉴定了922个纯合突变的差异抗病相关基因。结合基因定位结果，在子预44中鉴定了一个新的抗稻瘟病候选基因。【结论】研究结果为子预44中抗稻瘟病新基因的克隆提供了参考，对子预44广谱持久抗瘟分子机制的研究奠定了基础。     

Genetics and Improvement of Bacterial Blight Resistance of Hybrid Rice in China

Since 1980s,rice breeding for resistance to bacterial blight has been rapidly progressing in China. The gene Xa4 was mainly used in three-line indica hybrid and two-line hybrid rice. The disease has been ‘quiet’ for 20 years in China,yet in recent years it has gradually emerged and been prevalent in fields planted with newly released rice varieties in the Changjiang River valley. Under the circumstances,scientists inevitably raised several questions:what causes the resurgence and what should we do next? And...     

中国杂交水稻白叶枯病抗性的遗传改良

 20世纪80年代,中国杂交水稻抗白叶枯病育种进展迅速。在大面积种植的籼型三系和两系杂交稻中主要应用白叶枯病抗性基因Xa4,白叶枯病在生产中几乎“销声匿迹”了20年。但近年来,此病害却在长江流域一些种植新组合的稻田里又爆发了。什么原因引起它再度流行？品种的抗性是否还有效？抗病育种是否还是水稻改良的主要目标之一？通过哪些途径才能更有效地利用抗病基因培育出更为持久的抗性品种？为回答这些问题,就我国杂交水稻对白叶枯病抗性的改良,寄主与病菌群体的互动演变效应进行了分析,讨论了拓宽抗性遗传基础、白叶枯病菌毒性群体结构、基因轮换、合理利用基因等问题。 在采用了包括传统方法、分子标记辅助选择和转基因技术的综合策略后,杂交水稻抗白叶枯病育种研究将出现新的局面。     

浙江省主栽水稻品种抗稻瘟病基因的分子检测

稻瘟病是水稻的三大重要病害之一。利用稻瘟病抗病基因进行新品种培育是控制稻瘟病发生最经济有效的措施,对水稻安全生产具有重要意义。本研究在鉴定63个浙江省主栽水稻品种的叶瘟和穗颈瘟抗性水平的基础上,利用Pi1、Pi5、Pi2、Pi9、Pia、Pizt、Pigm、Pib、Pik和Pikh等10个已克隆抗稻瘟病基因的特异分子标记,对这63个品种的抗稻瘟病基因组成进行了鉴定,并进一步分析了抗病基因与稻瘟病抗性之间相关性。抗性鉴定结果显示,63个浙江省主栽水稻品种中,抗叶瘟品种占66.7%、抗穗瘟品种占50.8%。抗稻瘟病基因分析显示,有60个主栽品种聚合了多个抗病基因,含有Pizt和Pib基因的品种45个、含有Pi2和Pi5基因的品种43个,此外,抗病基因Pia、Pik、Pi1、Pi2、Pizt、Pikh、Pi5及Pib在浙江省主栽品种中分布频率整体较高,而Pigm和Pi9分布频率很低,仅3.2%。本研究揭示了浙江省主栽水稻品种抗稻瘟病基因的组成及抗稻瘟病基因对稻瘟病抗性提高的贡献,为浙江省抗病新品种的培育提供参考。     

黄淮海稻区水稻品种的穗颈瘟抗性

[目的]为明确黄淮海稻区育成水稻品种的稻瘟病抗性及其基因型,[方法]收集了来自本稻区5个不同地区的水稻品种,连续两年通过稻瘟病菌接种鉴定和田间病圃自然诱发对供试品种进行了稻瘟病抗性鉴定,并利用4个抗病基因Pita, Pib, Pi54和Pikm的分子标记,对水稻品种基因型进行了统计分析,并以抗性等级为表型值对品种资源进行聚类分析。[结果]黄淮海稻区水稻品种发病率较高,分别达 85.2%和 65.9%。Pi54和Pib检出率分别达 84%、65%。同时抗性聚类分析表明Pi54和Pib在本稻区的抗性贡献率却很低。[结论]黄淮海稻区水稻品种稻瘟病抗性弱,携带的抗瘟基因的抗性在不断减弱或丧失,需加强稻瘟病主效抗性的发掘、开发与利用。     

黄淮海稻区水稻品种的穗颈瘟抗性

【目的】为明确黄淮海稻区育成水稻品种的稻瘟病抗性及其基因型，【方法】收集了来自本稻区5个不同地区的水稻品种，连续两年通过稻瘟病菌接种鉴定和田间病圃自然诱发对供试品种进行了稻瘟病抗性鉴定，并利用4个抗病基因Pita, Pib, Pi54和Pikm的分子标记，对水稻品种基因型进行了统计分析，并以抗性等级为表型值对品种资源进行聚类分析。【结果】黄淮海稻区水稻品种发病率较高，分别达85.2%和65.9%。Pi54和Pib检出率分别达84%、65%。同时抗性聚类分析表明Pi54和Pib在本稻区的抗性贡献率却很低。【结论】黄淮海稻区水稻品种稻瘟病抗性弱，携带的抗瘟基因的抗性在不断减弱或丧失，需加强稻瘟病主效抗性的发掘、开发与利用。     

Pigm特异性选择标记的开发及其在粳稻穗颈瘟抗性育种中的利用

【目的】Pigm是一个广谱的稻瘟病抗性基因,源自持久抗性品种谷梅4号,与Piz、Piz-t、Pi2、Pi9和Pi40等互为复等位基因但抗谱存在差异。为了更好地在分子辅助选择育种中利用Pigm基因,开发与Pigm特异性标记具有重要意义。【方法】在已有文献报道定位结果的基础上,通过随机测序获得了一段谷梅4号基因组的特异序列,并据此开发了一组用于筛选Pigm基因的分子标记,进一步选取江淮稻区3个不同生态型代表性粳稻品种作为受体,利用分子标记辅助选择结合对抗性基因的背景检测将Pigm基因导入受体品种。【结果】Pigm-4标记位于Pigm基因簇内部,与抗病功能元件PigmR紧密连锁,对不同类型的品种检测发现该标记特异性强,且利用该标记可将Pigm与Piz、Piz-t、Pi2、Pi9以及Pi40区分开来。对受体亲本稻瘟病抗性基因的检测和接种结果分析发现江淮稻区粳稻品种虽然携带了Pib、Pi54、Pita、Pb1中的2~3个基因,但是对强毒力的稻瘟病小种抗性普遍不强,而3种代表性粳稻背景下导入Pigm基因均可显著提高其对穗颈瘟的抗性水平。【结论】Pigm可以作为抗稻瘟病粳稻育种的有利基因资源加以利用,而Pigm-4是分子标记辅助筛选Pigm的优异标记。     

水稻抗稻瘟病分子机制研究进展

稻瘟病是危害世界水稻生产最严重的真菌病害之一。稻瘟病菌生理小种变异快,水稻品种的抗性一般仅能维持3~5年。培育和种植抗性品种是目前最经济有效的措施。近年来,对稻瘟病菌致病机制和抗性基因分子机理的系统研究,加深了对该病原菌-宿主系统中病原相关分子模式诱导的免疫反应机制和病原菌效应蛋白诱导的免疫反应机制的了解。本文综述了水稻抗稻瘟病的两种天然免疫机制研究的最新进展,并对目前水稻抗稻瘟病分子机制研究中急需解决的问题和挑战进行探讨和展望。     

水稻抗稻瘟病分子机制研究进展

稻瘟病是危害世界水稻生产最严重的真菌病害之一。稻瘟病菌生理小种变异快，水稻品种的抗性一般仅能维持3~5年。培育和种植抗性品种是目前最经济有效的措施。近年来，对稻瘟病菌致病机制和抗性基因分子机理的系统研究，加深了对该病原菌-宿主系统中病原相关分子模式诱导的免疫反应机制和病原菌效应蛋白诱导的免疫反应机制的了解。本文综述了水稻抗稻瘟病的两种天然免疫机制研究的最新进展，并对目前水稻抗稻瘟病分子机制研究中急需解决的问题和挑战进行探讨和展望。     

Genetic Improvement of Rice for Bacterial Blight Resistance: Present Status and Future Prospects

The production and productivity of rice has been challenged due to biotic and abiotic factors. Bacterial blight (BB) disease, caused by Xanthomonas oryzae pv. oryzae, is one of the important biotic stress factors, which reduces rice production by 20%–50%. The deployment of host plant resistance is the most preferred strategy for management of BB disease, and breeding disease resistant varieties remains a very economical and effective option. However, it is difficult to develop rice varieties with durable broad-spectrum resistance against BB using conventional approaches alone. Modern biotechnological tools, particularly the deployment of molecular markers, have facilitated the cloning, characterization and introgression of BB resistance genes into elite varieties. At least 46 BB resistance genes have been identified and mapped from diverse sources till date. Among these, 11 genes have been cloned and characterized. Marker-assisted breeding remains the most efficient approach to improve BB resistance by introducing two or more resistance genes into target varieties. Among the identified genes, xa5, xa13 and Xa21 are being widely used in marker-assisted breeding and more than 70 rice varieties or hybrid rice parental lines have been improved for their BB resistance alone or in combination with genes/QTLs conferring tolerance to other stress. We review the developments related to identification and utilization of various resistance genes to develop BB resistant rice varieties through marker-assisted breeding.     

转hrf1基因水稻对稻瘟病多小种非专化的稳定抗性

以表达水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae）编码harpin广谱抗性激发子的hrf1基因的转基因系为材料,在温室和田间病圃鉴定它们对稻瘟病菌的抗性,分析转基因水稻抗稻瘟病的作用机制。研究结果显示,转hrf1基因水稻对稻瘟病菌ZC3、ZD1和ZG1小种表现高抗,对ZB13表现中抗,表明hrf1基因在水稻中表达可以产生非小种专化抗性。在稻瘟病圃中对水稻稻瘟病抗性鉴定结果显示,转hrf1基因水稻在T1、T3、T5和T7代对稻瘟病菌都表现很好的抗性,表明转hrf1基因水稻对稻瘟病菌的抗性能稳定遗传。 转hrf1基因抗病水稻中防卫反应基因OsPR1a、OsPR1b、PAL和Chia4a以及正向调控水杨酸介导信号传导的NPR1基因的表达显著增强。转基因抗病水稻中硅含量显著提高。Harpin编码基因在水稻中表达,可能通过激发水稻中防卫反应基因的表达,提高水稻中硅含量等,从而使水稻产生对稻瘟病菌的广谱抗性。     

超级早稻中早39稻瘟病抗性的系谱分析

【目的】研究常规超级早籼稻品种中早39持久高抗稻瘟病的遗传基础,【方法】利用田间抗性鉴定、稻瘟病抗性基因分子标记检测和基因测序的方法,分析中早39稻瘟病持久抗性的遗传基础。【结果】中早39、金早47和中组3号对7个稻瘟病抗性基因Piz、Pi9、Pib、Pi36、Pi64、Pi-d2和Pi-d3的分子标记均呈阳性条带;且中早39与金早47、中组3号对5个稻瘟病生理小种的抗性表现完全相同。中早39与金早47、中组3号无论是表型还是基因型都完全一致;序列分析结果表明中早39系谱中的供试材料均含有Pi-d2抗病等位基因。【结论】中早39的抗稻瘟病特性经中组3号,来源于金早47。含有Pi-d2的地谷与中早39、金早47和中组3号对5个稻瘟病生理小种的表型相同。     

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.     

应用系统聚类方法筛选持久抗瘟性水稻品种

 以Tetep、Moroberekan、B40为抗、感对照，用8个抗性组分，对47个云南地方稻种资源的抗瘟性进行了系统聚类分析。结果表明，可以较好地将1996~1999年的50、27、16、11个品种分别分成6、6、4、3类。持久抗病对照品种Tetep(感~抗)分布于1、2、4类内，但较集中于第2类内；持久抗性品种Moroberekan(感~中抗)集中于第2类内；云南地方品种大白谷、毫弄早、毫玉浪每年同聚于第1类品种中，经连续几年试验，均表现出比Tetep和Moroberekan较稳定和较强的抗瘟性。第3、5、6类为感病品种，其中第3类感病对照品种B40历年与其他品种间的距离较其他品种相互间聚类距离大，属高度感病。连续4年的试验结果表明，系统聚类分析可作为水稻持久抗瘟性品种筛选的方法。     

稻瘟病抗性基因对安徽省稻瘟病菌种群抗性的影响

为分析抗稻瘟病基因对安徽省稻瘟病菌株的抗性水平的影响,采用10个水稻抗稻瘟病单基因品系对安徽不同地区的稻瘟病菌株进行致病力测定,同时对201份水稻品种的抗稻瘟病基因Pi9进行扩增,结合田间抗性表现,分析抗性基因Pi9的利用价值。抗性基因Pi9、Pik、Pizt等对安徽地区的稻瘟病菌株具有较好的抗性,抗性频率分别为66%、60%和54%,其中携带Pi9抗性基因的水稻品种的田间抗性表现也较好,说明Pi9可直接作为分子标记辅助安徽地区水稻品种选育抗原基因。     

利用dsRNA干涉方法研究水稻WRKY转录因子的抗病性

 为了研究植物WRKY基因编码的转录因子在水稻上的功能，构建了包含WRKY保守结构域的dsRNA发夹结构干涉载体，用农杆菌介导法转野生型水稻中花11，得到9个有干涉表型的株系。PCR和Southern结果表明dsRNA已整合入中花11的基因组中，且多数为单拷贝。结合T-DNA插入的WRKY突变体植株T456，研究了其中的3个干涉苗和T456对稻瘟病和白叶枯病的抗性，发现3个干涉苗对这两种水稻主要病害的抗性均明显强于T456和中花11，且T456比中花11略抗稻瘟病和白叶枯病，表明dsRNA干涉是成功的，它可能干涉或抑制了某些OsWRKY家族中负向调控抗病基因的成员。     

Osa-mi R439 Negatively Regulates Rice Immunity Against Magnaporthe oryzae

Osa-miR439 is a rice-specific microRNA family. Here we showed that Osa-miR439 acted as anegative regulator in rice immunity against blast fungus Magnaporthe oryzae. Osa-miR439 differentiallyresponded to M. oryzae between susceptible and resistant rice accessions. The accumulation ofOsa-miR439 was constitutively more in the susceptible accession than in the resistant one. Transgeniclines overexpressing Osa-miR439a (OX439a) showed higher susceptibility associating with lower inductionof defense-related genes and less hydrogen peroxide (H2O2) accumulation at the infection sites than thecontrol plants. In contrast, transgenic lines expressing a target mimic of Osa-miR439 (MIM439) displayedcompromised susceptibility associating with increased H2O2 accumulation. Furthermore, we found thatthe expression of three predicted target genes was decreased in OX439a but increased in MIM439 incomparison to control plants, and this expression was differential in susceptible and resistant accessionsupon M. oryzae infection, indicating that Osa-miR439a may regulate rice blast resistance via these genes.Our results unveiled the role of Osa-miR439a in rice blast resistance and provided the potentiality toimprove the blast resistance via miRNA.