水稻抗衰老IPT基因与抗白叶枯病基因Xa23的聚合研究

以转抑制衰老有关的异戊烯基转移酶(IPT)基因株系GC-1、携带抗白叶枯病基因Xa23的“CBB23”和抗稻瘟病水稻品种“合系15号”为供体．采用分子标记辅助选择与生物学鉴定相结合的方法,将IPT基因与Xa23及抗稻瘟病基因进行聚合。在3个复交组合中获得了17株聚合IPT基因与Xa23基因的植株,用这些植株与两系杂交稻亲本9311、E32、培矮64S及W9834S进行杂交和回交,经PCR分子检测和抗白叶枯病、抗稻瘟病鉴定和细胞分裂素含量的测定,最终在4个BC,回交组合“(9311／／／合系15／CBB23／／GC-1)X9311”、“(E32／／／合系15／CBB23／／GC-1)XE32”、“(培矮64S／／／合系15／CBB23／／GC-1)X培矮64S”和“(GC-1／CBB23／／W9834S／合系15)XW9834S”中获得了17株携带IPT基因与Xa23基因的BC1F1植株,这些植株对来自北方稻区21个稻瘟病菌系全部表现为抗。携带IPT基因的抗病植株再与杂交稻亲本进行回交．在2个BC2回交组合“[(9311／／／合系15／CBB23／／GC-1)X9311]X9311’’和“[(E32／／／合系15／CBB23／／GC-1)XE32]XE32”中获得了7株携带IPT基因与Xa23基因的植株,这些植株再经过1—2代回交和自交,即可用于杂交稻育种。     

国际水稻所水稻新株系的抗性评价

为了进一步扩大我国稻种资源,丰富水稻育种材料,引进了165份国际水稻研究所在非洲进行穿梭育种的水稻新株系,于2011年和2012年在湖北生态条件下进行稻瘟病抗性、白叶枯病抗性和褐飞虱抗性的评价。评价结果表明,在165份新株系中有14份株系在宜昌和恩施2个稻瘟病病圃鉴定均表现抗或中抗稻瘟病,有40份株系同时高抗或抗白叶枯病菌株ZHE173和GD1358,有19份株系抗或中抗褐飞虱,有7份株系同时抗白叶枯病和稻瘟病,有8份株系同时抗白叶枯病和褐飞虱,有1份株系同时中抗稻瘟病、褐飞虱和白叶枯病。部分材料正在作为中间材料用于水稻育种。     

无选择标记和载体骨干序列的Xa21转基因水稻的获得

利用双右边界T-DNA载体通过根癌农杆菌介导法将水稻白叶枯病广谱抗性基因Xa21导入杂交稻重要恢复系C418中。T0代共获得27个独立转基因株系,通过田间抗性鉴定与PCR分析,有17个株系的Xa21基因分子鉴定为阳性,且对白叶枯病原菌P6生理小种具有抗性。通过对17个株系的后代植株进行田间抗性鉴定,分子标记辅助选择及Southern杂交分析,结果显示4个株系的T1代植株中能分离出无潮霉素标记基因的Xa21转基因植株。无选择标记Xa21转基因株系的获得率为15%。PCR检测还表明,这些无选择标记的Xa21转基因植株不带有载体骨架序列。通过对转基因后代进一步的抗性鉴定与PCR辅助选择,获得了无选择标记和载体骨架序列的转基因Xa21纯合的抗白叶枯病水稻。     

转cry1Ab基因抗虫水稻的田间试验

采用农杆菌介导的遗传转化技术, 将Bt基因cry1Ab导入三系恢复系明恢63获得了一批抗虫性及农艺性状较好的转基因纯合株系。研究中, 进一步检测了这些纯合株系的拷贝数、Bt蛋白含量、抗虫性及主要农艺性状。结果显示: 5个转基因纯合株系均为单拷贝; 纯合株系T_1Ab-10及其杂种的Bt蛋白含量最高; 人工接虫和自然感虫试验中, T_1Ab-10对二化螟、三化螟和稻纵卷叶螟均表现高度抗性; 喷药试验中, 阳性植株与阴性植株、对照植株的主要农艺性状均没有显著差异。这说明转cry1Ab基因纯合株系T_1Ab-10具商品化潜力, 可作基因聚合材料。     

经基因工程改良的抗白叶枯病水稻粳型恢复系“C418—Xa21”及其杂交稻

通过农杆菌介导的转化系统,将业已克隆的水稻抗白叶枯病基因Xa21导入重要的粳型杂交稻恢复系“C418”。PCR和抗性分析表明单拷贝整合的Xa21在T1代的分离比为3：1。在T2代通过PCR和抗性分析选择了Xa21纯合的转基因株系“C418－Xa21”。将选择的转基因纯合系“C418－Xa21”与常用的雄性不育系“屉锦A”杂交,产生了带有转基因Xa21的杂交稻“屉优418－Xa21”（简称转基因杂交稻）。分子分析表明转基因Xa21在杂交稻“屉优418－Xa21”中能稳定遗传,抗性分析表明转基因恢复系“C418－Xa21”和转基因杂交稻“屉优418－Xa21”对白叶枯病具有高度的广谱抗性,并保持了受体对照的优良农艺性状。另外我们还转基因杂交稻“屉优418－Xa21”对白叶枯病的抗性水平高于转基因恢复系“C418－Xa21”,这可能是遗传背景的差异所致,抗白叶枯病转基因粳型恢复系数 杂交稻的育成将有益于杂交稻在我国北方稻区的推广。     

水稻抗褐飞虱基因Bph3和Bph24(t)的聚合育种利用

在水稻(Oryza sativa)的生产实践中,常常会受到褐飞虱(Nilaparvatal lugens)等多种病虫害的威胁。聚合不同抗性基因,培育抗性品系,是应对各种生物胁迫最有效的策略。传统香稻由于其自身抗性等条件限制,无法大面积推广。本研究结合分子标记辅助选择(marker-assisted selection, MAS)技术,以含有抗褐飞虱基因Bph3或Bph24(t)的供体亲本与含有抗稻瘟病基因(Pi2、Pib或Pimh)、抗白叶枯病基因(Xa23)或香味基因(badh2)的优质三系杂交水稻保持系和恢复系进行多亲本复合杂交。将抗褐飞虱基因聚合到优良水稻品系中,筛选获得41个含有抗褐飞虱基因且与其他目的基因以不同组合方式相聚合的稳定品系。抗性鉴定、香味检测和农艺性状测定表明,各聚合品系的褐飞虱抗性水平较受体亲本均有明显提升,且具有双抗、三抗和/或香味等优良表型。这些新品系为多抗、优质水稻新品种的选育提供新的种质材料。     

抗稻瘟病水稻资源抗性基因Pita、Pib、Pi9以及Pikm的分布研究

利用抗稻瘟病水稻资源品种杂交,聚合多个抗性基因是培育持久抗稻瘟病水稻新品种的主要育种途径.利用分子标记技术对水稻抗性资源进行基因型鉴定是分子辅助聚合育种的基础.通过以亚华种业科学院稻瘟病病圃抗病水稻资源为材料,利用特异性分子标记对Pi9、Pita、Pib以及Pikm基因在水稻抗稻瘟病资源的分布进行了鉴定,初步建立了抗性基因数据库.同时对抗性基因及与抗性反应的相关性进行了探讨,结果表明以Pi9为主效基因,同时聚合Pita和Pib抗性基因能提高持久抗稻瘟病能力.     

来源于疣粒野生稻的白叶枯病新抗源的鉴定

疣粒野生稻对白叶枯病高抗甚至免疫。粳稻品种8411与疣粒野生稻体细胞杂交获得了2个高抗所有栽培稻白叶枯病抗性基因均不能抵御的国际广致病菌系P6的新种质SH5和SH76,遗传分析鉴定出新种质含有1个抗P6小种的显性基因。本研究以高感白叶枯病水稻品种IR24及携带不同抗性基因的16个材料为参照,对分蘖期、孕穗期的SH5和SH76分别接种11个白叶枯病小种,抗性分析表明SH5和SH76的抗谱广,与IRBB21(Xa21)抗谱一致,与IRBB5(xa5)、IRBB7(Xa7)和Asominori(Xa17)的较相近。用Xa21的分子标记pTA248和XA21检测,确定SH5和SH76不携带Xa21基因,前期研究结果证实新种质中不含xa5和Xa7;与Asominori的杂交试验表明其抗性基因与Xa17基因不等位。这些结果表明SH5和SH76中存在1个抗P6小种的新基因。     

云南野生稻抗白叶枯病类Xa21基因的鉴定

水稻白叶枯病是水稻生产上的主要细菌病害之一。从野生稻中发掘优异的水稻白叶枯病抗性材料,可以拓宽栽培稻抗白叶枯病遗传基础。经过温室接菌鉴定和PCR标记分析,对云南野生稻进行Xa21基因的检测鉴定。温室接菌鉴定表明,云南野生稻对广谱致病小种PX099及云南强致病菌Y8具有较好的抗性能力,特别是疣粒野生稻对致病菌株达到免疫程度;PCR标记分析表明,云南野生稻不含有Xa21基因,但含有与Xa21基因某些区域同源的片段。本研究结果为寻找新的抗源材料及快速发掘利用云南野生稻中的抗白叶枯病基因提供理论依据。     

云南野生稻中的抗白叶枯病基因Xa21的鉴定

水稻白叶枯病是水稻生产上的主要细菌病害之一。从野生稻中发掘优异的水稻白叶枯病抗性材料,可以拓宽栽培稻抗白叶枯病遗传基础。经过温室接菌鉴定和PCR标记分析,对云南野生稻进行Xa21基因的检测鉴定。温室接菌鉴定表明,云南野生稻对广谱致病小种PX099及云南强致病菌Y8具有较好的抗性能力,特别是疣粒野生稻对致病菌株达到免疫程度;PCR标记分析表明,云南野生稻不含有Xa21基因,但含有与Xa21基因某些区域同源的片段。本研究结果为寻找新的抗源材料及快速发掘利用云南野生稻中的抗白叶枯病基因提供理论依据。     

Marker-assisted introgression of the major bacterial blight resistance gene, <Emphasis Type="Italic">Xa21</Emphasis> and blast resistance gene, <Emphasis Type="Italic">Pi54</Emphasis> into RPHR-1005, the restorer line of the popular rice hybrid,DRRH3

The elite Indian rice hybrid, DRRH3 is highly susceptible to two major diseases, bacterial blight (BB) and blast, which limit its productivity significantly. In the present study, we have introgressed two major genes, viz., Xa21 and Pi54 conferring resistance against BB and blast, respectively into RPHR-1005, the male parent of DRRH3 through marker-assisted backcross breeding (MABB) and analyzed the backcross derived plants for their resistance against BB and blast. RPBio Patho-2 was used as a donor for both the resistance genes. Gene-specific markers were used for the foreground selection of Xa21 and Pi54 at each stage of backcrossing and markers specific for the major fertility restorer genes, Rf3 and Rf4 were used only at BC₁F₁ generation for foreground selection. Background selection was done using 62 polymorphic SSR markers and marker-assisted backcrossing was continued till BC₃ generation. At BC₃F₄, through intensive phenotype-based selections 15 promising lines (ABLs) possessing high level of resistance against BB and blast, high yield, fine-grain type, complete fertility restoration along with better panicle exsertion and taller plant type as compared to RPHR-1005 were identified and test crossed with APMS 6 A, the female parent of DRRH3. The newly derived hybrids (i.e. improved versions of DRRH3) were observed to possess high level of resistance against BB and blast along with medium-slender grain type and yield level better than or equivalent to that of DRRH3. Our study exemplifies the utility of MABB for targeted improvement of multiple traits in hybrid rice.     

水稻抗白叶枯病基因Xa-4的PCR标记研究

根据与水稻抗白叶枯病基因Ｘａ－４紧密连锁的分子标记Ｍ５５的序列设计引物,通过对国际水稻研究所育成的抗白叶枯病近等基因系和基因累加系的叶片ＤＮＡ、半粒种子提取物及Ｘａ－４基因的杂合体ＤＮＡ的ＰＣＲ特异扩增,初步建立了Ｘａ－４的ＰＣＲ标记体系。进而用该标记体系对我国籼型杂交水稻常用的亲本材料进行分析,揭示出了Ｘａ－４在这些材料中的分布情况。     

Marker-assisted breeding of Xa4, Xa21 and Xa27 in the restorer lines of hybrid rice for broad-spectrum and enhanced disease resistance to bacterial blight

Hybrid rice based on heterosis can significantly increase rice yield compared to inbred rice. Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive bacterial diseases that affect hybrid rice production. To breed a broad-spectrum and high disease resistance to BB in hybrid rice, we introduced the Xa4, Xa21 and Xa27 genes into the restorer lines of Mianhui 725 or 9311 genetic backgrounds and pyramided the three R genes in the progeny derived from the cross between the two lines. A near-isogenic line of the Xa27 gene in the genetic background of 9311 [9311(Xa27)] and another line with the Xa4 and Xa21 genes in the genetic background of Mianhui 725 (WH421) were firstly developed through marker-assisted selection. A new restorer line carrying Xa4, Xa21 and Xa27, designated as XH2431, was selected from the F8 progeny of the cross between 9311(Xa27) and WH421 through marker-assisted breeding and pedigree selection. XH2431 and II You 2431, the hybrids derived from cytoplasmic male-sterile line II-32A and restorer line XH2431, conferred high resistance to all 23 Xoo strains collected from 10 countries. XH2431 restored the fertility of II-32A to the normal level in the F1 generation. In addition, II You 2431 showed good agronomic traits under greenhouse conditions. The development of XH2431, 9311(Xa27) and WH421 provides a set of restorer lines with broad-spectrum and enhanced resistance to BB for hybrid rice.     

Pyramiding three bacterial blight resistance genes (xa5, xa13 and Xa21) using marker-assisted selection into indica rice cultivar PR106

Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in several countries. Three BB resistance genes, xa5, xa13 and Xa21, were pyramided into cv. PR106, which is widely grown in Punjab, India, using marker-assisted selection. Lines of PR106 with pyramided genes were evaluated after inoculation with 17 isolates of the pathogen from the Punjab and six races of Xoo from the Philippines. Genes in combinations were found to provide high levels of resistance to the predominant Xoo isolates from the Punjab and six races from the Philippines. Lines of PR106 with two and three BB resistance genes were also evaluated under natural conditions at 31 sites in commercial fields. The combination of genes provided a wider spectrum of resistance to the pathogen population prevalent in the region; Xa21 was the most effective, followed by xa5. Resistance gene xa13 was the least effective against Xoo. Only 1 of the BB isolates, PX04, was virulent on the line carrying Xa21 but avirulent on the lines having xa5 and xa13 genes in combination with Xa21. Received: 26 May 2000 / Accepted: 16 August 2000     

Two broad-spectrum blast resistance genes,<Emphasis Type="Italic"> Pi9</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) and<Emphasis Type="Italic"> Pi2</Emphasis>(<Emphasis Type="Italic">t</Emphasis>), are physically linked on rice chromosome 6

To understand the molecular basis of broad-spectrum resistance to rice blast, fine-scale mapping of the two blast resistance (R) genes, Pi9( t) and Pi2( t), was conducted. These two genes were introgressed from different resistance donors, previously reported to confer resistance to many blast isolates in the Philippines, and were mapped to an approximately 10-cM interval on chromosome 6. To further test their resistance spectrum, 43 blast isolates collected from 13 countries were used to inoculate the Pi2( t) and Pi9( t) plants. Pi9( t)-bearing lines were highly resistant to all isolates tested, and lines carrying Pi2( t) were resistant to 36 isolates, confirming the broad-spectrum resistance of these two genes to diverse blast isolates. Three RAPD markers tightly linked to Pi9( t) were identified using the bulk segregant analysis technique. Twelve positive bacterial artificial chromosome (BAC) clones were identified and a BAC contig covering about 100 kb was constructed when the Pi9( t) BAC library was screened with one of the markers. A high-resolution map of Pi9( t) was constructed using BAC ends. The Pi2( t) gene was tightly linked to all of the Pi9( t) markers in 450 F(2) plants. These data suggest that Pi9( t) and Pi2( t) are either allelic or tightly linked in an approximately 100-kb region. The mapping results for Pi9( t) and Pi2( t) provide essential information for the positional cloning of these two important blast resistance genes in rice.     

Introgression of Pi2 and Pi5 Genes for Blast (Magnaporthe oryzae) Resistance in Rice and Field Evaluation of Introgression Lines for Resistance and Yield Traits

Blast caused by Magnaporthe oryzae is the most devastating disease causing significant loss in rice production. The destructive nature of the disease is mainly due to the genetic plasticity of M. oryzae which complicates the breeding strategies. Blast can be effectively managed by the deployment of R genes. In this study, broad‐spectrum blast resistance genes Pi2 and Pi5 were introgressed independently into popular but blast susceptible rice variety, Samba Mahsuri (BPT5204) by applying marker‐assisted backcross breeding approach. Tightly linked markers AP5930 for Pi2 and 40N23r for Pi5 gene were used in foreground selection. Background selection helped to identify the lines with maximum recovery of recurrent parent genome (RPG). The RPG recovery in Pi2 introgression lines was up to 90.17 and 91.46% in Pi5 lines. Homozygous introgression lines in BC₃F₄ generation carrying Pi2 and Pi5 gene were field evaluated for blast resistance, yield per se and yield‐related traits. The lines showed resistance to leaf and neck blast in multilocation field evaluation. Improved BPT5204 lines with improvement for blast resistance were on par with original BPT5204 in terms of grain yield and grain features.     

国外引进水稻种质资源的稻瘟病抗性基因检测与评价

为了筛选出福建省水稻稻瘟病重发区育种中可利用的新抗性资源,在福建省上杭县对156份外引水稻种质资源进行了2年田间自然诱发鉴定,并对Pi2、Pi9、Pi5、Pi54、Pikm、Pita、Pia和Pib等8个稻瘟病抗性基因做了分子检测。结果表明:156份资源对苗瘟、叶瘟、穗颈瘟和综合抗性表现抗病的分别有10份、14份、29份和26份,且苗瘟抗性级别与叶瘟抗性级别(r=0.816,P<0.01)、苗瘟抗性级别与穗颈瘟抗性级别(r=0.347,P<0.01)、以及叶瘟抗性级别与穗颈瘟抗性级别(r=0.344,P<0.01),均呈极显著正相关。分子标记检测到携带稻瘟病抗性基因Pi9、Pi2、Pi54、Pikm、Pi5、Pib、Pia和Pita的水稻资源分别有1、6、20、22、37、88、101和106份,其中携带稻瘟病抗性基因Pi9和Pi2的水稻资源的抗性表现较好,表现抗病的超过60%,携带其他稻瘟病抗性基因的水稻资源表现抗病的均在50%以下;水稻资源携带0~6个稻瘟病抗性基因,随着携带抗性基因数目增加,抗病率呈上升趋势,综合抗性等级呈下降趋势。进一步研究发现,携带Pi9+Pi5+Pikm+Pia、Pi5+Pib+Pita+Pikm+Pia和Pi2+Pi54+Pib+Pita+Pikm+Pia等3个基因型的水稻资源,稻瘟病抗性较好。最后,筛选了8份稻瘟病抗性较好的材料,提供育种者参考、利用。     

The identification of Pi50(t), a new member of the rice blast resistance Pi2/Pi9 multigene family

The deployment of broad-spectrum resistance genes is the most effective and economic means of controlling blast in rice. The cultivar Er-Ba-Zhan (EBZ) is a widely used donor of blast resistance in South China, with many cultivars derived from it displaying broad-spectrum resistance against blast. Mapping in a set of recombinant inbred lines bred from the cross between EBZ and the highly blast-susceptible cultivar Liangjiangxintuanheigu (LTH) identified in EBZ a blast resistance gene on each of chromosomes 1 (Pish), 6 (Pi2/Pi9) and 12 (Pita/Pita-2). The resistance spectrum and race specificity of the allele at Pi2/Pi9 were both different from those present in other known Pi2/Pi9 carriers. Fine-scale mapping based on a large number of susceptible EBZ?×?LTH F(2) and EBZ?×?LTH BC(1)F(2) segregants placed the gene within a 53-kb segment, which includes Pi2/Pi9. Sequence comparisons of the LRR motifs of the four functional NBS-LRR genes within Pi2/Pi9 revealed that the EBZ allele is distinct from other known Pi2/Pi9 alleles. As a result, the gene has been given the designation Pi50(t).