水稻抗稻瘟病基因Pi-d(t)1、Pi-b、Pi-ta2的聚合及分子标记选择

冈46B(G46B)是水稻生产应用中的一个农艺性状十分优良的保持系,其主要的缺陷是稻瘟病抗性较弱,通过对地谷,BL-1,Pi-4号等三个分别含抗病基因Pi-d(t)1、Pi-b、Pi-ta2的稻瘟病抗性材料与G46B聚合杂交,并利用抗病基因连锁的分子标记对杂交后代进行辅助选择,在聚合杂交的F2代及B1C1代群体中共获得了15株含Pid(t)1、Pi-b、Pi-ta2等三个抗稻瘟病基因的材料,其可能的基因型分别为:三基因杂合体Pi-d(t)1pi-d(t)1/Pi-bpi-b/Pi-ta2pi-ta2 4株,双基因杂合体10株,其中Pi-d(t)1Pi-d(t)1/Pi-bpi-b/Pi-ta2pi-ta26株,Pi-d(t)1pi-d(t)1/Pi-bpi-b/Pi-ta2Pi-ta23株,Pi-d(t)1pi-d(t)1/Pi-bPi-b/Pi-ta2pi-ta21株,双基因纯合体Pi-d(t)1Pi-d(t)1/Pi-bpi-b/Pi-ta2Pi-ta2仅1株,这一研究结果为进一步改良G46B的稻瘟病抗性奠定了基础,同时这一研究结果表明利用分子标记可快速、有效地实现多个抗病基因的聚合,大大提高水稻抗病育种的效率.     

水稻抗稻瘟病基因Pi-d(t)1、Pi-b、Pi-ta2的聚合及分子标记选择

冈46B(G46B)是水稻生产应用中的一个农艺性状十分优良的保持系 ,其主要的缺陷是稻瘟病抗性较弱 ,通过对地谷 ,BL-1,Pi-4号等三个分别含抗病基因Pi-d(t)¹、Pi-b、Pi-ta² 的稻瘟病抗性材料与G4-6B聚合杂交 ,并利用抗病基因连锁的分子标记对杂交后代进行辅助选择 ,在聚合杂交的F2代及B1C1代群体中共获得了 15株含Pi-d(t)¹ 、Pi-b、Pi-ta² 等三个抗稻瘟病基因的材料 ,其可能的基因型分别为 :三基因杂合体Pi-d(t)¹ pi-d(t)¹ Pi-bpi-b-Pi-ta² pi-ta²4株 ,双基因杂合体 10株 ,其中Pi-d(t)¹ Pi-d(t)¹ Pi-bpi-b-Pi-ta² pi-ta²6株 ,Pi-d(t)¹ pi-d(t)¹ Pi-bpi-b-Pi-ta² Pi-ta² 3株 ,Pi-d(t)¹ pi-d(t)¹ Pi-bPi-b-Pi-ta² pi-ta² 1株 ,双基因纯合体Pi-d(t)¹ Pi-d(t)¹ Pi-bpi-b-Pi-ta² Pi-ta²仅1株 ,这一研究结果为进一步改良G46B的稻瘟病搞性奠定了基础,同时这一研究结果表明利用分子标记可快速、有效地实现多个抗病基因的聚合,大大提高水稻抗病育种的效率。     

黄淮区粳稻抗稻瘟病基因Pi-ta、Pi-b、Pi54、Pikm的分子检测

利用水稻稻瘟病抗病基因Pi-ta、Pi-b、Pi54 和Pikm的功能标记对2016年山东省水稻中晚熟组区试、机插秧组区试的32个参试品系及连云港农业科学院科企水稻联合体黄淮粳稻区试16个品系进行了分子标记检测,结合稻瘟病抗性接种鉴定,对基因型与表型进行相关性分析。结果表明48个品种中携带Pi-ta、Pi-b、Pi54和Pikm抗性基因的品种数分别为15个、25个、26个和21个,其中鲁资稻7号、连粳14JD24含有4个基因的抗性等位基因,YS-6-6、济稻1号、D400等13个品种分别含有3个基因的抗性等位基因,临稻10号、丰稻2号、天和糯303等8个品种不含抗性等位基因。稻瘟病鉴定结果表明,48份品种中,济稻1号、圣稻072、连粳14JD24等4个品种表现中抗（MR）;临稻10、YS-6-6、圣稻053等26个品种表现中感（MS）;晶稻180、临13-105、圣稻504等15个品种表现感病（S）;H11-15、润农9号、晶稻160表现高感（HS）。Pi-ta、Pi-b、Pi54、Pikm 4个抗性基因已在黄淮区粳稻抗稻瘟病育种中得到广泛应用。其中Pikm与稻瘟病抗性综合指数存在显著相关性（r=0.477 5,P<0.01）。     

江苏省稻瘟病菌的毒性多样性及水稻品种的抗病性

在13个已知日本抗病基因品种上检测1997－1999年采集自江苏省吴江,赣榆,通州,高邮和宜兴等5个代表地区的324个稻瘟病菌株的毒性,结果可将上述菌株划分为90种毒性类型,表明江苏省稻瘟病菌存在着丰富的毒性多样性,毒性类型组成在地区间存在较大的差异,并且随着时间的推移,稻瘟病菌毒性类型组成有差异加大的趋势,在已知抗病基因品种上测定江苏省稻瘟病菌的毒力,结果显示：Pi-k^3,Pi-ta,Pi-ta^2和Pi-sh等抗病基因对江苏省的稻瘟病菌的抗谱很窄,而Pi-i,Pi-z,Pi-z^t和Pi-b等抗病基因的抗谱比较宽,可作为抗源加以利用,用6个代表性毒性类型菌株接种江苏省80筱水稻主载品种和新育成品种,品种抗性分析表明,上述水稻品种中的籼稻和杂交稻对江苏省稻瘟病菌具有较高的抗性,而粳稻品种的抗性较差,上述研究结果为利用水稻品种抗性多样性控制稻瘟病提供了依据。     

太湖流域粳稻地方品种黑壳子粳对稻瘟病抗性的遗传分析

太湖流域粳稻地方品种黑壳子粳对稻瘟病菌表现抗谱广,抗性强的特点,利用黑壳子粳与感病的云南稻地方品种丽江新团黑谷杂交获得的F1、F2和RIL群体,在苗期喷雾接种研54-04和北1两个日本稻瘟病鉴别菌系,根据抗感反应分析亲本的抗病基因组成,结果表明,黑壳子粳对菌系北1的抗性由一对显性基因控制,对菌系研54-04的抗性由两对互为独立遗传的显性基因控制,等位性测定结果和重组自交系的抗感反应表明：黑壳子粳对菌系北1的抗病基因兼抗菌系研54-04,该抗病基因与Pi-k,Pi-z,Pi-ta,Pi-b,Pi-t等5个已知抗病基因座呈非等位关系。也不是Pi-i和Pi-a基因,推断是一个未知的新基因;另一个抗病基因抗菌系研54-04,感菌系北1。     

抗稻瘟病基因Pi-ta、Pi-b、Pi54和Pikm在黄淮稻区水稻品种资源中的抗性分布

为了探明黄淮稻区4个抗稻瘟病基因Pi-ta、Pi-b、Pi54和Pikm在品种资源中的分布及其不同基因型组合的抗病效性。本研究利用上述4个抗病基因的功能标记,对88个黄淮稻区育成品种进行抗稻瘟病基因型检测,结果表明:检出最多的抗稻瘟病基因组合是Pib+Pi54,占总检测材料的60.2%。经过2016-2017年连续两年的人工接种鉴定和田间自然鉴定,稻瘟病感病材料比例分别达到87.5%、77.3%,感病材料的比例增高说明抗性基因的抗性作用在逐渐降低。在检测品种中,抗性较好的4个基因型或基因型组合分别是Pi-ta、Pi-ta+Pi-b、Pi-ta+Pi-b+Pi54和Pi-ta+Pi-b+Pi54+Pikm,但检出率很低。本研究结果说明,黄淮稻区品种中,携带Pi-ta、Pi-ta+Pi-b、Pi-ta+Pi-b+Pi54和Pi-ta+Pi-b+Pi54+Pikm基因型的抗病性较好,应得到加强利用。本研究结果对育成品种抗稻瘟病基因或基因组合跟踪检测和抗病性评价是抗稻瘟病育种的有效途径。     

利用微卫星标记鉴定水稻的稻瘟病抗性

应用水稻稻瘟病抗性基因Pid(t)紧密连锁的微卫星标记RM262对含有该抗病基因的品种地谷与感病品种江南香糯和8987的杂交F2群体进行遗传分析和抗性鉴定,结果表明,RM262的PCR扩增物在抗、感品种之间的多态性较好;在2个F2群体中,RM262和抗病基因间的重组率分别为5.74%和8.17%,应用该标记的抗性纯合和杂合带型选择抗性植株,其准确率可达98%以上。此外,还就分子标记辅助育种进行了讨论。     

XA21和PI-D2激酶蛋白质在酿酒酵母中的表达及其自我磷酸化研究

白叶枯病和稻瘟病是最主要的水稻病害。Xa21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质。在前期研究中,曾系统地研究了细菌中表达XA21激酶蛋白质的生化活性。在此实验中利用真核表达系统酿酒酵母对Xa21和Pi-d2编码的蛋白激酶进行了表达、纯化及自我磷酸化活性分析,为进一步的生化分析、蛋白质-蛋白质相互作用研究、底物筛选等奠定了基础。     

水稻白叶枯病抗性基因Xα21和稻瘟病抗性基因Pi-d2激酶蛋白质在毕赤酵母中的表达及条件优化

[目的]白叶枯病和稻瘟病是最主要的水稻病害,Xα21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质.本研究旨在毕赤酵母系统中表达XA21和PI-D2激酶蛋白质.[方法]用Xα21和Pi-d2的激酶区PCR产物,构建了pPICZαA-Xα21K、pPICZαA-Pi-d2K重组质粒,酶切及测序验证后,将重组质粒线性化,转化到毕赤酵母菌株中,系统地比较了不同酵母菌株(KM71、GS115、X33),不同甲醇浓度(1%、2%、3%),不同pH(pH5、pH6、pH7、pH8)值,不同诱导时间(24 h、48 h、72 h)条件下激酶蛋白质的表达情况.[结果]XA21和PI-D2激酶蛋白质可以在毕赤酵母中表达,但表达的蛋白质不能分泌到培养基上清中,而只能在菌体中检测到,对表达条件的系统比较发现,毕赤酵母菌株KM71和X33、2%的甲醇诱导浓度、pH5和48 h以上的诱导时间有利于激酶蛋白质的表达,最后我们在酵母裂解物上清中获得了纯化的考染可见的激酶蛋白质.[结论]在毕赤酵母中表达了XA21和PI-D2激酶蛋白质,为下一步生化特性研究奠定了基础.     

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。     

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.     

Detection of novel blast resistance genes, Pi58(t) and Pi59(t), in a Myanmar rice landrace based on a standard differential system

The use of broad-spectrum R genes is an effective way to achieve durable resistance against rice blast (Magnaporthe oryzae Couch, anamorph: Pyricularia oryzae Cavara) in rice (Oryza sativa L.). We previously surveyed the diversity of blast resistance in 948 rice varieties and found a Myanmar rice landrace, Haoru (International Rice Research Institute genebank acc. no. IRGC33090), with broad-spectrum resistance against the standard differential blast isolates. Here, we examined the genetic basis of Haoru’s broad-spectrum resistance by using the standard blast differential system consisting of the standard isolates and differential varieties. For genetic analysis, we used a BC1F1 population and BC1F2 lines derived from crosses of Haoru with a susceptible variety, US-2. Co-segregation analysis of the reaction pattern in the BC1F1 population against the 20 standard isolates suggested that Haoru harbors three R genes. By using bulk-segregant and linkage analysis, we mapped two of the three R genes on chromosomes 12 and 6, and designated them as Pi58(t) and Pi59(t), respectively. Pi58(t) and Pi59(t) were differentiated from other reported R genes using the standard differential system. The estimated resistance spectrum of Pi58(t) corresponded with that of Haoru, suggesting that Pi58(t) is primarily responsible for Haoru’s broad-spectrum resistance. In addition, Pi59(t) and the third gene were also proven to be new and useful genetic resources for studying and improving blast resistance in rice.     

辽宁地区水稻资源抗稻瘟病基因的检测分析

为了明确辽宁地区水稻资源中抗稻瘟病基因的分布情况及抗病效应,选取辽宁地区水稻资源176份,鉴定了抗稻瘟病基因pi21、Pi36、Pi37、Pita、Pid2、Pid3、Pi5及Pib在这些材料中的分布情况,并接种鉴定了这些材料对稻瘟病的抗性。结果表明:176份供试材料中,83份对稻瘟病表现抗病,栽培稻、杂草稻及农家种中抗病品种所占的比率分别为41.48%、1.14%及4.54%。抗稻瘟病基因pi21、Pi36和Pi37在所有参试材料中均未检测到,且分别有74份、49份、47份、52份及89份材料携带Pita、Pid2、Pid3、Pi5及Pib的抗病等位基因。抗病基因绝大部分分布在栽培种中,农家种和杂草稻中分布较少。不含有抗稻瘟病基因和只携带单个抗病基因的材料对稻瘟病的抗性均较差,而抗病基因聚合可不同程度提高材料的抗性。经检测,不含有本试验鉴定的pi21等8个已克隆抗病基因的材料共32份,其中表现抗病的占21.87%;只携带1个抗稻瘟病基因的材料为52份,表现抗病的占17.31%;携带2个抗稻瘟病基因的材料为39份,表现抗病的占69.23%,其中以携带Pita+Pi5的材料最多(14份),且均表现抗病;携带3个抗稻瘟病基因的材料为31份,表现抗病的占77.42%,以携带Pita+Pid3+Pi5的材料抗性最强;携带4个抗稻瘟病基因的水稻材料22份,表现抗病的占72.73%,携带5个抗病基因的水稻材料未检测到。     

Identification of Two Blast Resistance Genes in a Rice Variety, Digu

Blast, caused by Magnaporthe grisea is one of most serious diseases of rice worldwide. A Chinese local rice variety, Digu, with durable blast resistance, is one of the important resources for rice breeding for resistance to blast (M. grisea) in China. The objectives of the current study were to assess the identity of the resistance genes in Digu and to determine the chromosomal location by molecular marker tagging. Two susceptible varieties to blast, Lijiangxintuanheigu (LTH) and Jiangnanxiangnuo (JNXN), a number of different varieties, each containing one blast resistance gene, Pik^s, Pia, Pik, Pi‐b, Pi‐k^p, Pi‐ta², Pi‐ta, Pi‐z, Pi‐i, Pi‐k^m, Pi‐z^t, Pi‐t and Pi‐11, and the progeny populations from the crosses between Digu and each of these varieties were analysed with Chinese blast isolates. We found that the resistance of Digu to each of the two Chinese blast isolates, ZB13 and ZB15, were controlled by two single dominant genes, separately. The two genes are different from the known blast resistance genes and, therefore, designated as Pi‐d(t)1 and Pi‐d(t)2. By using bulked segregation method and molecular marker analysis in corresponding F₂ populations, Pi‐d(t)1 was located on chromosome 2 with a distance of 1.2 and 10.6 cM to restriction fragment length polymorphism (RFLP) markers G1314A and G45, respectively. And Pi‐d(t)2 was located on chromosome 6 with a distance of 3.2 and 3.4 cM to simple sequence repeat markers RM527 and RM3, respectively. We also developed a novel strategy of resistance gene analogue (RGA) assay with uneven polymerase chain reaction (PCR) to further tag the two genes and successfully identified two RGA markers, SPO01 and SPO03, which were co‐segregated toPi‐d(t)1 and Pi‐d(t)2, respectively, in their corresponding F₂ populations. These results provide essential information for further utilization of the Digu's blast resistance genes in rice disease resistance breeding and positional cloning of these genes.     

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.     

Genetic control of rice blast resistance in the durably resistant cultivar Gumei 2 against multiple isolates

To further our understanding of the genetic control of blast resistance in rice cultivar Gumei 2 and, consequently, to facilitate the utilization of this durably blast-resistant cultivar, we studied 304 recombinant inbred lines of indica rice cross Zhong 156/Gumei 2 and a linkage map comprising 181 markers. An analysis of segregation for resistance against five isolates of rice blast suggested that one gene cluster and three additional major genes that are independently inherited are responsible for the complete resistance of Gumei 2. The gene cluster was located to chromosome 6 and includes two genes mapped previously, Pi25(t), against Chinese rice blast isolate 92-183 (race ZC15) and Pi26(t) against Philippine rice blast isolate Ca89 (lineage 4), and a gene for resistance against Philippine rice blast isolate 92330-5 (lineage 17). Of the two genes conferring resistance against the Philippine isolates V86013 (lineage 15) and C923-39 (lineage 46), we identified one as Pi26(t) and mapped the other onto the distal end of chromosome 2 where Pib is located. We used three components of partial blast resistance, percentage diseased leaf area (DLA), lesion number and lesion size, all measured in the greenhouse, to measure the degree of susceptibility to isolates Ca89 and C923-39 and subsequently identified nine and eight quantitative trait loci (QTLs), respectively. Epistasis was determined to play an important role in partial resistance against Ca89. Using DLA measured on lines susceptible in a blast nursery, we detected six QTLs. While different QTLs were detected for partial resistance to Ca89 and C923-39, respectively, most were involved in the partial resistance in the field. Our results suggest that the blast resistance in Gumei 2 is controlled by multiple major genes and minor genes with epistatic effects.     

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).