Association Analysis of SP-SNPs and Avirulence Genes in Puccinia striiformis f. sp. tritici, the Wheat Stripe Rust Pathogen

Puccinia striiformis f. sp. tritici (Pst) is one of the pathogenic fungi on wheat, caused stripe rust that is a great threat for wheat production all over the world. Intensive efforts have been made to study genetics of wheat resistance to this disease, but few on avirulence of the pathogen due mainly to the nature of obligate biotrophism and the lack of systems for studying its genetics and molecular manipulations. To overcome these limitations, a natural Pst population comprising 352 isolates representative of a diverse virulence spectrum was genotyped using 97 secreted protein-single nucleotide polymorphism (SP-SNP) markers to identify candidate avirulence genes using association analysis. Among avirulence genes corresponding to 19 resistance genes, significantly associated SP-SNP markers were detected for avirulence genes AvYr1, AvYr2, AvYr6, AvYr7, AvYr8, AvYr44, AvYrExp2, AvYrSP, and AvYrTye. These results indicate that association analysis can be used to identify markers for avirulence genes. This study has laid the foundation for developing more SP-SNPs for mapping avirulence genes using segregating populations that can be generated through sexual reproduction on alternate hosts of the pathogen.

Geographic Distribution of Avirulence Genes in Rice Blast Fungusin Yunnan Province,China

Knowledge of the geographic distribution and frequency of avirulence genes will contribute to the development of strategies to effectively use rice varieties that carry various resistances genes, including combinations of varieties in mixture cropping systems. Here, we analyzed the geographic distribution and frequencies of avirulence genes in rice blast fungus using samples collected from 11 prefectures across Yunnan province, China. A total of 467 single spore isolates were assayed for pathotypes based on their reaction to 20 rice blast resistance monogenic lines. The results revealed that frequencies of avirulence genes among 10 prefectures showed insignificant difference, but frequencies of avirulenee genes in Xishuangbanna showed significant differences compared to the remaining 10 prefectures. The avirulence genes Avr-Pi9, Avr-Piz and Avr-Pizt were observed at the highest frequency in blast isolates from the 11 prefectures; their average frequency was greater than 80%. Our results imply that the composition and distribution of rice genetic diversity are more important than climate and other environment conditions for formation and maintenance of rice blast fungus genetic diversity. Using average frequencies, the avirulence genes can be categorized into 4 groups. There were significant differences of frequencies of avirulence genes among different groups, while insignificant differences observed within any group. These results will provide useful information for evaluation of resistance genes and effective management of rice blast disease.

Induction of avirulence by AVR-Pita1 in virulent U.S. field isolates of Magnaporthe oryzae’

The AVR-Pita1 gene,from the Chinese isolate O-137 of Magnaporthe oryzae,is an effector that determines the efficacy of the Pi-ta rice blast resistance gene.In the present study,the avirulence function of AVR-Pita1 was induced by transformation of field isolates(TM2,ZN19,B2 and B8)that originally were collected from the U.S.and are virulent on Pi-ta-carrying rice cultivars.The presence of AVR-Pita1 from O-137 in independent transformants was detected by PCR using AVR-Pita1 specific primers and verified by DNA sequencing and Southern blot analysis using the AVR-Pita1 coding region as a probe.The results of pathogenicity assays showed that the AVR-Pita1-transformed isolates were not able to infect rice cultivars Katy and Drew carrying Pi-ta.Control isolates that were transformed with inserts lacking the AVR-Pita1gene remained virulent.Our findings demonstrate that AVR-Pita1 can be used to induce novel gene-specific blast resistance in nature.

Comments on “Determination of heterozygosity for avirulence/virulence loci through sexual hybridization of Puccinia striiformis f. sp. tritici”

Over the past decades Puccinia striiformis f.sp.tritici（Pst）has developed into one of the most,if not the most important fungal pathogen in wheat production worldwide.In China,Pst has caused numerous epidemics with partially devastating yield losses[1].The occurrence of the'warrior'race in Europe in 2011 also caused significant problems[2].Pst,like other obligate biotrophs,is characterized by a high degree of genetic variability,especially with respect

Genetic and physical mapping of AvrPi7, a novel avirulence gene of Magnaporthe oryzae using physical position-ready markers

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most devastating crop diseases worldwide. The avirulence gene corresponding to rice blast resistance gene Pi7 in field isolate CHL346 was inherited as a single gene, designated AvrPi7, in a segregating population consisting of 189 ascospore progenies derived from a cross between field isolates CHL346 and CHL42. In order to determine the chromosomal location of the AvrPi7 locus, a total of 121 simple sequence repeat (SSR) markers were developed based on the whole-genome sequence of reference isolate 70-15 of M. oryzae. Linkage analysis of the locus with these SSR markers showed that eight SSR markers on chromosome 1 were linked to the locus, among which the closest flanking markers MS1-9 and MS1-15 were 3.2 and 16.4 cM from the locus, respectively. For fine mapping, additional PCR-based makers including eight SSR markers and three candidate avirulence gene (CAG) markers were developed in the region flanking both markers. The AvrPi7 locus was genetically delimited within a 1.6-cM region flanked by markers MS1-21 and MS1-22, and co-segregated with the marker CAG2. To construct a physical map of the AvrPi7 locus, molecular markers linked to the Avr gene were mapped on the supercontigs of the ref-erence isolate 70-15 through bioinformation analysis (BIA). Consequently, the AvrPi7 locus was delim-ited to a 75-kb interval flanked by markers MS1-21 and MS1-22 based on the reference sequence. Merodiploids observed in this study are also discussed.

2021年广西稻瘟病菌致病性分化及其无毒基因分析

【目的】探究广西稻瘟病菌的致病力、优势种群和优势生理小种,分析其无毒基因,了解其生理小种及无毒基因组成与分布情况,为水稻抗性育种和品种推广提供参考。【方法】使用7个我国统一鉴别品种和26份已知抗病基因的近等基因系,采用室内苗期人工喷雾接种方法对2021年从广西南部(桂南)、中部(桂中)、北部(桂北)和高寒山区4个不同生态稻作区分离得到的128株稻瘟病单孢菌株进行致病性测定。【结果】60.16%供试稻瘟病菌菌株表现出强致病力,128株稻瘟病菌株被划分为7个种群26个生理小种,ZB群为优势种群,出现频率为69.53%,优势生理小种为ZB_(13)和ZB_9,出现频率分别为25.00%、14.84%。供试菌株对26个抗病基因的毒力频率为28.12%~100.00%,其中,供试病菌对Pik、Pikm、Pi1、Pi9基因的毒力频率较低,分别为28.12%、28.91%、35.94%、37.50%。供试的广西稻瘟病菌含有与测试抗病基因相对应的无毒基因,其中15个无毒基因在桂南、桂中、桂北和高寒山区4个不同稻作区均有分布,无毒基因Avr-Pia(1)、Avr-Pia(2)、Avr-Pii、Avr-Pik^(s)、Avr-Pib、Avr-Pit、Avr-Pish(2)、Avr-Pi3(t)、Avr-Pi5(t)、Avr-Pi12(t)、Avr-Pi19(t)出现频率均低于20.00%。携带有5、6、7、8、10个无毒基因组合的菌株较多,其在供试菌株中占比分别为19.53%、11.72%、11.72%、15.63%、10.16%。【结论】2021年广西稻瘟病菌致病力强,优势种群为ZB群,优势生理小种为ZB_(13)和ZB_9,无毒基因Avr-Pia(1)、Avr-Pia(2)、Avr-Pii、Avr-Pik^(s)、Avr-Pib、Avr-Pit、Avr-Pish(2)、Avr-Pi3(t)、Avr-Pi5(t)、Avr-Pi12(t)和Avr-Pi19(t)出现频率较低,在水稻抗病育种与品种布局中,与之对应的抗病基因应慎用。

The arms race between Magnaporthe oryzae and rice: Diversity and interaction of Avr and R genes

Rice blast disease, caused by Magnaporthe oryzae, threatens global food security. The rice blast pathosystem is a longstanding model system for understanding plant-microbe interactions. In order to elucidate the coevolution of the host and pathogen, and provide the appropriate methods for preventing or controlling rice blast disease, researchers have focused on the evolution of virulence factors and resistance genes. Thus far, more than 30 rice blast resistance（R） genes and 12 avirulence（Avr） genes have been cloned. This review summarizes the cloned rice blast R genes, cloned Avr genes of M. oryzae and the interaction between them. This discussion also considers some of the major unanswered questions concerning this pathosystem and the opportunities for future investigations.

Analysis of the diversity and function of the alleles of the rice blast resistance genes Piz-t, Pita and Pik in 24 rice cultivars

Understanding the sequence diversity of rice blast resistance genes is important for breeding new resistant rice cultivars against the rice blast fungusMagnaporthe oryzae. In this study, we selected 24 rice cultivars with different genetic back-grounds to study the alelic diversity of rice blast resistance genesPiz-t, Pitaand Pik. For Piz-t, a total of 17 alelic types were found within the 24 cultivars. Blast inoculations showed that most of the mutations can affect the function of the resistance gene. For Pita, except for the difference at the 918th amino acid, a majority of the 21 mutations were detected among the cultivars. Inoculations with blast isolates carryingAvr-Pita revealed that cultivars with mutations in other sites except for the 918th amino acid did not affect the function of thePita gene. ForPik, a total of six alelic types were found within the 24 cultivars, but ifve of them lost the function of the resistance gene. In addition, we found thatPiz-t, Pita and Pik were expressed constitutively in the 24 rice cultivars and the expression level was not related to resistance. Our results have provided the sequence diversity information of the resistance genesPiz-t, Pita and Pik among the popular rice cultivars grown in the northeast region of China. Keywords：resistance gene, avirulence gene, aleles, function, genetic evolution zae（M. oryzae）, is one of the most destructive diseases in rice production worldwide. Over the years, comprehensive studies on rice blast resistance have been conducted （Silue et al. 1992）. The resistance in newly cultivated rice cultivars to M. oryzae can be lost quickly due to the high level of instability in the genome of the fungus （Bonmanet al. 1992）. Previous studies show that cultivars with durable and broad-spectrum resistance againstM. oryzae carry multiple major resistance （R） and minor resistance genes （Liuet al. 2014）. An effective way to control rice blast disease is, therefore, to breed rice cultivars with multiple R and QTL genes. To date, over 83 rice blast R genes have been identiifed, and are distributed on 11 rice chromosomes except Received 22 May, 2015 Accepted 26 October, 2015 WANG Yan, E-mail： 8806wy@163.com; Correspondence LIU Zhi-heng, Tel： ＋86-24-23738857, E-mail： lzhh1954@163.com; ZHENG Wen-jing, Tel： ＋86-24-31021081, E-mail： zwj27@126. com ＊These authors contributed equaly to this study. ？ 2016, CAAS. Al rights reserved. Published by Elsevier Ltd. doi： 10.1016/S2095-3119（15）61207-2 1. Introduction Rice blast disease, caused by the fungusMagnaporthe ory-

黑龙江省和海南省稻瘟病菌中AVR-Pita家族的分布及变异分析

【目的】通过检测黑龙江省和海南省不同稻瘟病菌(Magnaporthe oryzae)菌株群体中AVR-Pita家族的分布情况和变异特征,了解其变异类型的致病表型,为区域内抗病种质的筛选与选育提供参考。【方法】通过参考NCBI中公布的AVR-Pita家族序列分别对启动子区和CDS区设计特异性引物共6对,对2020年采自黑龙江省和海南省不同地区的397个稻瘟病菌单孢菌株提取DNA,进行PCR扩增。通过电泳检测结果,分别选取囊括不同地区的代表菌株对扩增片段进行测序。测序结果与NCBI中相应的碱基与氨基酸序列进行比较分析,并利用水稻抗性单基因系,对不同变异类型的稻瘟病菌菌株进行无毒功能验证。【结果】在PCR电泳检测结果中,黑龙江省稻瘟病菌菌株携带所有待测基因,分布广泛且检出频率较高;而海南省稻瘟病菌菌株中仅携带AVR-Pita1和AVR-Pita2,并以低频率集中存在。无毒基因组成分析结果显示,黑龙江稻区的稻瘟病菌菌株复杂多样,携带的基因型种类较海南菌株丰富。PCR产物测序检测结果显示,AVR-Pita家族以点突变、插入和缺失为主要变异类型划分为19种,且不同稻瘟病菌群体来源的菌株,其变异类型具有特异性。AVR-Pita1检测出10种变异类型,其中AVR-Pita1-(1-5)为黑龙江省稻瘟病菌菌株独有的变异类型,AVR-Pita1-(6-10)为海南省稻瘟病菌菌株独有。对这10种变异类型经功能验证后发现,无毒功能均已丧失。AVR-Pita2检测出8种变异类型,其中AVR-Pita2-(1-4)为黑龙江省稻瘟病菌菌株独有,AVR-Pita2-(5-8)为海南省稻瘟病菌菌株独有。经功能验证后发现,无毒功能均已丧失。AVR-Pita3在黑龙江省仅检测出1种变异类型AVR-Pita3-1。【结论】黑龙江和海南稻瘟病菌群体中AVR-Pita家族均为突变后的等位基因型存在,经致病性鉴定后,检测出的所有突变类型均不能被相对应抗性基因Pi-ta和Pi-ta2所识别,表现为感病。因此,抗性基因Pi-ta和Pi-ta2在黑龙江省和海南省对稻瘟病的抗病育种与利用过程中可聚合其他抗性基因应用来保证品种抗病性。同时,不同地理来源的稻瘟病菌菌株群体中AVR-Pita家族的分布和变异类型具有特异性。

黑龙江省和海南省PWL基因家族在稻瘟病菌中的分布及变异

【目的】了解不同稻瘟病菌(Magnaporthe oryzae)菌株群体中PWL基因家族的分布和变异特征,为研究不同稻瘟病菌群体的遗传多样性及特异性提供依据。【方法】通过参考NCBI中公布的无毒基因序列分别对PWL基因家族的启动子区和CDS区设计特异性引物共8对,对2020年采自黑龙江省和海南省不同地区的397个稻瘟病菌单孢分离菌株提取DNA,进行无毒基因的PCR扩增并通过琼脂糖凝胶电泳检测,从检测结果中分别选取囊括不同地区的代表菌株对扩增片段进行测序分析。测序结果与NCBI中相应无毒基因启动子区和CDS区的碱基与氨基酸序列进行比较分析。【结果】在PCR电泳检测结果中,PWL1在所有菌株中均未检测出;PWL2、PWL3和PWL4在黑龙江省和海南省中均扩增出特异性片段,说明这3种基因在两省中均有分布并分别存在不同的分布频率与变异类型。其中PWL2在黑龙江省和海南省中分布频率最高,分别为98.14%和100%;PWL3和PWL4在两省中的分布频率具有显著差异,这2种基因在黑龙江菌株中频率分别为89.30%和82.79%,而在海南菌株中均为5.49%。通过对无毒基因组合进行分析,结果显示,组合类型可分为6种,分别为PWL(f)、PWL2、PWL3、PWL2+PWL3、PWL2+PWL4、PWL2+PWL3+PWL4。其中黑龙江菌株含有所有组合类型,海南菌株仅含有2种,说明黑龙江菌株与海南菌株相比无毒基因型种类较为丰富。通过对PWL基因家族的PCR产物测序发现,PWL基因家族在启动子区和CDS区变异位点丰富,以点突变和缺失为主要变异类型划分为9种,且不同群体来源菌株的变异类型具有特异性和一致性。其中PWL2检测出5种变异类型PWL2-(1,2,3,4,5),碱基序列变化导致氨基酸序列发生错义突变;PWL3和PWL4分别检测出2种变异类型,分别为PWL3-(1,2)和PWL4-(1,2),均发生移码突变使后面氨基酸发生变化。【结论】不同群体来源的稻瘟病菌菌株中PWL基因家族的分布和变异类型具有地域差异性,且变异位点丰富。

云南元江普通野生稻稻瘟病菌无毒基因鉴定与分析

为了明确分离自云南元江普通野生稻4个自然居群稻瘟病菌无毒基因的组成。以分离自元江普通野生稻上的52个单孢菌株和24个以丽江新团黑谷为背景的稻瘟病抗性单基因系为试材,用苗期喷雾接种的方法,对供试菌株进行无毒基因构成鉴定。结果显示,52个稻瘟病菌菌株组成的群体中,中等、弱和无致病力的菌株占比分别为34.69%、65.39%和1.92%,以弱致病力菌株占优势;52个菌株仅对含有Pi19、Pii、Pi3、Pib和Pish等5个抗性单基因系具有强或中等毒性,对持有其余19个抗性基因的单基因系具有弱致病性或无致病性;根据供试菌株对24个水稻抗性单基因系的致病性分析,无毒基因的组成可分为10种类型。研究结论认为云南元江普通野生稻稻瘟病菌对24个水稻单基因系的致病性普遍偏弱,但仍存在致病性分化的现象。

福建省稻瘟病菌田间种群无毒基因的变异检测

稻瘟病严重威胁福建省水稻生产。稻瘟病菌田间种群无毒基因的变异监测是实现优质抗稻瘟病水稻品种合理布局和轮换、有效预防因抗性丧失导致的稻瘟病爆发成灾的重要措施。本研究以福建省3个水稻主产区采集并分离的113个稻瘟病菌单孢菌株为材料,检测其无毒基因的变异情况。首先,利用24个已知抗性的水稻单基因系对它们进行致病性测定,分析致病频率、毒力频率、无毒基因的存在频率和无毒基因的组合频率。结果显示,福建省建阳、宁化和上杭3个水稻主产区的稻瘟病田间菌株对24个水稻单基因系的致病频率分别为12.50%~95.83%、29.17%~100%和4.55%~86.36%。3个水稻主产区菌株均对Pik^(s)、Pib、Pi3和Pi12表现为强毒性,而建阳和宁化菌株还对其他12个抗性基因表现为强毒力(毒力频率≥70%),包括Pia、Pii、Piz、Pita、Pit、Pis^(h)、Pi5、Pi7、Pi19、Pi20、Pita^(2)和Pi11。其次,本研究还设计了8个无毒基因Avr-Pita、Avr-Pib、Avr-Pik、Avr-Piz-t、Avr-Pii、Avr-Pi9、Avr-Pi54和Avr-Co39的特异性引物,对单孢菌株进行基因型鉴定。结果显示,所有菌株均未检测到Avr-Co39,表明这些稻瘟菌株中均不携带该无毒基因。此外,Avr-Pib的出现频率也很小,仅为37.17%,但是该比例仍远超致病性测定中推测的Avr-Pib所占比例,表明检测到的Avr-Pib中很大一部分存在突变。为此,对Avr-Pib基因部分扩增产物进行测序,发现这些菌株的Avr-Pib在启动子区发生了缺失、插入和替换等突变。此外,部分菌株的Avr-Pik或Avr-Piz-t也存在启动子单核苷酸变异,而上杭菌株Avr-Pi9和宁化菌株Avr-Piz-t则均存在编码区的突变。这些位点的突变均可能导致无毒基因功能的缺失。研究还表明,携带Pi1、Piz5、Pi9和Pik、Pik^(h)、Pik^(m)位点的水稻品种仍适合在福建地区种植。

大豆膜系统cDNA文库的构建及大豆疫霉效应子PsAvr3a互作蛋白的筛选

植物质膜是响应侵染的第一道屏障,其受体蛋白是感知病原菌入侵的雷达,是植物免疫系统的重要组成部分。大豆疫霉(Phytophthora sojae)通过分泌效应子干扰寄主免疫反应以促进自身侵染定殖。构建大豆膜系统酵母双杂交cDNA文库,为进一步探究大豆疫霉效应子与寄主膜蛋白靶标互作机制奠定基础。以大豆疫霉无毒效应子PsAvr3a为诱饵蛋白筛选文库,初步获得198个候选寄主靶标。酵母一对一验证试验表明,有5个候选靶标蛋白与PsAvr3a互作,包括酪氨酸分解酶、囊泡转运蛋白、乙烯合成调控酶、细胞代谢结合蛋白、抗逆的渗透蛋白等。通过双分子荧光互补试验(bimolecular fluorescence complementation, BIFC)与荧光素酶互补试验(luciferase complementation assay, LCA)验证得出GmACO、GmSec61与PsAvr3a均存在互作。确定2个寄主大豆膜蛋白乙烯前体ACC氧化酶GmACO、三聚体易位子GmSec61是大豆疫霉无毒效应子PsAvr3a的互作靶标。

黑龙江省稻瘟病菌无毒基因分析及抗病种质资源筛选

采用7个稻瘟病菌无毒基因的特异性引物,对177个来自黑龙江省的稻瘟病菌单孢菌株进行PCR检测,结果表明7个无毒基因在黑龙江省不同地区均有分布,但出现频率不同,其中频率最高的为ACE1,达到61.6%,最低的为Avr1-co39,只有31.6%;来源于不同水稻品种的菌株无毒基因组成不同。采用国际水稻研究所20个已知抗性基因的单基因系对48个黑龙江省稻瘟病菌进行毒力分析,与PCR检测无毒基因结果一致。同时,对20个抗病单基因系采用多菌株人工接种发现Pi-9在黑龙江6个地区对稻瘟病菌的抗谱为80%~100%,在育种中具有较高利用价值。

稻瘟菌对水稻品种梅雨明的无毒性的遗传分析和分子标记

将交配型为 a、并对水稻品种梅雨明不致病的稻瘟菌菌株 S152 2与对该品种致病的、交配型为A的稻瘟菌菌株 P131和 S159杂交 ,分别获得了 2 4和 56个单子囊孢子后代。致病性测定结果表明 :这些后代菌株在梅雨明上的致病个体与不致病个体的比例近等于 1∶ 1。由此推测 :稻瘟菌对水稻品种梅雨明的无毒性是由 1个基因控制的。进一步通过 RAPD比较了致病后代与不致病后代的 DNA多态型 ,获得了 1个与该无毒基因连锁的、长度为 10 0 0 bp的 RAPD标记 ,两者间的遗传距离为3.7c M。

番茄青枯雷尔氏菌的强致病力与无致病力菌株生长竞争关系的研究

在单独和混合培养条件下,研究了番茄青枯雷尔氏菌的强致病力(RV)与无致病力菌株(RA)的生长能力的差异.单独培养时,24h前无致病力菌株的菌体生长量超过强致病力菌株,24h后强致病力菌株菌体生长量超过无致病力菌株,12h时无致病力菌株的生长速率是强致病力菌株的5.6倍,60h时无致病力菌株的菌量仅为强致病力菌株的0.37倍.混合培养时,混合比例RV RA<1时,两种菌株都呈正增长,但后者比前者增长快,当混合比例RV RA≤1时,强致病力菌呈负增长(-119.57%～-33.57%),无致病力菌呈正增长(84.50%～285.50%).随着RV RA值升高,增长率呈升高的趋势.上述结论表明:无致病菌株在一定的时间内具有高于强致病力菌的生长能力,当与强致病力菌混合时,能较快成为优势种群,这可能是人工大量施用无致病力菌株时,能有效地保护番茄植株在生长初期免受青枯病的危害,在短期内起到一定的防治效果的原因所在.但随着时间的延长,强致病力菌株又重新成为优势种群,这可能是导致无致病力菌防效下降的原因之一.

辽宁省稻瘟病菌无毒基因型鉴定及分析

【目的】鉴定稻瘟病菌(Magnaporthe oryzae)的无毒基因型,了解无毒基因在不同地区流行菌株中的分布情况,为品种布局提供参考。【方法】根据已经克隆且与稻瘟病菌致病性相关的6个无毒基因序列设计引物,选取辽宁省稻瘟病常发区的26株稻瘟病菌单孢菌株,提取各菌株DNA样本作为模板,进行PCR扩增。通过琼脂糖凝胶电泳及PCR产物测序,对6个无毒基因PCR产物进行碱基和氨基酸序列的分析比较。对琼脂糖凝胶电泳未出条带的,设计不同引物进行验证性试验。【结果】在PCR产物电泳检测中,Avr1-CO39、Avr-pia和Avr-pii没有产物条带,对这3个无毒基因设计验证引物,其PCR产物电泳检测仍没有条带出现,其结果证明辽宁省各水稻主产区流行稻瘟病菌中多不携带Avr1-CO39、Avr-pia和Avr-pii;对于其他3个无毒基因AvrPiz-t、Avr-pik和Avr-pita则有特异性扩增产物,说明这3个无毒基因在各稻区稻瘟病菌中以不同突变类型及不同频率出现。其中,与Pi2、Pi9和Piz-t对应的AvrPiz-t,分别在22个菌株的中被检测到,且有21个菌株的序列与其序列一致,说明该基因遗传相对稳定,也间接证明携带Pi2、Pi9和Piz-t的水稻品种在辽宁地区的广谱抗性;与基因组序列不同的16号菌株,在DNA序列192 bp处发生一个单碱基C的缺失,从而导致移码突变,且碱基突变导致氨基酸序列至72位氨基酸时提前终止,而使该基因编码的蛋白质失去无毒基因的功能。与Pik、Pik-p、Pik-m和pik-s对应的Avr-pik,电泳检测结果表明各菌株均有特异性条带出现,经测序验证等位基因序列分为4种类型(B、D、F、G),其中12个菌株携带可为Pik或其等位基因Pik-m和pik-p所识别的D类型;9个菌株携带B类型,该等位基因曾被报道,但是否具有无毒基因的功能仍未验证;另有2个菌株携带F类型等位基因,该基因为首次发现,并分别出现在丹东和盘锦地区。其特点在于与D类型基因间存在143(A/G)的碱基差异,氨基酸序列翻译结果显示其为错义突变,即48(G/D);而其余3个菌株携带G类型等位基因,该基因亦属首次发现,且仅出现在抚顺新宾地区,碱基序列与D类型存在168(G/A)的差异,导致翻译提前终止,基因功能丧失。对于Avr-pita,26个菌株特异性扩增产物一致,但测序检测到5种等位基因类型,且均与Avr-pita有差异,碱基序列的变化多导致错义突变。这5种等位基因的氨基酸序列之间差异由3个氨基酸位点的差异所致,分别为83(D/N)、192(Y/C)和207(K/R),3处突变均在基因结构域范围内,几种等位基因均已见报道。【结论】辽宁稻区流行稻瘟病菌中Avr-pik、AvrPiz-t和Avr-pita分布较为广泛,选育及推广携带相应抗病基因的水稻品种可减轻稻瘟病的危害。

一个与稻瘟病菌无毒基因AVR-Pik^m连锁的SCAR标记的分离

本研究将以前在稻瘟病菌菌株S1522获得的与决定对水稻品种梅雨明无毒性的基因(AVR-Pik^m)相连锁的1个RAPD标记OPO12_(1000)进行了克隆和鉴定。核苷酸序列测定与分析结果表明:OPO12_(1000)的大小为946个碱基,不含有与已报道的稻瘟病菌Mg-SINE、Fosburry、Magyy、Grasshopper、Pot2以及Pot3等同源的重复序列。根据OPO12_(1000)的核苷酸序列,设计了1对24个核苷酸的特异引物,对无毒表型亲本S1522和毒性表型亲本S159、无毒表型群体基因池、毒性表型基因池以及有性杂交后代108个菌株进行了PCR扩增,所有无毒表型的菌株均能特异性地扩增出1条与OPO12_(1000)大小相同的DNA条带,而毒性表型的菌株除5个重组个体外,均不能扩增出这条特异带。此结果表明,与稻瘟病菌无毒基因AVR-Pik^m连锁的RAPD标记OPO12_(1000)被成功地转化为SCAR标记,为进一步通过染色体步移克隆该无毒基因奠定了基础。

转无毒基因马铃薯抗晚疫病的研究 (英文)

晚疫病菌 (Phytophthorainfestans)引起的马铃薯晚疫病是危害全球马铃薯生产的严重病害。通过基因工程方法把外源基因导入植物体内以增强抗病性被证明是一条行之有效的途径。应用植物基因工程技术将具有能激活植物自身防御系统的无毒基因与适合于植物背景、非专一性的病原物诱导启动子组合成嵌合基因构建到植物表达载体中。通过农杆菌或基因枪的介导转化植物 ,可筛选出高效广谱的抗真菌和细菌病害的转基因植株。本研究从病原细菌PseudomonassyringaePV .tomato中获得的无毒基因avrD (0·93kb)和从病原真菌Phytophthoraparasitica中获得的无毒基因Elicitin(0 2 94kb)分别与非专一性病原物诱导启动子Pill和BG组成含 2个嵌合基因 (Pill -avrD ,BG -Elicitin)的植物表达载体pYH144和pYHEt。通过农杆菌LBA4 4 0 4介导转化马铃薯 ,其中用pYH144载体转化 2个品种 (克新 1号 ,2号 ) ,用pYHEt载体转化 3个品种 (Desiree ,克新 2号 ,4号 ) ,通过组织培养分别获得潮霉素 (HygromycinB)标记的转基因马铃薯试管苗。将转基因试管苗扩繁 ,应用马铃薯脱毒微型种薯生产技术获得转无毒基因微型薯 ,在温室 (15～ 2 5℃和湿度高 )条件下 ,观察转无毒基因马铃薯植株中对晚疫病菌自然感染的抗性。 1998年和 1999年 (每年的 3-

利用RAPD分子标记定位2个水稻稻瘟病菌非致病性基因

选择日本鉴别品种新 2号和K5 9,对源于云南省的水稻稻瘟病菌可育性菌株CHL12 4(MAT1 1)和CHL12 5 (MAT1 2 )及其杂交后代 72个子囊菌株的非致病性 (无毒性 )进行了遗传分析。结果表明 ,菌株CHL12 4对新 2号 (含有效抗性基因Pish)和K5 9(Pit)的非致病性分别由非致病性基因Avr2 Pish和Avr2 Pit控制。通过对这 2个基因控制的非致病性反应进行列联卡方测验 ,显示这 2个基因连锁在一起 ,重组率为 2 6 .8%。进一步利用分离群体分析法 ,对这 2个基因进行了RAPD (随机扩增多态性DNA)分析。结果发现 ,2个RAPD标记OPW 0 6 64 5和OPW 1364 5与非致病性基因Avr2 Pish连锁 ,它们的重组率分别为 2 7.8%和 2 6 .5 %;另 1个RAPD标记OPR11517则与非致病性基因Avr2 Pit连锁 ,重组率为 2 3.5 %。