Genetic and physical mapping of the partial resistance gene, pi34, to blast in rice

ABSTRACT Partial resistance to rice blast in the Oryza sativa japonica group cv. Chubu 32 is controlled by Pi34, a major quantitative trait locus (QTL) on chromosome 11, and several uncharacterized QTLs. The objectives of the study were (i) high-resolution genetic and physical mapping of Pi34 and (ii) identification of new QTL imparting resistance to rice blast. Chubu 32 was crossed with a susceptible chromosomal segment substitution line (CSSL) of cv. Koshihikari. From 4,012 of segregating individuals, 213 recombinants in the Pi34 region were screened by using polymerase chain reaction-based markers and tested resistance in the field and greenhouse. The Pi34 locus is located in the 54.1-kb region on the genomic sequence of cv. Nipponbare. We constructed a bacterial artificial chromosome (BAC) library of Chubu 32, selected the clone containing Pi34, and sequenced it. The Pi34 locus consequently was located on an interval of 65.3 kb containing 10 predicted open reading frames (ORFs). Two of these ORFs were predicted only in Chubu 32 and encoded transposable elements. The other eight ORFs were found in both Chubu 32 and Nipponbare and one of them, which encoded an unknown protein, showed significantly different amino acid sequences between two cultivars. The new QTL, Piq6(t), was detected on the short arm of chromosome 6 and the genetic distance of flanking markers was 16.9 centimorgans in Nipponbare. Pi34 and Piq6(t) acted additively on resistance to rice blast but the effect of Piq6(t) was relatively small compared with Pi34.     

Mapping quantitative trait Loci for resistance to rice blast

Quantitative trait loci (QTLs) conferring resistance to rice blast, caused by Magnaporthe oryzae, have been under-explored. In the present study, composite interval mapping was used to identify the QTLs that condition resistance to the 6 out of the 12 common races (IB1, IB45, IB49, IB54, IC17, and ID1) of M. oryzae using a recombinant inbred line (RIL) population derived from a cross of the moderately susceptible japonica cultivar Lemont with the moderately resistant indica cultivar Jasmine 85. Disease reactions of 227 F(7) RILs were determined using a category scale of ratings from 0, representing the most resistant, to 5, representing the most susceptible. A total of nine QTLs responsive to different degrees of phenotypic variation ranging from 5.17 to 26.53% were mapped on chromosomes 3, 8, 9, 11, and 12: qBLAST3 at 1.9 centimorgans (cM) to simple sequence repeat (SSR) marker RM282 on chromosome 3 to IB45 accounting for 5.17%; qBLAST8.1 co-segregated with SSR marker RM1148 to IB49 accounting for 6.69%, qBLAST8.2 at 0.1 cM to SSR marker RM72 to IC17 on chromosome 8 accounting for 7.22%; qBLAST9.1 at 0.1 cM to SSR marker RM257 to IB54, qBLAST9.2 at 2.1 cM to SSR marker RM108, and qBLAST9.3 at 0.1 cM to SSR marker RM215 to IC17 on chromosome 9 accounting for 4.64, 7.62, and 4.49%; qBLAST11 at 2.2 cM to SSR marker RM244 to IB45 and IB54 on chromosome 11 accounting for 26.53 and 19.60%; qBLAST12.1 at 0.3 cM to SSR marker OSM89 to IB1 on chromosome 12 accounting for 5.44%; and qBLAST12.2 at 0.3 and 0.1 cM to SSR marker OSM89 to IB49 and ID1 on chromosome 12 accounting for 9.7 and 10.18% of phenotypic variation, respectively. This study demonstrates the usefulness of tagging blast QTLs using physiological races by composite interval mapping.     

水稻空间诱变育种抗稻瘟病研究

粤香占和青华占2个水稻品种的种子经过高空气球搭载后,选育出株型好、分蘖力强,产量提高的空间诱变高代(6代以上)品系.为了解空间诱变品系对稻瘟病的抗性变异情况,采用36个广东省稻瘟病菌不同致病型的代表菌株分别对粤香占和青华占及其空间诱变品系进行抗谱测定.研究结果表明:①空间诱变能使水稻品种产生稻瘟病抗性变异,有的诱变品系比原种抗病,有的则比原种感病.②空间诱变产生的抗病性变异比较复杂,不同品种的抗病性变异不一样.③空间诱变引起的稻瘟病抗性变异有可能是由多个抗病基因引起的.④空间诱变可以获得抗稻瘟病的特异种质.粤香占是感病品种,本研究测定的抗谱为25%,从其空间诱变品系中,获得高抗稻瘟病的2个品系YX-8和YX-9,抗谱达到94.4%.空间诱变抗病品系的穗颈瘟鉴定试验正在进行中.     

Transgenic rice plants that over-express the mannose-binding rice lectin have enhanced resistance to rice blast

Mannose-binding rice lectin (MRL), which is almost identical to the salt-induced protein SalT, binds to mannose and glucose residues. Expression of the MRL gene in response to infection with Magnaporthe oryzae, the rice blast fungus, was stronger in the incompatible interaction than in the compatible. Transgenic rice plants that constitutively over-expressed MRL strongly suppressed the growth of invasive hyphae of the fungus on leaf sheaths and the development of typical susceptible-type lesions on leaf blades, but did not affect penetration by the fungus in comparison with the wild-type. On a polycarbonate plate, purified recombinant MRL inhibited conidial attachment and appressorium formation but not conidial germination. These results suggest that MRL may play an essential role in disease resistance by suppressing development of M. oryzae in situ.     

三种水稻穗瘟抗性鉴定方法的比较

稻瘟病是由稻瘟病菌Magnaporthe oryzcae引起的水稻重要病害[1].在生产上准确地评估水稻品种对稻瘟病的抗性,特别是对穗瘟的抗性,对品种的选用、合理布局及病害预警具有重要的意义.孙国昌等[2]对稻瘟病的离体接种技术进行了研究,包括注射、涂抹、棉花球包穗和喷雾四种方法,但由于各自的某些缺陷,没有得到广泛的应用.     

3个水稻持久抗瘟性品种的抗性遗传初步研究

用广谱感瘟籼型品种Ｂ４０分别与３个持久抗瘟性籼型品种Ｔｅｔｅｐ、湘资３１５０和谷梅２号杂交。３个组合的Ｆ_１和Ｆ_２及双亲在苗期用Ｚｈ-２-１菌株喷雾接种。结果表明,Ｔｅｔｅｐ、谷梅２号和湘资３１５０均由２对显性基因控制对试验菌株的抗性,其中前２个品种的基因间表现重叠效应,后１个品种的基因间表现抑制效应。     

Pi34-AVRPi34: a new gene-for-gene interaction for partial resistance in rice to blast caused by Magnaporthe grisea

The japonica rice (Oryza sativa) cultivar Chubu 32 has a high level of partial resistance to blast, which is mainly controlled by a dominant resistance gene located on chromosome 11. The partial resistance to the rice blast fungus (Magnaporthe grisea) in Chubu 32 has isolate specificity; isolate IBOS8-1-1 is more aggressive on Chubu 32 than are other isolates. We hypothesized that the gene-for-gene relationship fits this case of a partial resistance gene in Chubu 32 against the avirulence gene in the pathogen. The partial resistance gene in Chubu 32 was mapped between DNA markers C1172 (and three other co-segregated markers) and E2021 and was designated Pi34. In the 32 F₃ lines from the cross between a chromosome segment substitution line (Pi34⁻) from Koshihikari/Kasalath and Chubu 32, the lines with high levels of partial resistance to the M. grisea isolate Y93-245c-2 corresponded to the presence of Pi34 estimated by graphic genotyping. This indicated that Pi34 has partial resistance to isolate Y93-245c-2 in compatible interactions. The 69 blast isolates from the F₁ progeny produced by the cross between Y93-245c-2 and IBOS8-1-1 were tested for aggressiveness on Chubu 32 and rice cultivar Koshihikari (Pi34⁻). The progeny segregated at a 1 : 1 ratio for strong to weak aggressiveness on Chubu 32. The results suggested that Y93-245c-2 has one gene encoding avirulence to Pi34 (AVRPi34), and IBOS8-1-1 is extremely aggressive on Chubu 32 because of the absence of AVRPi34. This is the first report of a gene-for-gene relationship between a fungal disease resistance gene associated with severity of disease and pathogen aggressiveness.     

Ultrastructural and cytochemical aspects of silicon-mediated rice blast resistance

ABSTRACT Although exogenous application of silicon (Si) confers efficient control of rice blast, the probable hypothesis underlying this phenomenon has been confined to that of a mechanical barrier resulting from Si polymerization in planta. However, in this study, we provide the first cytological evidence that Si-mediated resistance to Magnaporthe grisea in rice correlates with specific leaf cell reaction that interfered with the development of the fungus. Accumulation of an amorphous material that stained densely with toluidine blue and reacted positively to osmium tetroxide was a typical feature of cell reaction to infection by M. grisea in samples from Si+ plants. As a result, the extent of fungal colonization was markedly reduced in samples from Si+ plants. In samples from Si- plants, M. grisea grew actively and colonized all leaf tissues. Cytochemi-cal labeling of chitin revealed no difference in the pattern of chitin localization over fungal cell walls of either Si+ or Si- plants at 96 h after inoculation, indicating limited production of chitinases by the rice plant as a mechanism of defense response. On the other hand, the occurrence of empty fungal hyphae, surrounded or trapped in amorphous material, in samples from Si+ plants suggests that phenolic-like compounds or phytoalexins played a primary role in rice defense response against infection by M. grisea. This finding brings new insights into the complex role played by Si in the nature of rice blast resistance.     

OsDCL1基因沉默突变体诱导稻瘟病抗性的转录组分析

稻瘟病是严重危害水稻生长的真菌病害,每年给水稻生产带来重大经济损失。与野生型日本晴NPB相比,OsDCL1基因沉默突变体增强了水稻对稻瘟菌的广谱抗病性。为了解水稻OsDCL1-RNAi突变体的抗病机制,我们分析和比较了它和野生型日本晴NPB在稻瘟菌侵染前后转录组水平的变化。响应稻瘟菌侵染的7 129个差异表达基因(DEGs)中,NPB和OsDCL1-RNAi突变体分别有5 382和5 180个基因表达发生了变化,参与生物胁迫反应、信号通路、蛋白代谢和RNA调控4个通路的基因明显被富集。以野生型未受稻瘟菌侵染的基因表达为对照,在OsDCL1-RNAi突变体中共发现1 318个DEGs,且超过70%是上调的,其中很多基因参与了生物胁迫反应(26.9%)和信号通路(11%),上调表达基因中与生物胁迫反应相关的主要为PR基因和细胞壁相关基因。另外,还发现10个茉莉酸相关基因和11个水杨酸相关基因分别在OsDCL1-RNAi突变体中显著下调和上调表达。在OsDCL1-RNAi突变体中还发现WRKY和MYB等一些转录因子家族以及部分受体类激酶被诱导表达。用qRT-PCR方法验证部分差异表达的基因,其结果与DEGs基本一致。OsDCL1-RNAi突变体中转录组变化的分析,为深入了解该突变体抗瘟性增强的机制奠定了基础。     

Silicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrier

A series of experiments were performed to study the effects of silicon (Si) on rice blast development, H₂O₂ accumulation and lipid peroxidation in a controlled rice—Magnaporthe grisea pathosystem. Rice plants supplied with Si as a single dose immediately after pathogen inoculation (−/+Si) exhibited the same high protection against disease as plants treated continuously with Si for the whole growth period (+/+Si), with disease severity indices of 20.8% and 19.6%, respectively, which were significantly lower than that for the control treatment with no Si supplied (63.7%). A single application of Si to rice plants before inoculation (+/−Si) conferred partial protection (disease severity index of 33.3%) compared with the control treatment. Silicon induced a rapid but transient burst of H₂O₂ at 24 h after inoculation. The addition of Si to rice plants significantly altered the activities of catalase and lipoxygenase and the concentration of malodialdehyde (indicative of lipid peroxidation) in rice plants. We propose that rice plants may respond to Si by increased H₂O₂ accumulation and lipid peroxidation. In turn, these responses are linked to host defence mechanisms such as lignin production, oxidative cross-linking in the cell wall, phytoalexin production, and the hypersensitive reaction. Thus, the mechanisms of Si-stimulated plant disease protection may extend beyond its established role in physically strengthening cell walls.     

Assessment of partial resistance to Pyricularia oryzae in six rice cultivars

The partial resistance to blast of six rice cultivars was assessed in greenhouse experiments and in an upland nursery experiment. IR36. Milyang30 and Milyang42, which have shown relatively little disease in farmers' fields for several years, had lower relative disease efficiency and smaller lesions with lower sporulation capacity than did IR50. Milyang 57 and Suweon 264. Latent period was the least important component of partial resistance. Using the area under disease progress curves as a measure of relative disease progress in the nursery, disease development on IR36 and Milyang 42 was slight and on Milyang 30 intermediate. By comparison, the three susceptible cultivars showed rapid increases in disease. Partial resistance may be associated with more durable blast resistance in certain cultivars.     

浙江省水稻新品种(系)对稻瘟病的抗性研究

用4个稻瘟病菌小种对浙江省280份水稻新品种(系)进行稻瘟病抗性分析,结果表明:浙江省水稻新品种(系)对稻瘟病菌具有较好的抗性,籼稻和粳稻对参试菌株的全抗率分别为21.62%和39.05%,说明粳稻具有较籼稻更广的抗谱;浙江省新品种(系)对ZB13小种具有较高的感病率,籼稻和粳稻的感病率分别为69.37%和52.07%。     

杂交稻对稻瘟病和稻白叶枯病的抗性鉴定

对广东省将推广的54份杂交稻组合对稻瘟病和白叶枯病的抗病性进行了鉴定。对稻瘟病的抗病性鉴定表明:抗病组合共49个,占90.7%;其中,高抗(抗性比≥91%)组合30个,占55.6%。对稻白叶枯病的抗病性鉴定表明:没有高抗(HR)和抗(R)的组合,仅有一个杂交稻组合(西胜2175)表现为中抗(mR),仅占1.9%,其余53个组合都表现感病,占98.1%。进一步分析表明:对稻瘟病表现高抗的30个杂交稻组合都不抗白叶枯病,而中抗白叶枯病的杂交稻组合西胜2175对稻瘟病表现为中抗,抗性比为74%。     

部分中国栽培稻资源对稻瘟病的抗性分析

以苗期16个稻瘟病生理小种的抗谱测定以及成株期叶瘟、穗颈瘟鉴定结果为依据,分析了75份中国栽培稻稻瘟病抗性资源的抗瘟性。结果显示,不同抗性品种的抗谱以及对成株期叶瘟和穗颈瘟的抗性反应差异较大。相关分析表明,苗期抗谱测定结果与成株期叶瘟抗性显著相关,而与穗颈瘟,以及叶瘟与穗颈瘟间相关不显著。根据聚类分析结果,抗性资源可分成9个类群,其中以第6类群包含品种最多,抗谱最广(81.3%～100%),并高抗A、B、E、G群生理小种和抗穗颈瘟,可作为水稻稻瘟病抗性育种优先利用的种质资源。     

湘资3150 微效抗瘟性基因鉴定

 以湘资3150 和CO39 为亲本建立F10 重组自交系群体为材料, 在桃江病圃应用自然诱发接种法对群体的田间叶瘟抗性表现进行了分析。结果表明, 在LOD 2. 5 的域值上,共检测到14 个有效的微效基因QTL 位点(LOD 值均大于2. 5),分别位于水稻第3、8 和10 染色体上,其表型变异贡献值差异比较大,介于11. 78% ~ 40. 57% 之间;表明可能控制不同抗性表型的QTL 紧密连锁或者同一个QTL 对不同的抗性表型均具有抗性贡献。     

黑龙江省水稻新品种（系）抗稻瘟病性鉴定及利用

1998-2009年采用稻瘟病人工接种和自然感病鉴定2种方法,共鉴定黑龙江省水稻新品种（系）485份（次）,鉴定出抗稻瘟病新品种（系）109份,12年间先后推广了‘空育131’、‘绥粳3号’、‘龙粳12’、‘龙粳14’、‘垦稻12’等49个新品种,推广了3个超级稻品种‘龙粳14’、‘龙粳18’、‘龙粳21’,18个新品种年种植面积超过6.67万hm2,获得了较高的经济效益和社会效益。     

吉林省主栽水稻品种抗瘟性评价及抗瘟基因型鉴定

 为明确吉林省主栽水稻品种的抗瘟性和抗瘟基因型,选用了一套已明确其无毒基因组成的稻瘟病菌标准菌株对吉林省68个主栽水稻品种进行喷雾和离体划伤接种试验。结果表明,68个品种的抗瘟能力存在较大差异,中抗以上抗病品种达到44个,占供试品种的64.7％;抗瘟基因推导结果结合特异性分子标记检测表明,Pita、Pia、Pish、Pita2、Pib和Pi9等6个基因为吉林省主栽水稻品种中主要的抗瘟基因,并且品种中含有的抗瘟基因数量与抗瘟能力呈正相关;根据结果推测,测试的所有品种中可能不含有Pi2、Pikh、Pikm、Pi11和Pi12基因。本研究揭示了吉林省近年主栽水稻品种的抗瘟能力和抗瘟基因组成,明确了吉林稻区主效抗瘟基因和基因聚合对稻瘟病的抗性贡献,为进一步培育广谱持久抗瘟品种和合理布局抗病品种提供了重要参考。     

Allelism of rice blast resistance genes in two Chinese rice cultivars, and identification of two new resistance genes

Blast, caused by Pyricularia grisea , is a major constraint on rice production. To broaden genetic diversity for resistance to this disease, two rice cultivars, GA20 and GA25 from Yunnan Province, China, were analysed for the genetic basis of their high resistance to blast. GA20 was crossed with 10 Japanese differential cultivars, and GA25 was crossed with nine of them and with the susceptible Chinese cultivar Lijiangxintuanheigu (LTH). The resistance of GA20 was governed by two dominant genes allelic to genes at the Pi-k and Pi-ta loci. The allele at the Pi-k locus was new, based on a reaction pattern different from known alleles at this locus. It could not be shown whether or not the allele at the Pi-ta locus is new, because races with virulence for Pi-ta were not tested. GA25 has one resistance gene, which is not allelic to genes at the loci Pi-a , Pi-k , Pi-z , Pi-ta , Pi-b , and Pi-t , but is linked to the Pi-i gene on chromosome 9 with a recombination frequency of 15.1 ± 2.8%.
The new allele at the Pi-k locus in GA20 is designated as Pi-k^g (t), and the new resistance gene in GA25 as Pi15(t) .