RFLP Mapping of Genes Conferring Complete and Partial Resistance to Blast in a Durably Resistant Rice Cultivar

Moroberekan, a japonica rice cultivar with durable resistance to blast disease in Asia, was crossed to the highly susceptible indica cultivar, CO39, and 281 F(7) recombinant inbred (RI) lines were produced by single seed descent. The population was evaluated for blast resistance in the greenhouse and the field, and was analyzed with 127 restriction fragment length polymorphism (RFLP) markers. Two dominant loci associated with qualitative resistance to five isolates of the fungus were tentatively named Pi-5(t) and Pi-7(t). They were mapped on chromosomes 4 and 11, respectively. To identify quantitative trait loci (QTLs) affecting partial resistance, RI lines were inoculated with isolate PO6-6 of Pyricularia oryzae in polycyclic tests. Ten chromosomal segments were found to be associated with effects on lesion number (P < 0.0001 and LOD > 6.0). Three of the markers associated with QTLs for partial resistance had been reported to be linked to complete blast resistance in previous studies. QTLs identified in greenhouse tests were good predictors of blast resistance at two field sites. This study illustrates the usefulness of RI lines for mapping a complex trait such as blast resistance and suggests that durable resistance in the traditional variety, Moroberekan, involves a complex of genes associated with both partial and complete resistance.     

Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

湖南桃江病圃稻瘟病菌的无毒基因及水稻抗瘟单基因联合抗性分析

【目的】鉴定湖南省桃江病圃稻瘟病菌无毒基因型,为合理搭配种植湖南省水稻抗瘟品种和抗病育种提供依据。【方法】在湖南桃江病圃采集水稻品种"丽江新团黑谷"(LTH)稻瘟菌病样,用单孢分离法分离稻瘟病菌单孢并纯化获得单孢菌株,用针刺离体法将菌株接种到以"LTH"为轮回亲本培育而成的24个含单抗瘟基因的水稻5叶期第5叶片上,对供试菌株进行无毒基因鉴定,并应用联合致病性系数和联合抗病性系数分析抗瘟基因组合间的互作。【结果】供试92个稻瘟病单孢菌株含有全部的24个无毒基因,对24个已知含单抗瘟基因的水稻材料表现出不同程度的毒力水平,含水稻抗瘟基因Pi-20对供试菌株抗菌频率最高,达54.35%;通过联合致病性系数和联合抗病性系数分析抗瘟基因组合间的互作,结果表明最佳搭配组合为Pi-20×Pi-k~s(RAC=0.28,PAC=0.23)。【结论】湖南省桃江病圃稻瘟病菌致病力较强,24个抗瘟基因多已感病化,含抗性基因Pi-20与Pi-k、Pi-k~s、Pi-3组合的水稻品种目前可在湖南省推广利用,但需研究引进新的抗瘟基因。     

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.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F₂ population derived from a cross between an introgression line, ‘IR71033-121-15’, from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, ‘Junambyeo’. Resistance to BPH (biotype 1) was evaluated using 190 F₃ families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.     

A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance

The completion of the genome sequences of both rice and Magnaporthe oryzae has strengthened the position of rice blast disease as a model to study plant-pathogen interactions in monocotyledons. Genetic studies of blast resistance in rice were established in Japan as early as 1917. Despite such long-term study, examples of cultivars with durable resistance are rare, partly due to our limited knowledge of resistance mechanisms. A rising number of blast resistance genes and quantitative trait loci (QTL) have been genetically described, and some have been characterized during the last 20 years. Using the rice genome sequence, can we now go a step further toward a better understanding of the genetics of blast resistance by combining all these results? Is such knowledge appropriate and sufficient to improve breeding for durable resistance? A review of bibliographic references identified 85 blast resistance genes and approximately 350 QTL, which we mapped on the rice genome. These data provide a useful update on blast resistance genes as well as new insights to help formulate hypotheses about the molecular function of blast QTL, with special emphasis on QTL for partial resistance. All these data are available from the OrygenesDB database.     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

Genome-wide analysis of defense-responsive genes in bacterial blight resistance of rice mediated by the recessive<Emphasis Type="Italic"> R</Emphasis> gene<Emphasis Type="Italic"> xa13</Emphasis>

Defense responses triggered by dominant and recessive disease resistance ( R) genes are presumed to be regulated by different molecular mechanisms. In order to characterize the genes activated in defense responses against bacterial blight mediated by the recessive R gene xa13, two pathogen-induced subtraction cDNA libraries were constructed using the resistant rice line IRBB13—which carries xa13 —and its susceptible, near-isogenic, parental line IR24. Clustering analysis of expressed sequence tags (ESTs) identified 702 unique expressed sequences as being involved in the defense responses triggered by xa13; 16% of these are new rice ESTs. These sequences define 702 genes, putatively encoding a wide range of products, including defense-responsive genes commonly involved in different host-pathogen interactions, genes that have not previously been reported to be associated with pathogen-induced defense responses, and genes (38%) with no homology to previously described functional genes. In addition, R -like genes putatively encoding nucleotide-binding site/leucine rich repeat (NBS-LRR) and LRR receptor kinase proteins were observed to be induced in the disease resistance activated by xa13. A total of 568 defense-responsive ESTs were mapped to 588 loci on the rice molecular linkage map through bioinformatic analysis. About 48% of the mapped ESTs co-localized with quantitative trait loci (QTLs) for resistance to various rice diseases, including bacterial blight, rice blast, sheath blight and yellow mottle virus. Furthermore, some defense-responsive sequences were conserved at similar locations on different chromosomes. These results reveal the complexity of xa13 -mediated resistance. The information obtained in this study provides a large source of candidate genes for understanding the molecular bases of defense responses activated by recessive R genes and of quantitative disease resistance.Electronic Supplementary Material Supplementary material is available in the online version of this article at The first two authors contributed equally to this workCommunicated by R. Hagemann     

Quantitative trait loci associated with leaf and neck blast resistance in recombinant inbred line population of rice (Oryza sativa).

Blast is an economically important disease of rice. To map genes controlling blast resistance, recombinant inbred lines (RIL) were developed from Khao Dawk Mali 105, an aromatic, blast-susceptible cultivar and the blast resistance donor, CT 9993-5-10-M (CT). A linkage map encompassing 2112 cM was constructed from 141 RILs using 90 restriction fragment length polymorphisms (RFLPs) and 31 simple sequence repeats (SSR). Virulent isolates of blast fungus were identified by screening differential host sets against 87 single-spore isolates collected from the north and northeast of Thailand. Fifteen virulent blast isolates were selected for leaf blast screening. Neck blast was evaluated both under natural conditions and controlled inoculations. Quantitative trait loci (QTLs) for broad resistance spectrum (BRS) to leaf blast were located on chromosomes 7 and 9. In particular, the QTL(ch9) was mapped near the Pi5(t) locus. The QTL(ch7) was located close to a previously mapped partial resistance QTL. Both loci showed significant allelic interaction. Genotypes having CT alleles at both QTL(ch7) and QTL(ch9) were the most resistant. Two neck-blast QTLs were mapped on chromosomes 5 and 6. The inconsistent map locations between the leaf and neck blast QTLs indicate the complexity of fixing both leaf and neck blast resistance. The coincidence of BRS and field resistance QTLs on chromosome 7 supports the idea that BRS may reflect the broad resistance spectrum to leaf blast in rice. These findings laid the foundation for the development of a marker-assisted scheme for improving Khoa Dawk Mali 105 and the majority of aromatic Thai rice varieties that are susceptible to blast.     

Creation of blast disease-resistant rice varieties with modern DNA-markers

Based on modern technologies of molecular DNA-markers, blast disease–resistance genes (Pi-ta, Pi-b, Pi-1, Pi-2, and Pi-33) were introgressed and pyramided into domestic rice varieties to give them longterm disease resistance. For that purpose, this case study uses SSR-markers closely linked to these genes, as well as intragenic markers of genes Pi-ta and Pi-b. Multiplex PCR systems were created for simultaneous identification of two resistance genes in the hybrid progeny for the following combinations: Pi-1 + Pi-2, Pi-ta + Pi-b, Pi-ta + Pi-33.     

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.     

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.Yiwen Deng and Xudong Zhu contributed equally to this work.     

Coincidence in map positions between pathogen-induced defense-responsive genes and quantitative resistance loci in rice

Quantitative disease resistance conferred by quantitative trait loci (QTLs) is presumably of wider spectrum and durable. Forty-four cDNA clones, representing 44 defense-responsive genes, were fine mapped to 56 loci distributed on 9 of the 12 rice chromosomes. The locations of 32 loci detected by 27 cDNA clones were associated with previously identified resistance QTLs for different rice diseases, including blast, bacterial blight, sheath blight and yellow mottle virus. The loci detected by the same multiple-copy cDNA clones were frequently located on similar locations of different chromosomes. Some of the multiple loci detected by the same clones were all associated with resistance QTLs. These results suggest that some of the genes may be important components in regulation of defense responses against pathogen invasion and they may be the candidates for studying the mechanism of quantitative disease resistance in rice.     

Coincidence in map positions between pathogen-induced defense-responsive genes and quantitative resistance loci in rice

Quantitative disease resistance conferred by quantitative trait loci (QTLs) is presumably of wider spectrum and durable. Forty-four cDNA clones, representing 44 defense-responsive genes, were fine mapped to 56 loci distributed on 9 of the 12 rice chromosomes. The locations of 32 loci detected by 27 cDNA clones were associated with previously identified resistance QTLs for different rice diseases, including blast, bacterial blight, sheath blight and yellow mottle virus. The loci detected by the same multiple-copy cDNA clones were frequently located on similar locations of different chromosomes. Some of the multiple loci detected by the same clones were all associated with resistance QTLs. These results suggest that some of the genes may be important components in regulation of defense responses against pathogen invasion and they may be the candidates for studying the mechanism of quantitative disease resistance in rice.     

QTL analysis of resistance to the rice blast pathogen in barley (Hordeum vulgare)

Barley is compatible with the rice blast pathogen (Pyricularia oryzae Cav.). Fiftyfour barley cultivars of diverse geographic origin and pedigree were inoculated with three isolates of the rice blast pathogen. All barley genotypes showed blast disease symptoms when inoculated at the seedling stage with each of the three isolates. However, one genotype showed quantitative resistance to all three isolates and three genotypes showed quantitative resistance to one or two of the isolates. By inoculating a set of doubled-haploid lines derived from the cross ’Harrington’ (susceptible) and ’TR306’ (resistant) with isolate Ken 54–20, we mapped quantitative trait loci (QTLs) determining seedling stage blast resistance. At all QTLs, TR306 contributed the resistance alleles. The four QTLs, when considered jointly, explained 43.6% of the phenotypic variation in blast symptom expression. A comparison of the blast resistance QTLs with other disease resistance QTLs reported in this population revealed a region on chromosome 4 (4H) with multiple disease resistance loci. It will be useful to capitalize on the syntenic relationship of rice and barley and to integrate information on species-specific resistance genes with information on the reaction of the two species to the same pathogen. Received: 7 January 2000 / Accepted: 22 September 2000     

水稻抗稻瘟病基因Pi-2(t)物理图谱的构建

应用ＢＡＣ文库,采用基于分子标记的染色体着陆（ｍａｒｋｅｒ－ｂａｓｅｄ ｃｈｒｏｍｏｓｏｍｅ ｌａｎｄｉｎｇ）和染色体步查（ｃｈｒｏｍｏｓｏｍｅ ｗａｌｋｉｎｇ）等手段,建立了包含有裟抗稻瘟病基因Ｐｉ－２（ｔ）的物理图谱,该物理图谱由２２个ＢＡＣ克隆组成,遗传跨度８ｃＭ,而物理距离为９２５ｋｂ,该物理图谱的构建不仅为进一步分离和克隆该基因打下了基础,同时也可为分子标记辅助选择育种选择抗稻瘟病新材料     

A novel gene, <Emphasis Type="Italic">Pi40(t)</Emphasis>, linked to the DNA markers derived from NBS-LRR motifs confers broad spectrum of blast resistance in rice

Rice blast disease caused by Magnaporthe grisea is a continuous threat to stable rice production worldwide. In a modernized agricultural system, the development of varieties with broad-spectrum and durable resistance to blast disease is essential for increased rice production and sustainability. In this study, a new gene is identified in the introgression line IR65482-4-136-2-2 that has inherited the resistance gene from an EE genome wild Oryza species, O. australiensis (Acc. 100882). Genetic and molecular analysis localized a major resistance gene, Pi40(t), on the short arm of chromosome 6, where four blast resistance genes (Piz, Piz-5, Piz-t, and Pi9) were also identified, flanked by the markers S2539 and RM3330. Through e-Landing, 14 BAC/PAC clones within the 1.81-Mb equivalent virtual contig were identified on Rice Pseudomolecule3. Highly stringent primer sets designed for 6 NBS-LRR motifs located within PAC clone P0649C11 facilitated high-resolution mapping of the new resistance gene, Pi40(t). Following association analysis and detailed haplotyping approaches, a DNA marker, 9871.T7E2b, was identified to be linked to the Pi40(t) gene at the 70 Kb chromosomal region, and differentiated the Pi40(t) gene from the LTH monogenic differential lines possessing genes Piz, Piz-5, Piz-t, and Pi-9. Pi40(t) was validated using the most virulent isolates of Korea as well as the Philippines, suggesting a broad spectrum for the resistance gene. Marker-assisted selection (MAS) and pathotyping of BC progenies having two japonica cultivar genetic backgrounds further supported the potential of the resistance gene in rice breeding. Our study based on new gene identification strategies provides insight into novel genetic resources for blast resistance as well as future studies on cloning and functional analysis of a blast resistance gene useful for rice improvement.