SSRs for Marker-Assisted Selection for Blast Resistance in Rice (Oryza sativa L.)

Rice blast caused by the fungus Magnaporthe oryzae is one of the most devastating diseases of rice in nearly all rice growing areas of the world including Malaysia. To develop cultivars with resistance against different races of M. oryzae, availability of molecular markers along with marker-assisted selection strategies are essential. In this study, 11 polymorphic simple sequence repeat (SSR) markers with good fit of 1:2:1 ratio for single gene model in F₂ population derived from the cross of Pongsu seribu 2 (Resistant) and Mahsuri (Susceptible) rice cultivars were analysed in 296 F₃ families derived from individual F₂ plants to investigate association with Pi gene conferring resistance to M. oryzae pathotype. Parents and progeny were grouped into two phenotypic classes based on their blast reactions. Chi-square test for the segregation of resistance and susceptibility in F₃ generation fitted a ratio of approximately 3:1. Association of SSR markers with phenotypic trait in F₃ families was identified by statistical analysis. Four SSR markers (RM413, RM5961, RM1233 and RM8225) were significantly associated with blast resistance to pathotype 7.2 of M. oryzae in rice (p ≤ 0.01). These four markers accounted for about 20% of total phenotypic variation. So, these markers were confirmed as suitable markers for use in marker-assisted selection and confirmation of blast resistance genes to develop rice cultivars with durable blast resistance in Malaysian rice breeding programmes.     

Mapping of the quantitative trait locus (QTL) conferring partial resistance to rice leaf blast disease

Malaysian rice, Pongsu Seribu 2, has wide-spectrum resistance against blast disease. Chromosomal locations conferring quantitative resistance were detected by linkage mapping with SSRs and quantitative trait locus (QTL) analysis. For the mapping population, 188 F₃ families were derived from a cross between the susceptible cultivar, Mahsuri, and a resistant variety, Pongsu Seribu 2. Partial resistance to leaf blast in the mapping population was assessed. A linkage map covering ten chromosomes and consisting of 63 SSR markers was constructed. 13 QTLs, including 6 putative and 7 putative QTLs, were detected on chromosomes 1, 2, 3, 5, 6, 10, 11 and 12. The resulting phenotypic variation due to a single QTL ranged from 2 to 13 %. These QTLs accounted for approx. 80 % of the total phenotypic variation within the F₃ population. Therefore, partial resistance to blast in Pongsu Seribu 2 is due to combined effects of multiple loci with major and minor effects.     

Four QTL in Rice Associated with Broad Spectrum Resistance to Blast Isolates from Rice and Barley

Pyricularia grisea is the most destructive and cosmopolitan fungal pathogen of rice and it can also cause disease on other agriculturally important cereals. We determined the number, location and interaction of quantitative trait loci (QTL) associated with resistance to P. grisea isolates obtained from rice (THL142 and THL222) and barley (TH16 and THL80) grown in Thailand. The isolates showed a spectrum of virulence when used to inoculate a series of differentials. We used a reference blast resistance mapping population of rice (IR64 × Azucena). IR64 was highly resistant, and Azucena was highly susceptible, to all four isolates. The numbers of resistant vs. susceptible progeny suggest that the resistance of IR64 is determined by two or three genes with additive effects. The correlation coefficients for all pairwise comparisons of disease severity were high and highest between barley isolates and between rice isolates. Four QTL were detected, one on each of the following chromosomes 2, 8, 9 and 10. IR64 contributed resistance alleles at three of the QTL (chromosomes 2, 8 and 9). Azucena contributed the resistance allele at the QTL on chromosome 10 in response to inoculation with isolate THL142. The results of the QTL analysis support interpretation of the phenotypic frequency distributions regarding the number of genes determining resistance to the four isolates in this population. Our results are novel in adding blast isolates from barley to the catalogue of pathogen specificities to which a gene, or genes, from IR64 confer resistance.     

Analysis of simple sequence repeat markers linked with blast disease resistance genes in a segregating population of rice (Oryza sativa)

Among 120 simple sequence repeat (SSR) markers, 23 polymorphic markers were used to identify the segregation ratio in 320 individuals of an F(2) rice population derived from Pongsu Seribu 2, a resistant variety, and Mahsuri, a susceptible rice cultivar. For phenotypic study, the most virulent blast (Magnaporthe oryzae) pathotype, P7.2, was used in screening of F(2) population in order to understand the inheritance of blast resistance as well as linkage with SSR markers. Only 11 markers showed a good fit to the expected segregation ratio (1:2:1) for the single gene model (d.f. = 1.0, P < 0.05) in chi-square (χ(2)) analyses. In the phenotypic data analysis, the F(2) population segregated in a 3:1 (R:S) ratio for resistant and susceptible plants, respectively. Therefore, resistance to blast pathotype P7.2 in Pongsu Seribu 2 is most likely controlled by a single nuclear gene. The plants from F(2) lines that showed resistance to blast pathotype P7.2 were linked to six alleles of SSR markers, RM168 (116 bp), RM8225 (221 bp), RM1233 (175 bp), RM6836 (240 bp), RM5961 (129 bp), and RM413 (79 bp). These diagnostic markers could be used in marker assisted selection programs to develop a durable blast resistant variety.     

Genetic dissection of rice blast resistance by QTL mapping approach using an F3 population

Rice blast is one of the major fungal diseases that badly reduce rice production in Asia including Malaysia. There is not much information on identification of QTLs as well as linked markers and their association with blast resistance within local rice cultivars. In order to understanding of the genetic control of blast in the F₃ families from indica rice cross Pongsu seribu2/Mahsuri, an analysis of quantitative trait loci against one of the highly virulent Malaysian rice blast isolate Magnaporthe oryzae, P5.0 was carried out. Result indicated that partial resistance to this pathotype observed in the present study was controlled by multiple loci or different QTLs. In QTL analysis in F₃ progeny fifteen QTLs on chromosomes 1, 2, 3, 5, 6, 11 and 12 for resistance to blast nursery tests was identified. Three of detected QTLs (qRBr-6.1, qRBr-11.4, and qRBr-12.1) had significant threshold (LOD >3) and approved by both IM and CIM methods. Twelve suggestive QTLs, qRBr-1.2, qRBr-2.1, qRBr-4.1, qRBr-5.1, qRBr-6.2, qRBr-6.3, qRBr-8.1, qRBr-10.1, qRBr-10.2, qRBr-11.1, qRBr-11.2 and qRBr-11.3) with Logarithmic of Odds (LOD) <3.0 or LRS <15) were distributed on chromosomes 1, 2, 4, 5, 6, 8, 10, and 11. Most of the QTLs detected using single isolate had the resistant alleles from Pongsu seribu 2 which involved in the resistance in the greenhouse. We found that QTLs detected for deferent traits for the using isolate were frequently located in similar genomic regions. Inheritance study showed among F₃ lines resistance segregated in the expected ratio of 15: 1 for resistant to susceptible. The average score for blast resistance measured in the green house was 3.15, 1.98 and 29.95 % for three traits, BLD, BLT and % DLA, respectively.     

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.     

Enhancing disease resistances of Super Hybrid Rice with four antifungal genes

A plant expression vector harboring four antifungal genes was delivered into the embryogenic calli of ‘9311’, an indica restorer line of Super Hybrid Rice, via modified biolistic particle bombardment. Southern blot analysis indicated that in the regenerated hygromycin-resistant plants, all the four antifungal genes, including RCH10, RAC22, β-Glu and B-RIP, were integrated into the genome of ‘9311’, co-transmitted altogether with the marker gene hpt in a Mendelian pattern. Some transgenic R₁ and R₂ progenies, with all transgenes displaying a normal expression level in the Northern blot analysis, showed high resistance to Magnaporthe grisea when tested in the typical blast nurseries located in Yanxi and Sanya respectively. Furthermore, transgenic F₁ plants, resulting from a cross of R₂ homozygous lines with high resistance to rice blast with the non-transgenic male sterile line Peiai 64S, showed not only high resistance to M. grisea but also enhanced resistance to rice false smut (a disease caused by Ustilaginoidea virens) and rice kernel smut (another disease caused by Tilletia barclayana).     

A Simple and Accurate Resistance Identification Method of Rice to Neck Blast Disease In Vitro

Twelve rice cultivars with differential resistance to rice blast disease (Magnaporthe oryzae (Hebert) Barr), including Tetep (R), IR36 (MR) and Lijiangxituanhegu (HS), and nine locally planted rice cultivars in Jiangxi helped establish an identification method for rice resistance to neck blast. We describe a new technique of dropping a spore suspension on the panicle segment in vitro (DSSPS). This technique involved rice panicles that were initially 0.5–2 cm in length and then cut into a 7‐ to 8‐cm segment (i.e. an upper node of 1 cm and a lower node of 6–7 cm). The segment was placed into a Petri dish with a stack of sterile water saturated filter paper. The suspension (4 μl 1 × 10⁵spores/ml) was placed at each of three locations on the segment (with an approximate interval of 3 cm). Disease severity was then assessed according to a 0–9 scale after incubating for 9 days with a 12 h/12 h (light/day cycle) at 28°C. Choosing a suitable developmental stage of the rice panicle and blast strains was a key to evaluate resistance accurately. DSSPS is a simple and accurate method of identifying rice resistance to neck blast as compared to injecting the spore suspension into the rice panicle in vivo and resistance identification in natural nurseries. It is stressed that at least 20 single‐spore strains are needed to accurately assess rice resistance to neck blast. We tested 1005 rice cultivars for neck blast resistance in Jiangxi province during 2010–2015, which showed an accuracy of 85.77% by DSSPS as compared with natural nursery data.     

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.     

Analysis of the Relationship between Blast Resistance Genes and Disease Resistance of Rice Germplasm via Functional Molecular Markers

Rice blast disease is one of the most devastating diseases of rice (Oryza sativa L.) caused by the fungus Magnaporthe oryzae (M. oryzae), and neck blast is the most destructive phase of this illness. The underlying molecular mechanisms of rice blast resistance are not well known. Thus, we collected 150 rice varieties from different ecotypes in China and assessed the rice blast resistances under the natural conditions that favoured disease development in Jining, Shandong Province, China in 2017. Results showed that 92 (61.3%) and 58 (38.7%) rice varieties were resistant and susceptible to M. oryzae, respectively. Among the 150 rice varieties screened for the presence of 13 major blast resistance (R) genes against M. oryzae by using functional markers, 147 contained one to eight R genes. The relationship between R genes and disease response was discussed by analysing the phenotype and genotype of functional markers. The results showed that the rice blast resistance gene Pita was significantly correlated with rice blast resistance. Our results provided a basis for the further understanding of the distribution of 13 major R genes of rice blast in the germplasm resources of the tested rice varieties, and were meaningful for rice disease resistance breeding.     

Enhancing disease resistances of Super Hybrid Rice with four antifungal genes

A plant expression vector harboring four antifungal genes was delivered into the embryogenic calli of ‘9311’, an indica restorer line of Super Hybrid Rice, via modified biolistic particle bombardment. Southern blot analysis indicated that in the regenerated hygromycin-resistant plants, all the four anti-fungal genes, including RCH10, RAC22, β-Glu and B-RIP, were integrated into the genome of ‘9311’, co-transmitted altogether with the marker gene hpt in a Mendelian pattern. Some transgenic R1 and R2 progenies, with all transgenes displaying a normal expression level in the Northern blot analysis, showed high resistance to Magnaporthe grisea when tested in the typical blast nurseries located in Yanxi and Sanya respectively. Furthermore, transgenic F₁ plants, resulting from a cross of R₂ homozygous lines with high resistance to rice blast with the non-transgenic male sterile line Peiai 64S, showed not only high resistance to M. grisea but also enhanced resistance to rice false smut (a disease caused by Ustilaginoidea virens) and rice kernel smut (another disease caused by Tilletia barclayana).     

Population Structure of Magnaporthe grisea from North-western Himalayas and its Implications for Blast Resistance Breeding of Rice

Neutral and pathogenicity markers were used to analyse the population structure of Magnaporthe grisea rice isolates from the north‐western Himalayan region of India. Random amplified polymorphic DNA (RAPD)‐based DNA fingerprinting of 48 rice isolates of M. grisea with five primers (OPA‐04, OPA‐10, OPA‐13, OPJ‐06 and OPJ‐19) showed a total of 65 RAPD bands, of which 54 were polymorphic. Cluster analysis of 48 rice isolates of M. grisea on the basis of these 65 RAPD bands revealed the presence of high genotypic diversity and continuous DNA fingerprint variation in the pathogen population. No correlation was observed between RAPD patterns and virulence characteristics of the pathogen. The observed population structure contrasted with presumed clonal reproductive behaviour of the pathogen and indicated the possibility of ongoing genetic recombination in the pathogen population. Analysis of the virulence organization of five RAPD groups (RG1–RG5) using 20 rice genotypes comprising at least 15 resistance genes revealed that no combination of resistance genes would confer resistance against all RAPD fingerprint groups present in the M. grisea rice population. The possible implications of the observed population structure of M. grisea for blast resistance breeding have been discussed.     

转基因改良水稻抗稻瘟病的策略及其进展

稻瘟病是由子囊菌引起的广泛发生在世界各水稻产区的主要真菌病害。由于病原菌致病性的高度分化,使得对稻瘟病很难控制和防治。长期实践证明,培育抗病品种是稻瘟病抗病育种的主要目标。随着基因工程的发展,利用转基因技术导入外源基因改良稻瘟病抗性已成为一条新途径。现有研究表明,通过某些抗病基因、抗真菌蛋白基因、杀菌肽基因的克隆和转育,可以培育出获得对稻瘟病广谱抗性的水稻品种(系)。     

稻瘟病菌群体遗传结构的研究进展

稻梨孢引起的稻瘟病是世界水稻生产的最重要病害,严重影响水稻的产量和米质。文中综述了分子标记技术在稻瘟病菌群体遗传结构研究上的应用,分析了病原菌遗传宗谱的特点及其与致病谱的关系,探讨了导致稻瘟病菌群体遗传结构发生变化的相关因素。     

QTL analysis and mapping of pi21, a recessive gene for field resistance to rice blast in Japanese upland rice

Field resistance is defined as the resistance that allows effective control of a parasite under natural field conditions and is durable when exposed to new races of that parasite. To identify the genes for field resistance to rice blast, quantitative trait loci (QTLs) conferring field resistance to rice blast in Japanese upland rice were detected and mapped using RFLP and SSR markers. QTL analysis was carried out in F₄ progeny lines from the cross between Nipponbare (moderately susceptible, lowland) and Owarihatamochi (resistant, upland). Two QTLs were detected on chromosome 4 and one QTL was detected on each of chromosomes 9 and 12. The phenotypic variation explained by each QTL ranged from 7.9 to 45.7% and the four QTLs explained 66.3% of the total phenotypic variation. Backcrossed progeny lines were developed to transfer the QTL with largest effect using the susceptible cultivar Aichiasahi as a recurrent parent. Among 82 F₃ lines derived from the backcross, resistance segregated in the expected ratio of resistant 1 : heterozygous 2 : susceptible 1. The average score for blast resistance measured in the field was 4.2 ± 0.67, 7.5 ± 0.51and 8.2 ± 0.66, for resistant, heterozygous and susceptible groups, respectively. The resistance gene, designated pi21, was mapped on chromosome 4 as a single recessive gene between RFLP marker loci G271 and G317 at a distance of 5.0 cM and 8.5 cM, respectively. The relationship to previously reported major genes and QTLs conferring resistance to blasts, and the significance of marker-assisted selection to improve field resistance, are discussed. Received: 8 June 2000 / Accepted: 24 November 2000     

利用二代测序技术高通量分析水稻品种间抗病性差异的分子基础

稻瘟病和白叶枯病是由稻温病菌(Magnaporthe oryzae)和白叶枯病菌(Xanthomonas oryzae pv.oryzae)引起的两种主要水稻病害,也是制约中国水稻生产的主要病害。为了从DNA水平探索造成水稻感病品种‘丽江新团黑谷’(LTH)和高抗品种‘特特普’(Tetep,TTP)间抗病性差异的分子基础,该研究对其已知的3个抗稻瘟病基因和3个抗白叶枯基因所在DNA区段分别进行PCR扩增,将等量混合的PCR产物再与基因组重测序样品按Ct值差值(ΔCt)~10的比例混合,采用二代测序技术进行一次性测序和比较分析,并对有差异的基因区域进行常规传统测序验证,以确定这2个品种中抗性基因(R基因)的数目和结构与品种抗病或感病表型的关联性。实验结果表明,二代测序能够快速并准确地寻找到2个不同水稻品种中多个特定基因的序列差异,且差异位点与常规测序结果相符。从LTH和TTP这2种抗性不同水稻品种在多个抗性基因的DNA水平差异来看,有差异的抗性基因位点在高抗品种TTP中大都与原始抗性基因序列相同,而对应的普感品种LTH的抗性基因往往多表现为氨基酸突变,这些序列差异很可能就是导致TTP与LTH抗性差异的分子基础。     

Induced Pib Expression and Resistance to Magnaporthe grisea are Compromised by Cytosine Demethylation at Critical Promoter Regions in Rice

Pib is a well‐characterized rice blast‐resistance gene belonging to the nucleotide binding site (NBS) and leucine‐rich repeat (LRR) superfamily. Expression of Pib was low under non‐challenged conditions, but strongly induced by the blast‐causing fungal pathogen Magnaporthe grisea, thereby conferring resistance to the pathogen. It is generally established that cytosine methylation of the promoter‐region often plays a repressive role in modulating expression of the gene in question. We report here that two critical regions of the Pib promoter were heavily CG cytosine‐methylated in both cultivars studied. Surprisingly, induced expression of Pib by M. grisea infection did not entail its promoter demethylation, and partial demethylation by 5‐azacytidine‐treatment actually reduced Pib expression relative to wild‐type plants. Accordingly, the blast disease‐resistance was compromised in the 5′‐azaC‐treated plants relative to wild‐type. In contrast, the disease susceptibility was not affected by the 5′‐azaC treatment in another two rice cultivars that did not contain the Pib gene, ruling out effects of other R genes and non‐specific genotoxic effects by the drug‐treatment as a cause for the compromised Pib‐conditioned blast‐resistance. Taken together, our results suggest that promoter DNA methylation plays a novel enhancing role in conditioning high‐level of induced expression of the Pib gene in times of M. grisea infection, and its conferred resistance to the pathogen.     

SSR Markers Closely Linked to the Pi-z Locus are Useful for Selection of Blast Resistance in a Broad Array of Rice Germplasm

Pi-z is a disease resistance gene that has been effectively used to combat a broad-spectrum of races of the rice blast fungus Magnaporthe grisea. Although DNA markers have been reported for selection of the Pi2(t) and Pi-z resistance genes at the Pi-z locus, markers that are more tightly linked to the Pi-z locus would benefit rapid and effective cultivar development. Analysis of the publicly available genome sequence of Nipponbare near the Pi-z locus revealed numerous SSRs that could be converted into markers. Three SSRs on rice PAC AP005659 were found to be very tightly linked to the Pi-z locus, with one marker, AP5659-3, co-segregating with the Pi-z resistance reaction. The Pi-z factor conferring resistance to two races of blast was mapped to a 57 kb region on the physical map of Nipponbare in a location where the Pi2(t) gene was physically mapped. Two SSR marker haplotypes were unique for cultivars carrying the Pi-z gene, which indicates these markers are useful for selection of resistance genes at the Pi-z locus in rice germplasm.     

水稻稻瘟病抗性基因的克隆、育种利用及稻瘟菌无毒基因研究进展

稻瘟病是世界上影响水稻(Oryza sativa)粮食生产的主要病害之一, 抗病基因的发掘与利用是抗病育种的基础和核心。随着寄主水稻和病原菌稻瘟病菌(Magnaporthe oryzae)基因组测序和基因注释的完成, 水稻和稻瘟病菌的互作体系成为研究植物与真菌互作的模式系统。该文对稻瘟病抗病基因的遗传、定位、克隆及育种利用进行概述, 并通过生物信息学分析方法, 探讨了水稻全基因组中NBS-LRR类抗病基因在水稻12条染色体上的分布情况, 同时对稻瘟病菌无毒基因的鉴定及无毒蛋白与抗病蛋白的互作进行初步分析。最后对稻瘟病抗病基因研究存在的问题进行分析并展望了未来的研究方向, 以期为水稻抗稻瘟病育种发展和抗病机制的深入理解提供参考。