Identification of a major blast resistance gene in the rice cultivar 'Tetep'

Analysis of near‐isogenic lines (NILs) indicated the presence of a novel resistance gene in the indica rice cultivar ‘Tetep’ which was highly resistant to the rice blast fungus Magnaporthe grisea.‘Tetep’ was crossed to the widely used susceptible cultivar ‘CO39’ to generate the mapping population. A Mendelian segregation ratio of 3 : 1 for resistant to susceptible F₂ plants further confirmed the presence of a major dominant locus, in ‘Tetep’, conferring resistance to the blast fungal isolate B157, corresponding to the international race IC9. Simple sequence length polymorphism (SSLP) was used for molecular genetic analysis. The analysis revealed that the SSLP marker RM 246 was linked to a novel blast resistance gene designated Pi‐tp(t) in ‘Tetep’.     

A high-resolution map of the rice blast resistance gene Pi15 constructed by sequence-ready markers

The gene Pi15 for resistance of rice to Magnaporthe grisea was previously mapped to a ≈0.7-cM region on chromosome 9. To further define the chromosomal region of the Pi15 locus, a contig spanning the locus was constructed, in silico , through bioinformatics analysis using a reference sequence of the cultivar 'Nipponbare'. One simple sequence repeat marker adopted from the International Rice Microsatellite Initiative and six candidate resistance gene (CRG) markers, developed from gene annotation of the reference sequence of the contig, were used for linkage analysis in a mapping population consisting of 504 extremely susceptible F₂ plants. The Pi15 locus was delimited to a ≈0.5-cM region flanked by the markers CRG5 and CRG2 and co-segregated with the markers BAPi15₇₈₂, CRG3 and CRG4, which was physically converted to a 44-kb interval.     

Mapping of a blast field resistance gene Pi39(t) of elite rice strain Chubu 111

We investigated the mode of inheritance and map location of field resistance to rice blast in the elite rice strain Chubu 111, and yield under severe blast conditions. Chubu 111 carries the complete resistance gene Pii, although field testing showed this strain to be susceptible to infection. The level of field resistance of Chubu 111 was so high that chemicals used to control blast were not required, even in an epiphytotic area. Genetic analysis of field resistance to blast in 149 F₃ lines derived from a cross between Chubu 111 and the susceptible cultivar ‘Mineasahi’ suggested that field resistance is controlled by a dominant gene, designated Pi39(t), that cosegregates with the single sequence repeat marker loci RM3843 and RM5473 on chromosome 4. Comparative studies of polymorphism at RM3843 among Chubu 111 and six cultivars or lines in its pedigree suggested that the donor of the resistance gene was the Chinese cultivar ‘Haonaihuan’. Marker‐assisted selection of Pi39(t) should be useful in rice‐breeding programmes for field resistance to blast.     

Rapid survey for presence of a blast resistance gene Pi-ta in rice cultivars using the dominant DNA markers derived from portions of the Pi-ta gene

The Pi‐ta gene in rice confers resistance to strains of the blast pathogen Magnaporthe grisea (Herbert) Borr. (anamorph Pyricularia oryza Cav.) containing the corresponding avirulence gene AVR‐Pita in a gene‐for‐gene fashion. The Pi‐ta gene is a typical nucleotide‐binding site type resistance gene. Nucleotide sequences distinguishing the resistant Pi‐ta and susceptible pi‐ta alleles were previously identified and used for developing DNA markers for a resistant Pi‐ta haplotype and three susceptible pi‐ta haplotypes. In the present study, the existence of the Pi‐ta gene in 141 rice germplasm accessions was rapidly determined using these markers, and the results were confirmed by inoculating rice germplasm with an M. grisea strain containing AVR‐Pita. The Pi‐ta gene was found in accessions from several major rice producing countries, including China, Colombia, Japan, Vietnam, the Philippines, Iran and the United States. The usefulness of DNA markers for rapid determination of the genotype of rice germplasm was thus demonstrated. The Pi‐ta gene also was found in rice cultivar known to contain the Pi‐ta² gene, although the allelic relationship of these genes remains to be determined. The presence of the Pi‐ta gene in landrace cultivars in several different geographical locations, the Philippines and Vietnam, other indica rice cultivars in China and Colombia suggest that the Pi‐ta gene may have spontaneously originated in indica rice cultivars. These results are useful for incorporating the Pi‐ta gene into advanced breeding lines by marker‐assisted selection for rice breeding programmes worldwide.     

Dynamic analyses of rice blast resistance for the assessment of genetic and environmental effects

A doubled haploid population was employed to characterize the dynamic changes of the genetic components involved in rice blast resistance, including main-effect quantitative trait loci (QTLs), epistatic QTLs and QTL-by-environment interactions. The study was carried out at three different developmental stages of rice, using natural infection tests over 2 years. The number of main-effect QTLs, epistatic QTLs and their environmental interactions differed across the various measuring stages. One QTL ( d12 ) on chromosome 12 was detected at all stages, whereas most QTLs were active only at one or two stages in the population. These findings suggest that the unstable expression of most QTLs identified for blast resistance was influenced by the developmental status of the plants, epistatic effects between different loci and the environments in which they were grown. These findings demonstrate the complexity of expression of rice blast resistance and have important implications for durable resistance-breeding and map-based cloning of quantitative traits.     

Diversity analysis for resistance of rice (Oryza sativa L.) to blast disease [Magnaporthe grisea (Hebert) Barr.] using differential isolates from the Philippines

A wide variation in resistance to blast disease caused by Magnaporthe grisea (Hebert) Barr. ( Rossman et al. 1992 ) was found using 922 rice ( Oryza sativa L.) varieties collected mainly from Asia. These were classified into six varietal groups, termed clusters A–F, according to Ward's hierarchical cluster analysis, based on the reaction pattern to 20 standard differential blast isolates from the Philippines. The most susceptible two clusters, B and C, dominated in varieties from the Far East (Japan). Varieties from East Asia and Southeast Asia occurred less frequently in B and C clusters than those of Japan, and more frequently in E and F clusters, which were the most resistant of the cluster groups. Varieties from South Asia showed the widest variation, occurring in all clusters but less frequently in cluster B. The cluster B varieties dominated in Japan and showed a high frequency of isozyme type VI, corresponding to Japonica type. In contrast, the frequency of cluster B was low in the groups with isozyme types I, II, III and V, which dominated in South Asia. Isozyme type I corresponds to Indica type varieties. The distribution of resistance corresponded to the geographical distribution of rice varieties and might be related to differentiation into Indica and Japonica types. These findings will provide useful information for understanding the variation in blast resistance at the global level.     

Improving hybrid rice parental lines for blast resistance by introgression of broad-spectrum resistance genes Pi54 and Pi9 by marker-assisted selection

Hybrid rice technology offers a great promise to produce 15% to 20% more yield than pure line varieties. The success of hybrid rice hinges on developing superior parental lines. To improve the blast resistance of hybrid rice parental line RP5933-1-19-2R, crosses were made with donors of two major blast resistance genes namely, Pi54 (Tetep) and Pi9 (IR71033–121-15) and the resulting F₁s were confirmed for their hybridity by using Pi54MAS and NMSMPi9-1 genic markers. The confirmed F₁s were intercrossed to obtain ICF₁s and selected positive plants by markers were backcrossed to the recurrent parent, as well as selfed for advancing further to BC₁F₃ and ICF₄ generations. The segregating plants were phenotyped for blast resistance at Uniform Blast Nursery. The identified complete restorers namely, RP 6619-1, RP 6616-26, RP 6619-3 and RP 6619-11 with Pi9 and Pi54 genes would serve as donors for broad spectrum blast resistance. This could ultimately lead to the development of new rice hybrids with improved resistance to blast disease, which is crucial for sustainable rice production and food security.     

Mapping of leaf and neck blast resistance genes with resistance gene analog, RAPD and RFLP in rice

An F₈ recombinant inbred population was constructed using a commercial indica rice variety Zhong 156 as the female parent and a semidwarf indica variety Gumei 2 with durable resistance to rice blast as the male parent. Zhong 156 is resistant to the fungus race ZC₁₅ at the seedling stage but susceptible to the same race at the flowering stage. Gumei 2 is resistant to ZC₁₅ at both stages. The blast resistance of 148 recombinant inbred lines was evaluated using the blast race ZC₁₅. Genetic analysis indicated that the resistance to leaf blast was controlled by three genes and the presence of resistant alleles at any loci would result in resistance. One of the three genes did not have effects at the flowering stage. Two genes, tentatively assigned as Pi24(t) and Pi25(t), were mapped onto chromosome 12 and 6,respectively, based on RGA (resistance gene analog), RFLP and RAPD markers. Pi24(t) conferred resistance to leaf blast only, and its resistance allele was from Zhong 156. Pi25(t) conferred resistance to both leaf and neck blast, and its resistance allele was from Gumei 2. In a natural infection test in a blast hot-spot, Pi25(t) exhibited high resistance to neck blast, while Pi24(t) showed little effect. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development of cleaved amplified polymorphic sequence (CAPS) markers linked to a green rice leafhopper resistance gene, Grh3(t)

The chromosomal location of the resistance gene for green rice leafhopper (GRLH), an injurious insect for rice, has been determined and RFLP markers closely linked to this gene have been identified. The susceptible japonica rice variety ‘Nipponbare’ was crossed with a resistant japonica rice line ‘Aichi42’, in which green rice leaf hopper resistance had been introduced from an indica variety ‘Rantaj‐emas2’, and the 100 F₂ plants obtained were used for linkage analysis. The green rice leafhopper resistance gene, Grh3(t), was mapped between RFLP markers C288B and C133A on chromosome 6 and co‐segregated with C81. Of the RFLP markers tightly linked to Grh3(t), C81 was converted to a SCAR marker and C133A to a cleaved amplified polymorphic sequence marker that could distinguish the heterozygous genotype to establish an effective marker‐aided selection system for the GRLH resistance gene.     

Molecular mapping of the blast resistance gene Pi49 in the durably resistant rice cultivar Mowanggu

Rice blast, caused by the fungus Magnaporthe oryzae, is the most devastating fungal disease of rice. Mowanggu, a local japonica cultivar in Yunnan Province, China, confers broad-spectrum resistance to this pathogen. To identify the resistance gene(s) in Mowanggu, we obtained an F₂ population and 280 F₈ recombinant inbred lines (RILs) from a cross between Mowanggu and CO39, a highly susceptible indica cultivar. A linkage map with 145 simple sequence repeat (SSR) and single feature polymorphism markers over 12 chromosomes was constructed using the 280 RILs. The resistance evaluation of the F₂ and F₈ populations in both the growth chamber and in a natural rice blast nursery showed that a single dominant gene controls blast resistance in Mowanggu. Moreover, nine quantitative trait loci, which were responsible for different partial resistance components, were mapped on chromosomes 2, 3, 6, 8, 9, and 12, making contributions to the phenotypic variation ranging from 3.03 to 6.18 %. The dominant resistance gene, designated Pi49, was mapped on chromosome 11 with genetic distance of 1.01 and 1.89 cM from SSR markers K10 and K134, respectively. The physical distance between K10 and K134 is about 181 kb in the Nipponbare genome. The Pi49 gene accounted for the major phenotypic variation of disease severity in the growth chamber (where plants were inoculated with single blast isolates) and also accounted for most of the phenotypic variance of disease severity, lesion number, diseased leaf area, and lesion size in the blast nursery. Our study not only identified tightly linked markers for introgression of Pi49 into elite rice cultivars via marker-aided selection but also provides a starting point for map-based cloning of the new resistance gene.     

Molecular mapping of a new gene for resistance to rice blast (Pyricularia grisea Sacc.)

A single dominant blast resistance gene conferring resistance to a Korean rice blast isolate was identified in rice variety `Suweon 365'. We report the chromosomal localization and molecular mapping of this blast resistance gene designated as Pi-18, which confers resistance to Korean isolate `KI-313' of the blast pathogen. To know whether there is a relationship among genes conditioning resistance to location-specific isolates of the blast pathogen and thereby to identify linked markers to resistance gene for isolate KI-313 collected in Korea, RFLP markers previously reported to be linked to major blast resistance genes in different rice germplasm and other markers mapped to nearby regions were surveyed for polymorphism between a resistant (`Suweon 365') and a susceptible (`Chucheongbyeo') parent. Linkage associations of the RFLP markers with the resistance gene were verified using an F2 and F3 segregating population of known blast reaction. RFLP analysis showed that Pi-18 was located near the end of chromosome 11, linked to a single copy clone RZ536 at a distance of 5.4 centiMorgans (cM) and that this gene was different from Pi-1(t). An allelism test revealed that this gene was also different from Pi-k. Currently, a combination of RAPD and microsatellite primers is being employed to find additional markers in this region. Tightly linked DNA markers will facilitate selection for resistant genotypes in breeding programs and provide the basis for map based cloning of this new blast resistance gene. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular mapping of a new genic male-sterility gene causing chalky endosperm in rice ( Oryza sativa L.)

A genic male-sterility gene newly induced by chemical mutagenesis, tentatively designated as ms-h(t), was located on the molecular map of rice and tested for its effect on chalky endosperm. Bulked segregant analysis was used to determine the chromosomal location of the ms-h(t) locus by screening four to five RFLP markers per chromosome. After confirming that the gene was located on chromosome 9, twenty-four RFLP markers from chromosome 9 were surveyed for polymorphism in the parents of the mapping population. Of these, eleven markers were mapped around the ms- h(t) locus. RG451 and RZ404 flanked the ms-h(t) gene, at 2.5cM and 3.3cM, respectively. Heterozygous F2 to F4 progenies were tested for co- segregation of male-sterility and chalky endosperm and it was revealed that ms-h(t) might have a pleiotropic effect on chalky endosperm. This mutant would be a good biological material to characterize the biochemical mechanism of male sterility and related pleiotropic effects. Further studies should be needed to know the usefulness of this mutant for hybrid seed production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantitative trait loci for rice blast resistance detected in a local rice breeding population by genome-wide association mapping

Plant breeding programs aim to develop cultivars with high adaptability to the specific conditions in a local region. As a result, unique genes and gene combinations have been accumulated in local elite breeding populations during the long history of plant breeding. Genetic analyses on such genes and combinations may be useful for developing new cultivars with more-desirable agronomic traits. Here, we attempted to detect quantitative trait loci (QTL) for rice blast resistance (BR) using a local breeding rice population from Hokkaido, Japan. Using genotyping data on single nucleotide polymorphisms and simple sequence repeat markers distributed throughout the whole genomic region, we detected genetic regions associated with phenotypic variation in BR by a genome-wide association mapping study (GWAS). An additional association analysis using other breeding cultivars verified the effect and inheritance of the associated region. Furthermore, the existence of a gene for BR in the associated region was confirmed by QTL mapping. The results from these studies enabled us to estimate potential of the Hokkaido rice population as a gene pool for improving BR. The results of this study could be useful for developing novel cultivars with vigorous BR in rice breeding programs.     

水稻抗稻瘟病基因Pi-ta的分子标记辅助选择

利用已建立的水稻抗稻瘟病基因Pi-ta显性分子标记对30个品系和157个来自不同国家的一些水稻品种进行分子鉴定,并采用稻瘟病菌菌株ZN57（IC-17）和ZN61（IB-49）人工接种试验进行致病性测试。结果表明,大部分品系和少数水稻品种含抗病基因Pi-ta,且对稻瘟病菌菌株ZN57和ZN61表现抗病反应。除此之外,利用两对显性分子标记YL1     

24个单基因系对黑龙江省优势菌群的抗性及联合抗病性分析

为了明确24个抗稻瘟病基因在黑龙江省的抗性水平及其利用价值,将2006年黑龙江省优势菌株和强致病力菌株接种于以丽江新团黑谷为轮回亲本培育而成的含有24个抗瘟单基因系上。结果表明：水稻抗瘟基因Pi9(t)、pi-z5对优势和强致病力菌株抗性最强,是较好的抗源可以广泛的利用;对2个抗瘟基因的联合抗病性系数分析,结果表明基因两两搭配后的联合抗病系数最高、联合致病系数最低的组合是pi-9(t)*pi-z5 和pi-9(t)*pi-ta2 ;分析得出,黑龙江省稻瘟病菌致病力较强,大部分抗瘟基因都已失去抗性,急需引进新的抗瘟基因。     

High-resolution mapping revealed a 1.3-Mbp genomic inversion in Ssi1, a dominant semidwarf gene in rice (Oryza sativa)

Dwarf or semidwarf characters are an important trait for crop breeding as they provide lodging resistance. The rice mutant line DMF-1 has a dm-type semidwarf phenotype and high-lodging resistance controlled by the dominant gene, Short second internode 1 ( Ssi1 ). To elucidate the mechanism of reducing culm length in DMF-1, we sought to identify the Ssi1 gene by positional cloning using the chromosome segment substitution line as a crossing parent. As a result of high-resolution mapping, we found a 1.3-Mbp genomic inversion and a newly arranged gene in the Ssi1 locus. In this study, we report the high-resolution mapping and physical mapping of Ssi1 . We also discuss the possible function of a novel rearranged Ssi1 gene for the dominant dm-type semidwarf phenotype.     

Molecular mapping of the fertility-restoration gene Rf4 for WA-cytoplasmic male sterility in rice

The wild abortive cytoplasmic male sterility (CMS‐WA) system, an ideal type of sporophytic CMS in indica rice, is used for the large‐scale commercial production of hybrid rice. Searching for restorer genes is a good approach when phenotyping is very time‐consuming and requires the determination of spikelet sterility in testcross progeny. To establish more precisely the genetical and physical maps of the Rf4 gene, high‐resolution mapping of this locus was carried out using simple sequence repeat (SSR) markers and newly designed markers in a F₂ population. The genetic linkage analysis indicated that five SSR markers (RM6737, RM304, RM171, RM5841 and RM228) on the long arm of chromosome 10 were linked with the Rf4 gene. Rf4 was flanked by two SSR markers RM171 and RM6737 at distances of 3.2 and 1.6 cM, respectively. Also, within the region between Rf4 gene and RM171, a newly designed primer pair, AB443, produced two sterile‐specific markers, AB443‐400 and AB443‐500, 0.5 and 1.03 cM from the gene. The flanking markers identified give promise for their application in molecular marker‐assisted selection (MAS) and they are also suitable for starting chromosome walking to clone Rf4 gene in the near future.