Molecular mapping of a novel blast resistance gene Pi38 in rice using SSLP and AFLP markers

Blast, caused by Magnaporthe grisea, is the most devastating disease of rice worldwide. In this study, the main objective was to identify and map a new gene for blast resistance, in an indica rice cultivar ‘Tadukan’ against blast fungal isolate B157, using molecular tools. F₂ segregating population was derived from ‘CO39’ (susceptible) and ‘Tadukan’ (resistant), and molecular mapping of the blast resistance gene was carried out using simple sequence length polymorphism (SSLP) and amplified fragment length polymorphism (AFLP) methods. Two SSLP markers, RM206 and RM21 and three AFLP markers (AF1: E‐aca/M‐ctt; AF2: E‐aca/M‐cat and AF3: E‐acc/M‐cac2) were identified to be linked to the resistance gene. The co‐segregation analysis using SSLP markers implied that the blast resistance gene designated Pi38 resides on rice chromosome 11.     

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.     

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.     

Identification of two new genes conferring resistance to rice blast in the Chinese native cultivar 'Maowangu'

The Chinese native rice cultivar ‘Maowangu’ expresses a high level of resistance to many races of rice blast (Pyricularia grisea) collected from North China and Japan. ‘Maowangu’ was crossed with 10 Japanese differential cultivars and the susceptible Chinese cultivar ‘Lijiangxintuanheigu’ (LTH). Allelism tests were conducted in the F₂ populations with rice blast races. The resistance of ‘Maowangu’ was governed by two dominant genes which were non-allelic to the resistance genes at seven loci: Pi-a, Pi-i, Pi-k, Pi-z, Pi-ta, Pi-b, and Pi-t. To identify the two resistance genes, two F₃ lines of ‘Shin 2/Maowangu’ segregating 3R:1S were selected for linkage tests in 1994. One was linked to marker genes C and Amp-3 on chromosome 6 with recombination frequencies of 35.8 ± 6.4% and 42.1 ± 6.2%, respectively, and the other to Amp-1 on chromosome 2 with a recombination frequency of 37.6 ± 6.0%. To confirm these results, two F₃ lines of ‘LTH/Maowangu’ were selected for linkage tests in 1995. The one was linked to Amp-3, and other was linked to Amp-1, with recombination frequencies of 36.9 ± 3.1% and 34.3 ± 3.2%, respectively. The two genes on chromosomes 6 and 2 were designated Pi13(t) and Pi14(t), respectively.     

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.     

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.     

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.     

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。     

Identification of resistance gene analogs as markers of disease resistance loci in oats, using near-isogenic lines

Genomic sequences with features of the major class of disease resistance genes and which bear nucleotide‐binding leucine‐rich repeat sequences (resistance gene analogs; RGA) were tested as potential markers of crown rust resistance loci in hexaploid oats. Two collections of paired near‐isogenic lines carrying resistance to different isolates of crown rust, Puccinia coronata were screened. Two out of the four RGAs assayed showed restriction fragment length polymorphism (RFLP) between one line of each collection and its recurrent parent. The paired lines X466 and D494 were polymorphic for RGA III2.2 and the pair of lines X470 and D504 were polymorphic for RGA III2.18. The III2.18 polymorphism was located in the hexaploid map Avena byzantina cv. ‘Kanota’ × A. sativa cv. ‘Ogle’ in linkage group KO17 in a region previously associated with crown rust resistance. In addition, 220 random primers were used for random amplified polymorphic DNA (RAPD) analysis to screen the two sets of NILs. Only one polymorphic band was obtained that differentiated the paired lines X470 and D504 from their parents. The RAPD band was used as a probe and the relevant RFLP that differentiated the NILs X470 and D504 was found at 1.7 cM from the III2.18 marker in KO17. RFLP analysis using probes previously mapped in KO17 confirmed differences for X470 and D504 in the region around the III2.18 marker. These results suggest that the resistance locus shared by this pair of NILs is probably linked to the markers revealed by RGA III2.18. The use of RGAs as RFLP probes in the screening of NILs with differences in crown rust resistance has proved to be more effective than RAPDs for finding polymorphic markers possibly linked to resistance loci.     

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.     

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.     

Validation of a major QTL for scab resistance with SSR markers and use of marker-assisted selection in wheat

The objectives of this study were to validate the major quantitative trait locus (QTL) for scab resistance on the short arm of chromosome 3B in bread wheat and to isolate near‐isogenic lines for this QTL using marker‐assisted selection (MAS). Two resistant by susceptible populations, both using ‘Ning7840’ as the source of resistance, were developed to examine the effect of the 3BS QTL in different genetic backgrounds. Data for scab resistance and simple sequence repeat (SSR) markers linked to the resistance QTL were analyzed in the F_2:3 lines of one population and in the F_3:4 lines of the other. Markers linked to the major QTL on chromosome 3BS in the original mapping population (‘Ning7840’/‘Clark’) were closely associated with scab resistance in both validation populations. Marker‐assisted selection for the QTL with the SSR markers combined with phenotypic selection was more effective than selection based solely on phenotypic evaluation in early generations. Marker‐assisted selection of the major QTL during the seedling stage plus phenotypic selection after flowering effectively identified scab resistant lines in this experiment. Near‐isogenic lines for this 3BS QTL were isolated from the F₆ generation of the cross ‘Ning7840’/‘IL89‐7978’ based on two flanking SSR markers, Xgwm389 and Xbarc147. Based on these results, MAS for the major scab resistance QTL can improve selection efficiency and may facilitate stacking of scab resistance genes from different sources.     

Small differential interactions for partial resistance in rice cultivars to virulent isolates of the blast pathogen

Summary Six rice genotypes, differing in partial resistance, were exposed to three isolates of the blast pathogen. Of the variance due to host and pathogen genotypes, 39% was due to host genotype effects, 60% was due to isolate effects, and only 1% was due to host genotype × isolate interactions. Although small, this interaction variance was highly significant and mainly due to the IR50 × W6-1 and IR37704 × JMB8401-1 combinations. Although behaving largely as race-non-specific (large main effects only), the partial resistance cannot be classified as race-non-specific. The results suggest that minor genes for partial resistance operate in a gene for gene relationship with minor genes in the pathogen.     

Role of the rice blast resistance gene Pi-cd in rice (Oryza sativa) breeding programmes

A series of DNA markers for various agronomic traits may accelerate the success of marker-assisted selection in practical plant breeding programmes. Here, we developed DNA markers for the blast resistance gene Pi-cd. In this study, we examined the effects of the Pi-cd locus on not only blast resistance but also agronomic traits in agriculture. We developed three pyramiding lines (PLs) coupling Pi-cd with three blast resistance genes, pi21, Pi35 and Pi39. The effect of Pi-cd on blast resistance was dependent on the coupled resistance genes. Then, we evaluated the effects of Pi-cd on 13 agronomic traits. Amylose content and 1,000-grain weight showed significant differences between the PLs and current commercial varieties, which had no negative effects on agronomic trait values. Furthermore, we investigated the distribution of genotype for the Pi-cd locus among varieties of upland rice. The KT genotype specific to rice blast resistance may be predominant in the varieties. The results suggested that Pi-cd has the potential to be useful for improving blast resistance in rice breeding programmes.     

Effect of leaf age on components of partial resistance in rice to leaf blast

Summary Ten rice genotypes were inoculated with a virulent isolate of Magnaporthe grisea to study the effect of leaf age on components of partial resistance, and evaluate associations between these components. The number of sporulating lesions per cm² leaf declined with increase of leaf age in all genotypes. The number of lesions per cm² leaf area in one week old leaves was about 25% that of the number in very young leaves in the susceptible cultivar CO39, but less than 2% in the more resistant cultivars IR36, IR60 and IR62. Large differences between genotypes were found for the number of sporulating lesions that developed, and this factor was closely related to the period that leaves remained susceptible after appearance. The number of lesions in the most susceptible cultivar CO39 was about 7 times that in the cultivars IR60 and IR64. Differences between genotypes were also found for lesion size. The effect of aging on average lesion size was less pronounced than on lesion density. Lesion size and lesion density were positively correlated, but a rapid decline of density was not necessarily accompanied by a rapid decline of size. No apparent differences between genotypes were observed for latent period. Genotypes with leaves that became highly resistant soon after appearance expressed higher levels of partial resistance in the field.     

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.     

QTL analysis for brown spot resistance in American rice cultivar ‘Dawn’

Rice brown spot (BS), caused by Bipolaris oryzae, causes yield loss and deterioration of grain quality. Using single-nucleotide polymorphism (SNP) markers, we conducted quantitative trait locus (QTL) analysis of BS resistance in backcross inbred lines (BILs) from a cross between an American rice cultivar, ‘Dawn’ (resistant), and ‘Koshihikari’ (susceptible). Four QTLs for BS resistance were detected in a three-year field evaluation, and ‘Dawn’ contributed the resistance alleles at all QTLs. The QTL with the greatest effect, qBSR6-kd, explained 15.1% to 20.3% of the total phenotypic variation. Although disease score and days to heading (DTH) were negatively correlated in all three years, qBSR6-kd was located near a QTL for DTH at which the ‘Dawn’ allele promoted heading. Another BS resistance QTL (qBSR3.1-kd) was unlinked to the QTLs for DTH. Therefore, these two QTLs are likely to be useful for breeding BS-resistant varieties without delaying heading. The other two BS resistance QTLs (qBSR3.2-kd and qBSR7-kd) were located near DTH QTLs at which the ‘Dawn’ alleles delayed heading. The QTLs reported here will be good candidates for developing BS-resistant cultivars.