Identification of microsatellite markers linked to the blast resistance gene <Emphasis Type="Italic">Pi-1(t)</Emphasis> in rice

The present work was conducted to identify microsatellite markers linked to the rice blast resistance gene Pi-1(t) for a marker-assisted selection program. Twenty-four primer pairs corresponding to 19 microsatellite loci were selected from the Gramene database (www. gramene.org) considering their relative proximity to Pi-1(t) gene in the current rice genetic map. Progenitors and DNA bulks of resistant and susceptible families from F₃ segregating populations of a cross between the near-isogenic lines C101LAC (resistant) and C101A51 (susceptible) were used to identify polymorphic microsatellite markers associated to this gene through bulked segregant analysis. Putative molecular markers linked to the blast resistance gene Pi-1(t) were then used on the whole progeny for linkage analysis. Additionally, the diagnostic potential of the microsatellite markers associated to the resistance gene was also evaluated on 17 rice varieties planted in Latin America by amplification of the specific resistant alleles for the gene in each genotype. Comparing with greenhouse phenotypic evaluations for blast resistance, the usefulness of the highly linked microsatellite markers to identify resistant rice genotypes was evaluated. As expected, the phenotypic segregation in the F₃ generation agreed to the expected segregation ratio for a single gene model. Of the 24 microsatellite sequences tested, six resulted polymorphic and linked to the gene. Two markers (RM1233*I and RM224) mapped in the same position (0.0 cM) with the Pi-1(t) gene. Other three markers corresponding to the same genetic locus were located at 18.5 cM above the resistance gene, while another marker was positioned at 23.8 cM below the gene. Microsatellite analysis on elite rice varieties with different genetic background showed that all known sources of blast resistance included in this study carry the specific Pi-1(t) allele. Results are discussed considering the potential utility of the microsatellite markers found, for MAS in rice breeding programs aiming at developing rice varieties with durable blast resistance based on a combination of resistance genes. Centro Internactional de Agricultura Tropical (CIAT) institute where the research was carried out     

Durable resistance to rice blast disease-environmental influences

Summary Blast is one of the most serious diseases of rice worldwide. The pathogen,Pyricularia grisea, can infect nearly all parts of the shoot and is commonly found on the leaf blade and the panicle neck node. Host resistance is the most desirable means of managing blast, especially in developing countries. Rice cultivars with durable blast resistance have been recognized in several production systems. The durable resistance of these cultivars is associated with polygenic partial resistance that shows no evidence of race specificity. This partial resistance is expressed as fewer and smaller lesions on the leaf blade but latent period does not appear to be an important component. Partial resistance to leaf blast is positively correlated with partial resistance to panicle blast, although some cultivars have been found showing leaf-blast susceptibility and panicle-blast resistance. A diverse set of environmental factors can influence the expression of partial resistance, including temperature, duration of leaf-wetness, nitrogen fertilization, soil type, and water deficit. Because of the great diversity of rice-growing environments, resistance that proves durable in one system may or may not prove useful in another. In highly blast-conducive environments, other means of disease management must be applied to assist host-plant resistance.     

Genetic variation in resistance to blast disease (Pyricularia oryzae Cavara) in Japanese rice (Oryza sativa L.), as determined using a differential system

A total of 324 Japanese rice accessions, including landrace, improved, and weedy types were used to 1) investigate genetic variations in blast resistance to standard differential isolates, and 2) across the genome using polymorphism data on 64 SSR markers. From the polymorphism data, the accessions were classified into two clusters. Accessions from irrigated lowland areas were included mainly in cluster I, and upland and Indica types were mainly in cluster II. The accessions were classified into three resistance subgroups, A2, B1 and B2, based on the reaction patterns to blast isolates. The accessions in A2 were postulated to have at least two resistance genes Pish and Pik-s, whereas those in B1 had various combinations of the resistance genes Pish, Pia, Pii, Pi3, Pi5(t), and Pik alleles. The B2 accessions were resistant to almost all isolates, and many accessions of cluster II were included, and had Pish, Pia, Pii, Pi3, Pi5(t), certain Pik, Piz and Pita alleles, and unknown genes. The frequencies of accessions of B1 originating in Hokkaido, and those of B2 originating in the Kanto and Tohoku regions were remarkably higher than in the other regions.     

Molecular tagging of a gene for resistance to brown planthopper in rice (Oryza sativa L.)

An introgression line derived from an interspecific cross between Oryzasativa and Oryza officinalis, IR54741-3-21-22 was found to beresistant to an Indian biotype of brown planthopper (BPH). Genetic analysisof 95 F₃ progeny rows of a cross between the resistant lineIR54741-3-21-22 and a BPH susceptible line revealed that resistance wascontrolled by a single dominant gene. A comprehensive RAPD analysisusing 275 decamer primers revealed a low level of (7.1%) polymorphismbetween the parents.RAPD polymorphisms were either co-dominant (6.9%), dominant forresistant parental fragments (9.1%) or dominant for susceptible parentalfragments (11.6%). Of the 19 co-dominant markers, one primer,OPA16, amplified a resistant parental band in the resistant bulk and asusceptible parental band in the susceptible bulk by bulked segregantanalysis. RAPD analysis of individual F₂ plants with the primerOPA16 showed marker-phenotype co-segregation for all, with only onerecombinant being identified. The linkage between the RAPD markerOPA16₉₃₈ and the BPH resistance gene was 0.52 cM in couplingphase. The 938 bp RAPD amplicon was cloned and used as a probe on122 Cla I digested doubled haploid (DH) plants from aIR64xAzucena mapping population for RFLP inheritance analysis and wasmapped onto rice chromosome 11. The OPA16₉₃₈ RAPD markercould be used in a cost effective way for marker-assisted selection of BPHresistant rice genotypes in rice breeding programs.     

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.     

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.     

Partial resistance to neck blast influenced by stage of panicle development and rice genotype

Summary Neck nodes of eight rice genotypes were inoculated with a virulent isolate of the blast pathogen at four slightly different, increasingly older stages of panicle development shortly after flowering. Resistance to infection as well as resistance to growth of the pathogen after infection was assessed. Significant differences between genotypes were found for both components of resistance. Resistance to growth of the pathogen sharply increased with aging of the neck node as was indicated by a large reduction of the length of the lesions on the culm, but resistance to infection did not change much. The lesion length was closely related to yield loss. A small delay in time of infection can lead to a large decrease in yield reduction. Meaningful comparison of partial resistance to neck blast between genotypes therefore requires infection at an identical stage of panicle development. In the field, where panicles are in different stages of development, selection of genotypes that do show the symptoms of neck blast together with relatively little yield loss in infected panicles is recommended.     

RFLP/AFLP mapping of a brown planthopper (Nilaparvata lugens Stål) resistance gene Bph1 in rice

We have constructed a linkage map of the rice brown planthopper (BPH)resistance gene, Bph1. RFLP and AFLP markers were selected by thebulked segregant analysis and used in the mapping study of 262 F₂sthat were derived from a cross of `Tsukushibare', a susceptible japonica cultivar, and `Norin-PL3', an authentic japonicaBph1-introgression line. Twenty markers were mapped within a 28.9-cMregion containing the Bph1 locus on the long arm of rice chromosome12. Combining the result of segregation analysis of BPH resistance by themass seedling test and that of the markers, the Bph1 locus wasmapped within a 5.8-cM region between two flanking markers. The closestAFLP markers, em5814N and em2802N, was at 2.7 cM proximal to theBph1 locus. Together with the previously constructed high-resolutionmap of bph2 locating the locus at ca. 10 cM proximal to the Bph1 locus, this improved version of the linkage map would facilitatepyramiding these two important BPH resistance genes.     

Identification of quantitative trait loci (QTLs) for the characters of vascular bundles in peduncle related to indica-japonica differentiation in rice (Oryza sativa L.)

The number of vascular bundles in peduncle and the ratio of vascular bundles to primary rachis branches (V/R ratio)distinguishable between indica andjaponica, are the traits associated with the processes of differentiation between indica and japonica inrice (Oryza sativa L.). In this paper a doubled-haploid population derived from the F₁ hybrid of a cross between anindica cultivar and a japonicacultivar was used to map quantitative trait loci(QTLs) controlling numbers of vascular bundles in peduncle, primary rachis branches and the V/R ratio. For vascular bundles, three QTLs were detected and they collectively explained 58.8% of the total variation. Among them, the QTLqVB-8 with the largest effect,located on chromosome 8, individually accounted for 31.1% of the total variation. Two QTLs controlling primary rachis branches, located on chromosome 8and 10 respectively, were identified and they individually explained 10.5% and18.0% of the total variation respectively. Three QTLs for the V/R ratio, mapped on chromosome 1, 2 and 8, respectively,jointly explained 61.3% of the total variation. Of the three QTLs, the QTL qV/R-1 with the largest additive effect,explained 25.3% of the total variation,was located on chromosome 1 and found to be closely linked to the gene sh-2, a major gene underlying grain-shattering ability. In addition, four and two pairs of significant epistatic QTLs were detected for vascular bundles and the V/R ratio,respectively, but none for rachis branches. Our results suggested that the numbers of vascular bundles and primary rachis branches were independently controlled by different polygenic systems, but the two polygenic systems shared a fraction of quantitative trait loci. The present study also demonstrated that the chromosome region carrying the QTL qV/R-1 for the V/R ratio and the gene sh-2 might play an important role in the processes ofindica-japonica differentiation in rice (Oryza sativa L.). This revised version was published online in August 2006 with corrections to the Cover Date.     

QTL analysis of seed dormancy in rice (Oryza sativa L.)

Effective cumulative temperature (ECT) after heading would be a more reasonable parameter for seed sampling of pre-harvest sprouting/seed dormancy (SD) tests in segregating populations than the days after flowering. SD is an important agronomic trait associated with grain yielding, eating quality and seed quality. To identify genomic regions affecting SD at different grain-filling temperatures, and to further examine the association between SD and ECT during grain-filling, 127 double haploid (DH) lines derived from a cross between ZYQ8 (indica)/JX17 (japonica) by anther culture were analyzed. The quantitative trait loci (QTLs) and their digenic epistasis for SD were identified using a molecular linkage map of this population. A total of four putative QTLs for SD (qSD-3, qSD-5, 6 and 11) were detected on chromosomes 3, 5, 6 and 11, together explaining 41.4% of the phenotypic variation. Nine pairs of digenic epistatic loci were associated with SD on all but chromosome 9, and their contributions to phenotypic variation varied from 2.87%–8.73%. The SD QTL on chromosome 3 was identical to the QTLs found in other mapping populations with different genetic backgrounds, which could be a desirable candidate for gene cloning and marker-assisted selection in rice breeding.     

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.     

Effect on leaf photosynthetic rate by leaf blast for rice cultivars with different types and levels of resistance

Summary The effect of an inoculation with Pyricularia oryzae (isolate P06-6) on net leaf photosynthetic rate of rice (Oryza sativa) was studied with four cultivars. Measurements were taken on the sixth leaf of the main culm of plants in the early tillering stage. On cultivars CO39, IR50 and IR64 a susceptible infection type developed, but a clear difference in relative infection efficiency of the cultivars was observed. The highest number of lesions developed on leaves of CO39, whereas the lowest number was found on leaves of IR64. For all three cultivars the effect of a single lesion on the reduction in net leaf photosynthetic rate was found to be equal to a reduction in leaf area of three times the area occupied by the visible lesion. On IR68, a cultivar with complete resistance, brown specks of pinpoint size appeared without any effect on net leaf photosynthetic rate.     

Molecular mapping of S-c, an F1 pollen sterility gene in cultivated rice

Hybrids between indica and japonica rice varieties usually show partial sterility, and are a major limiting factor in the utilization of heterosis at subspecific level. When studying male-gamete (pollen) abortion, a possibly important cause for sterility, six loci (S-a, S-b, S-c, S-d, S-e and S-f) for F₁ pollen sterility were identified. Here we report genetic and linkage analysis of S-c locus using molecular markers in a cross between Taichung 65, a japonica variety carrying allele S-c ^j, and its isogenic line TISL5, carrying alleleS-c ^j. Our results show that pollen sterility occurring in the hybrids is controlled by one locus. We used 208 RFLP markers, as well as 500 RAPD primers, to survey the polymorphism between Taichung 65 and TISL5. Six RFLP markers located on a small region of chromosome 3, detected different RFLP patterns. Co-segregation analysis of fertility and RFLP patterns with 123 F₂ plants confirmed that the markers RG227, RG391, R1420 were completely linked with the S-c locus. The genetic distances between the markers C730, RG166 and RG369 and the S-c locus were 0.5 cM, 3.4 cM, and 3.4 cM respectively. Distorted F₂ ratios were also observed for these 4 RFLP markers in the cross. This result suggests that the `one locus sporo-gametophytic' model could explain F₁ hybrid pollen sterility in cultivated rice. RG227, the completely linked marker, has been converted to STS marker for marker-assisted selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

A new gene for resistance in rice to Asian rice gall midge (Orseolia oryzae Wood Mason) biotype 1 population at Raipur, India

The Asian rice gall midge, Orseolia oryzae Wood Mason (Diptera: Cecidomyiidae), is a major pest of rice in several South and South East Asian countries. The maggots feed internally on the growing tips of the tillers and transform them into tubular galls, onion leaf-like structures called ‘silver shoots’ resulting into severe yield loss to the rice crop. We studied the mode of inheritance and allelic relationships of the resistance genes involved in resistant donor Line 9, a sib of a susceptible cultivar ‘Madhuri’. The segregation behaviour of F₁, F₂ and F₃ populations of the cross between Line 9 and susceptible cultivar MW10 confirmed the presence of a single dominant gene for resistance. Tests of allelism with all the known genes giving resistance to this population indicated that Line 9 possessed a new gene which was designated Gm 9 This revised version was published online in July 2006 with corrections to the Cover Date.     

A dominant gene in rice for resistance to white-backed planthopper and its relationship to other plant characteristics

Summary The inheritance of resistance to white-backed planthopper, Sogatella furcifera Horvath, was investigated in a rice, Oryza sativa L., cultivar N22. Resistance to the white-backed planthopper in the cross IR30×N22 appears to be governed by a single dominant gene-designated Wbph. The classification for various characteristics of 397 F₃ families of the IR30×N22 cross confirmed earlier results about the monogenic dominant control of resistance to brown planthopper, green leafhopper, and bacterial leaf blight, and about the monogenic recessive control of short stature. Additionally, the genes governing plant height and resistance to white-backed planthopper, brown planthopper, green leafhopper, and bacterial leaf blight were found to segregate independently of each other in these 397 F₃ families.     

Evaluation of a simple technique for characterizing rice root systems in relation to drought resistance

Summary Drought is a constraint to rice yields in much of Asia's rainfed rice crop. Although genotypic variation for root system characteristics is well documented in this semiaquatic species, little improvement of rice for drought resistance has been attempted. No simple method to evaluate root systems exists. In this paper we report the development and testing of a simple device to measure the pulling force required to uproot rice seedlings. The force required to pull rice seedlings from the paddy soil was correlated with root weight, root branching and thick root number. The capability to separate rice cultivars by this technique and relationship of the root pulling force to other drought screening methods are illustrated.     

水稻圆粒基因RS的鉴定和定位

阐明水稻籽粒大小相关基因的遗传和分子机制对水稻产量形成具有重要意义。利用甲基磺酸乙酯(ethyl methanesulfonate, EMS)诱变粳稻品种宁粳3号筛选获得圆粒突变体round seed (rs)。遗传分析表明,突变体rs圆粒表型由单隐性核基因控制。颖壳扫描电镜观察发现,rs籽粒变圆主要是细胞数目改变导致的。在突变体rs中,细胞周期相关基因的表达较野生型显著升高。将RS定位在第3染色体短臂标记RM3413与N3-5之间,物理距离约589 kb。RS突变影响BR信号途径,改变了粒型相关基因的表达。本研究有助于阐明水稻籽粒发育的分子机制。     

Response to selection for high and low partial resistance to leaf blast in F2 populations of three rice crosses

Summary Divergent selection for higher and lower partial resistance to leaf blast was applied in F₂ populations of crosses between the rice cultivars IR36, IR64 and CO39 after exposure to a virulent isolate. IR36 and IR64 are partially resistant while CO39 is highly susceptible. As selection criteria the number of sporulating lesions in leaves of the main culm or the lesion density in the topmost leaf relative to other F₂ plants with the same stage of development were used. A highly significant and meaningful response to selection was obtained in most cases, but the heritability was low. Realized heritabilities varied from 0.14 to 0.25 depending on the cross and were similar for both selection criteria. Selection for improved partial resistance to leaf blast is possible as early as the F₂. The efficiency of selection is probably much higher if replicated tests could be made, and better results are therefore expected if selection among F₃ lines is carried out. The results indicated that the relatively low infection efficiency in IR36 and IR64 is oligo- or polygenically controlled.     

QTL mapping for biomass and physiological parameters linked to resistance mechanisms to ferrous iron toxicity in rice

Lowland rice is often subject to iron toxicity which may lead to yield reduction. In order to cope with this nutrient disorder, plants have developed resistance strategies. The aim of this research was to assess morphological and physiological parameters linked to iron toxicity resistance mechanisms and to identify quantitative trait loci (QTLs) involved in their genetic determinism. A segregating population consisting of 164 recombinant inbred lines (RILs) derived from a cross between Azucena and IR64 was tested twice in hydroponics at the vegetative stage at 0 and 250 mg Fe²⁺ l⁻¹. Morphological traits were measured on all 164 RILs. Physiological traits, which were too time-consuming to allow their measurement on all the population, were measured on the two parents and extreme individuals only, selected on the basis of their leaf bronzing index and shoot dry weight. A total of 24 putative QTLs was identified on chromosomes 1, 2, 3, 4, 7 and 11 for leaf bronzing index, shoot water content, shoot and root dry weight, relative variation of shoot and root dry weight, shoot iron concentration, stomatal resistance and chlorophyll content index. Several QTLs were detected in overlapping regions for different parameters. The pertinence of phenotyping extreme RILs only for a QTL analysis is discussed in this study. The QTL analysis allowed to better understand the physiological response of rice in the presence of an excess of ferrous iron, inclusive the relations existing between the stomata closure, the shoot water content reduction and the oxidative stress linked to these growth conditions.     

Inheritance of resistance to rice stripe virus in rice line `BL 1'

Rice stripe is the most serious virus disease in temperate rice-growing countries. The most economical and environmentally safe practice for controlling this disease is virus-resistant cultivars. ‘BL 1’ is an elite germplasm line with the blast resistance gene Pib, and has been used as a differential line for testing the pathogenicity of the blast fungus. We found that certain progenies from BL 1 showed resistance to both blast and rice stripe virus (RSV). The objectives of this study were to evaluate the RSV resistance in the field and under artificial conditions, to assess the reaction to the insect vector(small brown plant hopper, SBPH), and to examine its inheritance and its relationship to blast resistance in BL 1.BL 1 was susceptible to SBPH, but resistant to RSV in field and artificial inoculation tests. The inheritance of RSV resistance in F₃ lines from the cross Nipponbare (NPB)/BL 1 was studied using artificial inoculation with a population of viruliferous SBPH. A serological assay for RSV infection using an enzyme-linked immunosorbent assay (ELISA) was used. RSV resistance in BL 1 was controlled by a single major gene with incomplete dominance. The locus responsible for RSV resistance was genetically independent of the blast resistance gene Pib. The resistance gene for RSV infection in BL 1 was also independent of Stvb-i, a gene widely distributed in resistant Japanese cultivars. Resistance to RSV must be diversified in rice cultivars considering the potential for future emergence of new RSV strains. The new resistance gene identified in BL 1, which has improved plant type and blast resistance, is considered useful for breeding RSV-resistant cultivars in japonica rice. This revised version was published online in August 2006 with corrections to the Cover Date.