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.     

Allele-mining of rice blast resistance genes at AC134922 locus

The AC134922 locus is one of the most rapidly evolving nucleotide binding site-leucine-rich repeat (NBS-LRR) gene family in rice genome. Six rice blast resistance (R) genes have been cloned from this locus and other two resistance candidate genes, Pi34 and Pi47, are also mapped to this complex locus. Therefore, it seems that more functional R genes could be identified from this locus. In this study, we cloned 22 genes from 12 cultivars based on allele-mining strategy at this locus and identified 6 rice blast R genes with 4 of them recognizing more than one isolates. Our result suggests that gene stacking might be the evolutionary strategy for complex gene locus to interact with rapidly evolving pathogens, which might provide a potential way for the cloning of durable resistance genes. Moreover, the mosaic structure and ambiguous ortholog/paralog relationships of these homologous genes, caused by frequent recombination and gene conversion, indicate that multiple alleles of this complex locus may serve as a reservoir for the evolutionary novelty of these R genes.     

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

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

Identification of rice sheath blight and blast quantitative trait loci in two different O. sativa/O. nivara advanced backcross populations

Two accessions of Oryza nivara, a wild ancestral species of rice (O. sativa) identified as being moderately resistant to sheath blight and leaf blast disease, were used as donor parents to develop two advanced backcross populations with the US rice cultivar, Bengal, as the recurrent parent. The O. nivara donor parent for Wild-1 (252 BC2F1 lines) was acc. IRGC100898 and for Wild-2 (253 BC2F1 lines) was acc. IRGC104705. Both populations were genotyped with 131 simple sequence repeat markers and the linkage maps covered 1,567.5 cM (Wild-1) and 1,312.2 cM (Wild-2). Sheath blight (ShB) disease was evaluated in both inoculated greenhouse and field conditions. Days to heading (DH), plant height (PH), and plant type (PT), confounding factors for sheath blight disease under field conditions, were recorded. Leaf blast disease was rated under inoculated greenhouse conditions. Multiple interval mapping identified qShB6 with resistance to sheath blight disease attributed to the O. nivara parent in the greenhouse. In the field, qShB6 also was the most significant ShB quantitative trait locus (QTL) in all trials, with resistance attributed to O. nivara. In addition, qShB1 and qShB3 were identified in all trials but were not always attributed to the same parent. The qShB6 QTL is in the same region as the DH-QTL, qDH6, and qShB1 is in the same region as the major PH-QTL, qPH1, suggesting that these ShB-QTL may be confounded by other traits. Although qShB3 did not have as large an effect as other loci, it was not confounded by either DH or PH. For leaf blast, qBLAST8-1 was found in both populations providing resistance to races IB1 and IB49, whereas qBLAST12 providing resistance to both races, was only found in Wild-2. Resistance was attributed to O. nivara for both QTL, and blast resistance genes have been previously reported in these regions.     

Detection of quantitative trait loci controlling extremely early heading in rice

To clarify the genetic basis of extremely early heading in rice, we conducted quantitative trait locus (QTL) analyses using F₂ populations from two genetically wide cross combinations, Hayamasari/Kasalath (HaF₂) and Hoshinoyume/Kasalath (HoF₂). Hayamasari and Hoshinoyume are extremely early-heading japonica cultivars. Photoperiod sensitivity is completely lost in Hayamasari and weak in Hoshinoyume. Three QTLs, QTL(chr6), QTL(chr7), and QTL(chr8), for days-to-heading (DTH) in HaF₂ were detected on chromosomes 6, 7, and 8, respectively, and QTL(chr6) and QTL(chr7) were detected in HoF₂. On the basis of the chromosomal locations, QTL(chr6), QTL(chr7), and QTL(chr8) may be likely to be Hd1, Hd4, and Hd5, respectively, which had been detected previously as QTLs for DTH in an F₂ population of Nipponbare × Kasalath. Alleles of QTL(chr7) decreased DTH dramatically in both Hayamasari and Hoshinoyume, suggesting that QTL(chr7) has a major role in determining extremely early heading. In addition, allele-specific interactions were detected between QTL(chr6), QTL(chr7) and QTL(chr8). This result suggests that not only allelic differences but also epistatic interactions contribute to extremely early heading. QTL(chr8) was detected in HaF₂, but not in HoF₂, suggesting that it determines the difference in DTH between Hayamasari and Hoshinoyume. A major QTL was also detected in the region of QTL(chr8) in QTL analysis using an F₂ population of Hayamasari × Hoshinoyume. This result supports the idea that QTL(chr8) is a major factor that determines the difference in DTH between Hayamasari and Hoshinoyume, and is involved in photoperiod sensitivity.     

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F₂ progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.     

Mapping of the QTL (quantitative trait locus) conferring partial resistance to leaf blast in rice cultivar Chubu 32

The rice cultivar Chubu 32 possesses a high level of partial resistance to leaf blast. The number and chromosomal location of genes conferring this resistance were detected by restriction fragment length polymorphism (RFLP) linkage mapping and quantitative trait locus (QTL) analysis. For the mapping, 149 F₃ lines derived from the cross between rice cultivar Norin 29, with a low level of partial resistance, and Chubu 32 were used, and their partial resistance to leaf blast was assessed in upland nurseries. A linkage map covering six chromosomes and consisting of 36 RFLP markers was constructed. In the map, only one significant QTL (LOD>2.0) for partial resistance was detected on chromosome 11. This QTL explained 45.6% of the phenotypic variation. The segregation ratio of the F3 lines was 3:1 for partial resistance to susceptibility. These results suggest that the partial resistance in Chubu 32 is controlled by a major gene. Received: 15 March 2001 / Accepted: 13 August 2001     

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     

The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance.     

Accumulation of quantitative trait loci conferring broad-spectrum clubroot resistance in Brassica oleracea

Throughout the world, clubroot disease is one of the most damaging diseases affecting Brassica oleracea. To develop marker-assisted selection (MAS) that could assist the incorporation of durable clubroot resistance (CR) into cultivars, previous genetic analyses have identified several CR quantitative trait loci (CR–QTL). However, the independent and cumulative effects of each CR locus against various isolates have rarely been tested. Previously, we identified one major CR–QTL and four minor CR–QTL in the F2 plants from broccoli doubled haploid (DH) line × cabbage DH line of B. oleracea. In the present study, to clarify their effectiveness for controlling disease involving various isolates, inoculation testing was conducted in genotypes with various combinations of the CR genes, which were selected using the DNA markers closely associated with each CR–QTL. In exploring the overall disease incidence, it was apparent that a single involvement of the major CR gene located in the PbBo(Anju)1 locus, or accumulation of CR genes in the minor CR–QTL, is not enough to confer sufficient resistance. One major CR gene in the QTL PbBo(Anju)1 locus plus two to three minor CR genes conferred moderate resistance. The genotype in which all of the CR genes locating in the five QTL including PbBo(Anju)1 were accumulated showed the highest resistance, and it was broadly resistant against six isolates. Accumulation of several CR genes by MAS is necessary to conduct CR breeding in B. oleracea. Our developed DNA markers can be used efficiently to make selections of required loci for the acquisition of resistance, and use of these markers will be a powerful tool for CR breeding in B. oleracea.     

QTL mapping of panicle blast resistance in japonica landrace heikezijing and its application in rice breeding

The rice blast caused by Magnaporthe oryzae is one of the most devastating diseases worldwide, and the panicle blast could result in more loss of yield in rice production. However, the quantitative trait loci (QTLs) and genes related to panicle-blast resistance have not been well studied due to the time-consuming screening methodology involved and variation in symptoms. The QTLs for panicle blast resistance have been mapped in a population of 162 RILs (recombination inbreeding lines), derived from a cross between a highly blast-resistant rice landrace, Heikezijing, and a susceptible variety, Suyunuo. Two QTLs for panicle-blast resistance, qPbh-11–1 and qPbh-7-1, were identified, which were distributed on chromosomes 11 and 7. The QTL qPbh-11–1 was stably detected in three independent experiments, at Nanjing in 2013 and 2014 and at Hainan in 2014, located between the region of RM27187 and RM27381 on the distal end of chromosome 11 far from the reported resistant loci Pb1 and qPbm11 for panicle blast. The QTL qPbh-7-1 was detected only at Nanjing in 2013 and located between the region of M18 and RM3555 on chromosome 7. With marker-assisted selection (MAS) three introgression lines with the major panicle blast-resistance QTL qPbh-11–1 were developed from a recurrent parent Nanjing 44 (NJ44) and the panicle resistance of introgression lines was improved 46.36–55.47 % more than NJ44. Based on the results provided, Heikezijing appears to be a valuable source for panicle blast resistance.     

Genome‐wide association study identifies an NLR gene that confers partial resistance to Magnaporthe oryzae in rice

Because of the frequent breakdown of major resistance (R) genes, identification of new partial R genes against rice blast disease is an important goal of rice breeding. In this study, we used a core collection of the Rice Diversity Panel II (C‐RDP‐II), which contains 584 rice accessions and are genotyped with 700 000 single‐nucleotide polymorphism (SNP) markers. The C‐RDP‐II accessions were inoculated with three blast strains collected from different rice‐growing regions in China. Genome‐wide association study identified 27 loci associated with rice blast resistance (LABRs). Among them, 22 LABRs were not associated with any known blast R genes or QTLs. Interestingly, a nucleotide‐binding site leucine‐rich repeat (NLR) gene cluster exists in the LABR12 region on chromosome 4. One of the NLR genes is highly conserved in multiple partially resistant rice cultivars, and its expression is significantly up‐regulated at the early stages of rice blast infection. Knockout of this gene via CRISPR‐Cas9 in transgenic plants partially reduced blast resistance to four blast strains. The identification of this new non‐strain specific partial R gene, tentatively named rice blast Partial Resistance gene 1 (PiPR1), provides genetic material that will be useful for understanding the partial resistance mechanism and for breeding durably resistant cultivars against blast disease of rice.     

A “defeated” rice resistance gene acts as a QTL against a virulent strain of Xanthomonas oryzae pv. oryzae

The genetic components responsible for qualitative and quantitative resistance of rice plants to three strains (CR4, CXO8, and CR6) of Xanthomonas oryzae pv. oryzae (Xoo) were investigated using a set of 315 recombinant inbred lines (RILs) from the cross Lemont (japonica) × Teqing (indica) and a complete linkage map with 182 well distributed RFLP markers. We mapped a major gene (Xa4) and ten quantitative trait loci (QTLs) which were largely responsible for segregation of the resistance phenotype in the RILs. The Teqing allele at the Xa4 locus, Xa4 ^T , acted as a dominant resistance gene against CR4 and CXO8. The breakdown of Xa4 ^T -associated resistance mediated by the mutant allele at the avrXa4 locus in the virulent strain CR6 results from significant changes in both gene action (lose of dominance) and the magnitude of gene effect (≈50% reduction). Nevertheless, Xa4 ^T still acted as a recessive QTL with a significant residual effect against CR6. The mutant alleles at the avrXa4 locus in CXO8 and CR6 that lead to a reduction in effect, or “breakdown”, of Xa4 ^T were apparently accompanied by corresponding penalties for their fitness. The quantitative component of resistance to Xoo in the RILs was largely due to a number of resistance QTLs. Most resistance QTLs mapped to genomic locations where major resistance genes and/or QTLs for resistance to Xoo, blast and sheath blight were identified in the same cross. Most QTLs showed consistent levels of resistance against all three Xoo strains. Our results suggest that a high level of durable resistance to Xoo may be achieved by the cumulative effects of multiple QTLs, including the residual effects of “defeated” major resistance genes.     

ARCHIPELAGO: a dedicated resource for exploiting past, present, and future genomic data on disease resistance regulation in rice

Large amounts of expression data dealing with biotic stresses in rice have been produced in the past 5 years. Here, we extensively review approximately 70 publications and gather together information on more than 2,500 genes of the rice defense arsenal. This information was integrated into the OryGenesDB database. Several genes (e.g., metallothioneins and PBZ1) appear to be hallmarks of rice-pathogen interactions. Cross-referencing this information with the rice kinome highlighted some defense genes and kinases as possible central nodes of regulation. Cross referencing defense gene expression and quantitative trait loci (QTL) information identified some candidate genes for QTL. Overall, pathogenesis-related genes and disease regulators were found to be statistically associated with disease QTL. At the genomic level, we observed that some regions are richer than others and that some chromosomes (e.g., 11 and 12), which contain a lot of resistance gene analogs, have a low content of defense genes. Finally, we show that classical defense genes and defense-related genes such as resistance genes are preferentially organized in clusters. These clusters are not always coregulated and individual paralogs can show specific expression patterns. Thus, the rice defense arsenal has an ARCHIPELAGO-like genome structure at the macro and micro level. This resource opens new possibilities for marker-assisted selection and QTL cloning.     

Molecular mapping of four blast resistance genes using recombinant inbred lines of 93-11 and nipponbare

Molecular mapping of new blast resistance genes is important for developing resistant rice cultivars using marker-assisted selection. In this study, 259 recombinant inbred lines (RILs) were developed from a cross between Nipponbare and 93-11, and were used to construct a 1165.8-cM linkage map with 131 polymorphic simple sequence repeat (SSR) markers. Four major quantitative trait loci (QTLs) for resistance to six isolates of Magnaporthe oryzae were identified: qPi93-1, qPi93-2, qPi93-3, and qPiN-1. For the three genes identified in 93-11, qPi93-1 is linked with SSR marker RM116 on the short arm of chromosome 11 and explains 33% of the phenotypic variation in resistance to isolate CHE86. qPi93-2 is linked with SSR marker RM224 on the long arm of chromosome 11 and accounts for 31% and 25% of the phenotypic variation in resistance to isolates 162-8B and ARB50, respectively. qPi93-3 is linked with SSR marker RM7102 on chromosome 12 and explains 16%, 53%, and 28% of the phenotypic variation in resistance to isolates CHE86, ARB52, and ARB94, respectively. QTL qPiN-1 from Nipponbare is associated with SSR marker RM302 on chromosome 1 and accounts for 34% of the phenotypic variation in resistance to isolate PO6-6. These new genes can be used to develop new varieties with blast resistance via marker-aided selection and to explore the molecular mechanism of rice blast resistance.