Development of PCR-based SNP markers for rice blast resistance genes at the<Emphasis Type="Italic"> Piz</Emphasis> locus

We assessed the utility of single-nucleotide polymorphisms (SNPs) and small insertion/deletion polymorphisms (InDels) as DNA markers in genetic analysis and breeding of rice. Toward this end, we surveyed SNPs and InDels in the chromosomal region containing the Piz and Piz-t rice blast resistance genes and developed PCR-based markers for typing the SNPs. Analysis of sequences from a blast-susceptible Japanese cultivar and two cultivars each containing one of these genes revealed that SNPs are abundant in the Piz and Piz-t regions (on average, one SNP every 248 bp), but the number of InDels was much lower. The dense distribution of SNPs facilitated the generation of SNP markers in the vicinity of the genes. For typing these SNPs, we used a modified allele-specific PCR method. Of the 49 candidate allele-specific markers, 33 unambiguously and reproducibly discriminated between the two alleles. We used the markers for mapping the Piz and Piz-t genes and evaluating the size of DNA segments introgressed from the Piz donor cultivar in Japanese near-isogenic lines containing Piz. Our findings suggest that, because of its ability to generate numerous markers within a target region and its simplicity in assaying genotypes, SNP genotyping with allele-specific PCR is a valuable tool for gene mapping, map-based cloning, and marker-assisted selection in crops, especially rice.Communicated by D.J. Mackill     

Identification and fine mapping of a major R gene to Magnaporthe oryzae in a broad-spectrum resistant germplasm in rice

Identification of R genes and development of associated molecular markers will facilitate their application in the development of crop cultivars resistant to disease. We evaluated the resistance of a resistant germplasm ??D69??, 10 monogenic lines, and model cultivar ??Nipponbare?? to 56 M. oryzae isolates of blast disease in rice. The results demonstrated that only D69 exhibited full-spectrum resistance among the 12 investigated materials. Resistance inheritance in D69 was analyzed using a stable isolate GD08T13 with strong pathogenicity, collected from diseased panicles. A single dominant R gene was revealed and designated as Pi51(t). Through linkage analysis and the development of new markers, Pi51(t) was subsequently delimited to an interval of ~100.8?kb flanked by markers Ind306 and RM19818, where Pi2, Pi9, Piz, Piz-t, Pigm(t), and Pi40(t) reside. Different genotypes identified by linked markers pB8, Pi9-2, zt56591, and T845, and different pathotypes to the same set of isolates, distinguished Pi51(t) from Pi2, Pi9, Piz, and Piz-t. The origin of Pi40(t) in wild rice suggests that Pi51(t) and Pi40(t) are different. Comparison of resistance spectra suggests multiple R genes in D69, making its resistance durable and valuable in breeding programs. The results of this work will facilitate future studies on cloning and functional analysis of blast resistance genes for rice improvement.     

Identification of Two Blast Resistance Genes in a Rice Variety, Digu

Blast, caused by Magnaporthe grisea is one of most serious diseases of rice worldwide. A Chinese local rice variety, Digu, with durable blast resistance, is one of the important resources for rice breeding for resistance to blast (M. grisea) in China. The objectives of the current study were to assess the identity of the resistance genes in Digu and to determine the chromosomal location by molecular marker tagging. Two susceptible varieties to blast, Lijiangxintuanheigu (LTH) and Jiangnanxiangnuo (JNXN), a number of different varieties, each containing one blast resistance gene, Pik^s, Pia, Pik, Pi‐b, Pi‐k^p, Pi‐ta², Pi‐ta, Pi‐z, Pi‐i, Pi‐k^m, Pi‐z^t, Pi‐t and Pi‐11, and the progeny populations from the crosses between Digu and each of these varieties were analysed with Chinese blast isolates. We found that the resistance of Digu to each of the two Chinese blast isolates, ZB13 and ZB15, were controlled by two single dominant genes, separately. The two genes are different from the known blast resistance genes and, therefore, designated as Pi‐d(t)1 and Pi‐d(t)2. By using bulked segregation method and molecular marker analysis in corresponding F₂ populations, Pi‐d(t)1 was located on chromosome 2 with a distance of 1.2 and 10.6 cM to restriction fragment length polymorphism (RFLP) markers G1314A and G45, respectively. And Pi‐d(t)2 was located on chromosome 6 with a distance of 3.2 and 3.4 cM to simple sequence repeat markers RM527 and RM3, respectively. We also developed a novel strategy of resistance gene analogue (RGA) assay with uneven polymerase chain reaction (PCR) to further tag the two genes and successfully identified two RGA markers, SPO01 and SPO03, which were co‐segregated toPi‐d(t)1 and Pi‐d(t)2, respectively, in their corresponding F₂ populations. These results provide essential information for further utilization of the Digu's blast resistance genes in rice disease resistance breeding and positional cloning of these genes.     

Conventional Breeding and Molecular Markers for Blast Disease Resistance in Rice (<i>Oryza sativa</i> L.)

Monogenic lines, which carried 23 genes for blast resistance were tested and used donors to transfer resistance genes by crossing method. The results under blast nursery revealed that 9 genes from 23 genes were susceptible to highly susceptible under the three locations (Sakha, Gemmeza, and Zarzoura in Egypt); Pia, Pik, Pik-p, Piz-t, Pita, Pi b, Pi, Pi 19 and Pi 20. While, the genes Pii, Pik-s, Pik-h, Pi z, Piz-5, Pi sh, Pi 3, Pi 1, Pi 5, Pi 7, Pi 9, Pi 12, Pikm and Pita-2 were highly resistant at the same locations. Clustering analysis confirmed the results, which divided into two groups; the first one included all the susceptible genes, while the second one included the resistance genes. In the greenhouse test, the reaction pattern of five races produced 100% resistance under artificial inoculation with eight genes showing complete resistance to all isolates. The completely resistant genes: Pii, Pik-s, Piz, Piz-5 (=bi2) (t), Pita (=Pi4) (t), Pita, Pi b and Pi1 as well as clustering analysis confirmed the results. In the F₁ crosses, the results showed all the 25 crosses were resistant for leaf blast disease under field conditions. While, the results in F₂ population showed seven crosses with segregation ratio of 15 (R):1 (S), two cross gave segregated ratio of 3 R:1 S and one gave 13:3. For the identi- fication of blast resistance genes in the parental lines, the marker K3959, linked to Pik-s gene and the variety IRBLKS-F5 carry this gene, which was from the monogenic line. The results showed that four genotypes; Sakha 105, Sakha 103, Sakha 106 and IRBLKS-F5 were carrying Pik-s gene, while was absent in the Sakha 101, Sakha 104, IRBL5-M, IRBL9-W, IRBLTACP1 and IRBL9-W(R) genotypes. As for Pi 5 gene, the results showed that it was present in Sakha 103 and Sakha 104 varieties and absent in the rest of the genotypes. In addition, Pita-Pita- 2 gene was found in the three Egyptian genotypes (Sakha 105, Sakha 101 and Sakha 104) plus IRBLTACP1 monogenetic. In F2 generation, six populations were used to study the inheritance of blast resistance and specific primers to confirm the ratio and identify the resistance genes. However, the ratios in molecular markers were the same of the ratio under field evaluation in the most population studies. These findings would facilitate in breeding programs for gene pyramiding and gene accumulation to produce durable resistance for blast using those genotypes.     

国外引进水稻种质资源的稻瘟病抗性基因检测与评价

为了筛选出福建省水稻稻瘟病重发区育种中可利用的新抗性资源,在福建省上杭县对156份外引水稻种质资源进行了2年田间自然诱发鉴定,并对Pi2、Pi9、Pi5、Pi54、Pikm、Pita、Pia和Pib等8个稻瘟病抗性基因做了分子检测。结果表明:156份资源对苗瘟、叶瘟、穗颈瘟和综合抗性表现抗病的分别有10份、14份、29份和26份,且苗瘟抗性级别与叶瘟抗性级别(r=0.816,P<0.01)、苗瘟抗性级别与穗颈瘟抗性级别(r=0.347,P<0.01)、以及叶瘟抗性级别与穗颈瘟抗性级别(r=0.344,P<0.01),均呈极显著正相关。分子标记检测到携带稻瘟病抗性基因Pi9、Pi2、Pi54、Pikm、Pi5、Pib、Pia和Pita的水稻资源分别有1、6、20、22、37、88、101和106份,其中携带稻瘟病抗性基因Pi9和Pi2的水稻资源的抗性表现较好,表现抗病的超过60%,携带其他稻瘟病抗性基因的水稻资源表现抗病的均在50%以下;水稻资源携带0~6个稻瘟病抗性基因,随着携带抗性基因数目增加,抗病率呈上升趋势,综合抗性等级呈下降趋势。进一步研究发现,携带Pi9+Pi5+Pikm+Pia、Pi5+Pib+Pita+Pikm+Pia和Pi2+Pi54+Pib+Pita+Pikm+Pia等3个基因型的水稻资源,稻瘟病抗性较好。最后,筛选了8份稻瘟病抗性较好的材料,提供育种者参考、利用。     

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.Yiwen Deng and Xudong Zhu contributed equally to this work.     

辽宁地区水稻资源抗稻瘟病基因的检测分析

为了明确辽宁地区水稻资源中抗稻瘟病基因的分布情况及抗病效应,选取辽宁地区水稻资源176份,鉴定了抗稻瘟病基因pi21、Pi36、Pi37、Pita、Pid2、Pid3、Pi5及Pib在这些材料中的分布情况,并接种鉴定了这些材料对稻瘟病的抗性。结果表明:176份供试材料中,83份对稻瘟病表现抗病,栽培稻、杂草稻及农家种中抗病品种所占的比率分别为41.48%、1.14%及4.54%。抗稻瘟病基因pi21、Pi36和Pi37在所有参试材料中均未检测到,且分别有74份、49份、47份、52份及89份材料携带Pita、Pid2、Pid3、Pi5及Pib的抗病等位基因。抗病基因绝大部分分布在栽培种中,农家种和杂草稻中分布较少。不含有抗稻瘟病基因和只携带单个抗病基因的材料对稻瘟病的抗性均较差,而抗病基因聚合可不同程度提高材料的抗性。经检测,不含有本试验鉴定的pi21等8个已克隆抗病基因的材料共32份,其中表现抗病的占21.87%;只携带1个抗稻瘟病基因的材料为52份,表现抗病的占17.31%;携带2个抗稻瘟病基因的材料为39份,表现抗病的占69.23%,其中以携带Pita+Pi5的材料最多(14份),且均表现抗病;携带3个抗稻瘟病基因的材料为31份,表现抗病的占77.42%,以携带Pita+Pid3+Pi5的材料抗性最强;携带4个抗稻瘟病基因的水稻材料22份,表现抗病的占72.73%,携带5个抗病基因的水稻材料未检测到。     

Development and validation of co-dominant gene based markers for <Emphasis Type="Italic">Pi9</Emphasis>, a gene governing broad-spectrum resistance against blast disease in rice

Rice production and grain quality are severely affected by blast disease caused by the ascomycetous fungus Magnaporthe oryzae. Incorporation of genes that confer broad-spectrum resistance to blast has been a priority area in rice breeding programs. The blast resistance gene Pi9 sourced from Oryza minuta has shown broad spectrum and durable resistance to blast world-wide. In the present study co-dominant gene-based markers were developed for the precise marker-assisted tracking of Pi9 in breeding programs. The developed markers were validated across a diverse set of cultivars including basmati, indica and japonica varieties. Two markers, Pi9STS-1 and Pi9STS-2, effectively differentiated Pi9 donors from all the indicas and commercial basmati varieties tested. However, these markers were monomorphic between Pi-9 donors (IRBL9-W and Pusa 1637) and japonica type varieties. An additional gene-derived CAPS marker Pi91F_ 2R was developed to differentiate Pi9 donors from japonicas and traditional basmati lines. The co-dominant markers developed in the present study will be of immense utility to rice breeders for precise and speedy incorporation of Pi-9 into susceptible rice varieties through marker-assisted selection.     

Introgression of Pi2 and Pi5 Genes for Blast (Magnaporthe oryzae) Resistance in Rice and Field Evaluation of Introgression Lines for Resistance and Yield Traits

Blast caused by Magnaporthe oryzae is the most devastating disease causing significant loss in rice production. The destructive nature of the disease is mainly due to the genetic plasticity of M. oryzae which complicates the breeding strategies. Blast can be effectively managed by the deployment of R genes. In this study, broad‐spectrum blast resistance genes Pi2 and Pi5 were introgressed independently into popular but blast susceptible rice variety, Samba Mahsuri (BPT5204) by applying marker‐assisted backcross breeding approach. Tightly linked markers AP5930 for Pi2 and 40N23r for Pi5 gene were used in foreground selection. Background selection helped to identify the lines with maximum recovery of recurrent parent genome (RPG). The RPG recovery in Pi2 introgression lines was up to 90.17 and 91.46% in Pi5 lines. Homozygous introgression lines in BC₃F₄ generation carrying Pi2 and Pi5 gene were field evaluated for blast resistance, yield per se and yield‐related traits. The lines showed resistance to leaf and neck blast in multilocation field evaluation. Improved BPT5204 lines with improvement for blast resistance were on par with original BPT5204 in terms of grain yield and grain features.     

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

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

Identification and fine mapping of a resistance gene to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in a space-induced rice mutant

Finding novel sources of resistance (R) to rice blast disease should facilitate breeding for improved resistance. The objectives of the present study were to evaluate reactions to blast and identify in a space-induced mutant an R gene to a representative isolate of rice blast pathogen. The mutant H4, its parent and twelve monogenic lines were evaluated for their responses to 35 isolates collected from Guangdong Province, China. H4 was found to be resistant to more isolates than its parent and the twelve monogenic lines, suggesting newly acquired resistance may be a function of one or more R genes. A representative isolate GD0193 was used to identify and map the R gene from H4. Genetic analysis revealed that resistance to the isolate GD0193 was controlled by a single dominant gene, designated Pi46(t). Linkage analysis using susceptible F2 individuals showed that Pi46(t) was mapped between the markers RM224 and RM27360 within 1.04 and 1.2 cM on the long arm of chromosome 11. Subsequently, Pi46(t) was delimited to an interval of approximately 183.7 kb flanked by the markers K67 and T94. These results provide essential information for the cloning of the Pi46(t) gene and will facilitate marker-assisted selection in rice breeding.     

Identification of a new resistance gene Pi-Da(t) from Dacca6 against rice blast fungus (Magnaporthe oryzae) in Jin23B background

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, severely threatens rice production worldwide. A new resistance gene, Pi-Da(t), was found in Dacca6, a local upland rice variety from the Philippines. It was mapped into a region between RM5529 and RM211 on chromosome 2, where no blast resistance gene has been reported, by bulk segregant analysis (BSA) in a BC₁F₂ population from a cross between Dacca6 and Jin23B. The presence of Pi-Da(t) in Jin23B background, an elite parental line preferred for its good grain quality and widely adopted in China??s three-line hybrid rice breeding program over the past 20?years, was verified by BSA and graphical genotyping with additional eight BC₁F₂ bulks. This work presents an example of combining gene mapping work and gene introgression with BSA and graphical genotyping methods in a backcross (BC) breeding scheme. Both the resistant Jin23B line and the linked markers will provide useful information and materials for marker-assisted breeding against blast disease in rice.     

Identification and mapping of Pi41, a major gene conferring resistance to rice blast in the Oryza sativa subsp. indica reference cultivar, 93-11

The Oryza sativa subsp. indica reference cultivar (cv.), 93-11 is completely resistant to many Chinese isolates of the rice blast fungus. Resistance segregated in a 3:1 (resistance/susceptible) ratio in an F₂ population from the cross between 93-11 and the japonica reference cv. Nipponbare, when challenged with two independent blast isolates. The chromosomal location of this monogenic resistance was mapped to a region of the long arm of chromosome 12 by bulk segregant analysis, using 180 evenly distributed SSR markers. Five additional SSR loci and nine newly developed PCR-based markers allowed the target region to be reduced to ca. 1.8 cM, equivalent in Nipponbare to about 800 kb. In the reference sequence of Nipponbare, this region includes an NBS-LRR cluster of four genes. The known blast resistance gene Pi-GD-3 also maps in this region, but the 93-11 resistance was distinguishable from Pi-GD-3 on the basis of race specificity. We have therefore named the 93-11 resistance Pi41. Seven markers completely linked to Pi41 will facilitate both marker-assisted breeding and gene isolation cloning.     

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

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

A set of near-isogenic lines of Indica-type rice variety CO 39 as differential varieties for blast resistance

Twenty-seven near-isogenic lines (NILs) with the genetic background of a blast-susceptible variety, CO 39, were developed by repeated backcrossing as a first set of a large number of differential varieties (DVs) with Indica-type genetic background. The NILs included 14 resistance genes—Pish, Pib, Piz-5, Piz-t, Pi5(t), Pik-s, Pik, Pik-h, Pik-m, Pik-p, Pi1, Pi7(t), Pita, and Pita-2—derived from 26 donor varieties. The reaction patterns of NILs against 20 standard isolates from the Philippines were similar to those of blast monogenic lines with the same resistance gene, except for those against two isolates that are avirulent to Pia in the genetic background of CO 39. A genome-wide DNA marker survey revealed that chromosome segments were introgressed in the regions where each resistance gene was previously mapped and most of the other chromosome regions in each NIL were CO 39 type. Segregation analysis of resistance and co-segregation analysis between resistance and DNA markers using F3 populations derived from the crosses between each NIL and the recurrent parent, CO 39, revealed a single-gene control of resistance and association between resistance and target introgressed segments. The morphological characters of each NIL were almost the same as those of the recurrent parent except for some lines, suggesting that these NILs can be used even under tropical conditions where Japonica-type DVs are not suitable for cropping. Thus, these NILs are useful not only as genetic tools for blast resistance study but also as sources of genes for breeding of Indica-type rice varieties.     

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.     

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

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

Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice

Three major genes (Pi1, Piz-5 and Pita) for blast resistance on chromosomes 11, 6 and 12, respectively, were fine-mapped and closely linked RFLP markers identified. New markers for Pi1 and Pita were found that were flanking the genes. The three genes were pyramided using RFLP markers. A PCR-based SAP (sequence amplified polymorphism) marker was used to identify Piz-5 in the segregating population. The plants carrying the two- and three-gene combinations that were tested for resistance to leaf blast in the Philippines and India indicated that combinations including Piz-5 have enhanced resistance than when it is present alone. The genes from the pyramided lines are at present being deployed into agronomically superior ricevarieties by marker-aided selection (MAS). Received: 20 June 1997 / Accepted: 14 September 1999     

Development and validation of functional marker targeting an InDel in the major rice blast disease resistance gene Pi54 (Pik h )

Rice blast is one of the most devastating diseases affecting the rice crop throughout the world. In molecular breeding for host plant resistance, functional markers are very useful for enhancing the precision and accuracy in marker-assisted selection (MAS) of target gene(s) with minimum effort, time and cost. Pi54 (which was earlier known as Pik ^h ) is one of the major blast resistance genes and has been observed to show resistance against many isolates of the blast pathogen in India. The gene has been cloned through map-based strategy and encodes a nucleotide-binding site?Cleucine-rich repeat (NBS?CLRR) domain-containing protein. In the present study, we carried out allele mining for this gene and identified a 144-bp insertion/deletion (InDel) polymorphism in the exonic region of the gene. A PCR-based co-dominant molecular marker targeting this InDel, named Pi54 MAS, was developed. Pi54 MAS was observed to perfectly co-segregate with blast resistance in a mapping population with no recombinants. Validation of this marker in 105 genotypes which are either susceptible or resistant to rice blast disease showed that the marker is polymorphic in most of the resistant?Csusceptible genotype combinations and is more accurate than the earlier reported markers for Pi54. Hence this functional, co-dominant marker is suggested for routine deployment in MAS of Pi54 in breeding programs.     

Identification of SSR markers for a broad-spectrum blast resistance gene Pi20(t) for marker-assisted breeding

The Pi20(t) gene was determined to confer a broad-spectrum resistance against diverse blast pathotypes (races) in China based on inoculation experiments utilizing 160 Chinese Magnaporthe oryzae (formerly Magnaporthe grisea) isolates, among which isolate 98095 can specifically differentiate the Pi20(t) gene present in cv. IR24. Two flanking and three co-segregating simple sequence repeat (SSR) markers for Pi20(t), located near the centromere region of chromosome 12, were identified using 526 extremely susceptible F₂ plants derived from a cross of Asominori, an extremely susceptible cultivar, with resistant cultivar IR24. The SSR OSR32 was mapped at a distance of 0.2 cM from Pi20(t), and the SSR RM28050 was mapped to the other side of Pi20(t) at a distance of 0.4 cM. The other three SSR markers, RM1337, RM5364 and RM7102, co-segregated with Pi20(t). RM1337 and RM5364 were found to be reliable markers of resistance conditioned by Pi20(t) in a wide range of elite rice germplasm in China. As such, they are useful tags in marker-assisted rice breeding programs aimed at incorporating Pi20(t) into advanced rice breeding lines and, ultimately, at obtaining a durable and broad spectrum of resistance to M. oryaze. Wei Li and Cailin Lei contributed equally to this work.