Two broad-spectrum blast resistance genes,<Emphasis Type="Italic"> Pi9</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) and<Emphasis Type="Italic"> Pi2</Emphasis>(<Emphasis Type="Italic">t</Emphasis>), are physically linked on rice chromosome 6

To understand the molecular basis of broad-spectrum resistance to rice blast, fine-scale mapping of the two blast resistance (R) genes, Pi9( t) and Pi2( t), was conducted. These two genes were introgressed from different resistance donors, previously reported to confer resistance to many blast isolates in the Philippines, and were mapped to an approximately 10-cM interval on chromosome 6. To further test their resistance spectrum, 43 blast isolates collected from 13 countries were used to inoculate the Pi2( t) and Pi9( t) plants. Pi9( t)-bearing lines were highly resistant to all isolates tested, and lines carrying Pi2( t) were resistant to 36 isolates, confirming the broad-spectrum resistance of these two genes to diverse blast isolates. Three RAPD markers tightly linked to Pi9( t) were identified using the bulk segregant analysis technique. Twelve positive bacterial artificial chromosome (BAC) clones were identified and a BAC contig covering about 100 kb was constructed when the Pi9( t) BAC library was screened with one of the markers. A high-resolution map of Pi9( t) was constructed using BAC ends. The Pi2( t) gene was tightly linked to all of the Pi9( t) markers in 450 F(2) plants. These data suggest that Pi9( t) and Pi2( t) are either allelic or tightly linked in an approximately 100-kb region. The mapping results for Pi9( t) and Pi2( t) provide essential information for the positional cloning of these two important blast resistance genes in rice.     

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.     

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.     

Use of Pi5(t) markers in marker-assisted selection to screen for cultivars with resistance to Magnaporthe grisea

Identification of the PCR markers tightly linked to genes that encode important agronomic traits is useful for marker-assisted selection (MAS). The rice Pi5(t) locus confers broad-spectrum resistance to Magnaporthe grisea, the causal agent of rice blast disease. It has been hypothesized that the Pi5(t) locus carries the same gene as that encoded by the Pi3(t) and Pii(t) loci. We developed three PCR-based dominant markers (JJ80-T3, JJ81-T3, and JJ113-T3) from three previously identified BIBAC clones—JJ80, JJ81, and JJ113—that are linked to the Pi5(t) locus. PCR analysis of 24 monogenic lines revealed that these markers are present only in lines that carry Pi5(t), Pi3(t), and Pii(t). PCR and DNA gel-blot analysis of candidate resistance lines using JJ80-T3, JJ81-T3, and JJ113-T3 indicated that Tetep is the likely donor of Pi5(t). Of the 184 rice varieties tested, 34 carried the JJ80-T3-, JJ81-T3-, and JJ113-T3-specific bands. Disease evaluation of those 34 varieties revealed that all conferred resistance to PO6-6. The genomic structure of three of these resistant varieties (i.e., IR72, Taebaeg, Jahyangdo) is most similar to that of Pi5(t). Our results demonstrate the usefulness of the JJ80-T3, JJ81-T3, and JJ113-T3 markers for MAS for M. grisea resistance.G.Yi and S.-K. Lee contributed equally to this work.     

利用微卫星标记鉴定水稻的稻瘟病抗性

应用水稻稻瘟病抗性基因Pid(t)紧密连锁的微卫星标记RM262对含有该抗病基因的品种地谷与感病品种江南香糯和8987的杂交F2群体进行遗传分析和抗性鉴定,结果表明,RM262的PCR扩增物在抗、感品种之间的多态性较好;在2个F2群体中,RM262和抗病基因间的重组率分别为5.74%和8.17%,应用该标记的抗性纯合和杂合带型选择抗性植株,其准确率可达98%以上。此外,还就分子标记辅助育种进行了讨论。     

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.     

Genetic and physical mapping of <Emphasis Type="Italic">Pi36</Emphasis>(t), a novel rice blast resistance gene located on rice chromosome 8

Blast resistance in the indica cultivar (cv.) Q61 was inherited as a single dominant gene in two F₂ populations, F₂-1 and F₂-2, derived from crosses between the donor cv. and two susceptible japonica cvs. Aichi Asahi and Lijiangxintuanheigu (LTH), respectively. To rapidly determine the chromosomal location of the resistance (R) gene detected in Q61, random amplified polymorphic DNA (RAPD) analysis was performed in the F₂-1 population using bulked-segregant analysis (BSA) in combination with recessive-class analysis (RCA). One of the three linked markers identified, BA1126₅₅₀, was cloned and sequenced. The R gene locus was roughly mapped on rice chromosome 8 by comparison of the BA1126₅₅₀ sequence with rice sequences in the databases (chromosome landing). To confirm this finding, seven known markers, including four sequence-tagged-site (STS) markers and three simple-sequence repeat (SSR) markers flanking BA1126₅₅₀ on chromosome 8, were subjected to linkage analysis in the two F₂ populations. The locus was mapped to a 5.8 cM interval bounded by RM5647 and RM8018 on the short arm of chromosome 8. This novel R gene is therefore tentatively designated as Pi36(t). For fine mapping of the Pi36(t) locus, five additional markers including one STS marker and four candidate resistance gene (CRG) markers were developed in the target region, based on the genomic sequence of the corresponding region of the reference japonica cv. Nipponbare. The Pi36(t) locus was finally localized to an interval of about 0.6 cM flanked by the markers RM5647 and CRG2, and co-segregated with the markers CRG3 and CRG4. To physically map this locus, the Pi36(t)-linked markers were mapped by electronic hybridization to bacterial artificial chromosome (BAC) or P1 artificial chromosome (PAC) clones of Nipponbare, and a contig map was constructed in silico through Pairwise BLAST analysis. The Pi36(t) locus was physically delimited to an interval of about 17.0 kb, based on the genomic sequence of Nipponbare.     

Genetic and physical mapping of a rice blast resistance locus,Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea

We have identified, genetically mapped and physically delineated the chromosomal location of a new rice blast resistance locus, designated Pi-CO39(t). This locus confers resistance to Magnaporthe grisea isolates carrying the AVR1-CO39 avirulence locus. The AVR1-CO39 locus is conserved in non-rice (cereals and grasses)-infecting isolates of M. grisea, making Pi-CO39(t) useful for engineering M. grisea resistance in rice and other cereals. The resistance in the rice line CO39 was inherited as a single dominant locus in segregating populations derived from F(2) and F(3) crosses between disease-resistant (CO39) and susceptible (51583) rice genotypes. Microsatellite, RFLP and resistance gene analog (RGA) markers were used to map the Pi-CO39(t) locus to a 1.2-cM interval between the probenazole-responsive ( RPR1) gene (0.2 cM) and RFLP marker S2712 (1.0 cM) on the short arm of rice chromosome 11. RFLP markers G320 and F5003, and resistance gene analogs RGA8, RGA38 and RGACO39 were tightly linked to the Pi-CO39(t) locus (no recombination detected in a sample of ~2400 gametes). A large-insert genomic library of CO39 was constructed in the binary plant transformation vector pCLD04541. A library screen using RGA8, RGA38 and probes derived from the ends of CO39 clones, as well as BAC end probes from the corresponding locus in the rice cv. Nipponbare, resulted in the assembly of three CO39 contigs of 180 kb, 110 kb and 145 kb linked to the Pi-CO39(t) locus. A 650-kb contig was also constructed representing the susceptible locus, pi-CO39(t), in the Nipponbare genome. The two genomes are highly divergent with respect to additions, deletions and translocations at the Pi-CO39(t) locus, as revealed by the presence or absence of mapping markers.     

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.     

Quantitative trait loci associated with leaf and neck blast resistance in recombinant inbred line population of rice (Oryza sativa).

Blast is an economically important disease of rice. To map genes controlling blast resistance, recombinant inbred lines (RIL) were developed from Khao Dawk Mali 105, an aromatic, blast-susceptible cultivar and the blast resistance donor, CT 9993-5-10-M (CT). A linkage map encompassing 2112 cM was constructed from 141 RILs using 90 restriction fragment length polymorphisms (RFLPs) and 31 simple sequence repeats (SSR). Virulent isolates of blast fungus were identified by screening differential host sets against 87 single-spore isolates collected from the north and northeast of Thailand. Fifteen virulent blast isolates were selected for leaf blast screening. Neck blast was evaluated both under natural conditions and controlled inoculations. Quantitative trait loci (QTLs) for broad resistance spectrum (BRS) to leaf blast were located on chromosomes 7 and 9. In particular, the QTL(ch9) was mapped near the Pi5(t) locus. The QTL(ch7) was located close to a previously mapped partial resistance QTL. Both loci showed significant allelic interaction. Genotypes having CT alleles at both QTL(ch7) and QTL(ch9) were the most resistant. Two neck-blast QTLs were mapped on chromosomes 5 and 6. The inconsistent map locations between the leaf and neck blast QTLs indicate the complexity of fixing both leaf and neck blast resistance. The coincidence of BRS and field resistance QTLs on chromosome 7 supports the idea that BRS may reflect the broad resistance spectrum to leaf blast in rice. These findings laid the foundation for the development of a marker-assisted scheme for improving Khoa Dawk Mali 105 and the majority of aromatic Thai rice varieties that are susceptible to blast.     

A novel gene, <Emphasis Type="Italic">Pi40(t)</Emphasis>, linked to the DNA markers derived from NBS-LRR motifs confers broad spectrum of blast resistance in rice

Rice blast disease caused by Magnaporthe grisea is a continuous threat to stable rice production worldwide. In a modernized agricultural system, the development of varieties with broad-spectrum and durable resistance to blast disease is essential for increased rice production and sustainability. In this study, a new gene is identified in the introgression line IR65482-4-136-2-2 that has inherited the resistance gene from an EE genome wild Oryza species, O. australiensis (Acc. 100882). Genetic and molecular analysis localized a major resistance gene, Pi40(t), on the short arm of chromosome 6, where four blast resistance genes (Piz, Piz-5, Piz-t, and Pi9) were also identified, flanked by the markers S2539 and RM3330. Through e-Landing, 14 BAC/PAC clones within the 1.81-Mb equivalent virtual contig were identified on Rice Pseudomolecule3. Highly stringent primer sets designed for 6 NBS-LRR motifs located within PAC clone P0649C11 facilitated high-resolution mapping of the new resistance gene, Pi40(t). Following association analysis and detailed haplotyping approaches, a DNA marker, 9871.T7E2b, was identified to be linked to the Pi40(t) gene at the 70 Kb chromosomal region, and differentiated the Pi40(t) gene from the LTH monogenic differential lines possessing genes Piz, Piz-5, Piz-t, and Pi-9. Pi40(t) was validated using the most virulent isolates of Korea as well as the Philippines, suggesting a broad spectrum for the resistance gene. Marker-assisted selection (MAS) and pathotyping of BC progenies having two japonica cultivar genetic backgrounds further supported the potential of the resistance gene in rice breeding. Our study based on new gene identification strategies provides insight into novel genetic resources for blast resistance as well as future studies on cloning and functional analysis of a blast resistance gene useful for rice improvement.     

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。     

Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

Fine mapping of a strong QTL of field resistance against rice blast, <Emphasis Type="Italic">Pikahei-1</Emphasis>(t), from upland rice Kahei,utilizing a novel resistance evaluation system in the greenhouse

Field resistances (FR) against rice blast are highly evaluated by breeders for their durability, in contrast to the conspicuous but often less durable true resistances. However, lack of efficient systems for evaluation of resistance has delayed their practical application. Kahei, an upland domestic cv., is known for its very high FR against rice blast. We fine-mapped its highest quantitative trait loci (QTL), qBFR4-1, using residual heterozygosity of recombinant inbred lines (RILs) and our semi-natural rice blast inoculation/evaluation system in the greenhouse, with comparable accuracy to the true resistance genes. This system enabled reproducible high-density infection, and consequently allowed quantification of the resistance level in individual plants. The target region was first narrowed down to about 1 Mb around at 32 Mb from the top of chromosome 4 in the Nipponbare genome, with the upland evaluation system assessing the F(7) generation of Koshihikari (lowland, FR: very weak) x Kahei (upland, FR: very strong) RILs. Then, F(9) plants (4,404)-siblings of hetero F(8) plants at the region-were inoculated with rice blast in a greenhouse using the novel inoculation system, and individual resistance levels were diagnosed for fine QTL analysis and graphical genotyping. Thus, the resistance gene was fine-mapped within 300 kb at 31.2-31.5 Mb on chromosome 4, and designated Pikahei-1(t). By annotation analysis, seven resistance gene analog (RGA) ORFs of nucleotide-binding-site and leucine-rich-repeat (NBS-LRR)-type were found in the center of the region as the most likely candidate counterparts of the resistance gene. This is similar in structure to the recently reported Pik cluster region, suggesting that most of the other dominant QTLs of the FRs may have similar RGA structures.     

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.     

水稻抗稻瘟病基因Pi-d（t）^1、Pi-b、Pi-tα^2的聚合及分子标记选择

冈46B(G46B)是水稻生产应用中的一个农艺性状十分优良的保持系,其主要的缺陷是稻瘟病抗性较弱,通过对地谷,BL-1,Pi-4号等三个分别含抗病基因Pi-d(t)^1、Pi-b、Pi-tα^2的稻瘟病抗性材料与G46B聚合杂交,并利用抗病基因连锁的分子标记对杂交后代进行辅助选择,在聚合杂交的F2代及B1C1代群体中共获得了15株含Pi-d(t)^1、Pi-b、Pi-tα^2等三个抗稻瘟病基因的材料,其可能的基因型分别为：三基因杂合体Pi-d(t)^1pi-d(t)^1,Pi-bpi-b／Pi-tα^2 pi-tα^2 4株,双基因杂合体10株,其中Pi-d(t)^1Pi-d(t)^1／Pi-bpi-b／Pi-tα^2pi-tα^2 6株,Pi-d(t)^1pi-d(t)^1／Pi-bpi-b／Pi-tα^2Pi-tα^2 3株,Pi-d(t)^1pi-d(t)^1,Pi-bPi-6,Pi-tα^2 pi-tα^2 1株,双基因纯合体Pi-d(t)^1Pi-d(t)^1/Pi-bpi-b/Pi-tα^2Pi-tα^2仅1株,这一研究结果为进一步改良G46B的稻瘟病抗性奠定了基础,同时这一研究结果表明利用分子标记可快速、有效地实现多个抗病基因的聚合,大大提高水稻抗病育种的效率。     

Genetics of resistance to anthracnose and identification of AFLP and RAPD markers linked to the resistance gene in PI 320937 germplasm of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medikus)

Anthracnose, caused by Colletotrichum truncatum, is a major disease problem and production constraint of lentil in North America. The research was conducted to examine the resistance to anthracnose in PI 320937 lentil and to identify molecular markers linked to the resistance gene in a recombinant inbred line (RIL) population developed from a cross of Eston lentil, the susceptible parent, and PI 320937, the resistant parent. A total of 147 F(5:6) RILs were evaluated for resistance to anthracnose in the greenhouse using isolate 95B36 of C. truncatum. Bulked segregant analysis (BSA) strategy was employed and two contrasting DNA bulks were constructed based on greenhouse inoculation of F(5)-derived F(6) RILs. DNA from the parents and bulks were screened with 700 RAPD primers and seven AFLP primer combinations. Analysis of segregation data indicated that a major dominant gene was responsible for resistance to anthracnose while variations in the resistance level among RILs could be the influences of minor genes. We designate the major gene as LCt-2. MapMaker analysis produced two flanking RAPD markers OPEO6(1250) and UBC-704(700) linked to LCt-2 locus in repulsion (6.4 cM) and in coupling (10.5 cM), respectively. Also, three AFLP markers, EMCTTACA(350) and EMCTTAGG(375) in coupling, and EMCTAAAG(175) in repulsion, were linked to the LCt-2 locus. These markers could be used to tag the LCt-2 locus and facilitate marker-assisted selection for resistance to anthracnose in segregating populations of lentil in which PI 320937 was used as the source of resistance. Also, a broader application of the linked RAPD markers was also demonstrated in Indianhead lentil, widely used as a source of resistance to anthracnose in the breeding program at the Crop Development Centre, University of Saskatchewan. Further selection within the few F(5:6) lines should be effective in pyramiding one or several of the minor genes into the working germplasm of lentil, resulting in a more durable and higher level of resistance.     

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.     

A Pid3 allele from rice cultivar Gumei2 confers resistance to Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. Using map-based strategy and in silico approach we isolated a new rice (Oryza sativa L.) blast resistance allele of Pid3, designated Pi25, from a stable blast resistance cultivar Gumei2. Overexpression analysis and complementation test showed that Pi25 conferred blast resistance to M. oryzae isolate js001-20. Sequence analysis showed that Pi25 was an intronless gene of 2772 nucleotides with single nucleotide substitution in comparison to Pid3 at the nucleotide position 459 and predicatively encoded a typical coiled coil-nucleotide binding site-leucine rich repeat (CC--NBS--LRR) protein of 924 amino acid residuals with 100% identity to Pid3 putative protein. The susceptible allele pi25 in Nipponbare contained a nonsense mutation at the nucleotide position 2209 resulting in a truncated protein with 736 amino acid residuals. In addition, 14 nucleotide substitutions resulting in 10 amino acid substitutions were identified between Pi25 and pi25 upstream the premature stop codon in the susceptible allele. Although the mechanism of Pi25/Pid3-mediated resistance needs to be further investigated, the isolation of the allele would facilitate the utilization of Pi25/Pid3 in rice blast resistance breeding program via transgenic approach and marker assisted selection.     

The blast resistance gene Pi37 encodes a nucleotide binding site leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1

The resistance (R) gene Pi37, present in the rice cultivar St. No. 1, was isolated by an in silico map-based cloning procedure. The equivalent genetic region in Nipponbare contains four nucleotide binding site-leucine-rich repeat (NBS-LRR) type loci. These four candidates for Pi37 (Pi37-1, -2, -3, and -4) were amplified separately from St. No. 1 via long-range PCR, and cloned into a binary vector. Each construct was individually transformed into the highly blast susceptible cultivar Q1063. The subsequent complementation analysis revealed Pi37-3 to be the functional gene, while -1, -2, and -4 are probably pseudogenes. Pi37 encodes a 1290 peptide NBS-LRR product, and the presence of substitutions at two sites in the NBS region (V239A and I247M) is associated with the resistance phenotype. Semiquantitative expression analysis showed that in St. No. 1, Pi37 was constitutively expressed and only slightly induced by blast infection. Transient expression experiments indicated that the Pi37 product is restricted to the cytoplasm. Pi37-3 is thought to have evolved recently from -2, which in turn was derived from an ancestral -1 sequence. Pi37-4 is likely the most recently evolved member of the cluster and probably represents a duplication of -3. The four Pi37 paralogs are more closely related to maize rp1 than to any of the currently isolated rice blast R genes Pita, Pib, Pi9, Pi2, Piz-t, and Pi36.