Mapping of avirulence genes in the rice blast fungus, Magnaporthe grisea, with RFLP and RAPD markers

Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoi and AVR1-Ku86 were closely linked to telomere RFLPs such as marker TelG (6 cM from AVR1-MedNoi) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.     

Molecular mapping of two cultivar-specific avirulence genes in the rice blast fungus <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

Rice blast, caused by the fungus Magnaporthe grisea, is a globally important disease of rice that causes annual yield losses. The segregation of genes controlling the virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in the interaction of rice and M. grisea. The segregation of avirulence and virulence was studied in 87 M. grisea F₁ progeny isolates from a cross of two isolates, Guy11 and JS153, using resistance-gene-differential rice cultivars. The segregation ratio indicated that avirulence and virulence in the rice cultivars Aichi–asahi and K59, respectively, are controlled by single major genes. Genetic analyses of backcrosses and full-sib crosses in these populations were also performed. The χ² test of goodness-of-fitness for a 1:1 ratio indicated that one dominant gene controls avirulence in Aichi-asahi and K59 in this population. Based on the resistance reactions of rice differential lines harboring known resistance genes to the parental isolates, two genetically independent avirulence genes, AVR–Pit and AVR–Pia, were identified. Genetic linkage analysis showed that the SSR marker m355–356 is closely linked to AVR–Pit, on the telomere of chromosome 1 at a distance of approximately 2.3 cM. The RAPD marker S487, which was converted to a sequence-characterized amplified region (SCAR) marker, was found to be closely linked to AVR–Pia, on the chromosome 7 telomere at a distance of 3.5 cM. These molecular markers will facilitate the positional cloning of the two AVR genes, and can be applied to molecular-marker-assisted studies of M. grisea populations.     

Development of microsatellite markers and construction of genetic map in rice blast pathogen Magnaporthe grisea

Magnaporthe grisea is the most destructive fungal pathogen of rice and a model organism for studying plant-pathogen interaction. Molecular markers and genetic maps are useful tools for genetic studies. In this study, based on the released genome sequence data of M. grisea, we investigated 446 simple sequence repeat (SSR) loci and developed 313 SSR markers, which showed polymorphisms among nine isolates from rice (including a laboratory strain 2539). The number of alleles of each marker ranged 2-9 with an average of 3.3. The polymorphic information content (PIC) of each marker ranged 0.20-0.89 with an average of 0.53. Using a population derived from a cross between isolates Guy11 and 2539, we constructed a genetic map of M. grisea consisting of 176 SSR markers. The map covers a total length of 1247 cM, equivalent to a physical length of about 35.0 Mb or 93% of the genome, with an average distance of 7.1cM between adjacent markers. A web-based database of the SSR markers and the genetic map was established (http://ibi.zju.edu.cn/pgl/MGM/index.html).     

Magnaporthe Grisea Genes for Pathogenicity and Virulence Identified through a Series of Backcrosses

We have identified genes for pathogenicity toward rice (Oryza sativa) and genes for virulence toward specific rice cultivars in the plant pathogenic fungus Magnaporthe grisea. A genetic cross was conducted between the weeping lovegrass (Eragrostis curvula) pathogen 4091-5-8, a highly fertile, hermaphroditic laboratory strain, and the rice pathogen O-135, a poorly fertile, female-sterile field isolate that infects weeping lovegrass as well as rice. A six-generation backcrossing scheme was then undertaken with the rice pathogen as the recurrent parent. One goal of these crosses was to generate rice pathogenic progeny with the high fertility characteristic of strain 4091-5-8, which would permit rigorous genetic analysis of rice pathogens. Therefore, progeny strains to be used as parents for backcross generations were chosen only on the basis of fertility. The ratios of pathogenic to nonpathogenic (and virulent to avirulent) progeny through the backcross generations suggested that the starting parent strains differ in two types of genes that control the ability to infect rice. First, they differ by polygenic factors that determine the extent of lesion development achieved by those progeny that infect rice. These genes do not appear to play a role in infection of weeping lovegrass because both parents and all progeny infect weeping lovegrass. Second, the parents differ by simple Mendelian determinants, ``avirulence genes,' that govern virulence toward specific rice cultivars in all-or-none fashion. Several crosses confirm the segregation of three unlinked avirulence genes, Avr1-CO39, Avr1-M201 and Avr1-YAMO, alleles of which determine avirulence on rice cultivars CO39, M201, and Yashiro-mochi, respectively. Interestingly, avirulence alleles of Avr1-CO39, Avr1-M201 and Avr1-YAMO were inherited from the parent strain 4091-5-8, which is a nonpathogen of rice. Middle repetitive DNA sequences (``MGR sequences'), present in approximately 40-50 copies in the genome of the rice pathogen parent, and in very low copy number in the genome of the nonpathogen of rice, were used as physical markers to monitor restoration of the rice pathogen genetic background during introgression of fertility. The introgression of highest levels of fertility into the most successful rice pathogen progeny was incomplete by the sixth generation, perhaps a consequence of genetic linkage between genes for fertility and genes for rice pathogenicity. One chromosomal DNA segment with MGR sequence homology appeared to be linked to the gene Avr1-CO39. Finally, many of the crosses described in this paper exhibited a characteristic common to many crosses involving M. grisea rice pathogen field isolates. That is, pigment-defective mutants frequently appeared among the progeny.     

Genetic Mapping of Magnaporthe grisea Avirulence Gene Corresponding to Leaf and Panicle Blast Resistant QTLs in Jao Hom Nin Rice Cultivar

The avirulence characteristic of Magnaporthe grisea isolate TH16 corresponding to Jao Hom Nin (JHN) rice cultivar was studied by mapping population of 140 random ascospore progenies derived from the cross between B1-2 and TH16 isolates. Segregation analyses of the avirulence characteristic performing on JHN rice at the seedling and flowering stages were performed in this mapping population. We used the reference map of Guy11/2539 to choose microsatellite DNA markers for mapping the avirulence gene. The genetic map of this population was constructed from 39-microsatellite markers. The genetic map was spanned by covering seven chromosomes with an average distance of 11.9 cM per marker. In mapping population the distribution of pathogenic and non-pathogenic progenies on JHN rice were found to be fitted to 1 : 1 ratio for two of the rice stages, seedling and flowering stages. The Quantitative Trait Loci (QTL) analysis for avirulence genes corresponding to two rice stages were located at the same region on chromosome 2 between markers Pyms305 and Pyms435. The LOD score and percentage of phenotypic variance explained (PVE) on two rice stages were 5.01/16.69 and 6.73/20.26, respectively. These loci were designated as Avr-JHN(lb) and Avr-JHN(pb) corresponding to leaf and panicle blast characteristics. The findings of this study can be the initial step for positional cloning and identifying any function of avirulence genes corresponding to leaf and panicle blast characteristics.     

Establishment of a New Cross of the Rice Blast Fungus Derived from Japanese Differential Strain Ina168 and Hermaphroditic Rice Pathogen Guy11

Mating experiments between Magnaporthe grisea Japanese rice pathogens and Guy11, a hermaphroditic fertile rice pathogen, were done aimed at identification of avirulence genes. A cross named cross 2107 with thirty-six random progenies was obtained. Segregation analyses of genetic markers found that the cross was less suitable for genetic analysis. Backcrosses with cross 2107 progenies and Guy11 were done and another cross named cross 5307 with sixty-five progenies was obtained. A locus controlling kasugamycin resistance named Ksg1R was identified and used for a model case of genetic mapping. Bulked segregant analysis was done to find adjacent RAPD markers for mapping of the gene. Three adjacent markers to Ksg1R were obtained and a genetic map around the Ksg1R was made, but these markers were not located on a single chromosome. These results suggest that genetic analysis to identify a gene locus is available in cross 5307. Infection assay of parental strains of cross 5307 to Japanese differential rice cultivars suggested the possibility of genetic analysis of cultivar specificity toward four rice cultivars: Aichiasahi, Kusabue, Tsuyuake, and K59.     

Establishment of a new cross of the rice blast fungus derived from Japanese differential strain Ina168 and hermaphroditic rice pathogen Guy11

Mating experiments between Magnaporthe grisea Japanese rice pathogens and Guy11, a hermaphroditic fertile rice pathogen, were done aimed at identification of avirulence genes. A cross named cross 2107 with thirty-six random progenies was obtained. Segregation analyses of genetic markers found that the cross was less suitable for genetic analysis. Backcrosses with cross 2107 progenies and Guy11 were done and another cross named cross 5307 with sixty-five progenies was obtained. A locus controlling kasugamycin resistance named Ksg1R was identified and used for a model case of genetic mapping. Bulked segregant analysis was done to find adjacent RAPD markers for mapping of the gene. Three adjacent markers to Ksg1R were obtained and a genetic map around the Ksg1R was made, but these markers were not located on a single chromosome. These results suggest that genetic analysis to identify a gene locus is available in cross 5307. Infection assay of parental strains of cross 5307 to Japanese differential rice cultivars suggested the possibility of genetic analysis of cultivar specificity toward four rice cultivars: Aichi-asahi, Kusabue, Tsuyuake, and K59.     

RAPD和SSR两种标记构建的中国对虾遗传连锁图谱

利用RAPD和SSR分子标记结合拟测交策略,对中国对虾(Fenneropenaeuschinensis)“黄海1号”雌虾与野生雄虾作为亲本进行单对杂交产生的F1代,采用RAPD和SSR两种分子标记技术初步构建了中国对虾雌、雄遗传连锁图谱。对460个RAPD引物和44对SSR引物进行筛选,共选出61个RAPD引物和20对SSR引物,用于对父母本和82个F1个体进行遗传分析。共得到母本分离标记146个(RAPD标记128个,微卫星标记18个)和父本分离标记127个(RAPD标记109个,微卫星标记18个)。雌性图谱包括8个连锁群、9个三联体和14个连锁对,标记间平均间隔为11·28cM,图谱共覆盖1173cM,覆盖率为59·36%;雄性图谱包括10个连锁群、12个三联体和7个连锁对,标记间平均间隔为12·05cM,图谱共覆盖1144·6cM,覆盖率为62·01%。中国对虾遗传图谱的构建为其分子标记辅助育种、比较基因组作图及数量性状位点的定位与克隆奠定了基础。     

Analysis of the structure of the AVR1-CO39 avirulence locus in virulent rice-infecting isolates of Magnaporthe grisea

The AVR1-CO39 gene that came from a Magnaporthe grisea isolate from weeping lovegrass controls avirulence on the rice cultivar CO39. AVR1-CO39 was not present in the genome of the rice-infecting M. grisea isolate Guyll from French Guyana, suggesting that the gene had been deleted. Molecular analysis of the deletion breakpoints in the AVR1-CO39 locus revealed the presence of a truncated copy of a previously unknown retrotransposon at the left-hand border. At the right-hand border was a truncated copy of another repetitive element that is present at multiple locations in the genome of Guyll. The structures of avr1-CO39 loci were further examined in 45 rice-infecting isolates collected in Brazil, China, Japan, India, Indonesia, Mali, and the Philippines. Most isolates showed no hybridization signal with the AVR1-CO39 probe and had the same locus structure as Guyll. Some isolates from Japan showed a signal with the AVR1-CO39 probe, but the region specifying avirulence activity was rearranged. These findings suggest that widespread virulence to 'CO39' among rice-infecting M. grisea isolates is due to ancestral rearrangements at the AVR1-CO39 locus that may have occurred early in the evolution of pathogenicity to rice.     

Identification of a Cross Between Two Compatible Isolates of Magnaporthe grisea (Hebert) Barr and Genetic Analysis of Avirulence/Virulence of Rice

A cross was successful between two field isolates of Magnaporthe grisea Guy 11 and CD 128, both pathogenic to rice. One ascospore isolated in this cross could be backcrossed to the most fertile parental isolate Guy 11. Backcrosses to CD 128 were not compatible. All progenies were inoculated to seven rice varieties in order to analyze the genetic basis of avirulence. Avirulence to Kusabue was found to be controlled by one gene. Two other controls of avirulence to rice were shown: a) two independent genes involved in avirulence to Pi-n°4, both required by the isolates for virulence expression, b) two independent genes, each one leading to virulence on the rice varieties K 1 and Aichi asahi. Implications of these results to Flor's gene-for-gene hypothesis are discussed.     

Genetic and physical mapping of Avr1a in Phytophthora sojae

The interaction between soybean and the phytopathogenic oomycete Phytophthora sojae is controlled by host resistance (Rps) genes and pathogen avirulence (Avr) genes. We have mapped the Avr1a locus in F(2) populations derived from four different P. sojae races. Four RAPD and nine AFLP markers linked to Avr1a were initially identified. Nine markers were used to compare genetic linkage maps of the Avr1a locus in two distinct F(2) populations. Distorted segregation ratios favoring homozygous genotypes were noted in both crosses. Segregation analysis of all the markers in one F(2) population of 90 progeny generated a map of 113.2 cM encompassing Avr1a, with one marker cosegregating with the gene. The cosegregating DNA marker was used to isolate P. sojae BAC clones and construct a physical map covering 170 kb, from which additional DNA markers were developed. Three markers occurring within the BAC contig were mapped in an enlarged population of 486 F(2) progeny. Avr1a was localized to a 114-kb interval, and an average physical to genetic distance ratio of 391 kb/cM was calculated for this region. This work provides a basis for the positional cloning of Avr1a.     

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.     

Construction of a genetic linkage map in <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck) using RAPD and SSR markers

The primary genetic linkage maps of Fenneropenaeus chinensis (Osbeck) were constructed by using the “two-way pseudo-testcross” strategy with RAPD and SSR markers. Parents and F1 progeny were used as segregating populations. Sixty-one RAPD primers and 20 pairs of SSR primers were screened from 460 RAPD primers and 44 pairs of SSR primers. These primers were used to analyze the parents and 82 progeny of the mapping family. About 146 primers (128 RAPDs, 18 microsatellites) in the female and 127 primers (109 RAPDs, 18 microsatellites) in the male were segregating markers. The female linkage map included eight linkage groups, nine triplets and 14 doublets, spanning 1,173 cM with the average marker density of 11.28 cM, and the observed coverage was 59.36%. The male linkage map included 10 linkage groups, 12 triplets and seven doublets, spanning 1,144.6 cM with the average marker density of 12.05 cM, and the observed coverage was 62.01%. The construction of the F. chinensis genetic linkage maps here opened a new prospect for marker-assisted selection program, comparative genomics and quantitative trait loci (QTL) gene location and cloning.     

Tagging and mapping of rice sheath blight resistant gene

Sheath blight (Rhizoctonia solani Kühn) is one of the severe rice diseases worldwide. In this study, an F(2) population from a cross between "4011" and "Xiangzaoxian19" is used to identify molecular markers linked with the resistant trait. "4011" was a transgenic rice cultivar carrying a resistant gene to sheath blight, while "Xiangzaoxian19" is a highly susceptible one. As a result, five molecular markers, including three RFLP markers converted from RAPD and AFLP markers, and two SSR markers were identified to link with the sheath blight resistant gene. This dominant resistant gene was named as R sb 1 and mapped on rice chromosome 5. The linkage distance between the markers (E-AT:M-CAC(120), E-AT:M-CTA(230), OPN-16(2000), RM164(320) and RM39(300)) and R sb 1 was 1.6 cM, 9.9 cM, 1.6 cM, 15.2 cM and 1.6 cM, respectively.     

稻瘟菌无毒基因研究进展

稻瘟菌是引起水稻稻瘟病的病原物。水稻与稻瘟菌间存在广泛而特异的相互作用,是研究寄主与病原物互作的重要模式系统。本文对稻瘟菌与水稻互作最重要的激发子―无毒基因的研究现状进行了概括,讨论了无毒基因的定位、克隆方法以及已克隆无毒基因的功能及进化研究,同时对今后无毒基因研究的重要方向进行了探讨,为深入理解无毒基因的功能及与水稻可能的互作关系奠定了基础。     

Identification and fine mapping of AvrPi15, a novel avirulence gene of Magnaporthe grisea

Avirulence of Magnaporthe grisea isolate CHL346 on rice cultivar GA25 was studied with 242 ascospore progenies derived from the cross CHL346 × CHL42. Segregation analysis of the avirulence in the progeny population was in agreement with the existence of a single avirulence (Avr) gene, designated as AvrPi15. For mapping the Avr gene, we developed a total of 121 microsatellite DNA markers [simple sequence repeat (SSR)], which evenly distributed in the whole-genome of M. grisea through bioinformatics analysis (BIA) using the publicly available sequence. Linkage analysis of the AvrPi15 gene with these SSR markers showed that six markers on chromosome 6, MS6-1, MS6-2, MS6-3, MS6-7, MS6-8 and MS6-10, were linked to the AvrPi15 locus. To further define the chromosomal location of the AvrPi15 locus, two additional markers, MS6-17 and STS6-6, which were developed based on the sequences of telomeric region 11 (TEL11), were subjected to linkage analysis. The results showed that MS6-17 and STS6-6 were associated with the locus by 3.3 and 0.8 cM, respectively. To finely map the Avr gene, two additional candidate avirulence gene (CAG) markers, CAG6-1 and CAG6-2, were developed based on the gene annotation of the sequence of TEL 11. Linkage analysis of the Avr gene with these two markers revealed that both of them completely cosegregated with the AvrPi15 locus. Finally, this locus was physically mapped into ∼ 7.2-kb interval of the TEL11 by BIA using these sequence-ready markers. This is the key step toward positional cloning of the AvrPi15 gene.     

Inheritance and mapping of 11 avirulence genes in Phytophthora sojae

Two new crosses involving four races (races 7, 16, 17, and 25) of the soybean root and stem rot pathogen Phytophthora sojae were established (7/16 cross; 17/25 cross). An F2 population derived from each cross was used to determine the genetic basis of avirulence towards 11 different resistance genes in soybean. Avirulence was found to be dominant and determined by a single locus for Avr1b, 1d, 1k, 3b, 4, and 6, as expected for a simple gene-for-gene model. We also observed several cases of segregation, inconsistent with a single dominant gene being solely responsible for avirulence, which suggests that the genetic background of the different crosses can affect avirulence. Avr4 and 6 cosegregated in both the 7/16 and 17/25 crosses and, in the 7/16 cross, Avr1b and 1k were closely linked. Information from segregating RAPD, RFLP, and AFLP markers screened on F2 progeny from the two new crosses and two crosses described previously (a total of 212 F2 individuals, 53 from each cross) were used to construct an integrated genetic linkage map of P. sojae. This revised genetic linkage map consists of 386 markers comprising 35 RFLP, 236 RAPD, and 105 AFLP markers, as well as 10 avirulence genes. The map is composed of 21 major linkage groups and seven minor linkage groups covering a total map distance of 1640.4cM.     

Gain of virulence caused by insertion of a Pot3 transposon in a Magnaporthe grisea avirulence gene

The avirulence gene AVR-Pita in Magnaporthe grisea prevents the fungus from infecting rice cultivars carrying the disease resistance gene Pi-ta. Insertion of Pot3 transposon into the promoter of AVR-Pita caused the gain of virulence toward Yashiro-mochi, a rice cultivar containing Pi-ta, which demonstrated the ability of Pot3 to move within the M. grisea genome. The appearance of Pot3 in M. grisea seems to predate the diversification of various host-specific forms of the fungus.