Mapping and candidate gene screening of Cladosporium fulvum resistance gene Cf-12 in tomato (Solanum lycopersicum) by high-throughput sequencing

Tomato leaf mould disease is a serious foliar disease caused by Cladosporium fulvum, resulting in a significant reduction in yield and quality, especially under protected cultivation. Finding new resistant genes against leaf mould has become one of the important goals of tomato breeding. Cf-12-tomato showed good resistance to several C. fulvum races. The Cf-12 was mapped using re-sequencing and BSA methods. Allelism test for Cf-12, Cf-5 and Cf-9 was performed. The results showed that Cf-12 is an independently assigned C. fulvum resistance gene. About 67,853 high-quality polymorphic SNPs were found and 233 genes were annotated in three associated regions of 5.85 Mb on Chr. 6s. Three genes with typical Cf-type characters encoding the leucine-rich repeat receptor-like protein were screened out. Using qRT-PCR, structural and phylogenetic analysis, only the Solyc06g008265.1.1-CGN7495 was finally identified. An effective InDel marker, InD-3F/1R, was designed for MAS breeding. Our research provides new insights into the mapping of the Cf-12, and provides a basis for Cf-12 cloning and the Cf genetic evolution analysis.     

Genetic and molecular organization of the short arm and pericentromeric region of tomato chromsome 6

We have previously presented an integrated linkage map of tomato chromosome 6, that showed the position of restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers relative to a variety of classical markers. As for the short arm, map resolution has now been improved by crossing the chromosome 6 substitution line WSL6 to additional tester lines, carrying markers on the short arm. Molecular linkage analysis of the F₂ populations enabled us to produce an integrated linkage map showing the position of molecular markers relative to the classical markers Aps-1, yv, Mi, Cf-2/Cf-5, tl and pds. In order to incorporate the centromere into the integrated map, a radiation-induced deletion mapping strategy was applied, using irradiated pollen from L. pennellii LA716 in crosses to a L. esculentum line recessive for the markers yv and tl, that flank the centromere. Molecular analysis of the hemizygous yv-deletion and tl-deletion plants identified among the F₁ progeny, provided an estimate of the size of the respective deletions and, thus, of the position of the centromere relative to the molecular markers linked to yv and tl. This radiation mapping approach also provided evidence showing that, unlike published data, the root knot nematode resistance gene Mi as well as the Cladosporium fulvum resistance genes Cf-2/Cf-5 are located on the short arm.     

Recent experiences of breeding leaf mould resistant tomatoes

After the introduction, in 1961, of breeding lines of tomato with complete resistance to all races of Cladosporium fulvum known until then, it was found in 1963 that this resistance could be broken by the occurrence of new races. Resistance to the new races was shown to be also available and to be dependent on one dominant factor.Assumedly a new relation between host and parasite had occurred. Consequences for breeding are discussed.     

Genes for race-specific resistance against blackleg disease in Brassica napus L.

Specificity of interaction at the cotyledon stage was recently demonstrated between the blackleg pathogen, Leptosphaeria maculans, and Brassica napus. Three pathogenicity groups were distinguished, PG2 avirulent towards ‘Quinta’ and ‘Glacier’, PG3 avirulent towards ‘Quinta’, and PG4 virulent on the two cultivars. The genetic control of the interactions was investigated on both the pathogen and the plant. Tetrad analysis was performed following PG3 × PG4 and PG2 × PG4 crosses.‘Quinta’ and ‘Glacier’ were crossed with the susceptible winter oilseed rape cultivar ‘Score’. The analysis of F₁, F₂ and testcross populations suggested that the incompatible interaction between ‘Quinta’ and PG3 isolates is conditioned by the presence of the dominant single resistance allele Rlml in ‘Quinta’ and the matching avirulence gene AvrLml in L. maculans. Race-specific resistance of ‘Glacier’ to PG2 isolates was conditioned by the matching gene pair Rlm2/AvrLm2. Finally, the data suggest that two avirulence genes matching two dominant loci control the ‘Quinta’-PG2 interaction. The consequences of the occurrence of race-specific resistance in B. napus are discussed with respect to future breeding for blackleg resistance.     

New data on the specificity of the root-knot nematode resistance genes Me1 and Me3 in pepper

Root‐knot nematodes, Meloidogyne spp., cause severe damage on solanaceous crops, including tomato and pepper. The objective of this study was to test whether nematodes virulent against the tomato Mi resistance gene were able to overcome the Me1 and Me3 resistance genes from pepper. For that purpose, a collection of 22 Meloidogyne arenaria, Meloidogyne incognita and Meloidogyne javanica isolates, avirulent or virulent against the Mi gene, was assembled. The reproduction of each isolate was evaluated on both susceptible and resistant tomato and pepper genotypes in a growth chamber. The Me1 resistance gene controlled all the avirulent and Mi‐virulent nematodes tested, and therefore appears promising for pepper breeding. In contrast, one M. arenaria and two M. incognita virulent isolates were shown to overcome the pepper Me3 resistance gene. These results encourage the testing of a large number of Meloidogyne isolates to evaluate the plant‐nematode interaction, which could have important consequences for the use and management of resistance genes in the field with respect to specificity and durability of the resistance.     

Gene‐for‐gene resistance is expressed in cotyledons,leaves and pods,but not during late stages of stem colonization in the Leptosphaeria maculans–Brassica napus pathosystem

The blackleg fungus, Leptosphaeria maculans, interacts with canola (Brassica napus) in a gene‐for‐gene manner. These major resistance genes are well characterized in the seedling stage of development, but not in other plant organs. Cotyledons, leaves, pods and stems of plants of two cultivars of B. napus, each harbouring a different major resistance gene (Rlm1 and Rlm4), were inoculated with two individual L. maculans isolates with different alleles of the corresponding avirulence genes (AvrLm1, avrLm4 and avrLm1, AvrLm4), and the disease phenotype in terms of lesion development was determined. Major gene resistance was expressed in cotyledons, all leaves and during pod set, but not in the stems of the adult plant. This is the first time major gene resistance has been shown to be effective in B. napus pods.     

番茄叶霉菌无毒基因的研究进展

无毒基因是病原物遗传因子,其编码的产物激发病原物与植物特异性相互作用。病原物无毒基因与植物抗病基因产物间直接或间接相互作用导致产生的基因对基因抗性是植物抗病性的重要形式。番茄与叶霉病之间的特异互作被认为是遵循Flor的“基因对基因”假说的典型体系。绝大多数已克隆的无毒基因之间,及其与已知蛋白之间,均无显著的序列同源性。无毒基因具有双重功能：在含瓦补抗性基因植物中表现无毒效应,而在不含互补抗性基因植物中显示毒性效应。本文综述了番茄叶霉菌无毒基因的多样性、意义、结构及其功能等等,了解病原菌无毒基因的结构及功能,有助于了解病原物与植物的识别机制,对认识植物的抗病性,特别是非寄主植物对病原菌的广谱抗病性也具有重要意义。     

Analysis of rice blast resistance gene <Emphasis Type="Italic">Pi</Emphasis>-<Emphasis Type="Italic">z</Emphasis> in rice germplasm using pathogenicity assays and DNA markers

The Pi-z gene in rice confers resistance to a wide range of races of the rice blast fungus, Magnaporthe oryzae. The objective of this study was to characterize Pi-z in 111 rice germplasm accessions using DNA markers and pathogenicity assays. The existence of Pi-z in rice germplasm was detected by using four simple sequence repeat (SSR) markers (RM527, AP4791, AP5659-1, AP5659-5) closely linked to Pi-z, and was verified using pathogenicity assays with an avirulent strain (IE1k) and two virulent races (IB33 and IB49). Among 111 germplasm accessions evaluated, 73 were found to contain the Pi-z gene using both SSR markers and pathogenicity assays. The remaining 38 germplasm accessions were found to be inconsistent in their responses to the blast races IB33, IEIk and IB49 with expected SSR marker alleles, suggesting the presence of unexpected SSR alleles and additional R gene(s). These characterized germplasm can be used for genetic studies and marker-assisted breeding for improving blast resistance in rice.     

Screening and identification of resistance to Leptosphaeria maculans (stem canker) in Brassica napus accessions

The occurrence of race-specific resistance genes to the stem canker fungus, Leptosphaeria maculans, was analysed in 453 accessions of B. napus, mainly originating from the Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK) GeneBank. Major resistance genes Rlm1, Rlm2, Rlm4 and the putative RlmBBA gene were investigated using genetically improved strains of the fungus harbouring as few corresponding avirulence genes as possible. In addition, a screening with fully virulent isolates was used to uncover novel resistance sources. Major resistance genes were rarer in frequency and diversity in spring-type cultivars compared to winter types. In the former, 65.7% of the accessions were fully susceptible to all isolates, whereas only 12.2% of the winter types were devoid of at least one R gene. In spring cultivars, the most common R gene, Rlm4 was found in 26.6% of accessions, whereas the other R genes were rare. In winter cultivars, the most common R genes were Rlm2 (more than 45.9–54.0% of the accessions) and Rlm4 (26.4–27.7% of the genotypes). In winter types however, the improvement of the quality of oils, through the generation of single- and double-low genotypes improved the homogeneity of the cvs, whereas it impoverished R gene diversity, including the loss of complete resistance that was harboured by 18.4% of the less advanced accessions, and a reduction in the ratio of accessions harbouring Rlm1. Correlation between the R gene(s) present in the accessions and their field resistance is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sources of resistance to angular leaf spot (Phaeoisariopsis griseola) in common bean core collection, wild Phaseolus vulgaris and secondary gene pool

Angular leaf spot (ALS) is one of the most devastating diseases of common bean (Phaseolus vulgaris L.) in tropical and subtropical countries. The causal fungus, Phaeoisariopsis griseola(Sacc.) Ferr. is highly variable and a diverse source of resistance genes is required to manage this disease. We evaluated a common bean core collection,primary and secondary gene pools and lines derived from inter-specific crosses of P. vulgaris and P. coccineus or P. polyanthus (secondary gene pool) for resistance to angular leaf spot. Of the 1441 accessiones in the core collection, only 2.2% were resistant to both Andean and Mesoamerican races of P. griseola, 28% were resistant only to Andean and 9% to Mesoamerican races. Of the 32 resistant accessions, 68%originated from Bolivia, Colombia,Guatemala and Mexico. More accessions from these countries should be examined for P. griseola reaction. Very few wild P. vulgaris accessions (4%), were resistant to ALS. In contrast, high levels of resistance (62%) were found in the secondary gene pool. Among the 1010 lines from inter-specific crosses, 109 lines were highly resistant. These genotypes from the primary and secondary common bean gene pools resistant to Andean and Mesoamerican races of P. griseola offer a potential for developing broad and durable ALS resistance. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dynamics of changes in the races and virulence of wheat powdery mildew in Hungary between 1971 and 1999

Important microevolutional processes have taken place in the wheatpowdery mildew population over the last thirty years. There has been aconsiderable change in the race composition of the pathogen populationand in the prevalent races. Of the 78 races identified, only eleven have`lived' for more than 15 years. Many races were only isolated in one ortwo years. The number of virulence genes rose from 2.03 in 1973 to 5.63in 1993. On the basis of race composition and virulence the wheatpowdery mildew population between 1971 and 1999 can be divided intofour distinct groups. A large proportion of the powdery mildew isolates arevirulent to most resistance genes. Complete resistance is provided byresistance genes Pm4a (Khapli) and partial resistance by Pm2 + Mld (Halle st. 13471), Pm4b+ (TP 315/2) and Pm1 + 2 + 9 (Normandie). The majority of cultivated varieties carry theresistance gene Pm8 due to the presence of the 1B/1R translocation.     

Rapid survey for presence of a blast resistance gene Pi-ta in rice cultivars using the dominant DNA markers derived from portions of the Pi-ta gene

The Pi‐ta gene in rice confers resistance to strains of the blast pathogen Magnaporthe grisea (Herbert) Borr. (anamorph Pyricularia oryza Cav.) containing the corresponding avirulence gene AVR‐Pita in a gene‐for‐gene fashion. The Pi‐ta gene is a typical nucleotide‐binding site type resistance gene. Nucleotide sequences distinguishing the resistant Pi‐ta and susceptible pi‐ta alleles were previously identified and used for developing DNA markers for a resistant Pi‐ta haplotype and three susceptible pi‐ta haplotypes. In the present study, the existence of the Pi‐ta gene in 141 rice germplasm accessions was rapidly determined using these markers, and the results were confirmed by inoculating rice germplasm with an M. grisea strain containing AVR‐Pita. The Pi‐ta gene was found in accessions from several major rice producing countries, including China, Colombia, Japan, Vietnam, the Philippines, Iran and the United States. The usefulness of DNA markers for rapid determination of the genotype of rice germplasm was thus demonstrated. The Pi‐ta gene also was found in rice cultivar known to contain the Pi‐ta² gene, although the allelic relationship of these genes remains to be determined. The presence of the Pi‐ta gene in landrace cultivars in several different geographical locations, the Philippines and Vietnam, other indica rice cultivars in China and Colombia suggest that the Pi‐ta gene may have spontaneously originated in indica rice cultivars. These results are useful for incorporating the Pi‐ta gene into advanced breeding lines by marker‐assisted selection for rice breeding programmes worldwide.     

Resistance to late blight in potato. A putative gene that suppresses R genes and is elicited by specific isolates

Eleven progenies, ten bi-parental and one selfed, of tetraploid potato were assessed for phenotypic segregation ratios of resistance to susceptible individuals against two isolates of P. infestans, one avirulent to all 11 known potato R genes (avirulent pathotype) and one virulent to eight R genes (virulent pathotype). Assuming Mendelian inheritance of independent R genes, the numbers of individuals segregating for resistance to both races could not be explained sufficiently for all progenies. However, the assumed presence of dominant, specific suppressors of the R gene function that were elicited by specific isolates of the fungus and that segregated in the host independently from R genes explained all frequencies of resistant and susceptible individuals in each of the progenies studied. This revised version was published online in July 2006 with corrections to the Cover Date.     

An analysis of genes for resistance against Indian stem rust races in two bread wheat cultivars

Summary The genetic constitution of two bread wheat accessions from the International Spring Wheat Rust Nurseries (E 5883 and E 6032) has been studied for reaction to four Indian races of stem rust. Analysis of E 5883 has revealed that for each of the races 15C, 21 and 40 a single dominant gene operates for resistance. The dominant gene against race 15C was identified as Sr6. The dominant genes for resistance against races 21 and 40 were found to be different from the genes described so far. Resistance against race 122 is controlled by a single recessive gene producing characteristically a 2 type of reaction. This gene was identified as Sr8.The resistance of E 6032 against each of the races 15C, 21 and 40 is controlled by two genes, one dominant and one recessive, which act independently. Dominant genes effective against 15C, 21 and 40 were conclusively identified as Sr6, Sr5 and Sr9b, respectively. From the correlated behaviour against races 15C and 40 as well as from the phenotypes of the resistance reactions rhe same recessive gene, undescribed so far, operates against the two races. The second recessive gene operating against race 21 was also observed to be different from those so far designated. E 6032 was, however, found to be susceptible to races 122.The presence of Sr6 both in E 5883 and E 6032 against race 15C was further confirmed through F₂ and F₃ segregation data.     

Identification by genetic analysis of two races of Erysiphe polygoni DC. causing powdery mildew disease in mungbean

Powdery mildew disease is one of the important foliar diseases of mungbean. Earlier, resistance sources have been identified and their genetic nature studied, but there is no information on race identification of Erysiphe polygoni so far. Different genotypes having combinations of the host resistance genes Pm₁Pm₁Pm₂Pm₂‘TARM‐1’, Pm₁Pm₁pm₂pm₂ (S‐158‐16) and pm₁pm₁Pm₂Pm₂ (S‐2‐4‐1) and no resistance genes pm₁pm₁pm₂pm₂ (TPM‐1) were used in the present investigation. The genotypes were screened with four isolates from Akola, Trombay, Jabalpur and Gauribidanur centres in India. The interactions of resistant differentials with the Akola isolate were different from the other three isolates. The inheritance patterns of different resistance genes in various crosses also differed with respect to the Akola isolate. The variation observed in the Akola isolate could be explained as the result of mutation of the dominant avirulence Avr₁Avr₁ genes to virulence avr₁avr₁ genes corresponding to host resistance Pm₁Pm₁genes. Therefore, the Akola isolate is identified as a second race of E. polygoni.     

无毒基因在稻瘟病菌株007中的克隆鉴定及表达分析

为了能明确稻瘟病国际标准菌株race 007中包含的无毒基因及其可能的主效无毒基因,本研究设计5个常见的无毒基因特异性引物进行克隆鉴定,并分别检测液体培养条件下和侵染水稻过程中race 007菌株中5个无毒基因的表达情况。本研究发现,稻瘟病菌株race 007中含有PWL2、AvrPiz-t、Avr-pik、Avr-pita、ACE1 5个无毒基因,克隆鉴定的结果表明这5个无毒基因的变异不大。相比较于液体培养条件下,在侵染水稻的过程中,PWL2表达上调,ACE1、AvrPiz-t、Avr-Pik表达基本不变,而Avr-Pita基因表达出现了下调。因此PWL2基因表达变化较为显著,本研究进一步对PWL2启动子区域进行分析,结果表明该PWL2基因启动子区域存在着一些顺式作用元件来影响PWL2基因的表达。本研究表明,稻瘟病菌race 007含有5种常见的无毒基因且序列变异不大,而且PWL2基因的表达是受到调控并可能发挥主要的致病作用。     

New sources of major gene resistance in Lactuca to Bremia lactucae

Summary A total of 1789 accessions of several lettuce collections was screened to find new major gene resistance to the downy mildew fungus Bremia lactucae Regel. The accessions belonged to the species Lactuca sativa (N=1288), L. serriola (N=399), L. saligna (N=52) and L. virosa (N=50). A total of 20 races of B. lactucae were used, 14 of which were NL-races, isolated from cultivated lettuce in the Netherlands. The other six races were isolated from wild L. serriola in Czechoslovakia. The accessions were initially screened with two races: NL1 and NL3. Accessions with resistance to one or both of these races were tested with the other races. Phenotypes with new resistance were found in accessions of all four Lactuca species. Of L. sativa, four accessions were found with resistance phenotypes that could not be explained by combinations of known major genes. Many accessions of L. serriola had resistance phenotypes that indicated the presence of unknown resistance genes. All interactions between accessions of L. saligna and races of B. lactucae were incompatible in leaf disc tests, except for four accessions, which showed some sporulation with race NL6. Several accessions of L. virosa were resistant to all races used. Other accessions of L. virosa gave a race-specific interaction with B. lactucae.     

Inheritance of the Powdery Mildew Resistance Gene Pm9 in Relation to Pm1 and Pm2 of Wheat

The inheritance of the powdery mildew resistance gene Pm9 originating from the hexaploid spring wheat cultivar ‘Normandie’ was analyzed in relation to Pm1 and Pm2. Two leaf segments of individual P_1?, P_2?, F_1? and F₂-plants of the cross ‘Normandie’ (Pm1, 2, 9) בFederation’ (no known Pm gene) were inoculated separately with two powdery mildew isolates. Using powdery mildew isolate No. 6 virulent for Pm1 and Pm2 but avirulent for Pm9, a 1 resistant (r): 3 susceptible (s) F₂-segregation was found for the Pm9 gene. Using powdery mildew isolate No. 3 virulent for Pm1 and Pm9 but avirulent for Pm2, a 3 (r): 1 (s) F₂-segregation was found for the Pm2 gene. Combining the data of both experiments (leaf segments of identical plants had been used), a 9 (sr): 3 (ss): 3 (rr): 1 (rs) segregation resulted for the F₂ of this cross: therefore, independent inheritance of the genes Pm2 and Pm9 can be concluded. Similarly, the cross ‘Mephisto’ (Pm1, 2, 9) בAmor’ (no known Pm gene) was analyzed. The Pm9 gene again showed a monogenically recessive inheritance, whereas Pm1 showed a monogenically intermediate segregation upon inoculation with powdery mildew isolate No. 9a virulent for Pm2 and Pm9 but avirulent for Pm1. Combining the single gene segregations, linkage between both genes was found among the progenies. A distance of 8.5 cM was calculated. Analyzing a set of spring wheat cultivars with seven defined powdery mildew isolates, the presence of Pm1, Pm2 and Pm9 in these lines was verified; in most cases, Pm1 occurred together with Pm9.     

A genomic search for the gene conferring resistance to fusarium wilt in tomato

Fusarium wilt is an economically important disease of tomatoes, caused by the soil-born fungus Fusarium oxysporum f. sp. lycopersici. There are three host-specific races of this pathogen. The dominant tomato gene I-2 confers resistance to race 2. The I-2 fusarium resistance gene was mapped genetically to chromosome 11 of tomato, between the RFLP markers TG105 and TG36, 0.4 centiMorgan (cM) from TG105. A mean value of 43 kb for each cM was assigned in the vicinity of I-2. We have generated new RFLP markers in the region by chromosome walking from TG105 towards I-2 on lambda clones, and by subcloning a 350 kb long YAC clone (YAC 8) that contains TG105. These RFLP markers were mapped physically on YAC 8 by PFGE. The location of I-2 relative to these markers was genetically estimated using a recombinant inbred (RI) segregating population. The order of the markers according to the RI population is inconsistent with their order on the physical map. A cDNA clone, D14, that was isolated by YAC 8, turned out to be 53% similar to xanthine dehydrogenase from mammals and flies. Antibodies raised against a part of the protein encoded by D14 recognize cross reacting material of MW 80 kD, that is highly enriched in nodules of legumes, and seems to be induced by various environmental and pathogenic stress conditions.     

Resistance to Sunflower Rust and its Inheritance in Wild Sunflower Species

Resistance to the prevailing races of sunflower rust, Puccinia hehanthi Schw., is lacking in the commercial hybrids (Helianthus annuus L.). The objective of this study was to identify new sources of resistance to the four North American rust races in wild Helianthus species, and to determine their mode of inheritance. Seventy-eight accessions of H. annuus L., H. argophyllus Torrey and Gray, and H. petiolans Nutt. were evaluated in the greenhouse. Resistance to races 1, 2, 3, and 4 was observed in 25, 28, 15, and 26% of the plants, respectively, and 10% of the plants were resistant to all four races. Seven accessions that had a high percentage of resistant plants to all the four races were selected and one resistant plant from each accession was crossed with susceptible inbred line HA89. Three to four F₁ plants resistant to all four races from each cross were backcrossed with HA89. F₁ plants from PI-413118 × HA89 and PI 413175 × HA89 were resistant to all four races. The PI 413023 × HA89 F₁ plants were 100 % resistant to races 3 and 4 and segregated in a 3: 1 resistant (R) to susceptible (S) ratio to races 1 and 2. The other four F₁ combinations segregated 3R: IS ratios to all four races. Bc₁F₁ progenies revealed that plants from PI 413048, PI 413037, PI 413038, and PI 413171 used in the crosses possessed two dominant genes in heterozygous condition for resistance to each of the four races, whereas plants from PI 413023 possessed two dominant genes in heterozygous condition for resistance to each of races 1 and 2, and one dominant resistance gene in homozygous condition for each of races 3 and 4. Plants from PI 413118 and PI 413175 carried a single dominant gene in homozygous condition for resistance against each of the four races.