Molecular characerization of the interaction between the fungal pathogen Cladosporium fulvum and tomato

The fungus Cladosprium fulvum is a biotrophic leaf pathogen of tomato. The fungus develops in the intercellular space without forming specialized feeding structures and does not affect the leaf tissue. The outcome of the C. fulvum-tomato interaction can be described by the gene-for-gene model. Failure of infection is expressed by a hypersensitive response. Two fungal proteins, ECP1 and ECP2, have been isolated and their corresponding genes have been cloned. In a compatible interaction including many physiological races ECP1 and ECP2 are highly produced and a role in pathogenicity is suggestive. The ecp1 gene shows some homology with tumor necrosis factor receptors (TNFRs) while the ecp2 gene shows no homology with sequences known in data bases. However, disruption of one of the two genes showed no reduced pathogenicity of the fungus. Two race-specific elicitors, AVR4 and AVR9, have been isolated and their corresponding genes have been cloned. The avirulence genes Avr4 and Avr9 are only present in C. fulvum avirulent on Cf-4 and Cf-9 cultivars, respectively. The expression of these two genes is, like the expression of the ecp genes, highly induced when the fungus grows in planta. Disruption of the Avr9 gene in wild type avirulent races leads to virulence on tomato genotypes carrying the complementary resistance gene Cf-9. A single base-pair change in the avirulence gene Avr4 leads to virulence on tomato genotypes carrying the Cf-4 resistance gene. Isolation, characterization and possible function of ECP1, ECP2, AVR4, and AVR9 will be discussed.     

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.     

Molecular breeding for the BaMMV/BaYMV resistance gene ym4 in winter barley

The aim of this investigation was to develop a procedure for the largescale molecular breeding for ym4, allowing resistance to BaMMV/BaYMV to be fixed in early breeding generations of winter barley. A codominant STS marker derived from the restriction fragment length polymorphism marker MWG838 for the ym4 resistance gene was combined with a new and easy procedure for preparing leaf samples for polymerase chain reaction (PCR), theoretically allowing one person to extract DNA from 5000 samples in a single day. In the procedure for molecular breeding for ym4, all steps, including leaf sampling, DNA extraction, PCR amplification and digestion with restriction enzyme were assembled in microtitre plates allowing multipipetting throughout the procedure, including the loading of gels. The method is amenable to further automation with the aid of a robot arm. Double haploid (DH) lines, as well as F₂ and F₄ breeding lines were analysed and, based on markers, homozygous and heterozygous BaMMV/BaYMV resistant plants were identified for further breeding. The winter barley breeding programmes were modified to include marker-based selection for BaMMV/BaYMV resistance on DH or on F₂ individuals, which had been preselected for mildew and leaf rust resistance.     

Identification and molecular mapping of a leaf rust resistance gene in spelt wheat landrace Altgold

Leaf rust, caused by Puccinia triticina, is an important disease for wheat production, both in China and worldwide. In laboratory studies spelt wheat (Triticum aestivum ssp. spelta) landrace Altgold was resistant to P. triticina races THT and PHT and genetic analysis indicated that it possessed a dominant leaf rust resistance gene, temporarily designated LrAlt. F₆ recombinant inbred lines (RILs) derived from a cross with the susceptible common wheat cultivar Nongda 3338 were used to map LrAlt with SSR markers. The resistance gene was distal to SSR loci Xbarc212, Xwmc382, Xgwm636, and Xwmc407 on the short arm of chromosome 2A. The closest markers Xbarc212 and Xwmc382 which co-segregated were 1.8 cM away from LrAlt. The relationships of LrAlt and other wheat leaf rust resistance genes located on the short arm of chromosome 2A were discussed, suggesting that LrAlt might be a new leaf rust resistance gene.     

A leaf rust resistance gene, different from Lr34, associated with leaf tip necrosis in wheat

The gene Lr34 has contributed to durable resistance to leaf rust caused by Puccinia triticina in wheat worldwide. The closely associated leaf tip necrosis is generally used as the gene's marker. Lr34 has been postulated in many Indian bread wheat cultivars including ‘C 306’, based on the associated leaf tip necrosis and a few other field and glasshouse observations. The present study showed monogenic control of adult‐plant resistance in ‘C 306’ to leaf rust pathotype 77‐5 (121R63‐1). The F₂ segregation in the crosses between ‘C 306’ and the two known carriers of Lr34, ‘Line 897’ and ‘Jupateco 73’‘R’ fitted a digenic ratio. The F₃ families derived from the susceptible F₂ segregants were true breeding for susceptibility, proving the absence of Lr34 in ‘C 306’. The cross between ‘Line 897’ and ‘Jupateco 73’‘R’ did not segregate for susceptibility. Resistance in the cross ‘Agra Local’ (susceptible) × ‘C 306’ was associated with leaf tip necrosis, showing that the leaf rust resistance gene in ‘C 306’ was associated with leaf tip necrosis, but was different from Lr34. This gene is being temporarily designated as Lr‘C 306’. Hence, leaf tip necrosis cannot be considered as an exclusive marker for selecting Lr34 in wheat improvement.     

Genetics of leaf rust resistance in three Americano landrace-derived wheat cultivars from Uruguay

A genetic analysis of the landrace‐derived wheat accessions Americano 25e, Americano 26n, and Americano 44d, from Uruguay was conducted to identify the leaf rust resistance genes present in these early wheat cultivars. The three cultivars were crossed with the leaf rust susceptible cultivar ‘Thatcher’ and approximately 80 backcross (BC1) F₂ families were derived for each cross. The BC1F₂ families and selected BC1F₄ lines were tested for seedling and adult plant leaf rust resistance with selected isolates of leaf rust, Puccinia triticina. The segregation and infection type data indicated that Americano 25e had seedling resistance genes Lr3, Lr16, an additional unidentified seedling gene, and one adult plant resistance gene that was neither Lr12 nor Lr13, and did not phenotypically resemble Lr34. Americano 26n was postulated to have genes Lr11, Lr12, Lr13, and Lr14a. Americano 44d appeared to have two possibly unique adult plant leaf rust resistance genes.     

Common bean breeding for resistance against biotic and abiotic stresses: From classical to MAS breeding

Summary Breeding for resistance to biotic and abiotic stresses of global importance in common bean is reviewed with emphasis on development and application of marker-assisted selection (MAS). The implementation and adoption of MAS in breeding for disease resistance is advanced compared to the implementation of MAS for insect and abiotic stress resistance. Highlighted examples of breeding in common bean using molecular markers reveal the role and success of MAS in gene pyramiding, rapidly deploying resistance genes via marker-assisted backcrossing, enabling simpler detection and selection of resistance genes in absence of the pathogen, and contributing to simplified breeding of complex traits by detection and indirect selection of quantitative trait loci (QTL) with major effects. The current status of MAS in breeding for resistance to angular leaf spot, anthracnose, Bean common mosaic and Bean common mosaic necrosis viruses, Beet curly top virus, Bean golden yellow mosaic virus, common bacterial blight, halo bacterial blight, rust, root rots, and white mold is reviewed in detail. Cumulative mapping of disease resistance traits has revealed new resistance gene clusters while adding to others, and reinforces the co-location of QTL conditioning resistance with specific resistance genes and defense-related genes. Breeding for resistance to insect pests is updated for bean pod weevil (Apion), bruchid seed weevils, leafhopper, thrips, bean fly, and whitefly, including the use of arcelin proteins as selectable markers for resistance to bruchid seed weevils. Breeding for resistance to abiotic stresses concentrates on drought, low soil phosphorus, and improved symbiotic nitrogen fixation. The combination of root growth and morphology traits, phosphorus uptake mechanisms, root acid exudation, and other traits in alleviating phosphorus deficiency, and identification of numerous QTL of relatively minor effect associated with each trait, reveals the complexity to be addressed in breeding for abiotic stress resistance in common bean.     

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.     

Identification of a novel source of resistance to angular leaf spot disease of common bean within the secondary gene pool

Phaeoisariopsis griseola (Sacc.) Ferr., the agent of angular leaf spot disease of common bean, is a highly variable pathogen for which resistance gene diversification is required. This study analysed genetic resistance to this disease within genotypes of three Phaseolus species. Twenty-nine genotypes of Phaseolus vulgaris, Phaseolus coccineus and Phaseolus polyanthus were inoculated with 54 isolates of Phaeoisariopsis griseola. The genetic resistance was estimated according to the symptom intensity observed for each plant genotype-pathogen isolate combination. Globally, genotypes of the common bean secondary gene pool were resistant to a higher number of isolates than common bean varieties. Interactions between plant genotypes and pathogen isolates suggested vertical resistance genes within P. vulgaris, as well as within P. coccineus and P. polyanthus. The ‘NI666’accession (P. coccineus) showed resistance to all the fungal isolates inoculated while the variety ‘Aroana’(P. vulgaris) was susceptible to most of the isolates. Interspecific hybridization between these two genotypes gave F₁ hybrid plants which showed resistance to angular leaf spot disease.     

Chromosome and chromosomal arm locations of genes for resistance to Septoria glume blotch in wheat cultivar Cotipora

Summary Septoria glume blotch, caused by Stagonospora nodorum, is an important disease of wheat (Triticum aestivum). Separate genetic mechanisms were found to control flag leaf and spike resistance. Genes for resistance to S. nodorum were located on different chromosomes in the few wheat cultivars studied. These studies only partially agree on the chromosome locations of gene in wheat for resistance to S. nodorum, and chromosomal arm locations of such genes are not known. The objectives of this study were to determine the chromosome and chromosomal arm locations of genes that significantly influence resistance to S. nodorum in wheat cultivar Cotipora. Monosomic analysis showed that flag leaf resistance was controlled by genes on chromosomes 3A, 4A, and 3B whereas the spike resistance was controlled by genes on chromosomes 3A, 4A, 7A, and 3B (P=0.01). Additionally, genes on chromosomes 6B and 5A influenced the susceptibility of the flag leaf and spike reactions, respectively (P=0.01). Telocentric analysis showed that genes on both arms of chromosome 3A, and the long arms of chromosomes 4A and 3B were involved in the flag leaf resistance whereas genes on both arms of chromosome 4A, the short arm of chromosome 3A, and the long arm of chromosome 3B conferred spike resistance.     

Inheritance and characterization of the new and rare gene Rph25 conferring seedling resistance in Hordeum vulgare against Puccinia hordei

In this study, we characterized and mapped a new and rare resistance gene (RphFT) in the Chinese barley variety ‘Fong Tien’. RphFT, a dominant gene, was mapped to chromosome 5HL at a genetic position of 142.1 cM using DArT‐seq markers. The gene was also confirmed to be present in Australian cultivar ‘Yagan’ based on allelic tests, and likely ‘Lockyer’ based on multipathotype tests. The genetic studies also confirmed the presence of Rph12 in Australian cultivar ‘Baudin’. Rph12 is also located on chromosome 5HL close to RphFT, and the two loci were confirmed to be independent. Gene RphFT is of limited breeding value because it is effective to only one pathotype of P. hordei, 220P+ +Rph13 in Australia; nevertheless, it may play a role in controlling leaf rust if used in combination with other Rph genes. The locus symbol Rph25 is recommended for RphFT in accordance with the rules and numbering system of barley gene nomenclature.     

Postulation of leaf-rust resistance genes in Czech and Slovak barley cultivars and breeding lines

Leaf‐rust resistance (Rph) genes in 61 Czech and Slovak barley cultivars and 32 breeding lines from registration trials of the Czech Republic were postulated based on their reaction to 12 isolates of Puccinia hordei with different combinations of virulence genes. Five known Rph genes (Rph2, Rph3, Rph4, Rph7, and Rph12) and one unknown Rph gene were postulated to be present in this germplasm. To corroborate this result, the pedigree of the barley accessions was analysed. Gene Rph2, as well as Rph4, originated from old European cultivars. The donor of Rph3, which has been mainly used by Czech and Slovak breeders, is ‘Ribari’ (‘Baladi 16’). Rph12 originates from barley cultivars developed in the former East Germany. Rph7 in the registered cultivar ‘Heris’ originates from ‘Forrajera’. A combination of two genes was found in 10 cultivars. Nine heterogeneous cultivars were identified; they were composed of one component with an identified Rph gene and a second component without any resistance gene. No gene for leaf rust resistance was found in 17 of the accessions tested. This study demonstrates the utility of using selected pathotypes of P. hordei for postulating Rph genes in barley.     

‘CPAN 1842’: a new source of adult plant leaf rust resistance in bread wheat

There is worldwide interest in adult plant resistance (APR) because of greater durability of APR to the cereal rusts. Peruvian bread wheat genotype ‘CPAN (Coordinated Project Accession Number) 1842’ (LM 50–53) has shown leaf rust resistance in disease screening nurseries since its introduction in 1977. However, it is susceptible at the seedling stage to several Puccinia triticina (Pt) pathotypes including the widely prevalent 77‐5 (121R63‐1) that infects bread wheat. Inheritance studies showed that CPAN 1842 carried a dominant gene for APR to pathotype 77‐5, which was different from Lr12, Lr13, Lr22a, Lr34, Lr35, Lr37, Lr46, Lr48, Lr49 and Lr68, based on the tests of allelism; and from Lr67, based on genotyping with the closely linked SSR marker cfd71. This gene should also be different from Lr22b as the latter is totally ineffective against pathotype 77‐5. CPAN 1842 therefore appears to be a new promising source of leaf rust resistance. Also having resistance to stem rust and stripe rust, this line can contribute to breeding for multiple rust resistances in wheat.     

Molecular markers and allelic relationships of anthracnose resistance gene cluster B4 in common bean

Angular leaf spot is one of the major diseases of the common bean. The extensive genetic variability of this pathogen requires the constant development of new resistant cultivars. Different sources of resistance have been identified and characterized. For the State of Minas Gerais, Brazil, four main resistance sources were found: Mexico 54, AND 277, MAR 2 and Cornell 49-242. Independent characterization of these genotypes demonstrates that resistance in all four sources is dominant and monogenic. However, there are no studies on the relationship and independence of these genes. In the present work, allelism tests were carried out to understand the relationship among the resistance genes present in these four resistance sources. The data revealed a much higher complexity in the resistance inheritance of these genes than previously reported. It was demonstrated that Cornell 49-242 possesses a dominant gene (Phg-3); Mexico 54 possesses three genes, denominated Phg-2, Phg-5 and Phg-6. In MAR 2, two genes were found, one independent designated Phg-4 and the other, an allelic form of Phg-5, denominated of Phg-5². Allelic forms were also found in AND 277, Phg-2², Phg-3² and Phg-4². These results have special importance for breeding programs aiming to pyramid resistance genes.     

Development and characterization of common black bean lines resistant to anthracnose,rust and angular leaf spot in Brazil

Anthracnose, rust and angular leaf spot caused by Colletotrichum lindemuthianum, Uromyces appendiculatus and Pseudocercospora griseola, respectively, are economically important diseases affecting the common bean production in Brazil. The BIOAGRO/UFV bean breeding program developed Rudá-R, a dry bean line with ‘carioca’ seed type, containing the following disease resistance genes: Co-4, Co-6 and Co-10 (anthracnose); Ur-ON (rust) and Phg-1 (angular leaf spot). To transfer this combination of disease resistance genes present in Rudá-R to a black-seeded bean, a backcrossing program aided by molecular markers was conducted, involving Rudá-R (donor genitor) and Diamante Negro (recurrent genitor). Forty black-seeded BC₃F_3:6 lines were obtained with combinations of at least three markers linked to the indicated disease resistance genes. The lines were evaluated for resistance to the three mentioned pathogens. Eight of the lines were homozygous and resistant to all four evaluated races of C. lindemuthianum, but susceptible to race 2047. Four of the lines were homozygous and resistant to two races of U. appendiculatus. Twenty of the lines were homozygous and resistant to the two races of P. griseola tested. Grain yield of the BC₃F_3:6 lines was evaluated during the ‘winter’ season of 2006 and the ‘dry’ season of 2007. All lines had statistically equal or higher yields than Rudá-R and Diamante Negro. Lines were identified that not only were high yielding but also resistant to the three pathogens tested. These lines are potential genotypes for further testing and for release as new black common bean varieties.     

Genetic analysis of grain mould resistance in coloured sorghum genotypes

Grain moulds are a major constraint to sorghum production and to adoption of improved cultivars in many tropical areas. Information on the inheritance of grain mould reaction is required to facilitate breeding of resistant cultivars. The genetic control of grain mould reaction was studied in 7 crosses of 2 resistant sorghum genotypes. P₁, P₂, F₁, F₂, BC₁ and BC₂ families of each cross were evaluated under sprinkler irrigation for field grade and threshed grade scores and subjected to generation mean analysis. Frequency distributions for grain mould reaction were derived and F₂ and BC₁ segregation ratios were calculated. Grain mould reaction in crosses of coloured grain sorghum was generally controlled by two or three major genes. Resistance to grain moulds was dominant. Significant additive gene effects were also found in all cross/season combinations. Significant dominance effects of similar magnitude to additive effects were also observed in five out of ten cross/season combinations. Gene interactions varied according to the parents with both resistant and susceptible parents contributing major genes. Choice of parents with complementary resistance genes and mechanisms of resistance will be critical to the success of resistance breeding. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of resistance gene analogue markers closely linked to wheat powdery mildew resistance gene Pm31

Specific oligonucleotide primers, designed for the sequences of known plant disease resistance genes, were used to amplify resistance gene analogues (RGAs) from wheat genomic DNA. This method was applied in a bulked segregant analysis to screen for the RGA markers linked to the powdery mildew resistance gene Pm31, introgressed into common wheat from wild emmer. Two RGA markers (RGA200 and RGA390) were found to be closely linked to Pm31 and completely co‐segregating with the marker allele of Xpsp3029 linked to Pm31, with a genetic distance of 0.6 cM. These two RGA markers were then integrated into the formerly established microsatellite map of Pm31 region. The result showed the effectiveness of the RGA approach for developing molecular markers linked to disease resistance genes and demonstrated the efficiency of denaturing polyacrylamide‐gel electrophoresis for detecting polymerase chain reaction polymorphism.     

Selective genotyping to identify late blight resistance genes in an accession of the tomato wild species <Emphasis Type="Italic">Solanum pimpinellifolium</Emphasis>

Late blight (LB), caused by the oomycete Phytophtohra infestans, is one of the most destructive diseases of tomato (Solanum lycopersicum) and other Solanaceae species. Current disease control and prevention strategies are not sufficient to control the disease in tomato. Recent germplasm screening experiments led to the identification of a new source of resistance (PI 270443) in the tomato wild species S. pimpinellifolium. This study was conducted to identify genomic regions associated with LB resistance in this accession. A large F₂ population (n = 986) derived from a cross between PI 270443 and a LB-susceptible tomato breeding line (NCEBR-2) was screened for LB resistance using a highly aggressive isolate of P. infestans. Twenty-five of the most resistant and 29 of the most susceptible, but surviving F₂ individuals were identified based on disease evaluations conducted in the F₂ and F₃ progeny populations. The selected individuals were genotyped with 153 DNA markers located across the 12 tomato chromosomes. A selective genotyping approach led to the identification of two genomic regions on tomato chromosomes 1 and 10 associated with LB resistance in PI 270443. Identification of two genomic regions associated with resistance was consistent with a previous estimate of the number of LB resistance genes in this accession. Research is currently underway to fine map the two resistance genes and incorporate them into new tomato breeding lines and hybrid cultivars.     

Molecular genetics of race non-specific rust resistance in wheat

Over 150 resistance genes that confer resistance to either leaf rust, stripe rust or stem rust have been catalogued in wheat or introgressed into wheat from related species. A few of these genes from the ‘slow-rusting’ adult plant resistance (APR) class confer partial resistance in a race non-specific manner to one or multiple rust diseases. The recent cloning of two of these genes, Lr34/Yr18, a dual APR for leaf rust and stripe rust, and Yr36, a stripe rust APR gene, showed that they differ from other classes of plant resistance genes. Currently, seven Lr34/Yr18 haplotypes have been identified from sequencing the encoding ATP Binding Cassette transporter gene from diverse wheat germplasm of which one haplotype is commonly associated with the resistance phenotype. The paucity of well characterised APR genes, particularly for stem rust, calls for a focused effort in developing critical genetic stocks to delineate quantitative trait loci, construct specific BAC libraries for targeted APR genes to facilitate robust marker development for breeding applications, and the eventual cloning of the encoding genes.     

Genes conferring low seedling reaction to Mexican pathotypes of Puccinia recondita f. sp. tritici,and adult-plant responses of recent wheat cultivars from the former USSR

Fifty-five spring bread wheat (Triticum aestivum L.) cultivars, mostly released between 1975 and 1991 in eight leaf rust-prone spring wheat growing regions of the former USSR, were tested in the seedling growth stage for reaction to 15 Mexican pathotypes of Puccinia recondita f. sp. tritici. In total, seven known and at least two unknown genes were identified, either singly or in combinations: Lr3 (7 cultivars), Lr10 (14), Lr13 (5), Lr14a (1), Lr16 (1), Lr23 (3); the unknown genes were identified in 14 cultivars. The first unknown gene could be either Lr9, Lr19, or Lr25; however, the second unknown gene in 9 cultivars was different from any named gene. Twelve of the 15 pathotypes are virulent for this gene, hence its use in breeding for resistance will be limited. The cultivars were also evaluated at two field locations in Mexico with two pathotypes in separate experiments. The area under the disease progress curve and the final disease rating of the cultivars indicated genetic diversity for genes conferring adult plant resistance. based on the symptoms of the leaf tip necrosis in adult plants, resistance gene Lr34 could be present in at least 20 cultivars. More than half of the cultivars carry high to moderate levels of adult plant resistance and were distributed in each region.