SCAR,RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean

Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).The research was supported by the CNRS and the Ministère Français de l'Education Nationale     

Development of an STS marker tightly linked to <Emphasis Type="Italic">Yr26</Emphasis> against wheat stripe rust using the resistance gene-analog polymorphism (RGAP) technique

The gene Yr26 confers resistance to all races of Puccinia striiformis f. sp. tritici (PST), the casual pathogen of wheat stripe rust in China. Here, we report development of a molecular marker closely linked to Yr26 using a resistance gene-analog polymorphism (RGAP) technique. A total of 787 F₂ plants and 165 F₃ lines derived from the cross Chuanmai 42/Taichung 29 were used for linkage analysis. Eighteen near-isogenic lines (NILs) and 18 Chinese wheat cultivars and advanced lines with different genes for stripe rust resistance were employed for the validation of STS markers. A total of 1,711 RGAP primer combinations were used to test the parents and resistant and susceptible bulks. Five polymorphic RGAP markers were used for genotyping all F₂ plants. Linkage analysis showed that the five RGAP markers were closely linked to Yr26 with genetic distances ranging from 0.5 to 2.9 cM. These markers were then converted into STS markers, one, CYS-5, of which was located 0.5 cM to Yr26 and was closely associated with the resistance gene when validated over 18 NILs and 18 Chinese wheat cultivars and lines. The results indicated that CYS-5 can be used in marker-assisted selection targeted at pyramiding Yr26 and other genes for stripe rust resistance.     

An RFLP marker in tomato linked to the Fusarium oxysporum resistance gene I2

Summary The locus, I2, which in tomato confers resistance against Fusarium oxysporum f. sp. lycopersici race 2, was introgressed into Lycopersicon esculentum from the wild species L. pimpinellifolium (P.I. 126915). We searched for restriction fragment length polymorphisms (RFLPs) between nearly isogenic lines (NILs) in clones that map to the region introgressed from the wild species. Since I2 maps to chromosome 11, we used DNA clones from this chromosome as hybridization probes to Southern blots containing bound DNA of the NILs digested with 23 restriction enzymes. Of the 14 chromosome 11 clones, 9 exhibited polymorphism. These clones were further hybridized to verification filters that contained DNA from resistant and susceptible L. esculentum varieties digested with the enzymes that gave the polymorphism. One clone, TG105, was found to be associated with I2; 19 susceptible lines showed a different RFLP with this probe than 16 resistant lines, including the original L. pimpinellifolium accession used as a source for the resistance gene. These results together with our mapping analysis indicate that TG105 is closely linked to the resistance gene.     

Differentially expressed genes in cotton plant genotypes infected with Meloidogyne incognita

Meloidogyne incognita is a nematode responsible for huge losses of economically important crops. The control of this pathogen is heavily centered on chemical nematicides, which are toxic to humans and environment, besides being very expensive. Alternatively, resistant varieties of cotton generated from conventional breeding programs represent an attractive strategy for the control of M. incognita. In this context, the goal of the work reported here was to analyze the gene expression profile of one resistant and one susceptible cotton genotype infected with M. incognita aiming to understand the mechanisms involved in resistance. EST libraries of cotton in both resistant and susceptible to infection by M. incognita were constructed and sequenced, generating 2261 sequences that were assembled into 233 contigs and 1593 singlets. Genes differentially expressed were observed in both resistant and susceptible cotton. Twenty genes were found to be expressed exclusively in the resistant cotton genotype, with functions related to pathogen recognition, signal transduction, defense mechanisms and protein synthesis transport and activation. The coordinated action of these genes suggests the existence of a complex defense pathway towards nematode attack in cotton. Our data indicate some candidate genes for validation and use through transformation in other agronomically important plants.     

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.     

Molecular markers for wheat leaf rust resistance gene Lr41

Leaf rust, caused by Puccinia triticina Eriks., is an important foliar disease of common wheat (Triticum aestivum L.) worldwide. Pyramiding several major rust-resistance genes into one adapted cultivar is one strategy for obtaining more durable resistance. Molecular markers linked to these genes are essential tools for gene pyramiding. The rust-resistance gene Lr41 from T. tauschii has been introgressed into chromosome 2D of several wheat cultivars that are currently under commercial production. To discover molecular markers closely linked to Lr41, a set of near-isogenic lines (NILs) of the hard winter wheat cultivar Century were developed through backcrossing. A population of 95 BC₃F_2:6 NILs were evaluated for leaf rust resistance at both seedling and adult plant stages and analyzed with simple sequence repeat (SSR) markers using bulked segregant analysis. Four markers closely linked to Lr41 were identified on chromosome 2DS; the closest marker, Xbarc124, was about 1 cM from Lr41. Physical mapping using Chinese Spring nullitetrasomic and ditelosomic genetic stocks confirmed that markers linked to Lr41 were on chromosome arm 2DS. Marker analysis in a diverse set of wheat germplasm indicated that primers BARC124, GWM210, and GDM35 amplified polymorphic bands between most resistant and susceptible accessions and can be used for marker-assisted selection in breeding programs.     

Combining transgenic and natural resistance to obtain broad resistance to tospovirus infection in tomato (Lycopersicon esculentum mill)

This study was undertaken to develop tomato plants with broad resistanceto tospoviruses which are a major limiting factor to tomato productionworldwide. A nontransgenic tomato line Stevens-Rodale (S-R), six transgenictomato lines expressing the nucleocapsid (N) protein gene of the lettuceisolate of tomato spotted wilt virus (TSWV-BL), and progeny of the crosses between S-Rand three of the transgenic lines homozygous for the N gene were evaluated fortheir resistance to tospovirus infection in greenhouse inoculation tests. S-Rhas the Sw-5 gene that confers resistance to several TSWVisolates. The six transgenic lines showed high levels of resistance wheninoculated with either TSWV-BL or a tomato isolate from Hawaii (TSWV-H).However, these same plants were highly susceptible to the Brazilian isolate ofgroundnut ringspot virus (GRSV-BR). Plants with the Sw-5gene were resistant to TSWV-BL and GRSV-BR, but were susceptible to TSWV-H.When inoculated with any of the three viruses, the F₁ progeny of thecrosses exhibited a susceptible, tolerant, or resistant phenotype with a higherproportion of the plants being either tolerant or resistant. When F₂progeny from F₁ resistant plants of each cross were inoculated withany of the three viruses, a higher proportion of tolerant and resistant plantswas observed compared to the F₁ progeny. Our results show thepotential to obtain broad resistance to tospoviruses by combining transgenicand natural resistance in a single plant.     

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.     

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T₀, 2T₀, and 3T₀ showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T₁ progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T₁ plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T₁ plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T₂ progenies of 1T₀ conformed to dihybrid segregation of 15 resistant:1 susceptible plants.     

Inheritance of powdery mildew (Erysiphe polygoni DC) resistance in mungbean (Vigna radiata L. Wilczek)

Detached mungbean (Vigna radiata L.Wilczek) leaves were inoculated with a conidial suspension of a local isolate (TI-1) of the powdery mildew pathogen (Erysiphe polygoni DC) under controlled environment conditions. Based on the latent period and severity of the infection, a rating scale of 0–5 was used to classify the host pathogen interactions. Reactions 0, 1 and 2 were considered resistant and referred to as R0, R1 and R2 while 3, 4 and 5 were classified as susceptible (S). RUM lines (resistant to powdery mildew) and their derivatives are crossed with several susceptible (reaction types 3–5) genotypes and the inheritance of the resistance was studied in the F₁, F₂ and F₃ generations. The results showed that powdery mildew resistance in mungbean is governed by two dominant genes designated as Pm-1 and Pm-2. When both Pm-1 and Pm-2 were present, an R0 reaction was observed after inoculation with TI-1. The resistant reaction was R1 when only Pm-1 was present and R2 in the presence of Pm-2. In the absence of both Pm-1 and Pm-2, susceptible reactions 3, 4 and 5 were observed.     

Tomato resistance to Alternaria stem canker: localization in host genotypes and functional expression compared to non-host resistance

Summary The Alternaria stem canker resistance locus (Asc-locus), involved in resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici and in insensitivity to host-specific toxins (AAL-toxins) produced by the pathogen, was genetically mapped on the tomato genome. Susceptibility and resistance were assayed by testing a segregating F₂ population for sensitivity to AAL-toxins in leaf bioassays. Linkage was observed to phenotypic markers solanifolium and sunny, both on chromosome 3. For the Asc-locus, a distance of 18 centiMorgan to solanifolium was calculated, corresponding to position 93 on chromosome 3. This map position of the resistance locus turned out to be the same in three different resistant tomato accessions, one Dutch and two American, that are at least 40 years apart. AAL-toxin sensitivity in susceptible and resistant tomato genotypes was compared with AAL-toxin sensitivity in a non-host Nicotiana tabacum during different levels of plant cell development. In susceptible and resistant tomato genotypes, inhibitory effects were demonstrated at all levels, except for leaves of resistant genotypes. However, during pollen and root development, inhibitory effects on susceptible genotypes were larger than on resistant genotypes. In the non-host Nicotiana tabacum, hardly any effects of AAL-toxins were demonstrated. Apparently, a cellular target site is present in tomato, but not in Nicotiana tabacum. It was concluded that three levels of AAL-toxin sensitivity exist: (1) a susceptible host sensitivity, (2) a resistant host sensitivity, (3) a non-host sensitivity, and that the resistance mechanism operating in tomato is different from that operating in Nicotiana tabacum.     

Comparing single- vs. mixed-genotype infections of Mycosphaerella graminicola on wheat: effects on pathogen virulence and host tolerance

Mixed-genotype infections (infections of a host by more than one pathogen genotype) are common in plant-pathogen systems. However their impact on the course of the infection and especially on pathogen virulence and host response to infection is poorly understood. We investigated the effects of mixed-genotype infections on several parameters: host resistance and tolerance, as well as pathogen aggressiveness and virulence. For these purposes, we inoculated three wheat lines with three Mycosphaerella graminicola genotypes, alone or in mixtures, in a greenhouse experiment. For some of the mixtures, disease severity and virulence were lower than expected from infection by the same genotypes alone, suggesting that competition between genotypes was reducing their aggressiveness and virulence. One host line was fully resistant, but there were differences in resistance in the other lines. The two host lines that became infected differed slightly in tolerance, but mixed-genotype infections had no effect on host tolerance.     

The Sweet Pepper Ferredoxin-Like Protein (pflp) Conferred Resistance Against Soft Rot Disease in Oncidium Orchid

Genetic engineering to date has not been used to introduce disease resistance genes into the orchid gene pool. The ferredoxin-like protein gene originally isolated from sweet pepper is thought to function as a natural defense against infection due to its antimicrobial properties. Hence it was reasoned that introduction of this gene might produce Oncidium plants resistant to Erwinia carotovora, the causal agent for the soft rot disease. An expression vector containing sweet pepper ferredoxin-like protein (pflp) cDNA, hph and gusA coding sequence was successfully transformed into protocorm-like bodies (PLBs) of Oncidium orchid, using Agrobacterium tumefaciens strain EHA105. A total of 17 independent transgenic orchid lines was obtained, out of which six transgenic lines (-glucuronidase (GUS) positive) were randomly selected and confirmed by Southern, northern and western blot analyses. A bioassay was conducted on the transgenic lines. Transgenic plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest that pflp may be an extremely useful gene for genetic engineering strategies in orchids to confer resistance against soft rot disease.     

<Emphasis Type="Italic">CaMi</Emphasis>, a root-knot nematode resistance gene from hot pepper (<Emphasis Type="Italic">Capsium annuum</Emphasis> L.) confers nematode resistance in tomato

Several root-knot nematode (Meloidogyne spp.) resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, none of them has yet been cloned. In this study, a candidate root-knot nematode resistance gene (designated as CaMi) was isolated from the resistant pepper line PR 205 by degenerate PCR amplification combined with the RACE technique. Expression profiling analysis revealed that this gene was highly expressed in roots, leaves, and flowers and expressed at a lower level in stems and was not detectable in fruits. To verify the function of CaMi, a sense vector containing the genomic DNA spanning the full coding region of CaMi was constructed and transferred into root-knot nematode susceptible tomato plants. Sixteen transgenic plants carrying one to five copies of T-DNA inserts were generated from two nematode susceptible tomato cultivars. RT-PCR analysis revealed that the expression levels of CaMi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some transgenic lines compared to untransformed susceptible plants, and that the resistance was inheritable. Ultrastructure analysis showed that nematodes led to the formation of galls or root knots in the susceptible lines while in the resistant transgenic plants, the CaMi gene triggered a hypersensitive response (HR) as well as many necrotic cells around nematodes. Rugang Chen and Hanxia Li are contributed equally to this work.     

Proteomic analysis of rice mutants susceptible to Magnaporthe oryzae

To identify genes involved in rice Pi5-mediated disease resistance to Magnaporthe oryzae, we compared the proteomes of the RIL260 rice strain carrying the Pi5 resistance gene with its susceptible mutants M5465 and M7023. Proteins were extracted from the leaf tissues of both RIL260 and the mutant lines at 0, 24, and 48 h after M. oryzae inoculation and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified eight proteins that were differently expressed between the resistant and susceptible plants (three down- and five up-regulated proteins in the mutants). The down-regulated proteins included a triosephosphate isomerase (spot no. 2210), a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (no. 3611), and an unknown protein (no. 4505). In addition, the five up-regulated proteins in the mutants were predicted to be a fructokinase I (no. 313), a glutathione S-transferase (no. 2310), an atpB of chloroplast ATP synthase (no. 3616), an aminopeptidase N (no. 3724), and an unknown protein (no. 308). These results suggest that proteomic analysis of rice susceptible mutants is a useful method for identifying novel proteins involved in resistance to the M. oryzae pathogen.     

The determination of physiological race of Fusarium oxysporum f. sp. lycopersici of tomato in Zhejiang, China

A group of differential tomato lines was used to identify the races of Fusarium oxysporum f. sp. lycopersici in Zhejiang, China. Marmande verte carries no resistant genes and Marporum carries gene I-1. Both lines Motelle and Mogeor have Gene I-1 and I-2. Tomato seedlings of eighteen days after sowing were inoculated with an isolate of Fusarium oxysporum f. sp. lycopersici, No. 98-2 and kept in a growth chamber. The seedlings were evaluated at fourteen days after inoculation. Results showed that Marmande verte and Marporum were severely infected by the pathogen and established as susceptible. Motelle and Mogeor were not infected and established as resistant. These results indicated that the isolate No. 98-2 represented the race 2 of Fusarium oxysporum f. sp. lycopersici and gene I-2 is necessary for obtaining resistance to this pathogen in the Zhejiang region.     

The resistance of lettuce (Lactuca sativa L.) toNasonovia ribisnigri: Bionomics ofN. ribisnigri on near isogenic lettuce lines

The intrinsic rate of increase (r_m-values), mean relative growth rates and mortality ofNasonovia ribisnigri (Mosley) (Homoptera, Aphididae) on different lines of lettuce (Lactuca sativa L.) were determined. Near isogenic resistant and susceptible lines, plus their ancestors, were used. Bionomics ofN. ribisnigri on the resistant lines (NrNr) with the dominantNasonovia resistance gene (Nr-gene) differed clearly from the susceptible lines (nrnr). Mortality was high, no larvae reached adulthood, and no reproduction nor honeydew production was seen on the resistant lines. Transfer of aphids to susceptible plants after a period of 2 days on the resistant lines showed no signs of intoxication of aphids. Apparently there is no feeding on the resistant lines. It is not clear whether the aphids can not reach the phloem or do not accept it on the resistant line.     

Genetic transfer of resistance to powdery mildew and of an associated biochemical marker from Aegilops ventricosa to hexaploid wheat

Summary Resistance to powdery mildew, caused by the fungus Erysiphe graminis f.sp. tritici, has been transferred from Aegilops ventricosa (genomes D^vM^v) to hexaploid wheat (Triticum aestivum, ABD). In two transfer lines, H-93-8 and H-93-35, the resistance gene was linked to a gene encoding protein U-1, whereas one line, H-93-33, was resistant but lacked the molecular marker, and another line, H-93-1, was susceptible but carried the gene for U-1, indicating that the original M^v chromosome from Ae. ventricosa, carrying the two genes, had undergone recombination with a wheat chromosome in the last two lines.